Electroacupuncture Attenuates Cerebral Ischemia-Reperfusion Injury in Diabetic Mice Through Adiponectin Receptor 1-Mediated Phosphorylation of GSK-3β
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- Guo, F., Jiang, T., Song, W. et al. Mol Neurobiol (2015) 51: 685. doi:10.1007/s12035-014-8765-y
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Diabetes mellitus substantially increases the risk of stroke and enhances brain’s vulnerability to ischemia insult. Electroacupuncture (EA) pretreatment was proved to induce cerebral ischemic tolerance in normal stroke models. Whether EA could attenuate cerebral ischemia injury in diabetic mice and the possible underlying mechanism are still unrevealed. Male C57BL/6 mice were subjected to streptozotocin (STZ) for diabetic models. After inducing focal cerebral ischemia model, the levels of plasma and cerebral adiponectin (APN) were measured as well as the expression of cerebral adiponectin receptor 1 (AdipoR1) and 2 (AdipoR2). The neurobehavioral score, infarction volume, and cellular apoptosis were evaluated with or without AdipoR1 short interfering RNA (siRNA). The role of phosphorylation of glycogen synthesis kinase 3 beta (GSK-3β) at Ser-9 in the EA pretreatment was also assessed. EA pretreatment increased both plasma and cerebral APN levels and enhanced neuronal AdipoR1 in diabetic mice. In addition, EA reduced infarct size, improved neurological outcomes, and inhibited cell apoptosis after reperfusion. These beneficial effects were reversed by AdipoR1 knockdown. Furthermore, EA increased GSK-3β phosphorylation (p-GSK-3β) in the ipsilateral penumbra. Augmented p-GSK-3β induced neuroprotective effects similar to those of EA pretreatment. In contrast, dampened p-GSK-3β could reverse the neuroprotective effects of EA. In addition, the increase in p-GSK-3β by EA was abolished by AdipoR1 knockdown. We conclude that EA pretreatment increases the production of APN, which induce protective effects against cerebral ischemia-reperfusion injury through neuronal AdipoR1-mediated phosphorylation of GSK-3β in diabetic mice.
KeywordsDiabetesElectroacupunctureIschemia-reperfusion injuryAdiponectinAdiponectin receptorGlycogen synthesis kinase 3 beta
In 2008, there were about 347 million people worldwide who were suffering from diabetes mellitus (DM) . In China, the prevalence of DM and prediabetes is 9.7 and 15.5 %, respectively . Both types 1 and 2 DM substantially increase the risk for cerebral ischemia-reperfusion (I-R) injury [3, 4]. Cerebral I-R injury is a major cause of morbidity and mortality in diabetic patients, who demonstrated enhanced vulnerability to ischemia insult and resultant cell death . Importantly, DM, together with metabolic syndrome, could abolish or blunt the protective effects of some preconditioning methods [6, 7].
Adiponectin (APN) is an adipokine secreted by the adipose tissue and present at high levels in the serum . There are two subtypes of APN receptors: adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). Many studies investigated the relationship between APN and cardiovascular diseases. There are evidences suggesting that APN and AdipoRs are widely expressed in the brain and play an important role in ischemia injury [9, 10]. Moreover, APN supplementation and upregulation of AdipoR1 could reduce myocardial ischemic injury in a diabetic model . Therefore, APN and its receptors might play crucial roles in cerebral protection in DM patients.
Glycogen synthase kinase 3 beta (GSK-3β) has multiple functions, including regulating cellular development and tissue protection. GSK-3β is constitutively active in cells and its inactivation could be induced by phosphorylation at Ser-9 . Growing evidence implicated that GSK-3β is involved in brain tissue protection against ischemia injury or brain trauma [13, 14]. A selective GSK-3β inhibitor could cause the phosphorylation at Ser-9 and inactivate GSK-3β, thus provided a protective effect against transient cerebral ischemic injury . The inhibition of GSK-3β was effective for regulating metabolism disorders . Moreover, acute GSK-3β inhibition was proved to be a novel therapeutic strategy for acute myocardial infarction in a diabetic model . Thus, GSK-3β signaling pathway might be involved in cerebral ischemic injury in DM.
Electroacupuncture (EA) originates from the traditional Chinese medicine and has been shown to be effective in different models such as painful diabetic neuropathy and obesity [18, 19]. From our previous study, EA at the Baihui (GV 20) acupoint reduced neurological injury induced by transient middle cerebral artery occlusion . Additional studies have further explored the underlying mechanism and demonstrated that the endocannabinoid system as well as GSK-3β signaling pathway was involved in the neuroprotective effects of EA [21, 22]. On the other hand, EA has been shown to alleviate insulin resistance in diabetic animal model and the mechanism might be AMP-activated protein kinase related, which is a classic molecular one in APN-AdipoRs signaling pathway . However, the effect of EA against cerebral ischemic injury in diabetic model, as well as the role of APN in EA, is still not clear.
Therefore, we undertook the present study to determine whether EA could protect the brain against I-R injury in streptozotocin (STZ)-induced diabetic mice and to elucidate the hypothesis that APN, AdipoRs, and GSK-3β phosphorylation might be involved in the protection of EA pretreatment.
Materials and Methods
Induction of Diabetes
The experimental protocol was approved by the Ethics Committee for Animal Experimentation and was performed according to the Guidelines for Animal Experimentation of the Fourth Military Medical University and the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised in 1996. All efforts were made to minimize animal suffering and the number of animals used in this study. The animals were provided by the Experimental Animal Center of the Fourth Military Medical University. DM was induced in male C57/BL6J mice 8–12 weeks old, weighing 23–25 g, by intraperitoneal injection of streptozotocin (Sigma, St Louis, MO, USA) at a dose of 50 mg/kg dissolved in 100 mM citrate buffer pH 4.5 for five consecutive days, as previously reported . After 8 weeks, blood glucose levels were measured using Bayer’s BREEZE2 meter (Bayer HealthCare Pharmaceuticals, Montville, NJ, USA) by tail vein blood sampling. Mice with blood glucose levels of >250 mg/dl were used for the present study. Mice were housed under controlled condition with a 12-h light/dark cycle, a temperature of 21 ± 2 °C, and 60–70 % humidity. Mice were allowed free access to standard rodent diet and tap water.
To determine the effects of EA pretreatment on APN levels and AdipoR expression, diabetic mice were randomly divided into three groups: sham, middle cerebral artery occlusion (MCAO), and EA + MCAO.
To elucidate the role of AdipoR1 in EA-induced neuroprotection, diabetic mice were randomly divided into sham, MCAO, EA + MCAO, short interfering RNA (siRNA)-A (A stands for APN siRNA) + EA + MCAO, and siRNA-c (c stands for control siRNA) + EA + MCAO groups. AdipoR1 was silenced by siRNA.
To assess the role of GSK-3β in EA pretreatment, diabetic mice were randomly assigned to the EA + MCAO, wrt + EA + MCAO, and vehicle (W) + EA + MCAO groups. Wortmannin is a selective phosphatidylinositol 3-kinase (PI3K) inhibitor. This agent was injected into the right lateral ventricle at a dose of 3 μl (100 μM) in 10 % dimethyl sulfoxide (DMSO), 30 min before MCAO . The vehicle group received the same volume of DMSO alone. To further evaluate the effects of GSK-3β in EA pretreatment, we detected the neuroprotective effects of pretreatment with the selective GSK-3β inhibitor 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8). Diabetic mice were divided into three groups: MCAO, TDZD-8 + MCAO, and vehicle (T) + MCAO group. Mice from the TDZD-8 + MCAO group received an intraperitoneal injection of the drug at a dose of 1 mg/kg in 10 % DMSO, 1.5 h after reperfusion [15, 25], while the vehicle group received DMSO only.
To investigate the regulatory effects of AdipoR1 on GSK-3β phosphorylation, diabetic mice were randomly divided into MCAO, EA + MCAO, siRNA-A + EA + MCAO, and siRNA-c + EA + MCAO groups.
Transfection of siRNA into the Mice Brain
Transfection of siRNA into diabetic mice was conducted according to previously described methods . Mice were anesthetized with 40 mg/kg of chloral hydrate, followed by the stereotactic implantation of a stainless steel cannula in the unilateral cerebral ventricle. The stereotaxic coordinates were 0.4 mm posterior and 1.0 mm lateral to the bregma, at a depth of 2.5 mm from the surface of the skull. A 4-μl volume of 1 μg/μl AdipoR1-siRNA (sc-270168; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or control siRNA (sc-37007; Santa Cruz Biotechnology, Santa Cruz, CA, USA) of the diluted mixture was delivered into the ipsilateral lateral ventricle. After recovering from anesthesia, mice were returned to their cages and received ad libitum access to food and water. The protein expression of AdipoR1 was evaluated 3 days later by a Western blot assay.
EA pretreatment was performed as described in our previous study . Briefly, animals were anesthetized with 40 mg/kg of chloral hydrate (intraperitoneally) and inhaled oxygen by a face mask at a flow rate of 1 l/min. The acupoint “Baihui (GV 20),” which is located at the intersection of the sagittal midline and the line linking the mouse’s ears, was stimulated with an intensity of 1 mA and a frequency of 2/15 Hz for 30 min using the G6805-2 EA Instrument (Model No. 227033; Qingdao Xinsheng Ltd., China). The core temperature of all mice was maintained at 37.0 ± 0.5 °C during EA pretreatment by surface heating or cooling (Spacelabs Healthcare, Snoqualmie, WA, USA).
Transient Focal Cerebral Ischemia
Focal cerebral ischemia was induced by MCAO in mice using an intraluminal filament technique, as previously described [21, 26]. After 1 h of MCAO, the filament was withdrawn and regional cerebral blood flow was restored to normal. Regional cerebral blood flow (rCBF) was monitored through a disposable microtip fiber optic probe (diameter, 0.5 mm) connected through a master probe to a laser Doppler computerized main unit (PeriFlux 5000; Perimed AB, Jarfalla, Sweden). MCAO was considered adequate if rCBF sharply decreased to 30 % of the baseline level; otherwise, animals were excluded from analysis.
Neurobehavioral Evaluation and Infarct Assessment
Twenty-four hours after reperfusion, a six-point scoring scale with modifications was used for neurological assessment by a blinded observer . Then, animals were decapitated and 2-mm thick coronal sections from the whole brain were stained with 2 % 2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the infarct volume, as previously described . Infarction area was measured by subtracting the area of the non-infarcted ipsilateral hemisphere from that of the contralateral side. The volume of infarction was calculated by integration of the lesion areas.
Measurement of Serum and Tissue Adiponectin
Blood samples were kept at 25 °C for 30 min. Serum was separated after centrifugation at 3,000 g for 20 min, and the supernatant was stored at −80 °C until use. Samples of the ischemic penumbra were homogenized in cold radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Nantong, China) with 1× Roche complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany) and 1 mM phenylmethanesulfonyl fluoride (PMSF) on ice. Tissue extract was centrifuged at 12,000 g at 4 °C for 30 min, and the supernatant was stored at −80 °C until use. Total serum APN levels were measured by enzyme-linked immunosorbent assay (ELISA) (Westang Biotechnology, Shanghai, China).
Terminal Transferase-Biotinylated Deoxyuridine Triphosphate Nick-End Labeling
Samples from different groups were used for experiments (n = 5/group). Twenty-four hours after reperfusion, neuronal apoptosis was assessed in situ by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) staining. Brain sections were processed according to the manufacturer’s protocol (Roche Applied Science, Penzberg, Germany) . Briefly, pixels of 0.10 mm2 were visualized by light microscopy (×100 magnification) and the total number of positive cells in these pixels was counted and expressed as cells per millimeter squared.
Mice were anesthetized and decapitated 24 h after reperfusion. Brains were rapidly removed, and the penumbra of the ischemic side was homogenized in RIPA lysis buffer (Beyotime, Nantong, China) with 1× Roche complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany) and 1 mM PMSF on ice. Tissue extract was centrifuged at 12,000 g at 4 °C for 30 min. Western blotting was performed with standard procedures. Members were incubated with AdipoR1 monoclonal antibody (1:1,000 dilution; Epitomic, San Francisco, CA, USA), AdipoR2 polyclonal antibody (1:1,000 dilution; Abcam, Cambridge, MA, USA), B cell lymphoma 2 (Bcl-2) polyclonal antibody (1:1,000 dilution; Cell Signaling, Danvers, MA, USA), Bax monoclonal antibody (1:1,000 dilution; Epitomic, San Francisco, CA, USA), active caspase 3 polyclonal antibody (1:200 dilution; Abcam, Cambridge, MA, USA), GSK-3β phosphorylation (p-GSK-3β) (Ser-9) monoclonal antibody (1:1,000 dilution; Cell Signaling, Danvers, MA, USA), and GSK-3β monoclonal antibody (1:1,000 dilution; Cell Signaling, Danvers, MA, USA), followed with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse secondary antibody (1:1,000 dilution; Beyotime, Beijing, China) for 60 min at room temperature. Membranes were developed using an ECL technique. GAPDH or β-actin (1:10,000 dilution; CoWin Biotech, Beijing, China) was used as controls.
For analysis of AdipoR1/2, mice were anesthetized after 24 h of reperfusion and were transcardially perfused with PBS and 4 % paraformaldehyde. The brains were cut, prepared, and stained as previously described . Different brain areas in each section were observed and photographed using a microscope (Olympus, BX51, Japan) (×20) from the same part of the brain in each group.
SPSS 11.0 for Windows (SPSS, Inc., Chicago, IL, USA) was used for statistical analyses. All values, except for neurological scores, are presented as mean ± SEM and were analyzed by one-way analysis of variance (ANOVA), with post hoc Student-Newman-Keuls tests. The neurological deficit scores are expressed as median (range) and were analyzed with the Kruskal-Wallis test followed by the Mann-Whitney U test with Bonferroni correction. P values ≤0.05 were considered statistically significant.
EA Pretreatment Increased APN Plasma and Brain Tissue Levels in Diabetic Mice
EA Pretreatment Upregulated the Expression of Neuronal AdipoR1
APN binding to AdipoR1 can activate a signaling pathway leading to antioxidative and antiapoptosis effects. Using Western blot and immunofluorescence, we observed that AdipoR1 was significantly increased in ischemic mice and that the levels of the receptor were higher in EA-treated mice. Meanwhile, there was no change in AdipoR2 levels (Fig. 1c, d).
Neuroprotection of EA Pretreatment in Diabetic Mice Depended on AdipoR1
Physiologic variables of diabetic mice from all experimental groups
Blood glucose (mmol/l)
7.43 ± 0.01
105 ± 1.2
36 ± 0.8
22.8 ± 1.5
7.42 ± 0.01
106 ± 1.1
36 ± 0.6
25.2 ± 1.3
EA + MCAO
7.42 ± 0.01
107 ± 1.3
36 ± 0.6
25.2 ± 1.4
siRNA-A + EA + MCAO
7.43 ± 0.01
107 ± 0.9
34 ± 0.8
25.4 ± 1.3
siRNA-c + EA + MCAO
7.43 ± 0.01
106 ± 0.9
36 ± 0.4
22.8 ± 1.4
Onset of MCAO
7.39 ± 0.02
107 ± 1.6
39 ± 0.7
23.5 ± 1.5
7.40 ± 0.01
106 ± 1.9
39 ± 0.9
22.6 ± 1.3
EA + MCAO
7.41 ± 0.01
106 ± 1.4
38 ± 0.8
23.5 ± 1.0
siRNA-A + EA + MCAO
7.38 ± 0.01
104 ± 1.4
40 ± 0.8
25.0 ± 1.4
siRNA-c + EA + MCAO
7.39 ± 0.01
107 ± 0.8
39 ± 0.6
23.6 ± 1.5
Onset of reperfusion
7.39 ± 0.02
106 ± 1.2
31 ± 1.0
24.1 ± 1.4
7.40 ± 0.01
106 ± 1.3
30 ± 0.8
24.3 ± 1.3
EA + MCAO
7.40 ± 0.02
108 ± 1.0
36 ± 0.8
23.2 ± 1.0
siRNA-A + EA + MCAO
7.41 ± 0.02
107 ± 1.1
36 ± 0.8
23.2 ± 1.5
siRNA-c + EA + MCAO
7.41 ± 0.01
107 ± 1.5
36 ± 0.8
23.4 ± 1.3
EA Pretreatment Inhibited Cellular Apoptosis via AdipoR1
GSK-3β Phosphorylation Is Involved in the Neuroprotective Effects of EA Pretreatment
The infarct size and neurological scores 24 h after reperfusion in the presence or absence of wortmannin were also detected for further exploring the role of p-GSK-3β (Ser-9) in EA pretreatment. We found that wortmannin reversed the reduction in infarct size and abolished the improvement in neurological outcome of EA pretreatment compared with the EA + MCAO group (Fig. 4b). As expected, the vehicle (W) + EA + MCAO group did not show any statistically significant difference with the EA + MCAO group. We also found that TDZD-8 reduced the infarct size and improved neurological scores compared with the MCAO group (Fig. 4c), while the vehicle group showed no effect compared with the MCAO group, suggesting that the administration of TDZD-8 at an early phase could mimic the neuroprotection of EA pretreatment.
GSK-3β Phosphorylation by EA Pretreatment Was Mediated by AdipoR1
As an important part of Chinese traditional medicine, EA was effective against many different diseases, including cocaine addiction, osteoarthritis, and emesis [29–31]. One major finding of our past study is that EA pretreatment could efficiently reduce cerebral ischemic injury . Our current study indicated that EA pretreatment increased the production of APN, which elicited protective effects against cerebral I-R injury through neuronal AdipoR1-mediated phosphorylation of GSK-3β in diabetic mice.
APN is an abundant plasma protein secreted by the adipose tissue, contributes to glucose homeostasis, and possesses antioxidative and protective effects . Although the presence of APN and its role in the brain still remain controversial, APN was found to be present in cerebrospinal fluid and might play an important role in physiological functions of the CNS . AdipoR1 and AdipoR2 have been identified and exert distinct biological properties in different tissues . AdipoR1 is most abundant in skeletal muscles and has a high affinity to globular APN. AdipoR2 is primarily expressed in the liver and binds both full-length and globular forms of APN . Some studies showed that mouse cortical neurons, together with hypothalamus and endothelial cells, express both AdipoR1 and AdipoR2, with AdipoR1 expression being more pronounced than AdipoR2 [10, 37]. It has been reported that APN protected the brain against cerebral ischemic stroke . APN also activates its receptors, as well as downstream molecules, including PI3K and Akt, and plays important roles under physiological and pathological conditions [39, 40].
In our study, plasma APN levels were significantly elevated in the EA group, and the expression of AdipoR1, but not AdipoR2, was higher in EA-treated mice after cerebral I-R injury. The different expressions between AdipoR1 and AdipoR2 might be due to the fact that the former was more abundantly distributed in the cortex area. From our result, the AdipoR1 expression from MCAO group showed a slight upregulation, but not significant difference compared with sham group. We assume that the change might be due to the stress reaction and the result was partially consistent with that of Thundyil et al. . For further confirming the potential role of AdipoR1 in EA pretreatment, we observed that the intracerebroventricular administration of AdipoR1 siRNA attenuated the protective effects of EA pretreatment. The previous study demonstrated that acupuncture treatment increased APN levels in obese patients, consistent with our results . It has also been indicated that bolstering AdipoR1 expression and APN supplementation brought beneficial effects to myocardial ischemic injury in a diabetic model . Therefore, the elevation of APN levels could be a vital mechanism in the neuroprotective effects of EA.
Neuronal apoptosis could be induced and even worsen under pathological situations, such as hyperglycemia complicated by ischemia . Pro- and antiapoptotic proteins participate in the regulation of apoptosis. Bax and Bcl-2 belong to the Bcl-2 family and are both involved in ischemia-induced neuronal cell apoptosis. Bcl-2 is an antiapoptotic protein, while Bax is a proapoptotic one. In the current study, apoptotic events were revealed by TUNEL staining and the activation of caspase 3 as well as the ratio of Bax/Bcl-2. Our results suggest that with EA pretreatment, the upregulation of Bax after ischemia injury was inhibited, while Bcl-2 was enhanced. This finding is consistent with the decreased caspase 3 and apoptotic neuronal cell counting results. Meanwhile, the silencing of AdipoR1 abolished the antiapoptotic effects mentioned above. These findings provide the first evidence that EA may prevent cellular apoptosis following cerebral ischemia through an AdipoR1-mediated pathway in DM.
Emerging evidence suggests that GSK-3β participates in the regulation of metabolism disorders and cerebral ischemia injury. Inhibition of GSK-3β was recommended as a novel therapeutic target for DM treatment . TDZD-8 was protective against cerebral I-R injury by inhibiting GSK-3β activity  and could stimulate a protection against acute myocardial ischemic injury in a DM rat model . As Akt is widely accepted as the major mediator of the phosphorylation and subsequent inactivation of GSK-3β, GSK-3β seemed to be involved in EA treatment as the downstream intermediary. Therefore, we focused on GSK-3β and proved that the phosphorylation of GSK-3β was increased after EA pretreatment. Wortmannin, a PI3K inhibitor, reversed the protective effects of EA pretreatment, while TDZD-8, a GSK-3β inhibitor, reduced the infarct size and improved the neurological score. These results were consistent with previously published data showing that TDZD-8 reduced cerebral I-R injury in diabetic rats in the same way that EA pretreatment protected against I-R injury through phosphorylation of GSK-3β [22, 43]. Furthermore, with the administration of AdipoR1 siRNA, the phosphorylation of GSK-3β was inhibited. Thus, we concluded that GSK-3β is the key molecular effector of EA.
DM and ischemic stroke are common diseases that often arise together. On one hand, DM is a leading cause of cerebrovascular disorders . On the other hand, stroke is the second leading cause of long-term disability worldwide . People suffering from DM have more than a twofold risk of ischemic stroke compared with people without DM . The main pathological disorders include microvessel disease, cognitive decline, and dementia [47, 48]. Some preventive or therapeutic methods against DM cerebral ischemic injury, such as glucose-lowering treatment and thrombolysis, are still without strong supporting evidences . From the present study, EA might be a potential pretreatment method for ischemic stroke in DM.
Recently, rimonabant, the first selective cannabinoid-1 receptor (CB1R) blocker, was approved for clinical use and showed strong effects in inducing body weight loss and lowering cardiovascular disease risk factors in obese patients . Moreover, rimonabant directly stimulated APN gene expression in an adipocyte cell line . These results suggested that the endocannabinoid system exerts a strong negative effect on APN gene expression through the CB1R. However, from our previous study, EA was found to elicit protective effects against transient cerebral ischemia through the CB1R . To explain this apparent paradox, the present experiments were performed in streptozotocin-induced diabetic mice, which was neither normal nor typical type 2 diabetic animal models. Whether CB1R plays a positive or negative role in EA-induced neuroprotective effects still needs further experiments. Moreover, although we observed the expression changes of APN and AdipoR1, we could not conclude whether they were caused by the corresponding mRNA changes, or simply as one result of other reaction such as reactive oxygen species production. Future studies exploring the detail alteration of APN and its receptors during EA pretreatment are needed to support our findings.
In conclusion, the present study showed that EA pretreatment attenuated cerebral ischemic injury through an APN-mediated signaling pathway in streptozotocin-induced diabetic mice. These results suggest that EA may have a potential application and that APN and GSK-3β may be a promising target in preventing cerebrovascular events in DM.
This work was supported by the Overseas and Hong Kong, Macau 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 81072888 and 81171278).
Conflict of Interest
The authors declare that they have no conflict of interest.