c-kit pathway in diabetic rats.

Am. J. Chin. Med. 2013.41:1233-1249. Downloaded from www.worldscientific.com by 68.166.72.224 on 01/23/15. For personal use only.

The American Journal of Chinese Medicine, Vol. 41, No. 6, 1233–1249 © 2013 World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine DOI: 10.1142/S0192415X13500833

Electroacupuncture at ST36 Increases Contraction of the Gastric Antrum and Improves the SCF/c-kit Pathway in Diabetic Rats Yan Chen, Juanjuan Xu, Shi Liu and Xiaohua Hou Division of Gastroenterology, Union Hospital, Tongji Medical College Huazhong University of Science and Technology, Wuhan, China

Abstract: Electroacupuncture (EA) at ST36 is effective for improving gastric motility. However, the underlying mechanism remains poorly understood. The aim of this study was to investigate the effects of EA on gastric contraction and to determine whether interstitial cells of Cajal (ICCs) are involved. Rats were randomized into control, diabetic (DM), diabetic with sham EA (DMþSEA), diabetic with low frequency EA (DM þ LEA) and diabetic with high frequency EA (DMþHEA) groups. EA was performed everyday for four and eight weeks. Contractions in antrum strips were explored using the organ bath technique. Western blotting was employed to determine c-kit and transmembrane stem cell factor (M-SCF) expression in the gastric antrum, and levels of soluble stem cell factor (S-SCF) in serum were determined by enzyme-linked immunosorbent assay (ELISA). The distribution of ICCs was further assessed by immunohistochemistry. The results were as follows: (1) Contractions in the DM group were attenuated at four and eight weeks, but LEA and HEA restored the attenuated contraction. (2) ICCs were significantly decreased at eight weeks without alteration at four weeks in DM group, but were rescued in the LEA and HEA groups. (3) Whereas M-SCF and S-SCF in the DM group were slightly decreased at four weeks and were dramatically reduced at eight weeks, LEA and HEA markedly enhanced SCF at eight weeks. Collectively, the data suggest that in diabetic rats, LEA and HEA at ST36 could facilitate contraction of the gastric antrum, possibly by involving the SCF/c-kit pathway. Keywords: Electroacupuncture; Interstitial Cells of Cajal; Gastric Contraction; SCF/c-kit Pathway.

Correspondence to: Dr. Shi Liu, PhD, Division of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jie Fang Road, Wuhan 430022, Hubei, China. Tel: (þ86) 027-8572-6381, Fax: (þ86) 27-8572-6930, E-mail: [email protected]

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Introduction Patients with long-standing diabetes are often plagued by gastrointestinal motility disorders including early satiety, postprandial bloating, abdominal discomfort and recurrent vomiting. Nevertheless, the pathogenesis of gastrointestinal motility disorders is unclear and effective treatment is limited. In recent years, several studies have suggested that interstitial cells of Cajal (ICCs) play a critical physiological role in the coordination of gastric contractile activity and are an important aspect of gastric motility (Wang et al., 2005; Hirst and Edwards, 2006). In addition, losses of or deficiencies in ICCs have been observed in the stomach of diabetic animals and patients and are thought to contribute to the development of motility disorders (Ordög et al., 2000; Grover et al., 2012). Electroacupuncture (EA) is an advanced modification of traditional acupuncture and is widely accepted by Western countries for its reproducibility in clinical and research settings. Moreover, Zusanli (ST36) is a commonly-used acupoint to prevent and treat gastrointestinal disorders. Several studies have demonstrated that EA at ST36 can ameliorate abnormal gastric dysthymia, delay emptying and impair accommodation in patients with diabetes and animal models (Wang et al., 2008b; Yin et al., 2010). Recently, it has been documented that EA increased the expression of ICC in the colon of rats (Deng et al., 2011; Sun et al., 2011). Our recent studies consistently showed that EA at ST36 restored impaired ICCs in the colon of diabetic rats (Xu et al., 2012). However, the effect of EA on ICCs with regard to regulating gastric contraction has scarcely been investigated. It is likely that, c-kit proteins distributed on the surface of ICCs are activated via Kit receptor interactions with stem cell factor (SCF), which can exist both as a transmembrane protein (M-SCF) and a soluble protein (S-SCF). It is known that the SCF/c-kit signaling pathway is required for maintenance of ICC phenotype, survival, proliferation and differentiation (Torihashi et al., 1999; Tong et al., 2010). In addition, previous studies have documented that both SCF expression and c-kit expression are significantly reduced in diabetic animals (Horváth et al., 2006; Lin et al., 2010; Xu et al., 2012). Fortunately, several studies have shown that EA can increase the expression of SCF and c-kit in rat models (Wang et al., 2008b; Lu et al., 2013). Our recent study also provided evidence that EA at ST36 can regulate the SCF/c-kit pathway and restore expression of ICCs in the colon of diabetic rats (Xu et al., 2012). However, it is unclear how EA can ameliorate gastric contraction and whether the SCF/c-kit pathway is involved. The aim of this study was to evaluate the effects of EA at ST36 on gastric contractions, assess the effects of EA at ST36 on ICCs and determine whether the SCF/c-kit pathway is involved.

Materials and Methods Animal Subjects Adult male Sprague-Dawley rats (250–300 g) were obtained from the Experiment Animal Center of Tongji Medical College (Huazhong University of Science and Technology,


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Wuhan, China) and housed under standardized laboratory conditions (22 C, 12/12 h lightdark cycle). The rats were fed ad libitum with unrestricted access to water. After adaptation to the laboratory environment for one week, the animals were formally entered into the study and treated humanely. All experimental work was conducted strictly according to the ethical guidelines of the Animal Care and Use Committee of the university. Diabetic models were successfully established by a single intraperitoneal injection of streptozotocin (STZ, 60 mg/kg; Alexis Biochemical, San Diego, CA, USA) diluted in citrate buffer solution (pH 4.5; Sigma, St Louis, MO, USA). Age-matched rats treated with an equal volume of citrate buffer were used as normal controls. One week after the injection, blood concentration was measured using a drop of blood obtained through a tail vessel puncture. A diagnostic criterion for diabetes in rats was a blood glucose level that was elevated and sustained above 16.7 mmol/L. Blood glucose and body weight were also screened before the injection and in the 1st, 4th and 8th week after the injection. Experimental Protocols Rats were randomly placed into five equal groups (10 rats/group): the control, diabetic (DM), diabetic with sham EA (SEA, only acupuncture without electrical current), diabetic with low frequency EA (LEA, 10 Hz, 1–3 mA) and diabetic with high frequency EA groups (HEA, 100 Hz, 1–3 mA) groups. The electric current was determined by slight trembling of the hind limb. The EA parameters were selected according to preliminary experiments that suggested a stimulatory effect of gastric motility (Imai et al., 2008; Hu et al., 2013). Rats received the EA intervention between 8:00 and 8:30 AM every day for either four or eight weeks (Wang et al., 2008a) (Fig. 1). An electrical stimulator (G6805-2A; Shanghai Huayi Medical Instrument Factory, Shanghai, China) was used in the present study. Zusanli (ST36) is a bilateral point located 5 mm laterally, and below the anterior tubercle of the tibia in rats (Tang et al., 1997). Two acupuncture needles (13 mm in length, 0.3 mm in diameter; Suzhou Medical Appliance Factory, Jiangsu, China) were inserted 7 mm into the muscle layer at both ST36 acupoints. During the EA procedure, the rats were permitted to move freely in their own cages to eliminate the influence of restraining conditions. At the end of four and eight weeks, specimens of the gastric antrum were obtained for the mechanical contractility study, western blotting and immunohistochemistry. Contractile Activity of Gastric Longitudinal Muscle Strips Upon removal, the whole stomach was opened along the greater curvature and then cleaned with and immersed in Krebs solution with a gas saturation of 95% O2/5% CO2. The Krebs buffer (pH 7.4) comprised 118.3 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM K2HPO4, 2.5 mM CaCl2, 25 mM NaHCO3, and 11.1 mM D-glucose. After the mucosal layer was gently removed, longitudinal muscle strips measuring 7 mm by 2 mm were obtained from the distal stomach and were suspended between two L-shaped hooks in a 25 mL organ bath filled with Krebs buffer maintained at 37 C and continuously bubbled


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Figure 1. Schematic representation of the EA study protocol. After STZ injection, the diabetic rats were divided into six large groups depending on the frequency of EA: the SEA, LEA and HEA groups for four or eight weeks. EA was administered for 30 min day 1 , seven days week 1 during the whole course of the study. Abbreviations: EA: electroacupuncture; SEA, sham EA group; LEA, low-frequency EA group; HEA, high-frequency EA group; STZ, streptozotocin.

with 95% O2/5% CO2. The tension of muscle strips was monitored using isometric force transducers (Fort-10, WPI, Sarasota, FL, USA), which were linked to a chart recorder (MP-100 system) via an amplifier to record variations in isometric tension. The data were digitized and analyzed using the digital recording software Acknowledge 3.7.1 (Biopac Systems, Santa Barbara, CA). An optimal tension of 1 g was applied, and the tissue was prepared during a 60-min equilibration period. Concentrations from 10 7 to 10 3 mol/L of acetylcholine (ACH; Sigma, St. Louis, MO) were administered to activate the contractile response to a new plateau. Each concentration was maintained for a period of 5 min to ensure sufficient time for equilibration. Subsequently, the methylene blue and the illumination method was adopted to evaluate contractions of the strips without ICCs (Liu et al., 1994). To remove ICCs, the strips were maintained in 37 C Krebs buffer containing 50 mmol/L methylene blue and bubbled with 95% O2/5% CO2 away from light for 40 min and then immediately exposed to visible light (50 mW/cm) for 5 min. Equilibration was performed again, and the experiments were repeated using different ACH concentrations. Cumulative concentration-response curves were obtained. Area under the curve (AUC) analysis was employed to determine the contractile activity of longitudinal muscle strips. Contractile responses to ACH were calculated as the relative change in AUCACH at each concentration from a baseline spontaneous contraction and were expressed as “%Variation”, which reflected the effects of different interventions on


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each group. %Variation was calculated according to the following formula: %Variation ¼ ðAUCACH AUCbaseline Þ=AUCbaseline 100%: A comparative evaluation of AUCACH before and after removal of ICCs at the same concentration was performed, and the results were expressed as “%Decrease”, indicating the role of ICCs in the contraction. %Decrease was calculated according to the following formula:


%Decrease ¼ %Variationbefore ICC removed %Variationafter ICC removed :

Western Blotting Frozen antrum specimens were crushed and homogenized in RIPA Buffer (Upstate, Temecula, CA, USA) with protease inhibitor (Sigma Chemical Co., St Louis, MO, USA) and then incubated on ice for 30 min. The liquid part was centrifuged at 12,000 g for 10 min at 4 C, and the supernatants were considered the total protein. The protein concentration of each specimen was analyzed using the BCA reagent (Pierce, Rockford, IL, USA). Total proteins were gradually separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to NC membranes (Millipore, Bedford, MA, USA). These membranes were blocked in 5% skim milk for 60 min at room temperature and later reacted with anti-c-kit (1:200; Santa Cruz Biotechnology, Inc.), anti-SCF (1:400; Abcam, Cambridge, UK) and rabbit anti-mouse β-actin (1:400; Santa Cruz Biotechnology, Inc) primary antibodies overnight at 4 C. After the membranes were incubated with HRPlinked secondary antibody for 60 min at room temperature, the immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, and Piscataway, NJ). To quantify the expression of c-kit and M-SCF, the blotting images were analyzed using a densitometer. Immunohistochemistry The gastric antrum specimens were immediately fixed in 4% paraformaldehyde for 24 h at room temperature, dehydrated, paraffin embedded and cut at a thickness of 5–7 m. Paraffin sections underwent a process of de-paraffinization in xylene and hydration in a graded solution of ethanol. Endogenous peroxidase activity was inhibited using 3% hydrogen peroxide (H2O2) for 30 min and microwaving (750 W) for 5 min, and then the non-specific reaction was blocked by normal rabbit serum for 30 min. The primary antibody c-kit (1:150; Santa Cruz Biotechnology, Inc) was added to the sections in a moist chamber overnight at 4 C. After being rinsed 3 times with phosphate-buffered saline (PBS) at pH 7.4, the sections were incubated with secondary antibodies (rabbit anti-goat IgG; 1:200) for 60 min at 37 C. After being washed again with PBS and incubated with horseradish peroxidase-linked (HRP-linked) streptavidin for 30 min, the target protein was envisaged by incubating with a freshly prepared 3, 3-diaminobenzidine (DAB) solution. After being washed again with PBS and counterstained and dehydrated, the slides were mounted.

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A negative control (absence of a primary antibody) was used to confirm the specificity of the antibody. Two observers using an Olympus FV500 evaluated the immunohistochemistry results (Olympus, Tokyo, Japan).


Enzyme-Linked Immunosorbent Assay (ELISA) To study alterations in serum S-SCF levels, blood samples of approximately 1 to 2 ml were obtained from each animal. The serum concentration of S-SCF was determined using an enzyme-linked immunosorbent assay kit (CSB-E04720r; Cusabio Biotech Co, Ltd.). A standard curve was calculated for S-SCF by measuring the standard with known concentrations using a spectrophotometer at a wavelength of 450 2 nm, and the concentration of S-SCF in each sample was calculated by comparing the optical density (OD) of the samples with the standard samples. Statistical Analysis All values are presented as the mean S.E.M. One-way ANOVA was employed to evaluate the differences among multiple groups. The ACH-activated contractility of muscle strips was analyzed by applying two-way ANOVA with a Bonferroni post-hoc test. Pearson correlations and linear regressions were performed to determine the relationship between c-kit protein expression and SCF. p < 0:05 was adopted as the threshold of statistical significance. Statistical analysis was performed using SPSS 17.0 (SPSS Inc, Chicago, IL). Results Blood Glucose Levels and Body Weight Two rats in the DM group and one in the SEA group died by accident at eight weeks. The other STZ-induced diabetic rats successfully completed entire experiment. No significant differences were observed between the groups with respect to baseline blood glucose level (Fig. 2A). After the diabetic models were successfully induced, the blood glucose levels of the DM group was markedly higher than the levels of the control group at all times (all p ¼ 0:000). However, in response to the EA intervention, there were no significant changes in the SEA, LEA and HEA groups compared with the DM group (all p > 0:05). As shown in Fig. 2B, there was also no significant difference in the baseline body weight among the groups. In contrast to the normal controls, the body weight of the DM group was significantly decreased at the end of one, four and eight weeks ( p ¼ 0:009, 0.000 and 0.000, respectively). In the SEA group, the body weight was not altered remarkably compared with the DM group (all p > 0:05). Compared with the DM group, the body weight of the LEA group was significantly increased at eight weeks ( p ¼ 0:003), whereas the body weight of the HEA group was obviously improved at four and eight weeks ( p ¼ 0:003 and 0.000, respectively).



(A)

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(B)

Figure 2. Blood glucose level and body weight were monitored during the experiment. (A) The blood glucose levels of the diabetic rats were significantly increased compared with normal rats at all time points (all p ¼ 0:000). (B) Compared with the DM group, the LEA group gained weight at the end of eight weeks ( p ¼ 0:003). The body weight of the HEA group was increased compared with that of the DM group at both four and eight weeks (p ¼ 0:003, 0.000). *p < 0:05 compared with the DM group.

ACH-Activated Concentration-Dependent Contractions We hypothesized that the concentration-dependent contraction of gastric antrum longitudinal muscle strips was elicited by ACH (Fig. 3). When the concentration of ACH was 10 3 mol L 1 , the muscle strip achieved sufficient contractions in each group. At four weeks, %Variation before the removal of ICCs was significantly lower in the DM group than in the control group (73:21 11:56 and 123:02 5:66, respectively; p ¼ 0:035). However, %Variation was higher in the LEA and HEA groups when compared with the DM group (p ¼ 0:008 and p ¼ 0:021, respectively). At the same time, %Decrease was markedly reduced in the DM group compared with the control group (48:94 13:06 and 104:67 15:68, respectively; p ¼ 0:025). Similarly, %Decrease was also elevated in the LEA and HEA groups compared to the DM group (p ¼ 0:002 and p ¼ 0:053, respectively). No significant differences were noted in either %Variation or %Decrease between the SEA and DM groups (both p > 0:05). At eight weeks, %Variation before removal of ICCs was significantly decreased in the DM group compared with the control group (59:16 12:96 and 161:12 27:69, respectively; p ¼ 0:003). However, %Variation was elevated in the LEA and HEA groups compared with the DM group (p ¼ 0:005 and p ¼ 0:037, respectively). Moreover, %Decrease in the DM group was significantly smaller than that of the control group (48:05 13:35 and 138:42 30:58, respectively; p ¼ 0:009). However, %Decrease was obviously increased in the LEA and HEA groups when compared with the DM group ( p ¼ 0:01 and p ¼ 0:045). There was no statistical significance between the SEA and DM groups in either %Variation or %Decrease (both p > 0:05).


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(A)

(C)

(E)

(B)

(D)

(F)

Figure 3. ACH activated the concentration response of gastric antrum longitudinal muscle strips. (A)–(C) At four weeks, when the concentration of ACH was 10 3 mol L 1 , both %Variation and %Decrease were obviously lower in the DM group than in the normal control group (p < 0:05), but they were significantly elevated in the LEA and HEA groups compared with the DM group. (D)–(F) At eight weeks, when the ACH level was 10 3 mol L 1 , %Variation and %Decrease were decreased in the DM group compared with the normal control group (p < 0:05). Compared with the DM group, %Variation and %Decrease were significantly increased in the LEA and HEA groups. *p < 0:05 compared with the DM group.


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Western Blot Analysis of C-Kit and M-SCF As shown in Fig. 4, the expression of c-kit in the gastric antrum of the DM group was not obviously altered compared with the control group at four weeks (0:67 0:09 and 0:83 0:04, respectively; p ¼ 0:061), but it was significantly lowered at eight weeks (0:16 0:02 and 0:79 0:05, respectively; p ¼ 0:000). Compared with the DM group, the expression of c-kit at eight weeks was dramatically elevated in the LEA and HEA groups (both p ¼ 0:000). However, there was no significance between the SEA and DM groups at the two time points (p ¼ 0:764 and p ¼ 0:538, respectively). Compared with the control group, the expression of M-SCF in the DM group was slightly decreased at four weeks (0:66 0:03 and 0:79 0:03, respectively; p ¼ 0:04), but dramatically reduced at eight weeks (0:25 0:05 and 0:74 0:05, respectively; p ¼ 0:000).

(A)

(B)

(C)

(D) Figure 4. The expression of c-kit and M-SCF protein in the gastric antrum. At four weeks, c-kit protein expressed in the DM group was not significantly altered compared with that in the normal control group (A) and (B). At eight weeks, the expression of c-kit was significantly decreased in the DM group but markedly increased in the LEA and HEA groups (D) and (E). The expression of SCF in the DM group was slightly decreased at four weeks (C) and dramatically reduced at eight weeks (F). However, LEA and HEA markedly increased the expression of SCF at eight weeks. *p < 0:05 compared with the DM group.

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(E)

(F) Figure 4. (Continued )

At eight weeks, LEA and HEA markedly increased the expression of M-SCF compared to the DM group (p ¼ 0:001 and p ¼ 0:006, respectively), and the effect of LEA was more remarkable than that of HEA (p ¼ 0:008). At four and eight weeks, no significant difference was noted concerning M-SCF expression between the SEA and DM groups (p ¼ 0:647 and p ¼ 0:252, respectively). Immunohistochemical Staining of C-Kit As shown in Fig. 5, the distribution of ICCs in the gastric antrum was revealed by c-kit immunoreactivity in the different groups. At four weeks, large quantities of c-kit þ cells

Figure 5. Immunohistochemical staining of c-kit in the gastric antrum. At four and eight weeks, abundant c-kit þ cells in the normal control group were distributed in both the muscular layer and intermuscular layer (A) and (F). Compared with the normal control group, c-kit þ cells in the DM group were not significantly changed at four weeks (B) but were obviously decreased at eight weeks (G). A dramatic reduction in ICCs in the SEA group was observed at eight weeks (H). LEA and HEA increased the number of c-kit þ cells compared with the DM group at eight weeks (E) and (J). (Original magnification: 200).


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Table 1. Levels of S-SCF in Each Group S-SCF (pg/ml) 4 weeks 8 weeks

Control

DM

DMþSEA

DMþLEA

DMþHEA

24.47 1.41 22.80 1.18

17.79 2.01 # 13.61 0.95 #

20.20 1.82 15.15 1.03

21.69 1.39 27.86 1.83*

21.68 1.34 24.55 1.88*


Note: Values were expressed as mean S.E.M. # p < 0:05 comparation between Control and DM group, *p < 0:05 compared with DM group.

were observed in the muscular and intramuscular layers of non-diabetic rats. In the DM group, abundant c-kit þ cells were also distributed in the two layers. Compared with the DM group, no significant alteration was observed in the SEA, LEA and HEA groups. At eight weeks, c-kit þ cells in the gastric antrum of the DM group were very sparse in comparison with the control group. Similarly, those cells were rarely observed in the SEA group. However, following LEA and HEA treatment, c-kit expression was markedly enhanced in the gastric antrum of the DM group. S-SCF in Sera by ELISA As shown in Table 1, ELISA detected levels of S-SCF in serum during the experiment in each group. At four weeks, the average level of S-SCF in the DM group was significantly decreased compared with that in the control group (p ¼ 0:005). However, no significant differences were noted among the DM group and the three EA-treated groups (all p > 0:05). At eight weeks, the S-SCF level in the sera of the DM group was also obviously attenuated in comparison with that of the control group (p ¼ 0:000). There were no significant differences between the SEA and DM groups (p ¼ 0:946). However, LEA and HEA significantly elevated the serum level of S-SCF in the DM group ( p ¼ 0:000 and, p ¼ 0:002, respectively). Correlation and Regression Analysis As described above, S-SCF, M-SCF and c-kit varied in a similar fashion. To investigate the relationship between c-kit and SCF expression, Pearson correlation and linear regression analyses were performed. At four weeks, Pearson correlations among S-SCF, M-SCF and c-kit were calculated: S-SCF, r ¼ 0:327 (p ¼ 0:021); M-SCF, r ¼ 0:915 (p ¼ 0:000). Furthermore, in the linear regression analysis, S-SCF and M-SCF were entered as dependent variables (R 2 ¼ 0:884), and the results suggested that there was a significant and high positive correlation between M-SCF expression and c-kit expression ( p ¼ 0:000). A further stepwise regression analysis indicated that c-kit protein expression was only potentially determined by M-SCF expression (R 2 ¼ 0:838; p ¼ 0:000); the regression equation was Y ¼ 0:204 þ 1:303X (Fig. 6A). At eight weeks, the correlations among S-SCF, M-SCF and c-kit expression were as follows: S-SCF, r ¼ 0:281 ( p ¼ 0:048); M-SCF, r ¼ 0:809 (p ¼ 0:000). In the linear regression analysis, S-SCF and M-SCF were entered as dependent variables (R 2 ¼ 0:925), and a significant correlation was also found between M-SCF


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(A)

(B) Figure 6. Correlations between M-SCF expression and c-kit protein expression. At both four (A) and eight (B) weeks, a significant correlation between M-SCF and c-kit was observed (four weeks: R 2 ¼ 0:838, p ¼ 0:000; eight weeks: R 2 ¼ 0:892, p ¼ 0:000). Each point represents the value from an individual rat (four weeks: N ¼ 50; eight weeks: N ¼ 47).


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and c-kit ( p ¼ 0:000). Next, to exclude the effect of S-SCF, stepwise linear regression analysis was performed with M-SCF as a dependent variable (R 2 ¼ 0:892). Similarly, the outcome demonstrated that M-SCF expression was significantly correlated with c-kit protein expression (p ¼ 0:000), and the regression equation was Y ¼ 0:071 þ 1:234X (Fig. 6B).


Discussion As indicated previously, the present study demonstrated that contractions of the gastric antrum of diabetic rats were affected at four weeks and more seriously at eight weeks. A unique feature of low- and high-frequency EA at ST36 is that it improves contractions of the gastric antrum of diabetic rats; this effect of EA at ST36 was partly related to an enhanced SCF/c-kit pathway. A literature review on the use of EA to control blood glucose in diabetes suggested that blood glucose levels are lowered both in type 1 and type 2 diabetic rats in response to EA (15 Hz and 10 mA) at ST36 (Peplow and Baxter, 2012). However, blood glucose was mostly unchanged following EA at 20 Hz in type 1 diabetic rats. It was also unchanged at 15 Hz with 1–3 mA in type 2 diabetic rats (Tominaga et al., 2011; Peplow and Baxter, 2012). Recently, in high-fat and high-caloric diet-induced obese rats, body weight was not significantly changed following 10-Hz EA (2 mA) for 12 weeks (Gong et al., 2012). It is reported that high-frequency EA, at 100 Hz, can increase body weight in morphinedependent rats (Wu et al., 1999). Therefore, it is speculated that the different effects of EA may be dependent on different parameters and animal models. Similarly, in the present study, although different EA frequencies (10/100 Hz, 1–3 mA) were evaluated, the blood glucose level in STZ-induced diabetic rats was not significantly altered. By contrast, following treatment with low- or high-frequency EA, diabetic rats gradually increased in weight particularly high-frequency EA. Collectively, the results showed that low- or highfrequency EA can increase the body weight of diabetic rats but has no effect on reduction in the blood glucose level. Although EA has been proven to be effective in changing body weight (Ernst et al., 2011), it has been used to treat gastrointestinal disorders in Eastern countries for thousands of years (Lin and Chen, 2012). Particularly, treatment at Zusanli (ST36) has been confirmed to be effective in treating gastrointestinal disorders. Extensive studies have suggested that the clinical usefulness of EA at ST36 is derived from its immediate availability as a noninvasive, economical and extremely helpful procedure to recover gastric motility (Chen et al., 2008; Yin and Chen, 2010). Moreover, gastrointestinal motility disorders from functional dyspepsia to gastroparesis are reportedly common in individuals with long-standing diabetes. Recent studies have indicated that up to 75% of patients with diabetes mellitus have one or more gastrointestinal symptoms (Kort et al., 2012). Additionally, numerous studies have shown that delayed gastric emptying can be effectively accelerated by six weeks of EA in diabetic rats and two weeks of EA in patients with diabetes (Wang et al., 2008b; Yin et al., 2010). Because these studies were all performed in vivo, alterations in the motility of the gastric muscle due to EA and the underlying


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mechanism have scarcely been explored in vitro. It is noteworthy that with decreased gastric motility, loss of ICCs is observed in the stomach of diabetic animals at eight weeks but not at four weeks (Ordög et al., 2000; Choi et al., 2008). In addition, our previous study showed that impaired ICCs can be rescued by EA at eight weeks in the colon of diabetic rats, which is accompanied by improved contractions (Xu et al., 2012). In our study, the effects of EA on impaired gastric contractile activity were further investigated at different times and frequencies through a commonly accepted method that specifically destroys ICCs, with no effect on the enteric nerve system or smooth muscle cells (Liu et al., 1994). Our results showed that low- and high-frequency EA ameliorated the attenuation of contractile activity in antrum strips of untreated diabetic rats at four and eight weeks and failed to increase attenuated gastric contractions after destroying ICCs in diabetic rats, suggesting that ICCs were involved in the favorable effects of low- and high-frequency EA on the contractile activity of gastric muscle strips. It is worth noting that, without ICCs, the highest dose of ACH elicited only a small increase in contraction, particularly considering that the contractile effect of ACH mainly functions through the muscarinic receptors in smooth muscle cells. The expression of ICCs in the gastric antrum further confirmed by western blotting and immunohistochemistry was not significantly altered at four weeks but was obviously reduced at eight weeks in diabetic rats,which was consistent with previous findings in STZinduced diabetic rats (Choi et al., 2008; Wang et al., 2009; Mogami et al., 2013). Compared with the impaired contractions of the gastric muscle strips at four weeks, the enteric nerve system or smooth muscle cells were affected. However, at eight weeks, the decreased expression of ICCs further certified that impaired ICC expression was a main factor in the attenuation of contractions in the gastric muscle strips. In addition, it has been reported that ICC expression could be restored by EA in the colon of rats (Deng et al., 2011; Sun et al., 2011; Xu et al., 2012). Therefore, following intervention with low- and high-frequency EA, the number of ICCs was dramatically increased at eight weeks, suggesting that EA might dramatically ameliorate ICCs, resulting in improved gastric contractions. Frequently, with respect to the improvement in c-kit protein expression, stem cell factor (SCF) is considered to be involved in increasing the number of ICCs by EA. Previous studies have reported that a reduction in M-SCF in the antrum for 33 days and 60 days in diabetes-induced mice (Horváth et al., 2006; Jin et al., 2013). Likewise, alterations in S-SCF in diabetic animals were reported in a previously published paper at four and eight weeks (Li et al., 2009; Xu et al., 2012). The present results also demonstrated that both M-SCF expression and S-SCF expression in diabetic rats were slightly decreased at four weeks, with no dramatic alteration of c-kit, but were markedly reduced at eight weeks, suggesting that a reduction in SCF expression occurred earlier than a reduction in ICCs. Moreover, previous studies have demonstrated that EA at ST36 can increase the expression of SCF and c-kit in rat models (Deng et al., 2011; Xu et al., 2012; Lu et al., 2013). We also found that low- and high-frequency EA increased the expression of M-SCF and the level of S-SCF in diabetic rats and further determined that the effect of LEA was more significant than that of HEA, indicating that EA might improve SCF first and then enhanced SCF might increase the expression of c-kit.



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Together with ICC expression at four and eight weeks, SCF expression was mirrored by a similar change in c-kit expression, indicating that SCF might be involved in the regulation of ICC expression. Additionally, the data indicated that SCF expression, particularly M-SCF expression, induced more persistent activation and a longer c-kit life span (Miyazawa et al., 1995; Lin et al., 2010). In our previous study, M-SCF was described as a more effective agonist for c-kit receptors than S-SCF and effectively stimulated the c-kit receptors in colon tissue (Xu et al., 2012). In the present study, both S-SCF expression and M-SCF expression were significantly correlated with c-kit expression according to Pearson correlation analyses. However, the regression analyses suggested that M-SCF expression was the only potential determinant of c-kit protein expression at four and eight weeks. Consistent with the previous observation, it was found that higher M-SCF expression was accompanied by increased c-kit expression in gastric tissue. Therefore, enhancement of the M-SCF/c-kit pathway was speculated to be related to the improvement in gastric contractile activity in diabetic rats resulting from EA. In conclusion, the results of the present study show that EA at ST36 can normalize contractions of the gastric antrum of diabetic rats and can improve the effect of EA at ST36, partly by enhancing the SCF/c-kit pathway. EA may have therapeutic potential for the recovery of diabetic-induced abnormal gastric motility. Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (Nos. 30670775, 81270458). References Chen, J., G.Q. Song, J. Yin, T. Koothan and J.D. Chen. Electroacupuncture improves impaired gastric motility and slow waves induced by rectal distension in dogs. Am. J. Physiol. Gastrointest. Liver Physiol. 295: G614–620, 2008. Choi, K.M., S.J. Gibbons, T.V. Nguyen, G.J. Stoltz, M.S. Lurken, T. Ordög, J.H. Szurszewski and G. Farrugia. Heme oxygenase-1 protects interstitial cells of Cajal from oxidative stress and reverses diabetic gastroparesis. Gastroenterology 135: 2055–2064, 2008. Deng, J.J., X.R. Liu and Q. Yuan. Effect of acupuncture on gastrointestinal stem cell factor/kit system after colocolic anastomosis in rats. Zhen Ci Yan Jiu 36: 176–180, 2011. Ernst, E., M.S. Lee and T.Y. Choi. Acupuncture in obstetrics and gynecology: an overview of systematic reviews. Am. J. Chin. Med. 39: 423–431, 2011. Gong, M., X. Wang, Z. Mao, Q. Shao, X. Xiang and B. Xu. Effect of electroacupuncture on leptin resistance in rats with diet-induced obesity. Am. J. Chin. Med. 40: 511–520, 2012. Grover, M., C.E. Bernard, P.J. Pasricha, M.S. Lurken, M.S. Faussone-Pellegrini, T.C. Smyrk, H.P. Parkman, T.L. Abell, W.J. Snape, W.L. Hasler, R.W. McCallum, L. Nguyen, K.L. Koch, J. Calles, L. Lee, J. Tonascia, A. ”Unalp Arida, F.A. Hamilton and G. Farrugia. Clinicalhistological associations in gastroparesis: results from the gastroparesis clinical research consortium. Neurogastroenterol. Motil. 24: 531–539, e249, 2012. Hirst, G.D. and F.R. Edwards. Electrical events underlying organized myogenic contractions of the guinea pig stomach. J. Physiol. 576: 659–665, 2006.


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Hesperidin produces cardioprotective activity via PPAR-γ pathway in ischemic heart disease model in diabetic rats.

NF-κB Signaling Pathway in Type 2 Diabetic Rats.

GSK-3β signaling pathway.

Impaired contraction and relaxation in aorta from streptozotocin-diabetic rats: role of polyol pathway.

Snail Pathway in Diabetic Rats.

STAT signaling pathway.

Akt pathway.

Activation of the insulin-signaling pathway in sciatic nerve and hippocampus of type 1 diabetic rats.

Berberine inhibits hepatic gluconeogenesis via the LKB1-AMPK-TORC2 signaling pathway in streptozotocin-induced diabetic rats.

CNP-pGC-cGMP-PDE3-cAMP Signal Pathway Upregulated in Gastric Smooth Muscle of Diabetic Rats.

Association of the TLR4 signaling pathway in the retina of streptozotocin-induced diabetic rats.

STAT signaling pathway in experimental diabetic rats.

Antagonizing Wnt pathway in diabetic retinopathy.

endothelial NO synthase signaling pathway.

Progesterone ameliorates diabetic nephropathy in streptozotocin-induced diabetic Rats.

Anti-diabetic potential of chromium histidinate in diabetic retinopathy rats.

Carvedilol ameliorates early diabetic nephropathy in streptozotocin-induced diabetic rats.

Icariside II ameliorates diabetic nephropathy in streptozotocin-induced diabetic rats.

Tropisetron ameliorates early diabetic nephropathy in streptozotocin-induced diabetic rats.

Boldine prevents renal alterations in diabetic rats.

Eplerenone reduces arterial thrombosis in diabetic rats.

Hematological changes in opium addicted diabetic rats.

Myocardial phospholipid metabolism in alloxan diabetic rats.

Aminoguanidine ameliorates albuminuria in diabetic hypertensive rats.