JOURNAL OF NEUROCHEMISTRY
| 2015 | 132 | 266–275
doi: 10.1111/jnc.12973
*Department of Pathophysiology, Medical College of Qingdao University, Qingdao, China †Department of Pharmacy, College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao, China
Abstract The current study investigated the effects of nesfatin-1 in the hypothalamic paraventricular nucleus (PVN) on gastric motility and the regulation of the lateral hypothalamic area (LHA). Using single unit recordings in the PVN, we show that nesfatin-1 inhibited the majority of the gastric distention (GD)-excitatory neurons and excited more than half of the GD-inhibitory (GD-I) neurons in the PVN, which were weakened by oxytocin receptor antagonist H4928. Gastric motility experiments showed that administration of nesfatin-1 in the PVN decreased gastric motility, which was also partly prevented by H4928. The nesfatin-1 concentration producing a half-maximal response (EC50) in the PVN was lower than the value in the dorsomedial hypothalamic nucleus, while nesfatin-1 in the reuniens thalamic nucleus had no effect on
gastric motility. Retrograde tracing and immunofluorescent staining showed that nucleobindin-2/nesfatin-1 and fluorogold double-labeled neurons were observed in the LHA. Electrical LHA stimulation changed the firing rate of GD-responsive neurons in the PVN. Pre-administration of an anti- nucleobindin-2/nesfatin-1 antibody in the PVN strengthened gastric motility and decreased the discharging of the GD-I neurons induced by electrical stimulation of the LHA. These results demonstrate that nesfatin-1 in the PVN could serve as an inhibitory factor to inhibit gastric motility, which might be regulated by the LHA. Keywords: gastric distension responsive neurons, gastric motility, lateral hypothalamic area, nesfatin-1, paraventricular nucleus. J. Neurochem. (2015) 132, 266–275.
Nesfatin-1 is a newly discovered anorectic peptide, 82-amino acid derived from the precursor DNA binding/EF-hand/ acidic protein/nucleobindin-2 (NUCB2) (Oh-I et al. 2006). This peptide is located in the paraventricular nucleus (PVN), arcuate nucleus (ARC), lateral hypothalamic area (LHA), supraoptic nucleus, raphe pallidus, Edinger–Westphal nucleus, nucleus tractus solitarius (NTS), stomach and pancreas (Oh-I et al. 2006; Brailoiu et al. 2007; Foo et al. 2008; Zhang et al. 2010). Many studies have demonstrated that intracerebroventricular (i.c.v.) nesfatin-1 injections reduce feeding and weight in rats. Moreover, food deprivation significantly decreased nesfatin-1 in the PVN of rats, and reintroducing food induced c-Fos expression in nesfatin-1containing neurons in the PVN (Oh-I et al. 2006; Kohno et al. 2008). These findings suggest that nesfatin-1 could be integral in the regulation of food intake. It also has been shown that nesfatin-1 may act via a leptin-independent melanocortin signaling pathway, a central oxytocin system,
or a corticotropin releasing hormone/thyrotropin releasing hormone-histamine system. Both a melanocortin-3/4 receptor antagonist (SHU9119) or an oxytocin receptor antagonist
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Received January 14, 2014; revised manuscript received August 19, 2014; accepted October 10, 2014. Address correspondence and reprint requests to Luo Xu or Fei-fei Guo, Department of Pathophysiology, Medical College of Qingdao University, Qingdao 266021, China. E-mails: [email protected] or [email protected] Abbreviations used: Anti-Nn-Ab, anti-NUCB2/nesfatin-1 antibody; ARC, arcuate nucleus; CRH, corticotropin releasing hormone; DMH, dorsomedial hypothalamic nucleus; ES, electrical stimulation; FG, fluorogold; GD, gastric distention; GD-E, GD-excitatory; GD-I, GDinhibitory; LHA, lateral hypothalamic area; NR, normal rabbit serum; NS, normal saline; NTS, nucleus tractus solitarius; NUCB2, nucleobindin-2; POMC, proopiomelanocortin; PVN, paraventricular nucleus; Re, reuniens thalamic nucleus; SON, supraoptic nucleus; SS, sham stimulation; SubI, subincertal nucleus; TRH, thyrotropin releasing hormone; VMH, ventromedial hypothalamic nucleus.
© 2014 International Society for Neurochemistry, J. Neurochem. (2015) 132, 266--275
Effects of nesfatin-1 in PVN
(H4928 or vasotocin) blocked the anorexigenic effect of nesfatin-1 (Oh-I et al. 2006; Maejima et al. 2009; Yosten and Samson 2010; Gotoh et al. 2013; Stengel and Tache 2013). Recently, several groups also reported that i.c.v. administration of nesfatin-1 delays stomach emptying and suppresses gastric motility in rodents (Stengel et al. 2009, 2010; Atsuchi et al. 2010; Goebel-Stengel et al. 2011). Previous studies focused on the function of nesfatin-1 injected into the third cerebral ventricle. However, nesfatin1’s role in nuclei, such as the PVN, ARC, ventromedial hypothalamic nucleus, dorsomedial hypothalamic nucleus (DMH), and LHA remains unclear. Additionally, there is still debate as to whether these nuclei cooperate to regulate the energy homeostasis. The aims of the present study were as follows: (i) to explore the effects of exogenous nesfatin-1 in the PVN on gastric distention (GD)-responsive neurons and gastric motility/emptying; (ii) to study the projections of nesfatin-1 neurons from the LHA to the PVN, and (iii) to investigate the roles of the LHA in the regulation of the firing activity of neurons and gastric motility in the PVN.
Materials and methods Animals Male Wistar rats (250–300 g, Qingdao Institute for Drug Control, Shandong, China) were housed in a temperature-controlled room (25 2°C) and exposed to light from 08:00 a.m. to 08:00 p.m. Rats were fed with laboratory chow pellets and tap water ad libitum. Protocols have been approved by the Qingdao University Animal Care and Use Committee (Animal protocol number: 0014819). Experimental design The study was composed of three categories of experimental techniques, including electrophysiological experiments (A), gastric motility/emptying experiments (B), and retrograde tracing combining with immunohistochemistry staining experiments (C). Experiment A was used to record discharging of PVN neurons in two parts: Part IA was to investigate the GD-responsive neurons and their firing activity under the influence of nesfatin-1 (n=50 rats); Part IIA was to examine the influence of nesfatin-1 on GD-responsive neurons following electric stimulation of the LHA (n=80 rats). Experiment B consisted of two parts. Part IB was to observe the effects of nesfatin-1 injected into PVN on gastric motility (n = 160) and gastric emptying (GE; n = 48). The rats were randomly divided into six groups: (i) normal saline groups [NS was administrated into the PVN, the DMH, or the reuniens thalamic nucleus (Re)]; (ii) nesfatin-1 in the PVN groups (0.0001, 0.001, 0.01, 0.1 nmol or 1.0 nmol nesfatin-1 was administrated into the PVN, respectively); (iii) 1.0 nmol H4928 group; (iv) 0.01 nmol nesfatin-1 + 1.0 nmol H4928 group; (v) nesfatin-1 in the DMH groups (0.01, 0.1, 1.0, 10.0 nmol or 100 nmol nesfatin-1 was administrated into the DMH, respectively; (vi) nesfatin-1 in the Re group (10.0 nmol nesfatin-1 was injected into Re). Part IIB was to investigate the impacts on gastric motility by electrical stimulation (ES) in the LHA (n=90). This experiment had six treatment groups (n=15 per group): (i) LHA
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sham stimulation group (SS); (ii) LHA ES group; (iii) normal rabbit serum (NR) + SS; (iv) NR + ES; (v) anti-NUCB2/nesfatin-1 antibody (Anti-Nn-Ab) + SS; (vi) Anti-Nn-Ab + ES. For experiment C (n=10), the NUCB2/nesfatin-1 and fluorogold (FG) double-labeled neurons in LHA were visualized by combining retrograde tracing and immunohistochemistry staining. Surgery and electrophysiological recordings After fasting for 18 hours, rats were anesthetized with thiobutabarbital (100 mg/kg, i.p.; Sigma-Aldrich Chemical, St Louis, MO, USA). Balloon implantation into the stomach and cranial surgeries were carried out as described previously (Gong et al. 2013). Following a midline laparotomy, the fundus wall was opened with a small incision, and warm isotonic saline was used to wash out the gastric contents. Attached to a polyethylene tubing (PE-240), a latex balloon at a length of 3–4 cm was inserted into the gastric cavity through the incision and fixed on the edge of the incision. The pylorus was ligatured to avoid changes in gastric volume by duodenal reflux. Gastric distension was produced by air inflation of the balloon with volumes of 3–5 mL routinely at a rate of 0.5 mL/s and maintained for 10–30 s. The abdomen was then closed. After cranial surgery, with the aid of a hydraulic micropositioner, a four-barrel glass microelectrode with resistance at 5–15 MO and total tip diameter at 3–10 lm was inserted in an increment of 10 lm to the area of the PVN (1.6–1.9 mm caudal to the bregma, 0.1– 0.7 mm lateral to the middle line and 7.7–8.4 mm below the surface of the skull) according to the rat atlas of Paxinos and Watson (2007). The four-barrel glass microelectrode was used for electrophysiological recording and micropressure injection. The recording glass microelectrode was filled with 0.5 M sodium acetate and 2% Pontamine sky blue. The other three barrels connected with a fourchannel pressure injector (PM2000B; Micro Data Instrument Inc., Plainfield, NJ, USA) contained either: nesfatin-1 (Phoenix Pharmaceuticals, Burlingame, CA, USA), H4928 (an antagonist of oxytocin receptor; Sigma-Aldrich Chemical), or NS. With short pulse gas pressure (1500 ms, 5.0–15.0 psi), the drugs were injected on the surface of firing cells (Trudel et al., 2002). The drug concentrations were selected based on their ability to modulate cell firing activity. The injection volumes of drugs to the firing cells during extracellular recording were less than 1 nl. After a firing pattern had become stable, the unit was tested with a GD stimulus to determine whether there was input from gastric mechanoreceptors. A neuron was identified as a GD-responsive neuron if its mean firing frequency changed via gastric distension by at least 20% from the mean basal firing level. The GD-responsive neurons were divided into GD-excitatory (GD-E) and GD-inhibitory (GD-I) subcategories according to whether the spontaneous discharge increased or decreased with GD, respectively. The change in firing rate of GD-responsive neurons was calculated by 1009 (firing rate of GD-responsive neurons after treatment- firing rate of GDresponsive neurons before treatment)/(firing rate of GD-responsive neurons before treatment). Implantation of brain cannula Rats, fasted for 18 h, were anesthetized with chloral hydrate (400 mg/kg, i.p.; Sigma-Aldrich Chemical) and placed in a stereotaxic frame (Narashige SN-3, Tokyo, Japan). A stainless steel guide cannula (24 gauge) was implanted into the PVN
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(administration site, position described as above) or the LHA (1.8– 3.2 mm caudal to the bregma, 1.5–2.5 mm lateral to the middle line and 7.5–9.0 mm below the surface of the skull), respectively. The injection cannula (29 gauge) was connected to a syringe by a 10 cm piece of polyethylene tubing. A monopolar electrode was put into the cannula placed focally in the LHA for ES when necessary. Implantation of force transducer and gastric motility measurement The process of implanting a force transducer was conducted as previously described (Guo et al. 2011). Rats were returned to their original individual enclosures for recovery over 2 days. Rats were fasted overnight and acclimatized for 30 min at the recording area prior to gastric motility measurements. During recording, the rats were allowed to move freely. Water was provided, but food was not. A polygraph (3066–23; Chengdu Precision Instruments, Sichuan, China) was used to record baseline gastric motility for 30 min. After that, the drug of each group was slowly injected to the PVN via the cranial cannula. The recording period took 1–2 h per day for at least 2 days for each animal. The effects of peptides were evaluated by changes in the percentage motor index (%MI) of the motor activity in the antrum. Values of the %MI for a 5-min period in the antrum were calculated by 1009 (area under the manometric trace for each 5-min period after nesfatin-1 or vehicle injection)/(area under the manometric trace for the 5-min period immediately before nesfatin-1 or vehicle injection). Gastric emptying GE of a non-nutrient viscous solution was determined by the phenol red method as described previously (Czimmer et al. 2006). The rats were fasted for 18 h before the GE test. In the dark phase, rats were injected with nesfatin-1, H4928 or vehicle (5 lL, n = 8 per group) in PVN. After injection, rats were gavage fed 1.5 mL of a nonnutrient viscous solution. Twenty min later, animals were killed by cervical dislocation. The gastric contents were collected to check the phenol red concentration and to calculate the GE rate. Electrical stimulation A monopolar stimulation electrode (RH NE-100 01 9 50 mm; David Kopf Instruments, Tujunga, CA, USA), insulated with epoxy to within 200 lm of the tip, was inserted into the LHA (position described as above). Stimulation was from a stimulator with a radiofrequency output of square-wave current impulses, 20 lA in intensity, and 0.5 ms in duration, delivered for 10 s at 50 Hz (Sato-Suzuki et al. 1998). Histological verification To check the recording position of the glass microelectrode at the end of each experiment, an iron deposit of Pontamine sky blue was formed at the recording site by a direct current (10 lA, 20 min) passing through the electrode. To check the target of drug administration in the PVN and stimulation electrode in the LHA, 0.5 lL of Pontamine sky blue was microinjected through the cannula after gastric motility recording. At the end of experiment, each rat brain was perfused and fixed. Frozen coronal sections were cut at a thickness of 50 µm from PVN or LHA, and the sections with incorrect locations were discarded. Approximately 7.7% of animals
had misplaced microelectrodes in the PVN for electrophysiological recording (such as in the Re) and 6.1% rats had misplaced cannulas in the PVN or the LHA for gastric motility experiment (such as in the Re and subincertal nucleus), which were excluded from analysis (Fig. 4d). Retrograde tracing and immunohistochemistry Ten rats were anesthetized with 10% chloral hydrate (3 mL/kg, i.p.). A single 0.2 lL 3% (w/v) FG (Sigma-Aldrich Chemical) was pressure injected into the PVN. After a 7-day recovery period, the rats were fixed by perfusion. The brain was removed, post-fixed for 4 h, and cryoprotected in 30% sucrose overnight. A series of 15-lm brain coronal sections were cut on a freezing microtome (Kryostat 1720, Leica, Germany). For the rats that receives the FG injection, tissue sections were incubated with a primary Anti-Nn-Ab (1 : 500, catalog #: H-00322, species: rats; Phoenix Pharmaceuticals) for 40 h at 4°C. After incubation with the Cy3-conjugated goat anti-rabbit IgG secondary antibody (1 : 1000, catalog #: 111-165-144; Jackson Immunoresearch, West Grove, PA, USA) for 2 h, the sections were mounted with Citifluor antifadent solutions (Citifluor, London, UK). All fluorophores were visualized and photographs were taken under a BX50 microscope and a DP50 digital camera (Olympus, Tokyo, Japan). An image analysis system (Jeda Science and Technology Company, Nanjing, China) was used to count the number of double-labeled cells on the 50 slides from the LHA of 10 rats. Double-labeled cells were counted from five fields spaced evenly throughout the extent of LHA. The percentage of double-labeled cells (%) = the numbers of double-labeled cells/the numbers of NUCB2/nesfatin-1 positive neurons 9 100%. Statistical analysis The results are presented as the mean SEM and processed with SPSS 17.0 statistics software (SPSS Inc, Chicago, IL, USA). Comparisons were made between groups by either Student’s t-test (two groups only) or a two-way analysis of variance with a post hoc Bonferroni test for comparison among means. Values of p < 0.05 were considered as statistically significant.
Results Effect of nesfatin-1 on the GD-responsive neurons in the PVN In the pre-experiment, we chose a series of different concentrations of nesfatin-1 including 0.05, 0.5, 1, 5, 10, 50, 100, and 150 nM to test the effects of nesfatin-1 on neuronal firing activity. We took 10 nM of nesfatin-1 which could produce half of the maximal response (EC50) as the dose for the experiment. We recorded single neuron discharges in the PVN to identify GD-responsive neurons when stomachs were distended with 3–5 mL water for 20 s. Once a neuron was identified as responsive to GD, 1 nL nesfatin-1 (10 nM) was ejected on the surface of the neuron to study the effect of nesfatin-1 on the firing rate.
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Effects of nesfatin-1 in PVN
About 140 out of 218 (64.2%) tested neurons in the PVN responded to GD. The major responsive neurons (86/140, 61.4%) were excited by GD (GD-E), and the neuronal discharging frequency markedly increased from 1.97 0.12 to 3.94 0.23 Hz (p < 0.01, Fig. 1a), which changed 113.9 11.8% (Fig. 1c). Nesfatin-1 significantly decreased the firing rate of more than half GD-E neurons (66.3%, 57/ 86) to 1.15 0.07 Hz (p < 0.01, Fig. 1a), with a change rate of 43.6 9.8% (Fig. 1c). The firing rate of 16 GD-E (18.6%) neurons was elevated and 13 GD-E (15.1%) neurons had no change. About 54 of 140 (38.6%) neurons were inhibited by GD (GD-I), exhibiting a lower firing rate from 1.74 0.10 to 0.96 0.06 Hz (p < 0.01, Fig. 1b), and the change rate was 49.3 6.2% (Fig. 1c). For the 54 GD-I neurons, nesfatin-1 remarkably increased 57.4% of GD-I (31/54) neurons discharging frequency to 3.04 0.18 Hz, with a change rate of 78.1 15.3% (p < 0.01) (Fig. 1c). Fourteen GD-I (25.9%) neurons were decreased and nine GD-I (16.7%) neurons had no change. The effect of nesfatin-1 on GD-responsive neurons was partly abolished by the pre-treatment with oxytocin receptor antagonist H4928, which was administrated 2 min before nesfatin-1 (GD-E neurons: 1.38 0.08 Hz, p < 0.05 vs. nesfatin-1; GD-I neurons: 2.49 0.15 Hz, p < 0.05 vs. nesfatin-1). The change in firing rate of GD-E and GD-I neurons after the H4928 and nesfatin-1 administration was significantly higher than that of nesfatin-1 treatment (p < 0.05). H4928 alone had no effect on the activity of the GD-responsive neurons. A control injection of 0.9% NaCl was applied to confirm the specificity of the responses to nesfatin-1 (Fig. 1a–c). Effect of nesfatin-1 administration into the PVN on gastric motility and emptying The gastric motility of fasted rats was significantly inhibited by PVN microinjection of different doses (0.0001 nmol ~ 1.0 nmol/0.5 lL) of nesfatin-1, the EC50 was 0.012 0.003. The gastric %MI decreased significantly during the 10–20 min period after the administration of nesfatin-1 [p < 0.05 ~ 0.01 vs. NS group, Fig. 2a (ii–iv); Fig. 2b]. The three doses of nesfatin-1 (0.001, 0.01 nmol, or 0.1 nmol) did reach maximum efficacy at 10 min after administration of nesfatin-1 with the %MI of 90.9 2.9% (p < 0.05 vs. NS group), 84.0 5.3% (p < 0.01 vs. NS group) or 78.6 8.1% (p < 0.01 vs. NS group; p < 0.05 vs. 0.001 nmol group). Fifteen min after the administration of nesfatin-1, three doses of nesfatin-1 (0.001, 0.01 nmol, or 0.1 nmol/0.5 lL) decreased %MI of gastric contraction, respectively to 96.7 1.46% (p < 0.05 vs. NS group), 87.0 4.3% (p < 0.01 vs. NS group; p < 0.05 vs. 0.001 nmol group), 80.0 7.3% (p < 0.01 vs. NS group; p < 0.01 vs. 0.001 nmol group), which was in a dose-dependent manner
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(Fig. 2b). The %MI of 0.5 lL mixture of 0.01 nmol nesfatin-1 and 1 nmol H4928 group was 96.2 1.7% (p < 0.05 vs. 0.01 nmol group, Fig. 2b). H4928 alone had no effect on the gastric motility. In order to observe whether nesfatin-1 selectively mediated gastric motility in the PVN, different doses of nesfatin-1 were injected into a PVN-adjacent nucleus, such as DMH or Re. Results showed that the EC50 of nesfatin-1 in the DMH for modulating the gastric motility was significantly higher than that in the PVN (1.87 0.52 vs. 0.012 0.003, p < 0.05). The administration of 10 nmol nesfatin-1 into the Re had no effect on gastric motility. Nesfatin-1 (0.001, 0.01 nmol, or 0.1 nmol/0.5 lL) also dose-dependently reduced the 20-min GE of a non-nutrient viscous solution to 62.5 14.9% (p < 0.05), 51.9 12.9% (p < 0.05) and 40.5 8.8% (p < 0.01), respectively. This was compared with 73.5 18.4% in the NS group, as measured during the dark phase after i.c.v. injection. The GE of 0.5 lL mixture of 0.01 nmol nesfatin-1 and 1 nmol H4928 group was 63.2 13.7% (p < 0.05 vs. 0.01 nmol nesfatin-1) (Fig. 3). The retro-tracing of nesfatin-1 neuron projection from the LHA to the PVN As a feeding-regulating center, the PVN receives many neural projections from other nuclei (Morton et al. 2006). The retro-tracing technique was used to research the neural connections between the PVN and the LHA. After injection of 0.2 lL 3% FG in the PVN, FG-labeled neurons were detected in the LHA on the 7th day (Fig. 4a), which indicated that some LHA neurons projected axons to the PVN. Then the same slices were stained with the Anti-NnAb (Fig. 4b). Interestingly, some nesfatin-1 immunoreactive neurons in the LHA were FG-labeled (Fig. 4c), which implies that some nesfatin-1 positive neurons in the LHA project their axons to the PVN. Cell counting showed there were about 28.13% nesfatin-1-positive neurons co-localized with FG-labeled cells in the LHA. Effect of ES of the LHA on the GD neurons in the PVN A total of 232 GD-responsive neurons of the PVN were identified as 144 GD-E and 88 GD-I. ES of the LHA activated 50 (50/144, 34.7%) PVN GD-E neurons with firing rates increased from 2.06 0.12 to 4.23 0.21 Hz (p < 0.01, Fig. 5a). ES of the LHA inhibited 28 (28/144, 19.4%) and had no effect on 66 (66/144, 45.8%) GD-E neurons. For GD-I neurons, ES in the LHA increased the firing rate of 32 (32/88, 36.4%) neurons of the PVN from 1.61 0.09 to 4.83 0.25 Hz, p < 0.01, Fig. 5b), inhibited 20 (20/88, 22.7%), and had no effect on 36 (36/88, 40.9%) GD-I neurons. For the GD-E neurons, the pre-treatment of the Anti-Nn-Ab in the PVN further promoted the increase in the firing rate caused by ES in the LHA (12/50, 24.0%; 4.91 0.25 Hz vs. 4.23 0.21 Hz, p < 0.05; Fig. 5a). In GD-I neurons, the
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(a)
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Fig. 1 Effect of nesfatin-1 on the firing rate of GD-responsive neurons in the PVN. (a) GD-E neurons were inhibited by nesfatin-1, (b) GD-I neurons were excited by nesfatin-1 in the PVN. (c) The change in firing rate (%) of GD-responsive neurons in the PVN induced by nesfatin-1. Pre-treatment with the oxytocin receptor antagonist, H4928, weakened the effect of nesfatin-1 on the GD-responsive neurons. NS or H4928 alone did not significantly change the firing rate of neurons in the PVN. **p < 0.01 versus NS and #p < 0.05 versus nesfatin1. Data are showed as mean SEM. GDE, gastric distension-excitatory; GD-I, gastric distension-inhibitory; NS, normal saline; PVN, paraventricular nucleus; Bar, 60 s.
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increase in the firing rate was partly blocked (6/32, 18.8%; 4.15 0.18 Hz vs. 4.83 0.25 Hz, p < 0.05; Fig. 5b). The change in the GD neurons firing rate in the PVN is shown in Fig. 5c. Injection of the Anti-Nn-Ab in the PVN alone did not significantly change the firing activity of the GD-responsive neurons (GD-E neurons: 2.14 0.15 Hz vs. 2.25 0.23 Hz; GD-I neurons: 2.06 0.19 Hz vs. 1.92 0.17 Hz, p > 0.05, Fig. 5a and b). Effect of LHA ES on gastric motility via the PVN Our results showed that ES of the LHA notably accelerated gastric motility with a latency of approximately 5 min, and the maximum %MI of ES (131.0 31.6% vs. SS group, p < 0.05), NR + ES (135.0 39.9% vs. NR + SS group, p < 0.05), or Anti-Nn-Ab + ES (154.2 46.3% vs. AntiNn-Ab + SS group, p < 0.05) group appeared at 15 min after stimulation (Fig. 6b). The results of Anti-Nn-Ab + ES group showed that the Anti-Nn-Ab significantly strengthened the effect of ES in the LHA on gastric motility compared to that of the NR + ES group at 20 min after stimulation (150.2 38.5% vs. 120.0 27.4%, p < 0.05). Additionally, there was no significant difference in the gastric motility among the SS, NR + SS, and Anti-Nn-Ab + SS groups (Fig. 6a and b).
Discussion Our study demonstrated that administration of nesfatin-1 into the PVN regulated the GD-responsive neurons in the PVN and reduced gastric motility, which were partly blocked by pre-treatment with the oxytocin receptor antagonist, H4928. This study also showed that ES of the LHA increased the firing activities of GD-responsive neurons in the PVN and promoted the gastric motility. Pre-treatment with the AntiNn-Ab in the PVN considerably increased the firing rate of a few GD-E neurons and decreased significantly the firing rate of GD-I neurons activated by electrically stimulating the LHA. The Anti-Nn-Ab significantly strengthened the effect of ES of the LHA on gastric motility. Moreover, NUCB2/ nesfatin-1 and FG double-labeled neurons were identified in the LHA, indicating that nesfatin-1 in the PVN could play a pivotal role in the central control of gastric motility and that the LHA may participate in the regulatory process. Since nesfatin-1 is a newly discovered anorexigenic gutbrain peptide, little is known about its function as a neurotransmitter in CNS. In the present study, different doses of nesfatin-1 influenced the firing rate of major neurons in the PVN in a bell-shaped dose–response relationship. According to the change in the PVN neurons firing rate,
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(a) (i) (ii) (iii) (iv) (v) (vi)
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Fig. 2 Effect of nesfatin-1 on the gastric motility in the PVN. (a) Recording curves of gastric motility: Gastric motility was dosedependently decreased by administration of nesfatin-1 into the PVN at the doses of 0.001, 0.01, 0.1 nmol (ii–iv). However, the inhibitory effects induced by nesfatin-1 were weakened by synchronous microinjection of 1 nmol H4928 (vi). Administration of H4928 alone did not significantly change the gastric motility in PVN (v). (b) The percentage gastric motor index (%MI): Ten min after injection of nesfatin-1 into the PVN, %MI was decreased significantly compared to that of the NS group. Nonetheless, the mixture solution of 0.01 nmol nesfatin-1 and 1 nmol H4928 in PVN induced a weaker and shorter change in %MI compared to the effect of 0.01 nmol nesfatin-1 alone (*p < 0.05, **p < 0.01 vs. NS group. Dp < 0.05, DDp < 0.01 vs. 0.001 nmol nesfatin-1 group and #p < 0.05 vs. 0.01 nmol nesfatin-1. n = 10 animals per group). Data are showed as mean SEM. NS, normal saline; PVN, paraventricular nucleus.
10 nM nesfatin-1 was selected in the electrophysiology experiments. The results indicated that most GD-responsive neurons in the PVN were reactive to the administration of nesfatin-1. PVN is a vital center for regulating appetite, food intake, and gastric functions (Morrow et al. 1994; Morton et al. 2006; Ataka et al. 2008; Chaleek et al. 2012). The anorexigenic nesfatin-1 was highly expressed in the feeding related hypothalamus nuclei, such as the PVN, the ARC, and the LHA. While the function of nesftin-1 in the PVN has been
Fig. 3 Effect of nesfatin-1 on gastric emptying (GE) in the PVN. Nesfatin-1 reduced the 20-min GE of a non-nutrient viscous solution dose-dependently during the dark phase after intracerebroventricular (i.c.v.) injection. The GE showed a significant increase in 0.5 lL mixture of 0.01 nmol nesfatin-1 and 1 nmol H4928 group than that in 0.01 nmol nesfatin-1 alone group (*p < 0.05, **p < 0.01 vs. NS group. #p < 0.05 vs. 0.01 nmol nesfatin-1. n = 8 animals per group). Data are showed as mean SEM. NS, normal saline; PVN, paraventricular nucleus.
studied by many, little work has focused on the effect of nesfatin-1 on the firing rate of PVN neurons. We observed that administration of nesfatin-1 into the PVN mainly inhibited the GD-E neurons and excited the GD-I neurons. Gastric distension delayed gastric motility (Bozkurt et al. 1999). Atsuchi et al. (2010) showed that i.c.v. administration of nesftin-1 inhibited gastric motility. Thus, it has been suggested nesfatin-1 may regulate gastrointestinal motility via GD-responsive neurons in the PVN. Based on the above hypothesis, gastric motility was investigated further. In our study, microinjections of nesfatin1 into the PVN dose-dependently reduced the %MI of the stomach. In addition, GE was decreased dramatically by nesfatin-1. Several experiments also had shown that i.c.v. injection of nesfatin-1 reduces stomach emptying in rodents (Stengel et al. 2009; Goebel-Stengel et al. 2011) and suppressed gastroduodenal motility in mice (Atsuchi et al. 2010). Our data suggest nesfatin-1 modulates the gastric function via changing the activity of the PVN neurons. Because of lack of the specific receptor of NUCB2/ nesfatin-1 in the rat, it is still not possible to completely block the nesfatin-1 pathway (Stengel et al. 2013). Until now, there were varying opinions about the anorectic neural pathways of nesfatin-1. Oh-I et al. (2006) showed that satiety by nesfatin-1 was abolished by an antagonist of melanocortin-3/4 receptor, SHU9119. Maejima et al. (2009) revealed that centrally administered nesfatin-1 activated nesfatin-1 neurons to stimulate oxytocin neurons in the PVN, driving oxytocinergic signaling to the NTS proopiomelanocortin neurons and causing melanocortin-dependent anorexia. Yosten and Samson (2010) reported that nesfatin-1 activated the central melanocortin system, which acts through the central oxytocin system to inhibit food and water intake. Taken together, oxytocin or melanocortin
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Fig. 4 The projection of nesfatin-1 neurons in the LHA to the PVN. (a) Distribution of FG in the LHA, (b) Expression of nesfatin-1 in the LHA, (c) Merge of A and B to indicate coexist of nesfatin-1 and FG. (d) The PVN, DMH, or Re map. Dots indicate the locations of misplaced microelectrodes of PVN, and triangles declare the misplaced cannulas for microinjection of PVN or LHA and for electrical stimulation of LHA. FG, fluorogold; LHA, lateral hypothalamic area; PVN, paraventricular nucleus; DMH, dorsomedial hypothalamic nucleus; Re, reuniens thalamic nucleus; Bars, 25 lm.
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Fig. 5 Effect of electrical stimulation (ES) of the LHA on the GD neurons in the PVN. ES of the LHA activated the GD-E (a) and GD-I (b) neurons in the PVN. Nonetheless, preadministration of an anti-nucleobindin-2 (NUCB2)/nesfatin-1 antibody into the PVN enhanced the effect of ES of the LHA on minor GD-E neurons (a), and reduced the effect of ES of the LHA on major GD-I neurons (b). The change in firing rate (%) of GD-responsive neurons in the PVN induced by ES of the LHA was shown in (c) (#p < 0.05 vs. ES group). Data are showed as mean SEM. GD-E, gastric distension-excitatory; GD-I, gastric distension-inhibitory; LHA, lateral hypothalamic area; PVN, paraventricular nucleus; Bar, 60 s.
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system are two proposed mechanisms of action of nesfatin-1. In this experiment, we used the oxytocin receptor antagonist, H4928, to block the effect of nesfatin-1 on GD-responsive
neurons and gastric motility via PVN administration. The data showed that H4928 reduced the discharges of GDresponsive neurons and gastric motility caused by nesfatin-1.
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(iii) (iv) (v)
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Fig. 6 Effect of electrical stimulation (ES) of the LHA on the gastric motility. (a) The recording curves of gastric motility: ES of the LHA could enhance gastric motility (ii and iv). The administration of antinucleobindin-2 (NUCB2)/nesfatin-1 antibody into the PVN elevated the excitatory effects of ES of LHA on gastric motility (vi). There was no change in gastric motility in SS, NR + SS, and Anti-Nn-Ab + SS groups (i, iii and v). (b) The percentage gastric motor index (%MI): After a latency of approximately 5 min, ES of the LHA notably accelerated gastric motility of ES or NR + ES group. Moreover, the administration of anti-NUCB2/nesfatin-1 antibody significantly strengthened the effect of ES of the LHA on gastric motility. However, there was no significant difference in the gastric motility among the SS, NR + SS, and Anti-NnAb + SS groups (*p < 0.05 vs. SS group. #p < 0.05 vs. NR + SS group. Dp < 0.05 vs. anti-Nn-Ab + SS group and †p < 0.05 vs. NR + ES group. n = 15 animals per group). Data are showed as mean SEM. Anti-Nn-Ab, Anti-NUCB2/nesfatin-1 antibody; ES, electrical stimulation; LHA, lateral hypothalamic area; NR, normal rabbit serum; PVN, paraventricular nucleus; SS, sham stimulation.
Further work is needed to tease out the action of nesfatin-1 through the oxytocin pathway or the nesfatin-1 receptor using oxytocin receptor antagonists. To explore if the effect of nesfatin-1 in the PVN on the gastric motility is different from that in the other nuclei,
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nesfatin-1 was injected into adjacent nuclei of PVN, such as Re and DMH. Re, as one of the largest midline thalamic nuclei, is located on the upper rear of the PVN. Receiving extensive limbic inputs, Re provides a bridge linking the hippocampus and the medial prefrontal cortex (McKenna and Vertes 2004). Re influences memory consolidation for spatial learning and generalization of fear conditioning (Eleore et al. 2011; Xu and S€ udhof 2013). DMH is located below the rear of the PVN and has long been associated with termination of motivated behaviors such as feeding and intracranial selfstimulation (Porrino et al. 1983). In these studies, we compared nesfatin-1 efficacy in the three nuclei. We showed a higher EC50 in the DMH and a invalidity in Re, suggesting that nesfatin-1 has a more potent effect in the PVN to regulate gastric motility. As an important integrated nucleus, the PVN receives afferent inputs from the other sites including the LHA, ARC, subfornical organ, organum vasculosum of the lamina terminalis, medial septum/diagonal band of broca, medial preoptic area, and suprachiasmatic nucleus (Larsen et al. 1994; Hermes et al. 2006). The LHA, consisting of several distinct nuclei, is considered as one of the most extensively interconnected areas of the hypothalamus (Berthoud and M€ unzberg 2011). The LHA can receive and consolidate a vast array of interoceptive and exteroceptive information and widely project to several areas containing the amygdala, hippocampal formation, arcuate, dorsomedial as well as the paraventricular (Ter Horst and Luiten 1987; Simerly 1995; Goto et al. 2005). It has been observed that microinjection of ghrelin into the LHA increased feeding and wakefulness (Szentirmai et al. 2007) as well as induced c-fos immunoreactivity in the LHA and in other regions involved in feeding control (Olszewski et al. 2003). Our previous data also showed that ghrelin administration into the LHA promoted gastric motility in a dose-dependent manner (Gong et al. 2013). All of these findings revealed that LHA may be another vital center for the mediation of gastric motility. Additionally, the neural connectivity between the PVN and the LHA has been demonstrated (Date et al. 1999). In the current study, the retrograde tracing and immunofluorescent trials showed NUCB2/nesfatin-1 and FG double-labeled neurons in the LHA after intra-PVN injection of the FG, implying the NUCB2/ nesfatin-1 neural projections from the LHA to the PVN. Subsequently, the function of projection from the LHA to the PVN was investigated with electrical physiological techniques. As expected, ES of the LHA considerably increased the discharges of GD-responsive neurons in the PVN and accelerated gastric motility. Nonetheless, pretreatment with the Anti-Nn-Ab in the PVN increased the discharges of some GD-E neurons but decreased the discharges of GD-I neurons induced by ES of the LHA. Additionally, pre-administration of the Anti-Nn-Ab to the PVN strengthened gastric motility induced by ES of the
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LHA. These results revealed that the LHA had a role in the regulation of PVN on gastric motility. As a corollary, the data described here suggest that nesfatin-1 is one of the inhibitory neurotransmitters involved in the mediation on the gastric motility via the LHA-PVN pathway. In conclusion, the study demonstrated that intra-PVN injection of nesfatin-1 could inhibit gastric motility, which was regulated by the nesfatin-1 projections from the LHA to the PVN. However, further investigations are warranted to confirm such a potentially important pathway for the regulation of gastric motility.
Acknowledgments and conflict of interest disclosure We particularly thank Dr. Yong-ming Yu, our dean of Medical College of Qingdao University, for his support and encouragement to complete this work. This work was supported by the National Natural Science Foundation of China (Nos 81470815, 30670780, 81100260, 81270460 and 1300281), Qingdao Municipal Science and Technology Commission (13-1-4-170-jch and 14-2-3-3-nsh) and Shandong Province Health Department (No. 2013WS0263). The authors declare that they have no conflicts of interest. All experiments were conducted in compliance with the ARRIVE guidelines.
Contributions Both authors Luo Xu and Fei-fei Guo were responsive for the conception, design, and revision of the article. Authors Feifei Guo, Sheng-li Gao, Xiang-rong Sun, and Zhi-ling Li acquired the data. Authors Yan-ling Gong and Zhi-ling Li undertook the statistical analysis and interpretation of data, and author Fei-fei Guo wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.
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| 2015 | 132 | 266–275
doi: 10.1111/jnc.12973
*Department of Pathophysiology, Medical College of Qingdao University, Qingdao, China †Department of Pharmacy, College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao, China
Abstract The current study investigated the effects of nesfatin-1 in the hypothalamic paraventricular nucleus (PVN) on gastric motility and the regulation of the lateral hypothalamic area (LHA). Using single unit recordings in the PVN, we show that nesfatin-1 inhibited the majority of the gastric distention (GD)-excitatory neurons and excited more than half of the GD-inhibitory (GD-I) neurons in the PVN, which were weakened by oxytocin receptor antagonist H4928. Gastric motility experiments showed that administration of nesfatin-1 in the PVN decreased gastric motility, which was also partly prevented by H4928. The nesfatin-1 concentration producing a half-maximal response (EC50) in the PVN was lower than the value in the dorsomedial hypothalamic nucleus, while nesfatin-1 in the reuniens thalamic nucleus had no effect on
gastric motility. Retrograde tracing and immunofluorescent staining showed that nucleobindin-2/nesfatin-1 and fluorogold double-labeled neurons were observed in the LHA. Electrical LHA stimulation changed the firing rate of GD-responsive neurons in the PVN. Pre-administration of an anti- nucleobindin-2/nesfatin-1 antibody in the PVN strengthened gastric motility and decreased the discharging of the GD-I neurons induced by electrical stimulation of the LHA. These results demonstrate that nesfatin-1 in the PVN could serve as an inhibitory factor to inhibit gastric motility, which might be regulated by the LHA. Keywords: gastric distension responsive neurons, gastric motility, lateral hypothalamic area, nesfatin-1, paraventricular nucleus. J. Neurochem. (2015) 132, 266–275.
Nesfatin-1 is a newly discovered anorectic peptide, 82-amino acid derived from the precursor DNA binding/EF-hand/ acidic protein/nucleobindin-2 (NUCB2) (Oh-I et al. 2006). This peptide is located in the paraventricular nucleus (PVN), arcuate nucleus (ARC), lateral hypothalamic area (LHA), supraoptic nucleus, raphe pallidus, Edinger–Westphal nucleus, nucleus tractus solitarius (NTS), stomach and pancreas (Oh-I et al. 2006; Brailoiu et al. 2007; Foo et al. 2008; Zhang et al. 2010). Many studies have demonstrated that intracerebroventricular (i.c.v.) nesfatin-1 injections reduce feeding and weight in rats. Moreover, food deprivation significantly decreased nesfatin-1 in the PVN of rats, and reintroducing food induced c-Fos expression in nesfatin-1containing neurons in the PVN (Oh-I et al. 2006; Kohno et al. 2008). These findings suggest that nesfatin-1 could be integral in the regulation of food intake. It also has been shown that nesfatin-1 may act via a leptin-independent melanocortin signaling pathway, a central oxytocin system,
or a corticotropin releasing hormone/thyrotropin releasing hormone-histamine system. Both a melanocortin-3/4 receptor antagonist (SHU9119) or an oxytocin receptor antagonist
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Received January 14, 2014; revised manuscript received August 19, 2014; accepted October 10, 2014. Address correspondence and reprint requests to Luo Xu or Fei-fei Guo, Department of Pathophysiology, Medical College of Qingdao University, Qingdao 266021, China. E-mails: [email protected] or [email protected] Abbreviations used: Anti-Nn-Ab, anti-NUCB2/nesfatin-1 antibody; ARC, arcuate nucleus; CRH, corticotropin releasing hormone; DMH, dorsomedial hypothalamic nucleus; ES, electrical stimulation; FG, fluorogold; GD, gastric distention; GD-E, GD-excitatory; GD-I, GDinhibitory; LHA, lateral hypothalamic area; NR, normal rabbit serum; NS, normal saline; NTS, nucleus tractus solitarius; NUCB2, nucleobindin-2; POMC, proopiomelanocortin; PVN, paraventricular nucleus; Re, reuniens thalamic nucleus; SON, supraoptic nucleus; SS, sham stimulation; SubI, subincertal nucleus; TRH, thyrotropin releasing hormone; VMH, ventromedial hypothalamic nucleus.
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(H4928 or vasotocin) blocked the anorexigenic effect of nesfatin-1 (Oh-I et al. 2006; Maejima et al. 2009; Yosten and Samson 2010; Gotoh et al. 2013; Stengel and Tache 2013). Recently, several groups also reported that i.c.v. administration of nesfatin-1 delays stomach emptying and suppresses gastric motility in rodents (Stengel et al. 2009, 2010; Atsuchi et al. 2010; Goebel-Stengel et al. 2011). Previous studies focused on the function of nesfatin-1 injected into the third cerebral ventricle. However, nesfatin1’s role in nuclei, such as the PVN, ARC, ventromedial hypothalamic nucleus, dorsomedial hypothalamic nucleus (DMH), and LHA remains unclear. Additionally, there is still debate as to whether these nuclei cooperate to regulate the energy homeostasis. The aims of the present study were as follows: (i) to explore the effects of exogenous nesfatin-1 in the PVN on gastric distention (GD)-responsive neurons and gastric motility/emptying; (ii) to study the projections of nesfatin-1 neurons from the LHA to the PVN, and (iii) to investigate the roles of the LHA in the regulation of the firing activity of neurons and gastric motility in the PVN.
Materials and methods Animals Male Wistar rats (250–300 g, Qingdao Institute for Drug Control, Shandong, China) were housed in a temperature-controlled room (25 2°C) and exposed to light from 08:00 a.m. to 08:00 p.m. Rats were fed with laboratory chow pellets and tap water ad libitum. Protocols have been approved by the Qingdao University Animal Care and Use Committee (Animal protocol number: 0014819). Experimental design The study was composed of three categories of experimental techniques, including electrophysiological experiments (A), gastric motility/emptying experiments (B), and retrograde tracing combining with immunohistochemistry staining experiments (C). Experiment A was used to record discharging of PVN neurons in two parts: Part IA was to investigate the GD-responsive neurons and their firing activity under the influence of nesfatin-1 (n=50 rats); Part IIA was to examine the influence of nesfatin-1 on GD-responsive neurons following electric stimulation of the LHA (n=80 rats). Experiment B consisted of two parts. Part IB was to observe the effects of nesfatin-1 injected into PVN on gastric motility (n = 160) and gastric emptying (GE; n = 48). The rats were randomly divided into six groups: (i) normal saline groups [NS was administrated into the PVN, the DMH, or the reuniens thalamic nucleus (Re)]; (ii) nesfatin-1 in the PVN groups (0.0001, 0.001, 0.01, 0.1 nmol or 1.0 nmol nesfatin-1 was administrated into the PVN, respectively); (iii) 1.0 nmol H4928 group; (iv) 0.01 nmol nesfatin-1 + 1.0 nmol H4928 group; (v) nesfatin-1 in the DMH groups (0.01, 0.1, 1.0, 10.0 nmol or 100 nmol nesfatin-1 was administrated into the DMH, respectively; (vi) nesfatin-1 in the Re group (10.0 nmol nesfatin-1 was injected into Re). Part IIB was to investigate the impacts on gastric motility by electrical stimulation (ES) in the LHA (n=90). This experiment had six treatment groups (n=15 per group): (i) LHA
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sham stimulation group (SS); (ii) LHA ES group; (iii) normal rabbit serum (NR) + SS; (iv) NR + ES; (v) anti-NUCB2/nesfatin-1 antibody (Anti-Nn-Ab) + SS; (vi) Anti-Nn-Ab + ES. For experiment C (n=10), the NUCB2/nesfatin-1 and fluorogold (FG) double-labeled neurons in LHA were visualized by combining retrograde tracing and immunohistochemistry staining. Surgery and electrophysiological recordings After fasting for 18 hours, rats were anesthetized with thiobutabarbital (100 mg/kg, i.p.; Sigma-Aldrich Chemical, St Louis, MO, USA). Balloon implantation into the stomach and cranial surgeries were carried out as described previously (Gong et al. 2013). Following a midline laparotomy, the fundus wall was opened with a small incision, and warm isotonic saline was used to wash out the gastric contents. Attached to a polyethylene tubing (PE-240), a latex balloon at a length of 3–4 cm was inserted into the gastric cavity through the incision and fixed on the edge of the incision. The pylorus was ligatured to avoid changes in gastric volume by duodenal reflux. Gastric distension was produced by air inflation of the balloon with volumes of 3–5 mL routinely at a rate of 0.5 mL/s and maintained for 10–30 s. The abdomen was then closed. After cranial surgery, with the aid of a hydraulic micropositioner, a four-barrel glass microelectrode with resistance at 5–15 MO and total tip diameter at 3–10 lm was inserted in an increment of 10 lm to the area of the PVN (1.6–1.9 mm caudal to the bregma, 0.1– 0.7 mm lateral to the middle line and 7.7–8.4 mm below the surface of the skull) according to the rat atlas of Paxinos and Watson (2007). The four-barrel glass microelectrode was used for electrophysiological recording and micropressure injection. The recording glass microelectrode was filled with 0.5 M sodium acetate and 2% Pontamine sky blue. The other three barrels connected with a fourchannel pressure injector (PM2000B; Micro Data Instrument Inc., Plainfield, NJ, USA) contained either: nesfatin-1 (Phoenix Pharmaceuticals, Burlingame, CA, USA), H4928 (an antagonist of oxytocin receptor; Sigma-Aldrich Chemical), or NS. With short pulse gas pressure (1500 ms, 5.0–15.0 psi), the drugs were injected on the surface of firing cells (Trudel et al., 2002). The drug concentrations were selected based on their ability to modulate cell firing activity. The injection volumes of drugs to the firing cells during extracellular recording were less than 1 nl. After a firing pattern had become stable, the unit was tested with a GD stimulus to determine whether there was input from gastric mechanoreceptors. A neuron was identified as a GD-responsive neuron if its mean firing frequency changed via gastric distension by at least 20% from the mean basal firing level. The GD-responsive neurons were divided into GD-excitatory (GD-E) and GD-inhibitory (GD-I) subcategories according to whether the spontaneous discharge increased or decreased with GD, respectively. The change in firing rate of GD-responsive neurons was calculated by 1009 (firing rate of GD-responsive neurons after treatment- firing rate of GDresponsive neurons before treatment)/(firing rate of GD-responsive neurons before treatment). Implantation of brain cannula Rats, fasted for 18 h, were anesthetized with chloral hydrate (400 mg/kg, i.p.; Sigma-Aldrich Chemical) and placed in a stereotaxic frame (Narashige SN-3, Tokyo, Japan). A stainless steel guide cannula (24 gauge) was implanted into the PVN
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(administration site, position described as above) or the LHA (1.8– 3.2 mm caudal to the bregma, 1.5–2.5 mm lateral to the middle line and 7.5–9.0 mm below the surface of the skull), respectively. The injection cannula (29 gauge) was connected to a syringe by a 10 cm piece of polyethylene tubing. A monopolar electrode was put into the cannula placed focally in the LHA for ES when necessary. Implantation of force transducer and gastric motility measurement The process of implanting a force transducer was conducted as previously described (Guo et al. 2011). Rats were returned to their original individual enclosures for recovery over 2 days. Rats were fasted overnight and acclimatized for 30 min at the recording area prior to gastric motility measurements. During recording, the rats were allowed to move freely. Water was provided, but food was not. A polygraph (3066–23; Chengdu Precision Instruments, Sichuan, China) was used to record baseline gastric motility for 30 min. After that, the drug of each group was slowly injected to the PVN via the cranial cannula. The recording period took 1–2 h per day for at least 2 days for each animal. The effects of peptides were evaluated by changes in the percentage motor index (%MI) of the motor activity in the antrum. Values of the %MI for a 5-min period in the antrum were calculated by 1009 (area under the manometric trace for each 5-min period after nesfatin-1 or vehicle injection)/(area under the manometric trace for the 5-min period immediately before nesfatin-1 or vehicle injection). Gastric emptying GE of a non-nutrient viscous solution was determined by the phenol red method as described previously (Czimmer et al. 2006). The rats were fasted for 18 h before the GE test. In the dark phase, rats were injected with nesfatin-1, H4928 or vehicle (5 lL, n = 8 per group) in PVN. After injection, rats were gavage fed 1.5 mL of a nonnutrient viscous solution. Twenty min later, animals were killed by cervical dislocation. The gastric contents were collected to check the phenol red concentration and to calculate the GE rate. Electrical stimulation A monopolar stimulation electrode (RH NE-100 01 9 50 mm; David Kopf Instruments, Tujunga, CA, USA), insulated with epoxy to within 200 lm of the tip, was inserted into the LHA (position described as above). Stimulation was from a stimulator with a radiofrequency output of square-wave current impulses, 20 lA in intensity, and 0.5 ms in duration, delivered for 10 s at 50 Hz (Sato-Suzuki et al. 1998). Histological verification To check the recording position of the glass microelectrode at the end of each experiment, an iron deposit of Pontamine sky blue was formed at the recording site by a direct current (10 lA, 20 min) passing through the electrode. To check the target of drug administration in the PVN and stimulation electrode in the LHA, 0.5 lL of Pontamine sky blue was microinjected through the cannula after gastric motility recording. At the end of experiment, each rat brain was perfused and fixed. Frozen coronal sections were cut at a thickness of 50 µm from PVN or LHA, and the sections with incorrect locations were discarded. Approximately 7.7% of animals
had misplaced microelectrodes in the PVN for electrophysiological recording (such as in the Re) and 6.1% rats had misplaced cannulas in the PVN or the LHA for gastric motility experiment (such as in the Re and subincertal nucleus), which were excluded from analysis (Fig. 4d). Retrograde tracing and immunohistochemistry Ten rats were anesthetized with 10% chloral hydrate (3 mL/kg, i.p.). A single 0.2 lL 3% (w/v) FG (Sigma-Aldrich Chemical) was pressure injected into the PVN. After a 7-day recovery period, the rats were fixed by perfusion. The brain was removed, post-fixed for 4 h, and cryoprotected in 30% sucrose overnight. A series of 15-lm brain coronal sections were cut on a freezing microtome (Kryostat 1720, Leica, Germany). For the rats that receives the FG injection, tissue sections were incubated with a primary Anti-Nn-Ab (1 : 500, catalog #: H-00322, species: rats; Phoenix Pharmaceuticals) for 40 h at 4°C. After incubation with the Cy3-conjugated goat anti-rabbit IgG secondary antibody (1 : 1000, catalog #: 111-165-144; Jackson Immunoresearch, West Grove, PA, USA) for 2 h, the sections were mounted with Citifluor antifadent solutions (Citifluor, London, UK). All fluorophores were visualized and photographs were taken under a BX50 microscope and a DP50 digital camera (Olympus, Tokyo, Japan). An image analysis system (Jeda Science and Technology Company, Nanjing, China) was used to count the number of double-labeled cells on the 50 slides from the LHA of 10 rats. Double-labeled cells were counted from five fields spaced evenly throughout the extent of LHA. The percentage of double-labeled cells (%) = the numbers of double-labeled cells/the numbers of NUCB2/nesfatin-1 positive neurons 9 100%. Statistical analysis The results are presented as the mean SEM and processed with SPSS 17.0 statistics software (SPSS Inc, Chicago, IL, USA). Comparisons were made between groups by either Student’s t-test (two groups only) or a two-way analysis of variance with a post hoc Bonferroni test for comparison among means. Values of p < 0.05 were considered as statistically significant.
Results Effect of nesfatin-1 on the GD-responsive neurons in the PVN In the pre-experiment, we chose a series of different concentrations of nesfatin-1 including 0.05, 0.5, 1, 5, 10, 50, 100, and 150 nM to test the effects of nesfatin-1 on neuronal firing activity. We took 10 nM of nesfatin-1 which could produce half of the maximal response (EC50) as the dose for the experiment. We recorded single neuron discharges in the PVN to identify GD-responsive neurons when stomachs were distended with 3–5 mL water for 20 s. Once a neuron was identified as responsive to GD, 1 nL nesfatin-1 (10 nM) was ejected on the surface of the neuron to study the effect of nesfatin-1 on the firing rate.
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About 140 out of 218 (64.2%) tested neurons in the PVN responded to GD. The major responsive neurons (86/140, 61.4%) were excited by GD (GD-E), and the neuronal discharging frequency markedly increased from 1.97 0.12 to 3.94 0.23 Hz (p < 0.01, Fig. 1a), which changed 113.9 11.8% (Fig. 1c). Nesfatin-1 significantly decreased the firing rate of more than half GD-E neurons (66.3%, 57/ 86) to 1.15 0.07 Hz (p < 0.01, Fig. 1a), with a change rate of 43.6 9.8% (Fig. 1c). The firing rate of 16 GD-E (18.6%) neurons was elevated and 13 GD-E (15.1%) neurons had no change. About 54 of 140 (38.6%) neurons were inhibited by GD (GD-I), exhibiting a lower firing rate from 1.74 0.10 to 0.96 0.06 Hz (p < 0.01, Fig. 1b), and the change rate was 49.3 6.2% (Fig. 1c). For the 54 GD-I neurons, nesfatin-1 remarkably increased 57.4% of GD-I (31/54) neurons discharging frequency to 3.04 0.18 Hz, with a change rate of 78.1 15.3% (p < 0.01) (Fig. 1c). Fourteen GD-I (25.9%) neurons were decreased and nine GD-I (16.7%) neurons had no change. The effect of nesfatin-1 on GD-responsive neurons was partly abolished by the pre-treatment with oxytocin receptor antagonist H4928, which was administrated 2 min before nesfatin-1 (GD-E neurons: 1.38 0.08 Hz, p < 0.05 vs. nesfatin-1; GD-I neurons: 2.49 0.15 Hz, p < 0.05 vs. nesfatin-1). The change in firing rate of GD-E and GD-I neurons after the H4928 and nesfatin-1 administration was significantly higher than that of nesfatin-1 treatment (p < 0.05). H4928 alone had no effect on the activity of the GD-responsive neurons. A control injection of 0.9% NaCl was applied to confirm the specificity of the responses to nesfatin-1 (Fig. 1a–c). Effect of nesfatin-1 administration into the PVN on gastric motility and emptying The gastric motility of fasted rats was significantly inhibited by PVN microinjection of different doses (0.0001 nmol ~ 1.0 nmol/0.5 lL) of nesfatin-1, the EC50 was 0.012 0.003. The gastric %MI decreased significantly during the 10–20 min period after the administration of nesfatin-1 [p < 0.05 ~ 0.01 vs. NS group, Fig. 2a (ii–iv); Fig. 2b]. The three doses of nesfatin-1 (0.001, 0.01 nmol, or 0.1 nmol) did reach maximum efficacy at 10 min after administration of nesfatin-1 with the %MI of 90.9 2.9% (p < 0.05 vs. NS group), 84.0 5.3% (p < 0.01 vs. NS group) or 78.6 8.1% (p < 0.01 vs. NS group; p < 0.05 vs. 0.001 nmol group). Fifteen min after the administration of nesfatin-1, three doses of nesfatin-1 (0.001, 0.01 nmol, or 0.1 nmol/0.5 lL) decreased %MI of gastric contraction, respectively to 96.7 1.46% (p < 0.05 vs. NS group), 87.0 4.3% (p < 0.01 vs. NS group; p < 0.05 vs. 0.001 nmol group), 80.0 7.3% (p < 0.01 vs. NS group; p < 0.01 vs. 0.001 nmol group), which was in a dose-dependent manner
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(Fig. 2b). The %MI of 0.5 lL mixture of 0.01 nmol nesfatin-1 and 1 nmol H4928 group was 96.2 1.7% (p < 0.05 vs. 0.01 nmol group, Fig. 2b). H4928 alone had no effect on the gastric motility. In order to observe whether nesfatin-1 selectively mediated gastric motility in the PVN, different doses of nesfatin-1 were injected into a PVN-adjacent nucleus, such as DMH or Re. Results showed that the EC50 of nesfatin-1 in the DMH for modulating the gastric motility was significantly higher than that in the PVN (1.87 0.52 vs. 0.012 0.003, p < 0.05). The administration of 10 nmol nesfatin-1 into the Re had no effect on gastric motility. Nesfatin-1 (0.001, 0.01 nmol, or 0.1 nmol/0.5 lL) also dose-dependently reduced the 20-min GE of a non-nutrient viscous solution to 62.5 14.9% (p < 0.05), 51.9 12.9% (p < 0.05) and 40.5 8.8% (p < 0.01), respectively. This was compared with 73.5 18.4% in the NS group, as measured during the dark phase after i.c.v. injection. The GE of 0.5 lL mixture of 0.01 nmol nesfatin-1 and 1 nmol H4928 group was 63.2 13.7% (p < 0.05 vs. 0.01 nmol nesfatin-1) (Fig. 3). The retro-tracing of nesfatin-1 neuron projection from the LHA to the PVN As a feeding-regulating center, the PVN receives many neural projections from other nuclei (Morton et al. 2006). The retro-tracing technique was used to research the neural connections between the PVN and the LHA. After injection of 0.2 lL 3% FG in the PVN, FG-labeled neurons were detected in the LHA on the 7th day (Fig. 4a), which indicated that some LHA neurons projected axons to the PVN. Then the same slices were stained with the Anti-NnAb (Fig. 4b). Interestingly, some nesfatin-1 immunoreactive neurons in the LHA were FG-labeled (Fig. 4c), which implies that some nesfatin-1 positive neurons in the LHA project their axons to the PVN. Cell counting showed there were about 28.13% nesfatin-1-positive neurons co-localized with FG-labeled cells in the LHA. Effect of ES of the LHA on the GD neurons in the PVN A total of 232 GD-responsive neurons of the PVN were identified as 144 GD-E and 88 GD-I. ES of the LHA activated 50 (50/144, 34.7%) PVN GD-E neurons with firing rates increased from 2.06 0.12 to 4.23 0.21 Hz (p < 0.01, Fig. 5a). ES of the LHA inhibited 28 (28/144, 19.4%) and had no effect on 66 (66/144, 45.8%) GD-E neurons. For GD-I neurons, ES in the LHA increased the firing rate of 32 (32/88, 36.4%) neurons of the PVN from 1.61 0.09 to 4.83 0.25 Hz, p < 0.01, Fig. 5b), inhibited 20 (20/88, 22.7%), and had no effect on 36 (36/88, 40.9%) GD-I neurons. For the GD-E neurons, the pre-treatment of the Anti-Nn-Ab in the PVN further promoted the increase in the firing rate caused by ES in the LHA (12/50, 24.0%; 4.91 0.25 Hz vs. 4.23 0.21 Hz, p < 0.05; Fig. 5a). In GD-I neurons, the
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Fig. 1 Effect of nesfatin-1 on the firing rate of GD-responsive neurons in the PVN. (a) GD-E neurons were inhibited by nesfatin-1, (b) GD-I neurons were excited by nesfatin-1 in the PVN. (c) The change in firing rate (%) of GD-responsive neurons in the PVN induced by nesfatin-1. Pre-treatment with the oxytocin receptor antagonist, H4928, weakened the effect of nesfatin-1 on the GD-responsive neurons. NS or H4928 alone did not significantly change the firing rate of neurons in the PVN. **p < 0.01 versus NS and #p < 0.05 versus nesfatin1. Data are showed as mean SEM. GDE, gastric distension-excitatory; GD-I, gastric distension-inhibitory; NS, normal saline; PVN, paraventricular nucleus; Bar, 60 s.
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increase in the firing rate was partly blocked (6/32, 18.8%; 4.15 0.18 Hz vs. 4.83 0.25 Hz, p < 0.05; Fig. 5b). The change in the GD neurons firing rate in the PVN is shown in Fig. 5c. Injection of the Anti-Nn-Ab in the PVN alone did not significantly change the firing activity of the GD-responsive neurons (GD-E neurons: 2.14 0.15 Hz vs. 2.25 0.23 Hz; GD-I neurons: 2.06 0.19 Hz vs. 1.92 0.17 Hz, p > 0.05, Fig. 5a and b). Effect of LHA ES on gastric motility via the PVN Our results showed that ES of the LHA notably accelerated gastric motility with a latency of approximately 5 min, and the maximum %MI of ES (131.0 31.6% vs. SS group, p < 0.05), NR + ES (135.0 39.9% vs. NR + SS group, p < 0.05), or Anti-Nn-Ab + ES (154.2 46.3% vs. AntiNn-Ab + SS group, p < 0.05) group appeared at 15 min after stimulation (Fig. 6b). The results of Anti-Nn-Ab + ES group showed that the Anti-Nn-Ab significantly strengthened the effect of ES in the LHA on gastric motility compared to that of the NR + ES group at 20 min after stimulation (150.2 38.5% vs. 120.0 27.4%, p < 0.05). Additionally, there was no significant difference in the gastric motility among the SS, NR + SS, and Anti-Nn-Ab + SS groups (Fig. 6a and b).
Discussion Our study demonstrated that administration of nesfatin-1 into the PVN regulated the GD-responsive neurons in the PVN and reduced gastric motility, which were partly blocked by pre-treatment with the oxytocin receptor antagonist, H4928. This study also showed that ES of the LHA increased the firing activities of GD-responsive neurons in the PVN and promoted the gastric motility. Pre-treatment with the AntiNn-Ab in the PVN considerably increased the firing rate of a few GD-E neurons and decreased significantly the firing rate of GD-I neurons activated by electrically stimulating the LHA. The Anti-Nn-Ab significantly strengthened the effect of ES of the LHA on gastric motility. Moreover, NUCB2/ nesfatin-1 and FG double-labeled neurons were identified in the LHA, indicating that nesfatin-1 in the PVN could play a pivotal role in the central control of gastric motility and that the LHA may participate in the regulatory process. Since nesfatin-1 is a newly discovered anorexigenic gutbrain peptide, little is known about its function as a neurotransmitter in CNS. In the present study, different doses of nesfatin-1 influenced the firing rate of major neurons in the PVN in a bell-shaped dose–response relationship. According to the change in the PVN neurons firing rate,
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Fig. 2 Effect of nesfatin-1 on the gastric motility in the PVN. (a) Recording curves of gastric motility: Gastric motility was dosedependently decreased by administration of nesfatin-1 into the PVN at the doses of 0.001, 0.01, 0.1 nmol (ii–iv). However, the inhibitory effects induced by nesfatin-1 were weakened by synchronous microinjection of 1 nmol H4928 (vi). Administration of H4928 alone did not significantly change the gastric motility in PVN (v). (b) The percentage gastric motor index (%MI): Ten min after injection of nesfatin-1 into the PVN, %MI was decreased significantly compared to that of the NS group. Nonetheless, the mixture solution of 0.01 nmol nesfatin-1 and 1 nmol H4928 in PVN induced a weaker and shorter change in %MI compared to the effect of 0.01 nmol nesfatin-1 alone (*p < 0.05, **p < 0.01 vs. NS group. Dp < 0.05, DDp < 0.01 vs. 0.001 nmol nesfatin-1 group and #p < 0.05 vs. 0.01 nmol nesfatin-1. n = 10 animals per group). Data are showed as mean SEM. NS, normal saline; PVN, paraventricular nucleus.
10 nM nesfatin-1 was selected in the electrophysiology experiments. The results indicated that most GD-responsive neurons in the PVN were reactive to the administration of nesfatin-1. PVN is a vital center for regulating appetite, food intake, and gastric functions (Morrow et al. 1994; Morton et al. 2006; Ataka et al. 2008; Chaleek et al. 2012). The anorexigenic nesfatin-1 was highly expressed in the feeding related hypothalamus nuclei, such as the PVN, the ARC, and the LHA. While the function of nesftin-1 in the PVN has been
Fig. 3 Effect of nesfatin-1 on gastric emptying (GE) in the PVN. Nesfatin-1 reduced the 20-min GE of a non-nutrient viscous solution dose-dependently during the dark phase after intracerebroventricular (i.c.v.) injection. The GE showed a significant increase in 0.5 lL mixture of 0.01 nmol nesfatin-1 and 1 nmol H4928 group than that in 0.01 nmol nesfatin-1 alone group (*p < 0.05, **p < 0.01 vs. NS group. #p < 0.05 vs. 0.01 nmol nesfatin-1. n = 8 animals per group). Data are showed as mean SEM. NS, normal saline; PVN, paraventricular nucleus.
studied by many, little work has focused on the effect of nesfatin-1 on the firing rate of PVN neurons. We observed that administration of nesfatin-1 into the PVN mainly inhibited the GD-E neurons and excited the GD-I neurons. Gastric distension delayed gastric motility (Bozkurt et al. 1999). Atsuchi et al. (2010) showed that i.c.v. administration of nesftin-1 inhibited gastric motility. Thus, it has been suggested nesfatin-1 may regulate gastrointestinal motility via GD-responsive neurons in the PVN. Based on the above hypothesis, gastric motility was investigated further. In our study, microinjections of nesfatin1 into the PVN dose-dependently reduced the %MI of the stomach. In addition, GE was decreased dramatically by nesfatin-1. Several experiments also had shown that i.c.v. injection of nesfatin-1 reduces stomach emptying in rodents (Stengel et al. 2009; Goebel-Stengel et al. 2011) and suppressed gastroduodenal motility in mice (Atsuchi et al. 2010). Our data suggest nesfatin-1 modulates the gastric function via changing the activity of the PVN neurons. Because of lack of the specific receptor of NUCB2/ nesfatin-1 in the rat, it is still not possible to completely block the nesfatin-1 pathway (Stengel et al. 2013). Until now, there were varying opinions about the anorectic neural pathways of nesfatin-1. Oh-I et al. (2006) showed that satiety by nesfatin-1 was abolished by an antagonist of melanocortin-3/4 receptor, SHU9119. Maejima et al. (2009) revealed that centrally administered nesfatin-1 activated nesfatin-1 neurons to stimulate oxytocin neurons in the PVN, driving oxytocinergic signaling to the NTS proopiomelanocortin neurons and causing melanocortin-dependent anorexia. Yosten and Samson (2010) reported that nesfatin-1 activated the central melanocortin system, which acts through the central oxytocin system to inhibit food and water intake. Taken together, oxytocin or melanocortin
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Fig. 4 The projection of nesfatin-1 neurons in the LHA to the PVN. (a) Distribution of FG in the LHA, (b) Expression of nesfatin-1 in the LHA, (c) Merge of A and B to indicate coexist of nesfatin-1 and FG. (d) The PVN, DMH, or Re map. Dots indicate the locations of misplaced microelectrodes of PVN, and triangles declare the misplaced cannulas for microinjection of PVN or LHA and for electrical stimulation of LHA. FG, fluorogold; LHA, lateral hypothalamic area; PVN, paraventricular nucleus; DMH, dorsomedial hypothalamic nucleus; Re, reuniens thalamic nucleus; Bars, 25 lm.
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Fig. 5 Effect of electrical stimulation (ES) of the LHA on the GD neurons in the PVN. ES of the LHA activated the GD-E (a) and GD-I (b) neurons in the PVN. Nonetheless, preadministration of an anti-nucleobindin-2 (NUCB2)/nesfatin-1 antibody into the PVN enhanced the effect of ES of the LHA on minor GD-E neurons (a), and reduced the effect of ES of the LHA on major GD-I neurons (b). The change in firing rate (%) of GD-responsive neurons in the PVN induced by ES of the LHA was shown in (c) (#p < 0.05 vs. ES group). Data are showed as mean SEM. GD-E, gastric distension-excitatory; GD-I, gastric distension-inhibitory; LHA, lateral hypothalamic area; PVN, paraventricular nucleus; Bar, 60 s.
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system are two proposed mechanisms of action of nesfatin-1. In this experiment, we used the oxytocin receptor antagonist, H4928, to block the effect of nesfatin-1 on GD-responsive
neurons and gastric motility via PVN administration. The data showed that H4928 reduced the discharges of GDresponsive neurons and gastric motility caused by nesfatin-1.
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Fig. 6 Effect of electrical stimulation (ES) of the LHA on the gastric motility. (a) The recording curves of gastric motility: ES of the LHA could enhance gastric motility (ii and iv). The administration of antinucleobindin-2 (NUCB2)/nesfatin-1 antibody into the PVN elevated the excitatory effects of ES of LHA on gastric motility (vi). There was no change in gastric motility in SS, NR + SS, and Anti-Nn-Ab + SS groups (i, iii and v). (b) The percentage gastric motor index (%MI): After a latency of approximately 5 min, ES of the LHA notably accelerated gastric motility of ES or NR + ES group. Moreover, the administration of anti-NUCB2/nesfatin-1 antibody significantly strengthened the effect of ES of the LHA on gastric motility. However, there was no significant difference in the gastric motility among the SS, NR + SS, and Anti-NnAb + SS groups (*p < 0.05 vs. SS group. #p < 0.05 vs. NR + SS group. Dp < 0.05 vs. anti-Nn-Ab + SS group and †p < 0.05 vs. NR + ES group. n = 15 animals per group). Data are showed as mean SEM. Anti-Nn-Ab, Anti-NUCB2/nesfatin-1 antibody; ES, electrical stimulation; LHA, lateral hypothalamic area; NR, normal rabbit serum; PVN, paraventricular nucleus; SS, sham stimulation.
Further work is needed to tease out the action of nesfatin-1 through the oxytocin pathway or the nesfatin-1 receptor using oxytocin receptor antagonists. To explore if the effect of nesfatin-1 in the PVN on the gastric motility is different from that in the other nuclei,
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nesfatin-1 was injected into adjacent nuclei of PVN, such as Re and DMH. Re, as one of the largest midline thalamic nuclei, is located on the upper rear of the PVN. Receiving extensive limbic inputs, Re provides a bridge linking the hippocampus and the medial prefrontal cortex (McKenna and Vertes 2004). Re influences memory consolidation for spatial learning and generalization of fear conditioning (Eleore et al. 2011; Xu and S€ udhof 2013). DMH is located below the rear of the PVN and has long been associated with termination of motivated behaviors such as feeding and intracranial selfstimulation (Porrino et al. 1983). In these studies, we compared nesfatin-1 efficacy in the three nuclei. We showed a higher EC50 in the DMH and a invalidity in Re, suggesting that nesfatin-1 has a more potent effect in the PVN to regulate gastric motility. As an important integrated nucleus, the PVN receives afferent inputs from the other sites including the LHA, ARC, subfornical organ, organum vasculosum of the lamina terminalis, medial septum/diagonal band of broca, medial preoptic area, and suprachiasmatic nucleus (Larsen et al. 1994; Hermes et al. 2006). The LHA, consisting of several distinct nuclei, is considered as one of the most extensively interconnected areas of the hypothalamus (Berthoud and M€ unzberg 2011). The LHA can receive and consolidate a vast array of interoceptive and exteroceptive information and widely project to several areas containing the amygdala, hippocampal formation, arcuate, dorsomedial as well as the paraventricular (Ter Horst and Luiten 1987; Simerly 1995; Goto et al. 2005). It has been observed that microinjection of ghrelin into the LHA increased feeding and wakefulness (Szentirmai et al. 2007) as well as induced c-fos immunoreactivity in the LHA and in other regions involved in feeding control (Olszewski et al. 2003). Our previous data also showed that ghrelin administration into the LHA promoted gastric motility in a dose-dependent manner (Gong et al. 2013). All of these findings revealed that LHA may be another vital center for the mediation of gastric motility. Additionally, the neural connectivity between the PVN and the LHA has been demonstrated (Date et al. 1999). In the current study, the retrograde tracing and immunofluorescent trials showed NUCB2/nesfatin-1 and FG double-labeled neurons in the LHA after intra-PVN injection of the FG, implying the NUCB2/ nesfatin-1 neural projections from the LHA to the PVN. Subsequently, the function of projection from the LHA to the PVN was investigated with electrical physiological techniques. As expected, ES of the LHA considerably increased the discharges of GD-responsive neurons in the PVN and accelerated gastric motility. Nonetheless, pretreatment with the Anti-Nn-Ab in the PVN increased the discharges of some GD-E neurons but decreased the discharges of GD-I neurons induced by ES of the LHA. Additionally, pre-administration of the Anti-Nn-Ab to the PVN strengthened gastric motility induced by ES of the
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LHA. These results revealed that the LHA had a role in the regulation of PVN on gastric motility. As a corollary, the data described here suggest that nesfatin-1 is one of the inhibitory neurotransmitters involved in the mediation on the gastric motility via the LHA-PVN pathway. In conclusion, the study demonstrated that intra-PVN injection of nesfatin-1 could inhibit gastric motility, which was regulated by the nesfatin-1 projections from the LHA to the PVN. However, further investigations are warranted to confirm such a potentially important pathway for the regulation of gastric motility.
Acknowledgments and conflict of interest disclosure We particularly thank Dr. Yong-ming Yu, our dean of Medical College of Qingdao University, for his support and encouragement to complete this work. This work was supported by the National Natural Science Foundation of China (Nos 81470815, 30670780, 81100260, 81270460 and 1300281), Qingdao Municipal Science and Technology Commission (13-1-4-170-jch and 14-2-3-3-nsh) and Shandong Province Health Department (No. 2013WS0263). The authors declare that they have no conflicts of interest. All experiments were conducted in compliance with the ARRIVE guidelines.
Contributions Both authors Luo Xu and Fei-fei Guo were responsive for the conception, design, and revision of the article. Authors Feifei Guo, Sheng-li Gao, Xiang-rong Sun, and Zhi-ling Li acquired the data. Authors Yan-ling Gong and Zhi-ling Li undertook the statistical analysis and interpretation of data, and author Fei-fei Guo wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.
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