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Research Report
Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia$ Fan Guoa,b,1, Wenying Songa,1, Tao Jianga, Lixin Liuc, Feng Wanga, Haixing Zhonga, Hong Yinb, Qiang Wanga,n, Lize Xionga,n a
Department of Anesthesiology, Xijing Hospital, Forth Military Medical University, Xi'an, China Department of Radiology, Xijing Hospital, Forth Military Medical University, Xi'an, China c Department of Anesthesiology, School of Medicine, Stony Brook University, NY 11794-8480, USA b
art i cle i nfo
ab st rac t
Article history:
We investigated the protective effect of electroacupuncture (EA) on cerebral ischemic injury in
Accepted 13 May 2014
diabetic mice, and explored the role of NADPH oxidase-mediated oxidative stress. Male C57BL/ 6 mice were injected streptozotocin to induce diabetes. The mice were pretreated with EA at
Keywords:
acupoint “Baihui” for 30 min. Two hours after the end of EA pretreatment, focal cerebral
Diabetes mellitus
ischemia was induced following 24 h reperfusion. The neurobehavioral scores and infarction
Cerebral ischemia-reperfusion
volumes, malondialdehyde (MDA), reactive oxygen species (ROS), and activation of NADPH
injury
oxidase were determined in the presence or absence of the NADPH oxidase inhibitor apocynin
Electroacupuncture
or activator tetrabromocinnamic acid (TBCA). EA pretreatment reduced infarct size and
Oxidative stress
improved neurological outcomes 24 h after reperfusion in the diabetic mice. EA also decreased
NADPH oxidase
cerebral MDA and ROS levels compared with the control group, and inhibited the NADPH oxidase activation. The beneficial effects were abolished by TBCA while pretreatment with apocynin mimicked the neuroprotective and anti-oxidative effects of EA. Our results demonstrated that EA attenuated cerebral ischemic injury by inhibiting NAPDH oxidase-mediated oxidative damage in diabetic mice. These results suggest a novel mechanism of EA pretreatment-induced tolerance in diabetic cerebral ischemia. & 2014 Elsevier B.V. All rights reserved.
1.
Introduction
Stroke is a major public health concern in China with 1.5–2 million deaths per year currently. It is the leading cause of death and disability worldwide, with an estimated 5.7 million
deaths (He et al., 2005; Strong et al., 2007). Diabetes mellitus is one of the most severe risk factors for stroke. Further, the outcome of ischemic cerebral damage is exacerbated in diabetic patients. The functional impairment, referred to as “diabetic encephalopathy”, is characterized by morphological
☆ This work was originated from Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. n Corresponding authors. Fax: þ86 29 8477 1262. E-mail addresses: [email protected] (Q. Wang), [email protected] (L. Xiong). 1 Fan Guo and Wenying Song have contributed equally to this work.
http://dx.doi.org/10.1016/j.brainres.2014.05.020 0006-8993/& 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Guo, F., et al., Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.020
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and electrophysiological changes (Allen et al., 2004). Unfortunately, effective intervention in ischemia reperfusion injury of diabetes is still under study. The possible mechanisms associated with severe damage caused by stroke in diabetic patients and animals may include disruption of blood-brain barrier (BBB), degeneration of endothelial cells, enhanced advanced glycation end products (AGEs) induced apoptosis, biochemical changes in cerebral capillaries, and alterations of neurotransmitter activity (Chen et al., 2006; Mooradian, 1997). Among all the factors, oxidative stress, characterized by excessive production of reactive oxygen species (ROS), was proved to play a central role in diabetic tissue damage (Nelson et al., 1992). Hyperglycemia per se, and together with ischemic injury, destroys the balance between ROS generation and clearance, leading to a massive accumulation of ROS aggravating cerebral ischemic damage (Aragno et al., 2000; Wohaieb and Godin, 1987). NADPH oxidase is the major source of pathological ROS generation during cerebral ischemic injury (Chrissobolis and Faraci, 2008; Kahles et al., 2007; Park et al., 2007). Therefore, a promising therapeutic strategy should target NADPH oxidation-mediated oxidative damage (Drummond et al., 2011). Electroaupuncture (EA) is based on traditional Chinese medicine, with proven efficacy in many health conditions, such as acute pain and addictive behavior (D’Alberto, 2004; Madsen et al., 2009; Margolin et al., 2002; Shukla et al., 2011). We have previously reported that EA protected the brain from
ischemia reperfusion injury by reducing infarct size and improving neurological scores (Wang et al., 2005). We also proved that EA suppressed apoptosis caused by ischemic reperfusion injury (Wang et al., 2009). Further, previous studies suggested that EA increased the activation of antioxidant enzymes such as thioredoxin (Siu et al., 2005), and inhibited oxidative stress in ischemic injury (Chen et al., 2012). Prior work also suggested that EA was associated with beneficial effects for brain cholinergic system and against insulin resistance in obese mice (Liang et al., 2011; Rocco et al., 2013). However, the effectiveness of EA in stroke, along with the underlying mechanisms needs further investigation. In the present study we investigated whether EA protected ischemic reperfusion injury in streptozotocin (STZ)-induced diabetic mice. We tested the hypothesis that EA pretreatment protected against cerebral ischemic injury via inhibition of NADPH oxidase-mediated oxidative stress.
2.
Results
2.1.
EA conferred neuroprotection in diabetic mice
Pretreatment of diabetic mice with EA 2 h before MCAO was used to determine protection of brain tissue from ischemic injury (Fig. 1A). Compared with Sham group, MCAO group showed strong neurological deficits at 24 h after reperfusion.
Fig. 1 – EA pretreatment alleviated cerebral ischemic injury in diabetic mice. (A) A schematic diagram illustrating the experimental design of drug treatment and assessment of ischemic injury. (B) Neurological scores 24 h after reperfusion in the diabetic mice with 60 min of MCAO. EA pretreatment significantly improved the neurological scores. (C) Infarct sizes at 24 h after reperfusion in the diabetic mice with 60 min of MCAO. Compared with MCAO group, EA pretreatment significantly inhibited the brain injury induced by ischemia reperfusion in the diabetic mice. Data are expressed as means7SEM (n ¼8 per group), nPo0.05 vs. Sham group; #Po0.05 vs. MCAO group. Please cite this article as: Guo, F., et al., Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.020
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Pretreatment with EA significantly improved the neurological scores compared with the MCAO group (Fig. 1B). Similarly, assessment of infarct sizes 24 h after reperfusion, showed a significantly smaller brain infarct volume in EAþMCAO group compared with the MCAO group (Fig. 1C).
2.2. EA decreased MDA content and ROS formation after reperfusion In this study, malondialdehyde (MDA) was used as a marker of lipid peroxidation. After 24 h of reperfusion, MDA content showed a lower level in EAþMCAO group compared with MCAO group (Fig. 2A). On the other hand, EA pretreatment efficiently prevented the formation of ROS, compared with MCAO group (Fig. 2B).
2.3. EA suppressed activation of NADPH oxidase after reperfusion The expression of NADPH oxidase subunits gp91phox and p47phox was evaluated to determine the activation of NADPH oxidase. Compared with Sham group, MCAO group showed increased activation of NADPH oxidase in the brain penumbra tissue. EA pretreatment significantly reduced the activation of NADPH oxidase (Fig. 3). Pretreatment with TBCA significantly reversed the beneficial effect of EA. In contrast,
3
apocynin pretreatment, which mimics EA pretreatment, significantly reduced the activation of NADPH oxidase (Fig. 4).
2.4.
NADPH oxidase involved in neuroprotection of EA
The infarct volume was significantly larger in the TBCAþEAþ MCAO group compared with EA-treated animals, accompanied by lower neurological scores. In contrast, apocynin pretreatment inhibited the activation of NADPH oxidase, improved the neurological scores and reduced the infarction volumes compared with the MCAO group (Fig. 5).
2.5.
NADPH oxidase reversed oxidation of EA
TBCA pretreatment reversed the anti-oxidative effect of EA while apocynin simulated the decrease of ROS production (Fig. 6A). Administration of apocynin prevented the formation of MDA compared with the MCAO group. TBCA pretreatment reversed the effect of EA in MDA reduction (Fig. 6B).
3.
Discussion
In China, the estimated overall prevalence of diabetes was 11.6% and the prevalence of pre-diabetes was 50.1% (Xu et al., 2013). Cerebral ischemic reperfusion injury is associated with enhanced vulnerability to ischemia and death in diabetic
Fig. 2 – Effect of EA pretreatment on MDA and ROS levels after reperfusion. EA significantly reduced ROS formation and MDA production 24 h after reperfusion. Values were expressed as means7SEM (n ¼5 per group), #Po0.05 vs. Sham group; nPo0.05 vs. MCAO group.
Fig. 3 – Effect of EA pretreatment on activation of NADPH oxidase. EA significantly inhibited NADPH oxidase activation 24 h after MCAO. Values were calculated as relative intensity normalized to GAPDH intensity, and expressed as means7SEM (n ¼ 5 per group), #Po0.05 vs. Sham group; nPo0.05 vs. MCAO group. Please cite this article as: Guo, F., et al., Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.020
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Fig. 4 – Effect of EA, apocynin and TBCA on activation of NADPH oxidase. EA and apocynin reduced the activation of NADPH oxidase 24 h after MCAO, compared with MCAO group, while TBCA, a novel agonist of NADPH oxidase, reversed the inhibition of EA on NADPH oxidase. Values were calculated as relative intensity normalized to GAPDH intensity, and expressed as means7SEM (n ¼4 per group), #Po0.05 vs. Sham group; nPo0.05 vs. MCAO group.
Fig. 5 – Role of NADPH oxidase in neuroprotection induced by EA. (A) Reduction in infarct size by EA was reversed by pretreatment with TBCA. The apocynin, an NADPH oxidase inhibitor, reduced infarct volumes. Infarct volumes were expressed as means7SEM (n ¼10 per group). (B) Neurological deficits were significantly improved in diabetic animals treated with EA and apocynin but reversed in TBCA-pretreatment group (n¼ 10 per group); Po0.05 vs. EAþ MCAO group.
patients. Diabetes is associated with 1.5 to 3 times higher risk of cerebrovascular disease (CVD) compared with normal individuals (Capes et al., 2001; Sarwar et al., 2010). In animal studies, hyperglycemic cerebral ischemia leads to severe neurological deficit mediated by oxidative stress (Kamada et al., 2007). Therefore, it is vital to reduce the incidence of cerebrovascular events and ameliorate cerebral ischemic injury in diabetic patients. EA is accepted as a viable clinical alternative in multiple scenarios (Chao et al., 2009; Zhao, 2008). In 2003, we first proposed that pretreatment with EA induced tolerance to cerebral ischemia (Xiong et al., 2003). One recent study indicated a beneficial effect of EA in insulin resistance (Liang et al., 2011). The evidences suggested that EA was a potential treatment for metabolic disorder and brain injury. However, whether EA was effective in cerebral ischemia in diabetes was still unknown. Therefore, we tested the effect of EA pretreatment in brain ischemia reperfusion injury in diabetic mice. We demonstrated that treatment with EA 2 h before the onset of MCAO significantly decreased the brain infarct volume and improved neurologic outcomes. The neuroprotection occurred without altering physiological parameters during ischemia.
Brain ischemia-reperfusion triggers oxidative stress, which is an imbalance between the generation and elimination of ROS in the biological system. Our current results suggest that the production of ROS and MDA was significantly increased after cerebral ischemic injury, while EA pretreatment effectively neutralized the damage. NADPH oxidase is a multi-subunit, membrane-associated protein. It includes membrane integrated subunits (p22phox and gp91phox), cytosolic subunits (p40phox, p47phox, and p67phox) and the G-protein (Rac 1) (Bedard and Krause, 2007). Nox2 oxidase is an isoform of NADPH oxidase family of ROS-generating enzymes. It is a major promoter of ROS production. It increases after cerebral ischemia injury. Previous studies demonstrated that Nox2 oxidase was involved in neuronal injury following ischemic stroke (Brait et al., 2010; De Silva et al., 2011; Walder et al., 1997). Several lines of evidence support the protective effects of the NADPH oxidase inhibition during ischemia reperfusion injury (Ostrowski et al., 2006; Wang et al., 2006), indicating that NADPH oxidase played an important role in cerebral ischemic injury. Inhibition of NADPH oxidase efficiently reduced complications of ischemic injury in diabetes (Podar and Tuomilehto, 2002).
Please cite this article as: Guo, F., et al., Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.020
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Fig. 6 – Role of NADPH oxidase in reversal of oxidation by EA pretreatment. EA and apocynin administration reduced ROS production (A) and MDA level (B) whereas TBCA reversed EA the protective effects. Values are expressed as mean7SEM (n ¼5 per group); Po0.05 vs. EAþMCAO group.
Therefore, we assumed that the NADPH oxidase might be involved in the protective effect of EA. Our results showed that EA pretreatment reversed the increased NADPH oxidase expression in MCAO group. Apocynin, a NADPH oxidase inhibitor, decreased the oxidative stress associated with cerebral ischemic injury. On the other hand, casein kinase 2 (CK2) was identified as a negative regulator of NAPDH oxidase (Kim et al., 2009). We demonstrated that TBCA, a novel CK2 regulator, reversed the protective effect of EA in the NADPH oxidase-driven neuronal injury in ischemic stroke. Based on the above results, we deduced that the anti-oxidative effect of EA was at least partially mediated by the inhibition of NADPH oxidase. Our results were consistent with experimental studies reporting that EA reduced production of nitric oxide, inhibited NADPH oxidase activity and thus attenuated lipid peroxidation in acute cerebral ischemic injury (Siu et al., 2004a; Xu et al., 1996). It was also proved that EA efficiently decreased oxidative stress in multiple diseases via NADPH oxidase-mediated signaling pathway (Kim et al., 2008; Ma et al., 2005). Studies indicated that EA pretreatment increased the activation of antioxidant enzymes such as thioredoxin, superoxide dismutase and glutathione peroxidase, thus attenuating the production of MDA in cerebral ischemic injury (Siu et al., 2004b, 2005). Intervention with EA was effective for diabetic complications, such as neuropathy and allodynia (Lee et al., 2013; Shi et al., 2013). Therefore, EA was a promising preventive treatment for cerebral injury clinically. However, the molecular mechanisms need to be further investigated. The present study suggests that EA pretreatment may be a promising strategy to reduce cerebral ischemia reperfusion injury in diabetic model. However, many challenges still remain. STZ-induced type I diabetic model is characterized by hyperglycemia, hypoinsulinemia and reduced body weight. Although type II diabetes is more common worldwide, the investigation of type I diabetes is still worthwhile as it is associated with a higher incidence of cerebrovascular events, and similar pathogenesis with type II diabetes. In conclusion, our study showed that electroacupuncture pretreatment attenuated cerebral ischemic injury in streptozotocininduced diabetic mice, which might be mediated at least in part
by inhibition of NADPH oxidase activity. The results suggest that EA may have a potential role in the prophylaxis of cerebrovascular disease in diabetes.
4.
Experimental procedure
The experimental protocol was approved by the Ethics Committee or Animal Experimentation and was performed according to the Guidelines for Animal Experimentation of the Fourth Military Medical University. The experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications no. 80-23) revised 1996. All efforts were made to minimize animal suffering and the number of animals used in this study.
4.1.
Induction of diabetes
The animals were provided by the Experimental Animal Center of the Fourth Military Medical University. We induced diabetes mellitus in 8-to-12 week-old male C57/BL6J mice, weighing 23 to 25 g, by intraperitoneal injection of streptozotocin (Sigma, St. Louis, Missouri) at a dose of 50 mg/kg dissolved in 100 mM citrate buffer (pH 4.5) for 5 consecutive days (Wang et al., 2009). After 8 weeks, blood glucose levels were measured using Bayer's BREEZE2 meter (Bayer Health Care LLC, Mishawaka, USA) by tail vein blood sampling. Mice that showed blood sugar values of 250 mg/dl were used for the study. Mice were housed under controlled conditions with a 12 h light/dark cycle, a temperature at 21 1C72 1C, and humidity in 60–70%. The mice were allowed free access to standard rodent diet and tap water.
4.2.
Electroacupuncture pretreatment
EA pretreatment was performed as described in our previous studies (Wang et al., 2009). Briefly, animals were anesthetized with 40 mg/kg chloralic hydras (intraperitoneally) and administered oxygen by face mask at a flow rate of 1 l/min. The acupoint “Baihui (GV 20),” which is located at the intersection
Please cite this article as: Guo, F., et al., Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.020
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of the sagittal midline and the line linking 2 mouse ears, was electrically stimulated with 1 mA intensity at density-sparse wave with a frequency of 2/15 Hz for 30 min, as described in our previous studies (Li et al., 2012; Wang et al., 2009), using the G6805–2 EA Instrument (Model No.227033; Qingdao Xinsheng Ltd.). The core temperature of all the mice was maintained (Spacelabs Medical Inc.) at 37.070.5 1C during EA pretreatment by surface heating or cooling.
4.3.
Drug injection
Tetrabromocinnamic acid (TBCA; EMD Chemicals, Gibbstown, NJ) has a high specificity of inhibition for CK2 and activates NADPH oxidase. In the present study, TBCA (20 nmol in 2 μl of 50% dimethyl sulfoxide [DMSO] in PBS) was injected intracerebroventricularly (i.c.v) 30 min before induction of ischemia (bregma: 1.0 mm lateral, 0.2 mm posterior, 3.1 mm deep) (Kim et al., 2009). Fifty percent DMSO in PBS was used as a vehicle. Apocynin (Sigma-Aldrich, St Louis, MO, USA), an NADPH oxidase inhibitor, was dissolved in DMSO and phosphatebuffered saline, and administered intravenously at 2.5 mg/kg body weight 15 min before onset of MCAO (Kim et al., 2009). In the first part of the study, male diabetic mice were randomly assigned to Sham, MCAO, EAþMCAO groups. In the second part, the animals were randomly assigned to Sham, MCAO, EAþMCAO, apocyninþMCAO, Vehicle(A)þMCAO, TBCAþEAþMCAO and Vehicle(T)þEAþMCAO groups.
4.4.
Transient focal cerebral ischemia
Animals were administered general anesthesia with 40 mg/kg chloralic hydras (1%, intraperitoneally). Oxygen was administered by face mask at a flow rate of 1 L/min. Rectal temperature was maintained between 36.5 1C and 37.0 1C before, during, and after the surgery until the animals had recovered fully from anesthesia. Focal cerebral ischemia was induced by MCAO in mice using an intraluminal filament technique as described previously (Hata et al., 1998; Wang et al., 2009). Briefly, we performed EA pretreatment 2 h before the MCAO, and after 1 h of MCAO, the filament was withdrawn and wounds were sutured. Regional cerebral blood flow (rCBF) was measured by transcranial laser Dopplar flowmetry (PeriFlux 5000; Perimed AB). MCAO was considered adequate if rCBF decreased to 30% of the baseline level; Reperfusion was accompanied with rCBF recovered up to 80% of baseline; otherwise, animals were excluded from analysis.
4.5.
Neurobehavioral evaluation and infarct assessment
Twenty-four hours after reperfusion, neurological assessment was performed with a modified six-point scoring scale by a blinded observer (Tatlisumak et al., 1998). The animals were decapitated and 2-mm thick coronal sections from the brain were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the infarct volume, as described previously (Wang et al., 2009). The area of infarction was measured by subtracting the area of the non-lesioned ipsilateral hemisphere from that of the contralateral side. The volume of infarction was calculated by integration of the lesion areas.
4.6. Measurement of lipid peroxidation (MDA) and reactive oxygen species (ROS) The ischemic penumbra was harvested and lipid peroxidation measured, according to the method described by Ohkawa et al. (1979) using a detection kit from Beyotime Institute of Biotechnology, Shanghai, China. The absorbance of the supernatant was measured by spectrophotometry at 532 nm. ROS levels were determined using detection kits from Western Tang Bio Institution, Shanghai, China and the absorbance of the supernatant was measured by spectrophotometry at 490 nm.
4.7.
Western blot
The mice were anesthetized and decapitated 24 h after reperfusion. The brains were rapidly removed and the number of positive cells was counted in penumbra of ischemic side. Samples were homogenized in RIPA lysis buffer (Beyotime, Nantong, China) with 1 Roche complete protease inhibitor cocktail and 1 mM PMSF on ice. Tissue extract was centrifuged at 12,000g at 4 1C for 30 min. An equal amount of total protein was separated on 12% gel and then transferred to nitrocellulose filter membrane. The membranes were blocked with 5% non-fat dry milk in Tris buffer saline in 0.05% Tween-20 at 4 1C, and incubated with NADPH gp91phox and p47phox monoclonal antibody (1:500 dilution, Epitomics, USA) followed by the horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibody (1:1000, Sigma, USA), respectively, for 60 min at room temperature. Membranes were developed by an ECL-technique. Quantitative analysis for immune blotting was performed after scanning of the X-ray film with Quantitative-One software (version 4.60, Bio-Rad, USA). GAPDH (1:10000 dilution, CWBIO, China) served as the loading control.
4.8.
Statistical analysis
The software, SPSS 14.0 for Windows (SPSS Inc, Chicago, Ill), was used to conduct statistical analyses. All values, except for neurological scores, were expressed as means7SEM and analyzed by one-way analysis of variance. The betweengroup differences were detected with post hoc Student– Newman–Keuls test. The neurological deficit scores were expressed as median (range) values and analyzed with Kruskal–Wallis test followed by Mann–Whitney U test with Bonferroni correction. Values of Pr0.05 were considered statistically significant.
Acknowledgments This work was supported by the Overseas, Hong Kong & Macao Scholars Collaborated Researching Fund (Grant 81228022), the Program for Changjiang Scholars and Innovative Research Team in University (Grant IRT 1053), and the National Natural Science Foundation of China (Grants 81271342, 81171278).
Please cite this article as: Guo, F., et al., Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.020
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Shi, L., Zhang, H.H., Xiao, Y., Hu, J., Xu, G.Y., 2013. Electroacupuncture suppresses mechanical allodynia and nuclear factor kappa B signaling in streptozotocin-induced diabetic rats. CNS Neurosci. Ther. 19, 83–90. Shukla, S., Torossian, A., Duann, J.R., Leung, A., 2011. The analgesic effect of electroacupuncture on acute thermal pain perception – a central neural correlate study with fMRI. Mol. Pain 7, 45. Siu, F.K., Lo, S.C., Leung, M.C., 2004a. Effectiveness of multiple pre-ischemia electro-acupuncture on attenuating lipid peroxidation induced by cerebral ischemia in adult rats. Life Sci. 75, 1323–1332. Siu, F.K., Lo, S.C., Leung, M.C., 2004b. Electroacupuncture reduces the extent of lipid peroxidation by increasing superoxide dismutase and glutathione peroxidase activities in ischemicreperfused rat brains. Neurosci. Lett. 354, 158–162. Siu, F.K., Lo, S.C., Leung, M.C., 2005. Electro-acupuncture potentiates the disulphide-reducing activities of thioredoxin system by increasing thioredoxin expression in ischemiareperfused rat brains. Life Sci. 77, 386–399. Strong, K., Mathers, C., Bonita, R., 2007. Preventing stroke: saving lives around the world. Lancet Neurol. 6, 182–187. Tatlisumak, T., Takano, K., Carano, R.A., Miller, L.P., Foster, A.C., Fisher, M., 1998. Delayed treatment with an adenosine kinase inhibitor, GP683, attenuates infarct size in rats with temporary middle cerebral artery occlusion. Stroke 29, 1952–1958. Walder, C.E., Green, S.P., Darbonne, W.C., Mathias, J., Rae, J., Dinauer, M.C., Curnutte, J.T., Thomas, G.R., 1997. Ischemic stroke injury is reduced in mice lacking a functional NADPH oxidase. Stroke 28, 2252–2258.
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Please cite this article as: Guo, F., et al., Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.020
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Research Report
Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia$ Fan Guoa,b,1, Wenying Songa,1, Tao Jianga, Lixin Liuc, Feng Wanga, Haixing Zhonga, Hong Yinb, Qiang Wanga,n, Lize Xionga,n a
Department of Anesthesiology, Xijing Hospital, Forth Military Medical University, Xi'an, China Department of Radiology, Xijing Hospital, Forth Military Medical University, Xi'an, China c Department of Anesthesiology, School of Medicine, Stony Brook University, NY 11794-8480, USA b
art i cle i nfo
ab st rac t
Article history:
We investigated the protective effect of electroacupuncture (EA) on cerebral ischemic injury in
Accepted 13 May 2014
diabetic mice, and explored the role of NADPH oxidase-mediated oxidative stress. Male C57BL/ 6 mice were injected streptozotocin to induce diabetes. The mice were pretreated with EA at
Keywords:
acupoint “Baihui” for 30 min. Two hours after the end of EA pretreatment, focal cerebral
Diabetes mellitus
ischemia was induced following 24 h reperfusion. The neurobehavioral scores and infarction
Cerebral ischemia-reperfusion
volumes, malondialdehyde (MDA), reactive oxygen species (ROS), and activation of NADPH
injury
oxidase were determined in the presence or absence of the NADPH oxidase inhibitor apocynin
Electroacupuncture
or activator tetrabromocinnamic acid (TBCA). EA pretreatment reduced infarct size and
Oxidative stress
improved neurological outcomes 24 h after reperfusion in the diabetic mice. EA also decreased
NADPH oxidase
cerebral MDA and ROS levels compared with the control group, and inhibited the NADPH oxidase activation. The beneficial effects were abolished by TBCA while pretreatment with apocynin mimicked the neuroprotective and anti-oxidative effects of EA. Our results demonstrated that EA attenuated cerebral ischemic injury by inhibiting NAPDH oxidase-mediated oxidative damage in diabetic mice. These results suggest a novel mechanism of EA pretreatment-induced tolerance in diabetic cerebral ischemia. & 2014 Elsevier B.V. All rights reserved.
1.
Introduction
Stroke is a major public health concern in China with 1.5–2 million deaths per year currently. It is the leading cause of death and disability worldwide, with an estimated 5.7 million
deaths (He et al., 2005; Strong et al., 2007). Diabetes mellitus is one of the most severe risk factors for stroke. Further, the outcome of ischemic cerebral damage is exacerbated in diabetic patients. The functional impairment, referred to as “diabetic encephalopathy”, is characterized by morphological
☆ This work was originated from Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. n Corresponding authors. Fax: þ86 29 8477 1262. E-mail addresses: [email protected] (Q. Wang), [email protected] (L. Xiong). 1 Fan Guo and Wenying Song have contributed equally to this work.
http://dx.doi.org/10.1016/j.brainres.2014.05.020 0006-8993/& 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Guo, F., et al., Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.020
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and electrophysiological changes (Allen et al., 2004). Unfortunately, effective intervention in ischemia reperfusion injury of diabetes is still under study. The possible mechanisms associated with severe damage caused by stroke in diabetic patients and animals may include disruption of blood-brain barrier (BBB), degeneration of endothelial cells, enhanced advanced glycation end products (AGEs) induced apoptosis, biochemical changes in cerebral capillaries, and alterations of neurotransmitter activity (Chen et al., 2006; Mooradian, 1997). Among all the factors, oxidative stress, characterized by excessive production of reactive oxygen species (ROS), was proved to play a central role in diabetic tissue damage (Nelson et al., 1992). Hyperglycemia per se, and together with ischemic injury, destroys the balance between ROS generation and clearance, leading to a massive accumulation of ROS aggravating cerebral ischemic damage (Aragno et al., 2000; Wohaieb and Godin, 1987). NADPH oxidase is the major source of pathological ROS generation during cerebral ischemic injury (Chrissobolis and Faraci, 2008; Kahles et al., 2007; Park et al., 2007). Therefore, a promising therapeutic strategy should target NADPH oxidation-mediated oxidative damage (Drummond et al., 2011). Electroaupuncture (EA) is based on traditional Chinese medicine, with proven efficacy in many health conditions, such as acute pain and addictive behavior (D’Alberto, 2004; Madsen et al., 2009; Margolin et al., 2002; Shukla et al., 2011). We have previously reported that EA protected the brain from
ischemia reperfusion injury by reducing infarct size and improving neurological scores (Wang et al., 2005). We also proved that EA suppressed apoptosis caused by ischemic reperfusion injury (Wang et al., 2009). Further, previous studies suggested that EA increased the activation of antioxidant enzymes such as thioredoxin (Siu et al., 2005), and inhibited oxidative stress in ischemic injury (Chen et al., 2012). Prior work also suggested that EA was associated with beneficial effects for brain cholinergic system and against insulin resistance in obese mice (Liang et al., 2011; Rocco et al., 2013). However, the effectiveness of EA in stroke, along with the underlying mechanisms needs further investigation. In the present study we investigated whether EA protected ischemic reperfusion injury in streptozotocin (STZ)-induced diabetic mice. We tested the hypothesis that EA pretreatment protected against cerebral ischemic injury via inhibition of NADPH oxidase-mediated oxidative stress.
2.
Results
2.1.
EA conferred neuroprotection in diabetic mice
Pretreatment of diabetic mice with EA 2 h before MCAO was used to determine protection of brain tissue from ischemic injury (Fig. 1A). Compared with Sham group, MCAO group showed strong neurological deficits at 24 h after reperfusion.
Fig. 1 – EA pretreatment alleviated cerebral ischemic injury in diabetic mice. (A) A schematic diagram illustrating the experimental design of drug treatment and assessment of ischemic injury. (B) Neurological scores 24 h after reperfusion in the diabetic mice with 60 min of MCAO. EA pretreatment significantly improved the neurological scores. (C) Infarct sizes at 24 h after reperfusion in the diabetic mice with 60 min of MCAO. Compared with MCAO group, EA pretreatment significantly inhibited the brain injury induced by ischemia reperfusion in the diabetic mice. Data are expressed as means7SEM (n ¼8 per group), nPo0.05 vs. Sham group; #Po0.05 vs. MCAO group. Please cite this article as: Guo, F., et al., Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.020
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Pretreatment with EA significantly improved the neurological scores compared with the MCAO group (Fig. 1B). Similarly, assessment of infarct sizes 24 h after reperfusion, showed a significantly smaller brain infarct volume in EAþMCAO group compared with the MCAO group (Fig. 1C).
2.2. EA decreased MDA content and ROS formation after reperfusion In this study, malondialdehyde (MDA) was used as a marker of lipid peroxidation. After 24 h of reperfusion, MDA content showed a lower level in EAþMCAO group compared with MCAO group (Fig. 2A). On the other hand, EA pretreatment efficiently prevented the formation of ROS, compared with MCAO group (Fig. 2B).
2.3. EA suppressed activation of NADPH oxidase after reperfusion The expression of NADPH oxidase subunits gp91phox and p47phox was evaluated to determine the activation of NADPH oxidase. Compared with Sham group, MCAO group showed increased activation of NADPH oxidase in the brain penumbra tissue. EA pretreatment significantly reduced the activation of NADPH oxidase (Fig. 3). Pretreatment with TBCA significantly reversed the beneficial effect of EA. In contrast,
3
apocynin pretreatment, which mimics EA pretreatment, significantly reduced the activation of NADPH oxidase (Fig. 4).
2.4.
NADPH oxidase involved in neuroprotection of EA
The infarct volume was significantly larger in the TBCAþEAþ MCAO group compared with EA-treated animals, accompanied by lower neurological scores. In contrast, apocynin pretreatment inhibited the activation of NADPH oxidase, improved the neurological scores and reduced the infarction volumes compared with the MCAO group (Fig. 5).
2.5.
NADPH oxidase reversed oxidation of EA
TBCA pretreatment reversed the anti-oxidative effect of EA while apocynin simulated the decrease of ROS production (Fig. 6A). Administration of apocynin prevented the formation of MDA compared with the MCAO group. TBCA pretreatment reversed the effect of EA in MDA reduction (Fig. 6B).
3.
Discussion
In China, the estimated overall prevalence of diabetes was 11.6% and the prevalence of pre-diabetes was 50.1% (Xu et al., 2013). Cerebral ischemic reperfusion injury is associated with enhanced vulnerability to ischemia and death in diabetic
Fig. 2 – Effect of EA pretreatment on MDA and ROS levels after reperfusion. EA significantly reduced ROS formation and MDA production 24 h after reperfusion. Values were expressed as means7SEM (n ¼5 per group), #Po0.05 vs. Sham group; nPo0.05 vs. MCAO group.
Fig. 3 – Effect of EA pretreatment on activation of NADPH oxidase. EA significantly inhibited NADPH oxidase activation 24 h after MCAO. Values were calculated as relative intensity normalized to GAPDH intensity, and expressed as means7SEM (n ¼ 5 per group), #Po0.05 vs. Sham group; nPo0.05 vs. MCAO group. Please cite this article as: Guo, F., et al., Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.020
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Fig. 4 – Effect of EA, apocynin and TBCA on activation of NADPH oxidase. EA and apocynin reduced the activation of NADPH oxidase 24 h after MCAO, compared with MCAO group, while TBCA, a novel agonist of NADPH oxidase, reversed the inhibition of EA on NADPH oxidase. Values were calculated as relative intensity normalized to GAPDH intensity, and expressed as means7SEM (n ¼4 per group), #Po0.05 vs. Sham group; nPo0.05 vs. MCAO group.
Fig. 5 – Role of NADPH oxidase in neuroprotection induced by EA. (A) Reduction in infarct size by EA was reversed by pretreatment with TBCA. The apocynin, an NADPH oxidase inhibitor, reduced infarct volumes. Infarct volumes were expressed as means7SEM (n ¼10 per group). (B) Neurological deficits were significantly improved in diabetic animals treated with EA and apocynin but reversed in TBCA-pretreatment group (n¼ 10 per group); Po0.05 vs. EAþ MCAO group.
patients. Diabetes is associated with 1.5 to 3 times higher risk of cerebrovascular disease (CVD) compared with normal individuals (Capes et al., 2001; Sarwar et al., 2010). In animal studies, hyperglycemic cerebral ischemia leads to severe neurological deficit mediated by oxidative stress (Kamada et al., 2007). Therefore, it is vital to reduce the incidence of cerebrovascular events and ameliorate cerebral ischemic injury in diabetic patients. EA is accepted as a viable clinical alternative in multiple scenarios (Chao et al., 2009; Zhao, 2008). In 2003, we first proposed that pretreatment with EA induced tolerance to cerebral ischemia (Xiong et al., 2003). One recent study indicated a beneficial effect of EA in insulin resistance (Liang et al., 2011). The evidences suggested that EA was a potential treatment for metabolic disorder and brain injury. However, whether EA was effective in cerebral ischemia in diabetes was still unknown. Therefore, we tested the effect of EA pretreatment in brain ischemia reperfusion injury in diabetic mice. We demonstrated that treatment with EA 2 h before the onset of MCAO significantly decreased the brain infarct volume and improved neurologic outcomes. The neuroprotection occurred without altering physiological parameters during ischemia.
Brain ischemia-reperfusion triggers oxidative stress, which is an imbalance between the generation and elimination of ROS in the biological system. Our current results suggest that the production of ROS and MDA was significantly increased after cerebral ischemic injury, while EA pretreatment effectively neutralized the damage. NADPH oxidase is a multi-subunit, membrane-associated protein. It includes membrane integrated subunits (p22phox and gp91phox), cytosolic subunits (p40phox, p47phox, and p67phox) and the G-protein (Rac 1) (Bedard and Krause, 2007). Nox2 oxidase is an isoform of NADPH oxidase family of ROS-generating enzymes. It is a major promoter of ROS production. It increases after cerebral ischemia injury. Previous studies demonstrated that Nox2 oxidase was involved in neuronal injury following ischemic stroke (Brait et al., 2010; De Silva et al., 2011; Walder et al., 1997). Several lines of evidence support the protective effects of the NADPH oxidase inhibition during ischemia reperfusion injury (Ostrowski et al., 2006; Wang et al., 2006), indicating that NADPH oxidase played an important role in cerebral ischemic injury. Inhibition of NADPH oxidase efficiently reduced complications of ischemic injury in diabetes (Podar and Tuomilehto, 2002).
Please cite this article as: Guo, F., et al., Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.020
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Fig. 6 – Role of NADPH oxidase in reversal of oxidation by EA pretreatment. EA and apocynin administration reduced ROS production (A) and MDA level (B) whereas TBCA reversed EA the protective effects. Values are expressed as mean7SEM (n ¼5 per group); Po0.05 vs. EAþMCAO group.
Therefore, we assumed that the NADPH oxidase might be involved in the protective effect of EA. Our results showed that EA pretreatment reversed the increased NADPH oxidase expression in MCAO group. Apocynin, a NADPH oxidase inhibitor, decreased the oxidative stress associated with cerebral ischemic injury. On the other hand, casein kinase 2 (CK2) was identified as a negative regulator of NAPDH oxidase (Kim et al., 2009). We demonstrated that TBCA, a novel CK2 regulator, reversed the protective effect of EA in the NADPH oxidase-driven neuronal injury in ischemic stroke. Based on the above results, we deduced that the anti-oxidative effect of EA was at least partially mediated by the inhibition of NADPH oxidase. Our results were consistent with experimental studies reporting that EA reduced production of nitric oxide, inhibited NADPH oxidase activity and thus attenuated lipid peroxidation in acute cerebral ischemic injury (Siu et al., 2004a; Xu et al., 1996). It was also proved that EA efficiently decreased oxidative stress in multiple diseases via NADPH oxidase-mediated signaling pathway (Kim et al., 2008; Ma et al., 2005). Studies indicated that EA pretreatment increased the activation of antioxidant enzymes such as thioredoxin, superoxide dismutase and glutathione peroxidase, thus attenuating the production of MDA in cerebral ischemic injury (Siu et al., 2004b, 2005). Intervention with EA was effective for diabetic complications, such as neuropathy and allodynia (Lee et al., 2013; Shi et al., 2013). Therefore, EA was a promising preventive treatment for cerebral injury clinically. However, the molecular mechanisms need to be further investigated. The present study suggests that EA pretreatment may be a promising strategy to reduce cerebral ischemia reperfusion injury in diabetic model. However, many challenges still remain. STZ-induced type I diabetic model is characterized by hyperglycemia, hypoinsulinemia and reduced body weight. Although type II diabetes is more common worldwide, the investigation of type I diabetes is still worthwhile as it is associated with a higher incidence of cerebrovascular events, and similar pathogenesis with type II diabetes. In conclusion, our study showed that electroacupuncture pretreatment attenuated cerebral ischemic injury in streptozotocininduced diabetic mice, which might be mediated at least in part
by inhibition of NADPH oxidase activity. The results suggest that EA may have a potential role in the prophylaxis of cerebrovascular disease in diabetes.
4.
Experimental procedure
The experimental protocol was approved by the Ethics Committee or Animal Experimentation and was performed according to the Guidelines for Animal Experimentation of the Fourth Military Medical University. The experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications no. 80-23) revised 1996. All efforts were made to minimize animal suffering and the number of animals used in this study.
4.1.
Induction of diabetes
The animals were provided by the Experimental Animal Center of the Fourth Military Medical University. We induced diabetes mellitus in 8-to-12 week-old male C57/BL6J mice, weighing 23 to 25 g, by intraperitoneal injection of streptozotocin (Sigma, St. Louis, Missouri) at a dose of 50 mg/kg dissolved in 100 mM citrate buffer (pH 4.5) for 5 consecutive days (Wang et al., 2009). After 8 weeks, blood glucose levels were measured using Bayer's BREEZE2 meter (Bayer Health Care LLC, Mishawaka, USA) by tail vein blood sampling. Mice that showed blood sugar values of 250 mg/dl were used for the study. Mice were housed under controlled conditions with a 12 h light/dark cycle, a temperature at 21 1C72 1C, and humidity in 60–70%. The mice were allowed free access to standard rodent diet and tap water.
4.2.
Electroacupuncture pretreatment
EA pretreatment was performed as described in our previous studies (Wang et al., 2009). Briefly, animals were anesthetized with 40 mg/kg chloralic hydras (intraperitoneally) and administered oxygen by face mask at a flow rate of 1 l/min. The acupoint “Baihui (GV 20),” which is located at the intersection
Please cite this article as: Guo, F., et al., Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.020
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of the sagittal midline and the line linking 2 mouse ears, was electrically stimulated with 1 mA intensity at density-sparse wave with a frequency of 2/15 Hz for 30 min, as described in our previous studies (Li et al., 2012; Wang et al., 2009), using the G6805–2 EA Instrument (Model No.227033; Qingdao Xinsheng Ltd.). The core temperature of all the mice was maintained (Spacelabs Medical Inc.) at 37.070.5 1C during EA pretreatment by surface heating or cooling.
4.3.
Drug injection
Tetrabromocinnamic acid (TBCA; EMD Chemicals, Gibbstown, NJ) has a high specificity of inhibition for CK2 and activates NADPH oxidase. In the present study, TBCA (20 nmol in 2 μl of 50% dimethyl sulfoxide [DMSO] in PBS) was injected intracerebroventricularly (i.c.v) 30 min before induction of ischemia (bregma: 1.0 mm lateral, 0.2 mm posterior, 3.1 mm deep) (Kim et al., 2009). Fifty percent DMSO in PBS was used as a vehicle. Apocynin (Sigma-Aldrich, St Louis, MO, USA), an NADPH oxidase inhibitor, was dissolved in DMSO and phosphatebuffered saline, and administered intravenously at 2.5 mg/kg body weight 15 min before onset of MCAO (Kim et al., 2009). In the first part of the study, male diabetic mice were randomly assigned to Sham, MCAO, EAþMCAO groups. In the second part, the animals were randomly assigned to Sham, MCAO, EAþMCAO, apocyninþMCAO, Vehicle(A)þMCAO, TBCAþEAþMCAO and Vehicle(T)þEAþMCAO groups.
4.4.
Transient focal cerebral ischemia
Animals were administered general anesthesia with 40 mg/kg chloralic hydras (1%, intraperitoneally). Oxygen was administered by face mask at a flow rate of 1 L/min. Rectal temperature was maintained between 36.5 1C and 37.0 1C before, during, and after the surgery until the animals had recovered fully from anesthesia. Focal cerebral ischemia was induced by MCAO in mice using an intraluminal filament technique as described previously (Hata et al., 1998; Wang et al., 2009). Briefly, we performed EA pretreatment 2 h before the MCAO, and after 1 h of MCAO, the filament was withdrawn and wounds were sutured. Regional cerebral blood flow (rCBF) was measured by transcranial laser Dopplar flowmetry (PeriFlux 5000; Perimed AB). MCAO was considered adequate if rCBF decreased to 30% of the baseline level; Reperfusion was accompanied with rCBF recovered up to 80% of baseline; otherwise, animals were excluded from analysis.
4.5.
Neurobehavioral evaluation and infarct assessment
Twenty-four hours after reperfusion, neurological assessment was performed with a modified six-point scoring scale by a blinded observer (Tatlisumak et al., 1998). The animals were decapitated and 2-mm thick coronal sections from the brain were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the infarct volume, as described previously (Wang et al., 2009). The area of infarction was measured by subtracting the area of the non-lesioned ipsilateral hemisphere from that of the contralateral side. The volume of infarction was calculated by integration of the lesion areas.
4.6. Measurement of lipid peroxidation (MDA) and reactive oxygen species (ROS) The ischemic penumbra was harvested and lipid peroxidation measured, according to the method described by Ohkawa et al. (1979) using a detection kit from Beyotime Institute of Biotechnology, Shanghai, China. The absorbance of the supernatant was measured by spectrophotometry at 532 nm. ROS levels were determined using detection kits from Western Tang Bio Institution, Shanghai, China and the absorbance of the supernatant was measured by spectrophotometry at 490 nm.
4.7.
Western blot
The mice were anesthetized and decapitated 24 h after reperfusion. The brains were rapidly removed and the number of positive cells was counted in penumbra of ischemic side. Samples were homogenized in RIPA lysis buffer (Beyotime, Nantong, China) with 1 Roche complete protease inhibitor cocktail and 1 mM PMSF on ice. Tissue extract was centrifuged at 12,000g at 4 1C for 30 min. An equal amount of total protein was separated on 12% gel and then transferred to nitrocellulose filter membrane. The membranes were blocked with 5% non-fat dry milk in Tris buffer saline in 0.05% Tween-20 at 4 1C, and incubated with NADPH gp91phox and p47phox monoclonal antibody (1:500 dilution, Epitomics, USA) followed by the horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibody (1:1000, Sigma, USA), respectively, for 60 min at room temperature. Membranes were developed by an ECL-technique. Quantitative analysis for immune blotting was performed after scanning of the X-ray film with Quantitative-One software (version 4.60, Bio-Rad, USA). GAPDH (1:10000 dilution, CWBIO, China) served as the loading control.
4.8.
Statistical analysis
The software, SPSS 14.0 for Windows (SPSS Inc, Chicago, Ill), was used to conduct statistical analyses. All values, except for neurological scores, were expressed as means7SEM and analyzed by one-way analysis of variance. The betweengroup differences were detected with post hoc Student– Newman–Keuls test. The neurological deficit scores were expressed as median (range) values and analyzed with Kruskal–Wallis test followed by Mann–Whitney U test with Bonferroni correction. Values of Pr0.05 were considered statistically significant.
Acknowledgments This work was supported by the Overseas, Hong Kong & Macao Scholars Collaborated Researching Fund (Grant 81228022), the Program for Changjiang Scholars and Innovative Research Team in University (Grant IRT 1053), and the National Natural Science Foundation of China (Grants 81271342, 81171278).
Please cite this article as: Guo, F., et al., Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.020
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