Electroacupuncture Pretreatment Attenuates Cerebral Ischemia-Reperfusion Injury through Transient Receptor Potential Vanilloid 1-Mediated Anti-Apoptosis via Inhibiting NF-kB Signaling Pathway

Our previous study showed that electroacupuncture (EA) pretreatment elicited protective effect on cerebral ischemia-reperfusion injury in rats, at least partly, which was associated with transient receptor potential vanilloid 1 (TRPV1)-regulated anti-oxidant stress and anti-inammation. In this study, we further investigated the possible contribution of TRPV1-mediated anti-apoptosis in EA pretreatment-evoked neuroprotection. After EA pretreatment at Baihui (GV20), bilateral Shenshu (BL23) and Sanyinjiao (SP6) acupoints, transient focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion for 6 h in rats. Then, infarct volume, nerve cell injury, neuronal apoptosis, NF-κB signaling activation, and expression of TRPV1 were evaluated by TTC staining, hematoxylin-eosin staining, transmission electron microscopy, immunochemistry, immunouorescence, and Western blot, respectively. The presented data showed that EA pretreatment signicantly reduced infarct volume, relieved nerve cell injury, decreased the expression of pro-apoptotic proteins Bax and cleaved caspase-3, increased the level of anti-apoptotic protein Bcl-2, inhibited NF-κB (p65) transcriptional activity, and curbed TRPV1 expression in MCAO rats. By contrast, enhancement of TRPV1 expression accompanying capsaicin application, the specic TRPV1 agonists, markedly accelerated nerve cell damage, aggravated neuronal apoptosis, prompted nuclear translocation of NF-κB (p65), resulting in the reversion of EA pretreatment-evoked neuroprotective effect in MCAO rats. Thus, we conclude that EA pretreatment-induced downregulation of neuronal TRPV1 expression plays an anti-apoptosis role through inhibiting NF-κB signaling pathway, thereby protecting MCAO rats from cerebral ischemia-reperfusion injury. and TRPV1 expression in MCAO rats, and further claried the relationship between EA pretreatment-evoked neuroprotection and TRPV1-mediated anti-apoptosis. Our results provide a new evidence that EA pretreatment exerts its benecial action to improve cerebral ischemia-reperfusion injury in MCAO rats, at least partly, attributing to TRPV1-mediated anti-apoptosis through inhibiting NF-κB signaling pathway. GV20, bilateral BL23 acupoints decreased TRPV1 expression in hippocampus of rat. On the one hand, our previous study reported that downregulation of TRPV1 inhibited CIRI-induced mitochondrial damage and P38 MAPK phosphorylation, resulting in subsequent suppression of oxidative injury and inammation. On the other hand, accompanying reduction of TRPV1 expression in the current study, EA pretreatment signicantly prevented NF-κB transcriptional activity and regulated apoptosis-related protein expression, such as decreasing Bax and cleaved caspase-3 as well as increasing Bcl-2, which contributes to the anti-apoptosis activity of EA pretreatment in MCAO rats. current ndings shed light on the involvement of TRPV1-mediated anti-apoptosis in EA pretreatment-evoked neuroprotection against cerebral ischemia-reperfusion injury via inhibiting NF-κB signaling pathway.


Introduction
Stroke, a sudden circulatory disturbance of cerebrovascular blood ow, is de ned as a neurological disease with high morbidity, disability, and mortality. Generally, stroke is broadly divided into two main categories: ischemic stroke and hemorrhagic stroke, and the former accounts for about 80% of stroke cases [1]. Accompanying occlusion caused by a thrombus or embolus in cerebrovascular circulation, ischemic stroke always occurs due to decreased or blocked blood ow to brain tissues [2]. Although important role in the progression of CIRI based on the interconnection of cascades reactions [6]. The extrinsic apoptotic pathway is associated with the activation of death receptors that bind speci c ligands and transmit apoptotic signals. However, the intrinsic apoptotic pathway is triggered by intracellular stimuli such as DNA damage, hypoxia, and oxidative stress, which consequently lead to outer mitochondrial membrane permeabilization, contributing to cellular degradation of apoptosis [7]. Inhibition of apoptosis, including the downregulation of pro-apoptosis protein caspases 3, 6, 9 and Bax as well as the upregulation of anti-apoptosis protein Bcl-2, prevents neuronal damage and death, therefore regulation of apoptosis represents a valuable research direction for stroke rehabilitation [8].
As a branch of traditional Chinese medicine, acupuncture has been widely accepted in Eastern and Western countries with incomparable advantages, including simple operation, convenience, affordable cost, good repeatability, e cacy, safe, and no side effects [9]. Electroacupuncture (EA) pretreatment, application in advance through inserting a metal needle at speci c acupoints on the body surface with stimulation delivery via electric pulses, has been reported to be effective for treating CIRI or ischemic stroke through different mechanisms, such as inhibition of in ammation, suppression of oxidant stress, regulation of autophagy, attenuation of neuronal apoptosis [10,11]. Our recent study showed that EA pretreatment elicited neuroprotective effect on CIRI in rats, the mechanism of which was related to transient receptor potential vanilloid 1 (TRPV1)-regulated anti-oxidant stress and anti-in ammation [12].
Previous studies also indicated the involvement of TRPV1 channels and apoptosis in CIRI or ischemic stroke [13][14][15]. However, whether TRPV1-mediated anti-apoptosis contributes to EA pretreatment-elicited neuroprotection is still unclear.
In this study, we rstly investigated the effects of EA pretreatment on neuronal apoptosis and TRPV1 expression in MCAO rats, and further clari ed the relationship between EA pretreatment-evoked neuroprotection and TRPV1-mediated anti-apoptosis. Our results provide a new evidence that EA pretreatment exerts its bene cial action to improve cerebral ischemia-reperfusion injury in MCAO rats, at least partly, attributing to TRPV1-mediated anti-apoptosis through inhibiting NF-κB signaling pathway.
EA pretreatment A Han's Acupoint Nerve Stimulator (HANS-200A, Nanjing Jisheng Medical Technology Co., Ltd., Nanjing, China) was applied to "Baihui" (GV20, located at the intersection of sagittal midline and the line linking two rat ears), bilateral "Shenshu" (BL23, on both sides of the 2nd lumbar vertebra, and the distance was 6 mm from the dorsmedian) and "Sanyinjiao" (SP6, the tip of the medial malleolus of the hind limb is straight up 10mm) acupoints ( Fig. 1a and b). BL23 and SP6 acupoints on the same side were used together as pairs, and a shallow needle was prickled at 0.5 cm near Baihui point as an auxiliary electrode to form a pair with GV20 acupoint. The needles (diameter 0.25 mm and length 25 mm) (Beijing Zhongyan Taihe Medical Instrument Co., Ltd., Beijing, China) were inserted into the skin. Then, a dilatational wave at 2/100 Hz and 1 mA with electrical stimulation was used for 10 min, and kept the needles alone without electrical stimulation for 5 min. The process was continually repeated 4 times for 1 h in total as EA pretreatment.
Middle cerebral artery occlusion (MCAO) model Brie y, rats received intraperitoneal anesthesia with 5% chloral hydrate (0.6 mL/100 g). A mono lament nylon suture with a rounded tip coated with silicone rubber (2032/2432A5, Beijing Cinontech Co., Ltd., Beijing, China) was inserted into the internal carotid artery 18-20 mm to block the blood ow. When ischemia was reached for 2 h, the middle cerebral artery was re-perfused by withdrawing the silicone rubber coated mono lament to induce the MCAO model of focal cerebral ischemia-reperfusion injury in rats.

Experimental protocols
Experiment I 72 Rats were randomly distributed to four groups (n = 18 for each group): middle cerebral artery occlusion model group (MCAO), sham-operation group with surgery but no MCAO (Sham), EA pretreated-MCAO group (EA + MCAO), and sham EA pretreated-MCAO group (N-EA + MCAO). For sham EA pretreatment (N-EA), the needles were inserted vertically into ve acupoints only about 2-3 mm deep under the skin without electricity output, but procedures, electrode placements, and other treatment settings were the same as EA pretreatment. The sensations of "de qi" were not achieved in the sham EA pretreatment. As shown in Fig. 1C, rats were pretreated with EA stimulation at GV20, bilateral BL23 and SP6 acupoints for 1 h. Next, the rats were yield to unilateral right middle cerebral artery occlusion for 2 h, and then re-perfused for 6 h. Lastly, rats were sacri ced to collect the samples.

Measurement of infarct volume
After rats were sacri ced, the brains were collected and infarct volume was measured according to previous description [17]. The brain tissue was sectioned into six coronal blocks with an approximate thickness of 2 mm. Then, the sections were stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC) (T8877, Sigma-Aldrich, St. Louis, MO, USA) for 20 min and immersed in 4% paraformaldehyde overnight. Normal area presented red and infarct area kept white. The percentage of infarct volumes were calculated following the formula: Infarct volumes (%) = (Vc-V L ) / Vc × 100% Vc = volumes of normal gray matter in the control hemisphere V L = volumes of normal gray matter in the s lesioned hemisphere

Histological analysis
At the end of the experiment, brain tissues of rats were removed and xed in 4% paraformaldehyde. Fixed tissues were embedded in para n and serially sectioned to a thickness of 5 μm. Then, the sections were stained with hematoxylin and eosin (H&E) and examined for histological analysis. A minimum of three sections per animal experimentation was examined, and ve visual elds of each sample were randomly selected to observe nerve cell injury in a blinded manner.
Transmission electron microscopy Fresh brain tissues were taken from the ischemic cerebral hippocampus, cut into 1mm 3 size cubes and xed in 2.5% glutaraldehyde for 24 h. Next, the samples were xed in 1% osmium tetroxide for 2 h and dehydrated in graded ethanol and embedded in araldite. Then, the sections were cut at 50 nm and stained with uranyl acetate and lead citrate. Finally, the ultrastructure of neurons and mitochondria were observed with Tecnai G2 20 200kV transmission electron microscope (FEI/Philips Electron Optics, Eindhoven, Netherlands).

Immunohistochemistry
For immunohistochemical analysis, the para n sections of brain tissues were depara nized, blocked with 5% BSA, and treated with rabbit anti-rat Bax (ab32503), Bcl-2 (ab194583) (Abcam, Cambridge, Massachusetts, USA), cleaved caspase-3 (#9661) (Cell Signaling Technology Inc., Beverly, Massachusetts, USA) and TRPV1 (PA5-77317) (Invitrogen, Carlsbad, CA, USA) at 37°C for 2 h, separately. The sections were incubated with second antibody (ab6720, Abcam) at 37°C for 30 min. Then, SABC complex was added and the slides were stained with DAB solution. Finally, the slides were observed with a microscope (Olympus BX40, Tokyo, Japan). Under ×400 magni cation, the morphometric examination was performed in a blinded manner by two independent investigators. Cells with brown granule staining of the membrane/cytoplasm were considered positive. For each section, ve visual elds were chosen at random and mean number of the positive cells were represented.

Immuno uorescence
For dual immuno uorescence labeling, the slides of brain tissues were rinsed in 0.

EA pretreatment prevents rats from cerebral ischemia-reperfusion injury
In this study, GV20, bilateral BL23 and SP6 acupoints were used ( Fig. 1a and b) according to the experimental progress presented in Fig. 1c (Experiment I). Compared with MCAO group, infarct regions (white) (Fig. 1d) and the percentage of infarct volume were signi cantly decreased in EA + MCAO group (Fig. 1e). Simultaneously, EA pretreatment relieved nerve cell injury in the hippocampus of MCAO rats, such as reactive hyperplasia of glial cells, nuclear pyknosis, and even cell death (Fig. 2a). Under transmission electron microscope, neuronal nucleus in the hippocampus turned into smaller and transformed into pyknosis, deformation and even lysis in MCAO group. Additionally, the mitochondrial ultrastructure of neurons presented degenerative signs, such as vacuolation and swelling (Fig. 2b). EA pretreatment signi cantly relieved neurons and mitochondria injury accompanying alleviation of nerve cell damage as described above (Fig. 2), suggesting neuroprotective activity of EA pretreatment in MCAO rats.

EA pretreatment inhibits hippocampal neuronal apoptosis
Next, we assessed the expression of apoptosis-related proteins via immunohistochemical staining, including Bax, Bcl-2, and cleaved caspase-3. As shown in Fig. 3a, the numbers of Bax-and cleaved caspase-3-positive cells were both markedly decreased in EA + MCAO group in comparison with MCAO group, while Bcl-2-positive cells were increased in MCAO rats with EA application in advance (Fig. 3b-d).
The inhibitive effect of EA pretreatment on apoptosis-related protein expression was also con rmed by Western blot analysis (Fig. 4a and c), and the similar trends, i.e., reduction of Bax/Bcl-2 and cleaved caspase-3/GAPDH ration in EA + MCAO group compared with MCAO group, was presented in Fig. 4b and d.

EA pretreatment suppresses NF-κB signaling activation in MCAO rats
To explore molecular mechanism of the anti-apoptosis effect of EA pretreatment in MCAO rats, we further investigated the protein expression associated with NF-κB signaling pathway, including IκBα, p-IκBα, NF-κB (p65), and p-NF-κB (p65). Compared with Sham group, increasing cytosolic phosphorylation of IκBα and nuclear translocation of p-NF-κB (p65) as well as decreasing cytosolic NF-κB (p65) subunit were showed in MCAO rats (Fig. 5a and c). In contrast to the MCAO group, relative protein expression of p-IκBα/IκBα and p-NF-κB (p65)/NF-κB (p65) were both reduced in EA + MCAO group ( Fig.  5b and d).
EA pretreatment reduces TRPV1 expression in the hippocampus of MCAO rats As shown in Fig. 6a, immunohistochemistry showed the number of TRPV1-positive cells was upregulated in MCAO group compared with that in Sham group (Fig. 6b). EA pretreatment in MCAO rats reduced the TRPV1-positive cells and downregulated protein expression of TRPV1 in comparison with MCAO group (Fig. 6a-d). Furthermore, double immuno uorescence labeling indicated colocalization expression of TRPV1 and NeuN in the hippocampal neurons. In comparison with the MCAO group, EA pretreatment decreased the colocalization of TRPV1 and NeuN (Fig. 7), accompanying the increment of TRPV1positive cells and decrement of NeuN-positive cells. Colocalization analysis suggests that functional effects of TRPV1 contribute more to EA pretreatment-evoked neuroprotection relative to recovery of the number of neurons.
Capsaicin abrogates EA pretreatment-evoked neuroprotection in MCAO rats To further con rm the role of TRPV1 in the neuroprotective bene ts of EA pretreatment, capsaicin, the speci c TRPV1 agonists, was used in MCAO rats according to the schematic diagram of Experiment II described in Fig. 8a. As shown in Fig. 8b and c, capsaicin application obviously promoted TRPV1 expression at the protein level. Importantly, capsaicin signi cantly abrogated the inhibitive effect of EA pretreatment on TRPV1 expression in MCAO rats (Fig. 8d and e). Capsaicin administration reversed the neuroprotective effect of EA pretreatment in MCAO rats, which was emphasized by the increment of infarct regions and the augmentation of infarct volume ( Fig. 8f and g).

Capsaicin abolishes anti-apoptosis activity of EA pretreatment in MCAO rats
Following early application of capsaicin, the protective effect of EA pretreatment on nerve cell injury of the hippocampus in MCAO rats was abolished, such as increment of reactive hyperplasia of glial cells, development of cells with nuclear pyknosis, and appearance of dead cells (Fig. 9a). Compared with EA + MCAO group, the numbers of Bax-and cleaved caspase-3-positive cells were both increased as well as Bcl-2-positive cells decreased in capsaicin + EA + MCAO group (Fig. 9b-e). Simultaneously, capsaicin signi cantly raised the relative expression of Bax/Bcl-2 and cleaved caspase-3/GAPDH in MCAO rats with EA pretreatment at the protein level ( Fig. 10a-d).
Capsaicin revoked the suppressive effect of EA pretreatment on NF-κB signaling activation in MCAO rats Lastly, we examined NF-κB signaling activation in MCAO rats in response to capsaicin application. Compared with those in EA + MCAO group, phosphorylation of cytoplasmic IκBα and nuclear translocation of p-NF-κB (p65) (Fig. 11a and c), as well as relative protein expression of p-IκBα/IκBα and p-NF-κB (p65)/NF-κB (p65), were both enhanced in capsaicin + EA + MCAO group ( Fig. 11b and d). These ndings highlight the involvement of TRPV1-mediated anti-apoptosis in EA pretreatment-evoked neuroprotection via inhibiting NF-κB signaling activation.

Discussion
Prior to cerebral ischemia, certain "pretreatment" stimulation can develop resistance or tolerance to protect against subsequent injury such as promoting cell survival, which highlights the viewpoint of disease prevention in modern medicine [18]. EA pretreatment re ects the theory of "Zhi wei bing" (treating before sick) in traditional Chinese medicine, wherefore it may be clinically applicable for prevention [19], and not just as a complementary therapy against ischemic stroke [20]. According to our current knowledge, the concept of "EA pretreatment" was rst proposed in 2003, indicating that pretreatment with repeated EA at Baihui acupoint (GV20) attenuated transient focal cerebral ischemic injury in MCAO rats [21]. Besides GV20 [22], EA pretreatment in CIRI or ischemic stroke is always associated with acupuncture prescriptions of single acupoint or acupoints combination, such as Shuigou (GV26) [23], Sanyinjiao (SP6), Neiguan (PC6) [24], Dazhui (GV14) [25], Zusanli (ST36), and Quchi (LI11) [26], etc. Although the neuroprotective effect of EA pretreatment has been widely reported and con rmed, there is currently no uniform standard for the selection and combination of acupoints because of the complicated issues, such as acupoint indications, clinical syndrome differentiation, and individualization of treatment. Under the guide of "Biao ben pei xue" acupuncture prescription, in combination of clinical syndrome and acupoint indications, two or more acupoints are combined to enhance the collaborative effects of acupoints in order to achieve speci c e cacy and improve clinical outcomes [27]. In this study, GV20, bilateral BL23 and SP6 acupoints were selected and used according to our previous publication [12]. EA pretreatment reduced infarct volume, relieved neuronal and mitochondrial damage, and protects hippocampal neuronal from CIRI ( Figs. 1 and 2), which further emphasized the neuroprotective activity of EA pretreatment in CIRI and ischemic stroke.
During ischemic stroke, molecular cascades that contribute to neuronal cell death are diverse and have been subdivided into three main types, i.e., necrosis, apoptosis, and autophagy [28,29]. Due to the overlap between some forms of cell death and between cell death and other processes, it is hard to clearly distinguish the difference among the three under pathological conditions. Generally, apoptosis is diagnosed based on the following characteristics, including DNA fragmentation, changes of Bcl-2 family proteins, caspase activation, or phosphatidylserine exposure [30]. Increasing evidences has shown that regulation of apoptosis plays an essential role in the neuroprotection of EA pretreatment, which usually is related to upregulation of Bcl-2/Bax and downregulation of cleaved caspase-3 [31]. Simultaneously, mediation of signaling pathway is also associated with the anti-apoptosis process of EA pretreatment in CIRI and ischemic stroke [32]. A recent study showed that EA pretreatment elicited tolerance to CIRI through inhibiting GluN2B/m-calpain/p38 MAPK proapoptotic pathway [24]. Utilizing MCAO rat model, EA pretreatment exerted the neuroprotective effects by inhibiting neuronal apoptosis and autophagy via activating PI3K/AKT/mTOR signaling pathway after ischemic stroke [33]. Other publication reported that EA pretreatment exerted neuroprotection against ischemic injury through Notch pathway-mediated upregulation of hypoxia inducible factor-1α [34]. Consistent with previous publications, EA pretreatment increased the level of anti-apoptotic protein Bcl-2 as well as decreased the expression of pro-apoptotic protein Bax and cleaved caspase-3 (Figs. 3 and 4). Different with above reports, inhibition of NF-κB signaling activation participated in the anti-apoptosis activity of EA pretreatment (Fig. 5). Whether other signaling pathways (such as PI3K/Akt, P38 MAPK, and Notch) or transcription factors (HMGB1, HIF-1α, and CREB) get involved in the neuroprotection of EA pretreatment against ischemic stroke, further studies are needed to shed light on the details. TRPV1 is a nonselective calcium-permeable cation channel and are mainly expressed in the hippocampus and dorsal root ganglion [35]. There is accumulating evidence indicating the potential involvement of TRPV1 channels in ischemia-reperfusion injury [36]. Ischemic stroke incurred activation of TRPV1 in brain of mice, where neurological de cits and infarct volumes in TRPV1-KO mice were lower than those in WT mice [37]. Additionally, the mRNA expression of TRPV1 was signi cantly increased in the brain tissues of MCAO rats [38]. Utilizing a rat model of stroke, administration of AMG9810 (a TRPV1 antagonist) signi cantly reduced the infarct volume and decreased the neurological de cits. However, capsaicin (a TRPV1 agonist) increased the infarct volume after the induction of permanent MCAO [39]. Collectively, these studies suggest that TRPV1 exerts a deleterious effect on brain ischemic injury. Although accumulating data indicates the effects of EA on TRPV1 in the different experimental conditions, such as pain [40][41][42], spared nerve injury [43] and obesity [44], few studies focused on the relationship between EA treatment/pretreatment and TRPV1 in CIRI or stroke. A previous study reported that TRPV1 receptors were signi cantly increased in the hippocampal CA1 areas of MCAO rats, and the phenomenon was reversed by EA treatment at GV20 [45]. EA pretreatment exerted neuroprotection in MCAO rats with CIRI through TRPV1-mediated anti-oxidant stress and anti-in ammation via inhibiting P38 MAPK activation [12]. In the current study, TRPV1 expression was increased in the brain of MCAO rats, and EA pretreatment reduced the increment of TRPV1 in rat hippocampus (Figs. 6 and 7). Importantly, capsaicin signi cantly abrogated EA pretreatment-triggered anti-apoptosis and inhibition of NF-kB nuclear translocation (Fig. 8-11), which highlighted the involvement of TRPV1-mediated antiapoptosis and suppression of NF-κB signaling activation in EA pretreatment-evoked neuroprotection.
In conclusion, EA pretreatment at GV20, bilateral BL23 and SP6 acupoints decreased TRPV1 expression in the MCAO rats, thereby curbed NF-κB transcriptional activity and played an anti-apoptosis role, contributing to the neuroprotective effect of EA pretreatment in CIRI (Fig. 10). These ndings suggest the involvement of TRPV1-mediated anti-apoptosis in EA pretreatment-evoked neuroprotection via inhibiting NF-κB signaling activation, which provide a theoretical basis for the clinical application of EA pretreatment in the prevention of cerebral ischemia-reperfusion injury or ischemic stroke. Figure 1 EA pretreatment attenuates cerebral ischemia-reperfusion injury in MCAO rats. (a) Location of GV20, BL23 and SP6 acupoints in rat. (b) EA stimulation in SD rats. Red circles indicated the corresponding acupoints as mentioned above. (c) Schematic diagram of the experiment I as described in experimental protocols. (d) Representative TTC staining showed noninfarct (red) and infarct (white) regions of brain tissue section in the different groups at 6 h after reperfusion. (e) Quanti cation of infarct volume among the four groups was statistically analyzed (***P < 0.001 vs. MCAO; n = 8/group, data were expressed as mean ± SD).     was assessed by Western blot and (d) statistical analysis was shown (**P < 0.01 and ***P < 0.001 vs. MCAO; n = 5/group, data were expressed as mean ± SD).

Figure 7
Colocalization of TRPV1 and NeuN in the hippocampal neurons. Immuno uorescence images of brain tissue sections showed colocalization of TRPV1 (green) and NeuN (red) in the hippocampal neurons. Cell nuclei were stained with DAPI (blue). Merge panels were overlay images presenting triple-labeled cells (yellow). Zoom panels were magni ed images of the merge panels. Scar bar = 100 μm.

Figure 8
Capsaicin abrogates EA pretreatment-evoked neuroprotection against cerebral ischemia-reperfusion injury in MCAO rats. (a) Schematic diagram of the experiment II as described in experimental protocols.
(b, c) Representative Western blot showed TRPV1 expression in rats with or without capsaicin (TRPV1 agonists) application and statistical analysis was shown. (d) TRPV1 expression in the different groups were detected by Western blot. (e) Relative protein expression of TRPV1/GAPDH was statistically analyzed. (f) Representative TTC staining showed noninfarct (red) and infarct (white) regions of brain tissue section in the different groups. (g) Quantitative analysis of infarct volume in the four groups of rats was shown (***P < 0.001 vs. Control, #P < 0.05 and ##P < 0.01 vs. EA + MCAO; n = 8/group, data were expressed as mean ± SD). and cleaved caspase-3-positive cells in the different groups was shown, separately (##P < 0.01 and ###P < 0.001 vs. EA + MCAO; n = 5/group, data were expressed as mean ± SD).

Figure 11
Capsaicin revoked the suppressive effect of EA pretreatment on NF-κB signaling activation in MCAO rats.

Figure 12
Proposed mechanisms for neuroprotection of EA pretreatment against cerebral ischemia-reperfusion injury in rats. EA pretreatment at GV20, bilateral BL23 and SP6 acupoints decreased TRPV1 expression in hippocampus of the MCAO rat. On the one hand, our previous study reported that downregulation of TRPV1 inhibited CIRI-induced mitochondrial damage and P38 MAPK phosphorylation, resulting in subsequent suppression of oxidative injury and in ammation. On the other hand, accompanying reduction of TRPV1 expression in the current study, EA pretreatment signi cantly prevented NF-κB transcriptional activity and regulated apoptosis-related protein expression, such as decreasing Bax and cleaved caspase-3 as well as increasing Bcl-2, which contributes to the anti-apoptosis activity of EA pretreatment in MCAO rats. The current ndings shed light on the involvement of TRPV1-mediated antiapoptosis in EA pretreatment-evoked neuroprotection against cerebral ischemia-reperfusion injury via inhibiting NF-κB signaling pathway.