| Objective The epidemiological survey shows that the morality from stroke preponderates over the tumor and cardiovasculor dieases in China,which is a leading cause of death.In this study,we imitate the Middle Cerebral Artery Occlusion(MCAO)of Rat model.According to the principle of tip-root acupoint combination,we choose the following three acupoint: Baihui(GV20),Shenshu(BL23)and Sanyinjiao(SP6).The tip acupoint is GV20,which can induce resuscitation and the root acupoint are BL23 and SP6,which can nourish liver and kidney,remove channel and active collaterals.To observe the influence on electroacupuncture pretreatment on inflammation、oxidative stress and apoptosis in hippocampus in MCAO,and explore the balance mechanism of reglating the interior milier homeostasis via MAPK pathway,which offer the important theory foundation and guidelines for the application of the principle of tip-root acupoint combination in the clinic,and the electroacupuncture pretreatment for Cerebral Ischemia and Reperfusion Injury(CIRI)and its related disease.Methods 128 Specific-pathogen-free adult male Sprague Dawley rats wew housed with free access to food and water.After 1 week,these rats were randomly divided in Control group、Sham group、MCAO group and EA+MCAO group.Rats in MCAO group were given the MCAO model by the method of thread tying.The sham group had the same operation as the rats with MCAO group except uninserted silicone thread to block the vessels.The Control group received no treatment.1.Neurological function assessment : neurological function scores were assessed at 3h,6h,12 h and 24 h after reperfusion.2.Morphological observation: rats were sampled 6h after reperfusion,TTC staining was used to detect the volume of cerebral infarction,HE staining was used to observe the microstructure of neurons in the hippocampus,and transmission electron microscopy was used to observe the ultrastructure of neurons in the hippocampus.3.Detection of cytokine: rats were sampled 6h after reperfusion,and the expression of TNF-α in the hippocampus was detected by immunohistochemistry,and the TNF-α and IL-1β in the hippocampus were detected by Elisa.4.Detection of oxidative stress indicators: MCAO model rats were sampled 6h after reperfusion,and the ultrastructure of hippocampal neuron mitochondria was observed by transmission electron microscopy.The expression of Cyt-c in the hippocampal was detected by immunohistochemistry,and the expression of SOD,MDA and GSH in the hippocampal brain tissue was detected by Elisa.5.Detection of apoptosis: the expression of active-caspase-3,Bax and Bcl-2 in the hippocampus was detected by immunohistochemistry,and the expression of active-caspase-3,Bax and Bcl-2 in the hippocampus was detected by WB.6.Detection of the expression of MAPK family proteins: WB detected the expression of MAPK pathway-related proteins in the hippocampus,including ERK,JNK,P38 MAPK and phosphorylated p-ERK,p-JNK,p-P38 MAPK,and calculated the ratio of non-phosphorylation to phosphorylation.Results 1.Electropuncture pretreatment had a most notable intervention effect on the MCAO model rats in the reperfusion group of 6h The neurological function of rats were evaluated after reperfusion 3h,6h,12 h respectively.Neurological function scores showed that,compared with MCAO group of rats at the same time point,electropuncture pretreatment had a most notable intervention effect on the MCAO model rats in the reperfusion group of 6h.Hence,in follow-up experiments,we chose 6h as the observation point in the research of the mechanism of electropuncture treatment.2.Electropuncture pretreatment alleviated the morphological abnormities of hippocampal neurons in MCAO model rats ⑴Electropuncture pretreatment reduced the volume of cerebral infarction.Normal brain tissue is normal due to dehydrogenase activity,and TTC staining presents dark red,indicating that it is normal active tissue,while necrotic tissue is lost due to dehydrogenase activity,TTC staining is white,and the size of white area represents the size of infarct volume.Stained images showed no obvious infarcted areas in the Control group and in the sham group.Compared with MCAO group,the infarct volume of EA+MCAO group was significantly reduced(P < 0.05).⑵ Electropuncture preconditioning group can reduce hippocampal neuron damage.Histopathological abnormities of hippocampal neurons were observed by HE staining,and no obvious abnormalities were observed in the Control group and in the sham group.In the rat brain of Control group,neurons in the hippocampus were disordered,a large number of vacuoles were observed in the cells,the nuclei were dissolved or even disappeared,and a large number of cells were degenerative necrosis.In EA+MCAO group,the neurons in the hippocampus were arranged unneatly,the vacuoles in the cells were significantly reduced,and the nuclei were pinched.⑶ Electropuncture pretreatment can reduce the damage of hippocampal neuron organelles.The cell membrane boundary was clear and the morphology was normal in the Control group and the sham group.In MCAO group,cytolysis,cell membrane disruption,a large number of vacuoles in the cytoplasm,nuclear condensation,and nuclear chromatin gradually gathered to the center into irregular clumps,or even karyolysis were observed.In EA+MCAO group,the cell membrane was blurred,a small number of tiny vacuoles appeared in the cytoplasm,the endoplasmic reticulum pool was enlarged,and the changes of chromatin in the nucleus were not evident.3.Detection of inflammatory indicators ⑴ The expression of TNF-α and IL-1β were detected by Elisa There was no significant difference between the Control group and the sham group(P>0.05).TNF-α and IL-1β were increased in MCAO group compared with the Control group and the sham group(P<0.05).In EA+MCAO group,there was a significant decrease in the level of TNF-α and IL-1β,and the difference was statistically significant(P<0.05).⑵ The expression of TNF-α was detected by immunohistochemistry There was no significant difference between Control group and the sham group(P>0.05).Compared with the Control group and the sham group,there was a significant decrease in the level of TNF-α and IL-1β in EA+MCAO group,and the difference was statistically significant(P <0.05).The number of TNF-α positive cells in EA+MCAO group were significantly lower than that in MCAO group(P<0.05).4.Detection of oxidative stress related indicators ⑴ Electropuncture pretreatment reduced the mitochondrial swelling and vacuolation rate of hippocampal neurons in model rats Mitochondria are the structural units in which cells conduct electronic oxidation and generate energy.No obvious abnormalities were found in the mitochondria of the Control group and in the sham group.In the MCAO group of rats,the characteristic signs of mitochondrial damage appeared,such as the lack of typical tubular or oval shape,the disappearance of double-membrane structure,vacuolation,mitochondrial crest interruption and even disappearance;There were also obvious changes in EA+MCAO group,which showed that the mitochondria were round and slightly swollen,and the mitochondrial ridge was swollen.⑵ Electropuncture pretreatment reduced the expression of Cyt-c in the hippocampal area of model rats Cyt-c is an electron transporter in the process of biological oxidation.With the increase of electron transport,it will consume a large number of oxygen atoms and generate a large number of oxygen free radicals.Immunohistochemistry showed no difference in the positive expression of Cyt-c between the Control group and the sham group,while there was a significant increase in the expression of Cyt-c in rats of MCAO group,which was statistically significant compared with the Control group and the sham group(P < 0.05).The expression of Cyt-c was also significantly increased in EA+MCAO group,and the difference was statistically significant compared with the Control group and the sham group(P<0.05),but decreased compared with MCAO group(P<0.05).⑶ Electropuncture pretreatment regulated the expression of MDA,SOD and GSH in the hippocampal tissues of MCAO rats There was no significant difference in MDA between the Control group and the sham group(P>0.05).Compared with the Control group and the sham group,there was a significant increase in the expression of MDA in rats of MCAO group,and the difference was statistically significant(P<0.05).In EA+MCAO group,there was a significant decreasein the expression of MDA in rats of MCAO group,,and the difference was statistically significant(P<0.05).The antioxidant factors SOD and GSH in the Control group and the sham group showed no statistical significance(P>0.05).SOD and GSH were decreased in MCAO group,compared with the Control group and the sham group(P <0.05).In EA+MCAO group,SOD and GSH were significantly increased in MCAO group(P<0.05).5.Expression of apoptosis-related indicators ⑴ Immunohistochemistry and WB were used to detect the expressions of Bax and Bcl-2 The expression of Bcl-2/Bax was no significant difference between the Control group and the sham group(P>0.05).Compared with the control group and the sham group,the expression of bax was increased,the expression of bcl-2 was decreased and the ratio of bcl-2 to bax was significantly decreased in the middle cerebral artery ischemia group,the difference was statistically significant(P<0.05).Compared with the middle cerebral artery ischemia group,the expression of bax was significantly decreased,the expression of bcl-2 and the ratio of bcl-2 to bax were increased in electropuncture pretreatment group,the difference was statistically significant(P<0.05).⑵ Immunohistochemistry and WB detection showed the expression of Cleaved caspase-3 There was no significant difference in the expression of cleaved caspase-3 between Control group and sham group(P>0.05).Compared with the control group and the sham group,the expression of Cleaved caspase-3 in the middle cerebral artery ischemia group was significantly increased,and the difference was statistically significant(P < 0.05).Compared with the middle cerebral artery ischemia group,the expression of Cleaved caspase-3 in EA+MCAO group was decreased,and the difference was statistically significant(P <0.05).6.Expression of MAPK family proteins ⑴ Compared with the sham group,the ratio of p-ERK and ERK in the cerebral ischemia group and EA+MCAO group was increased,and the difference was statistically significant(P <0.05).However,there was no significant difference between the middle cerebral artery ischemia group and EA+MCAO group(p > 0.05).⑵ There was no significant difference in the ratio of p-JNK and JNK between the four groups,and the difference was no statistically significant(P>0.05).⑶ p-P38/P38 was significantly increased in the middle cerebral artery ischemia group and EA+MCAO group compared with the control group and the sham group,and the difference was statistically significant(P<0.05).And The ratio of p-p38 to p38 in EA+MCAO group was significantly lower than that in the rat brain ischemia group(p <0.05).Conclusion 1.Electropuncture pretreatment can effectively reduce the volume of cerebral infarction in MCAO model rats and reduce the degree of neuron damage.2.Electropuncture pretreatment can inhibit the expression of pro-inflammatory factors TNF-α and IL-1,control the inflammatory response,and play a role in protecting neurons.3.Electropuncture pretreatment can reduce mitochondrial damage,inhibit the expression of Cyt-c,and play a neuroprotective role by regulating the imbalance between oxidation and antioxidant factors.4.Electropuncture pretreatment can regulate the imbalance of Bcl-2 /Bax,reduce the expression of active-caspase-3,and thereby inhibit the apoptosis of nerve cells to achieve the effect of nerve pro tection.5.Electropuncture pretreatment plays a neuroprotective role by regulating the expression of p-p38 mapk in MAPK family proteins. |
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