| ObjectiveAs a major disabling disease,cerebral ischemia is the third leading cause of death in North American,European and Asian populations.Clinically in Chinese medicine,EA can significantly improve cerebral ischemic injury.The World Health Organization(WHO)recommends acupuncture as an alternative/complementary therapy to treat and improve stroke.The results of clinical trials and meta-analysis have proved that the efficacy of acupuncture in improving neurological deficits and overall health after stroke,but its mechanism is not completely clear.Baihui(GV20)and Dazhui(GV14)are commonly used and recognized acupoints that can promote the repair of the nervous system.In this study,starting from the aspects of behavior and molecular biology,we observed the effects of EA at Baihui and Dazhui acupoints on the neurological function,cell morphology,and related proteins and pathways in cerebral ischemic modeling rats,to clarify the regulation of the EPO-JAK2-STAT5 signal pathway and the cumulative effects of EA,and to clarify the correlation between EA stimulation-signal regulation-nerve homeostasis initiation,and finally provides experimental basis for clinical application and promotion of EA at Baihui and Dazhui point.Methods310 SD rats(SPF grade),half male and half male,were randomly divided into sham group and model group.The middle cerebral artery was blocked by electrocoagulation to establish a focal cerebral ischemia rat model,and the rats that have been successfully modeled are randomly divided into model group,EA group,AG490 group,EA+AG490 group,and each group was divided into 5 subgroups:2 hours(h),12 hours(h),24 hour(h),48 hours(h)and 72 hours(h),12 rats in each group.The sham group was also divided into five subgroups(12 rats in each group)according to the above time points,which rats were only exposed the middle cerebral artery without clotting.The rats in the AG490 group and the EA+AG490 group were injected with AG490 into the lateral ventricle,and 20 mi later,the middle cerebral artery was electrocoagulated to create the focal cerebral ischemia model.The rats in the EA group and EA+AG490 group were treated with EA at Baihui and Dazhui acupoints for 30 min each time with a sparse density wave,20 times/min,2-3 V.After 2 h,12 h,24 h,48 h,72 h ischemia,the rats were scored and recorded according to the Zea-Longa 5-point system.After scoring,the rats were anesthetized and then killed,and brain tissues were taken for examination.(1)Electron microscopy:Take the 1 mm×1 mm×1 mm cerebral cortex around the ischemic focus and observe the ultrastructure of nerve cells with JEM-1200EX transmission electron microscope;(2)HE staining and TUNEL staining:observe the damage and apoptosis of cerebral cortex tissue cells in ischemic focus;(3)Using RT-PCR and immunohistochemistry techniques to detect changes in the expression of cortical apoptosis-related genes/proteins and EPO-JAK2-STAT5 pathway-related genes/proteins at different periods of time.Results(1)The effects of EA on the neuromotor function of rats with focal cerebral ischemia.One-way analysis of variance between groups showed:the nerve function of the rats in the sham group was completely normal,and the nerve function defect score was 0.00±0.00.After electrocoagulation of the middle cerebral artery in rats,during the 12-72 h period of cerebral ischemia,the neurological deficit score of the model group remained at a high level,which was significantly higher than that of the sham group(P<0.05),but there is no significant difference between each time point(all P>0.05).After 24 h of EA treatment,the neurological deficit score had a downward trend,but there was no significant difference(P>0.05);after EA treatment for 48 h(including 72h),the neurological deficit scores were significantly decreased(P<0.05).After injection of AG490 into the lateral ventricle,the neurological deficit score was significantly higher than that of the model group(P<0.05).However,after combination of AG490 and EA treatment,the neurological deficit score was still at a high level.One-way analysis of variance at different time periods showed:Compared with 12 h,the scores of rats in the model group at 48 h and 72 h increased significantly.When EA treatment for 48 h and 72 h,the scores decreased significantly(P<0.05);after AG490 intracerebroventricular injection,compared with 12 h,the scores at 24 h,48 h and 72 h increased significantly,and compared with the previous period,there was a statistical difference(P<0.05).In the EA combined with AG490 group,the change trend of the neurological deficit score was similar to that of the AG490 group,and the score continued to increase(P<0.05).(2)The effects of EA on nerve tissue damage in rats with focal cerebral ischemia.HE staining observed that the neurons in the sham group were arranged regularly at each time point,and the nucleus was elliptical.With the prolongation of ischemia time(2 h-24 h),the neuronal damage in the cerebral ischemia model rats continued to increase.After 24 h(24 h-72 h)ischemia,the morphology of neurons in the model group did not change much.After EA treatment for 2h and 12h,the neurons morphology was similar to the model group.After EA treatment for 24 h(including 48 h and 72 h),the chromatin condensation phenomenon was gradually improved,the number of neurons gradually increased,and the arrangement was more orderly.However,after AG490 treatment for 2 h(including 12 h,24 h,48 h and 72 h),rats in the AG490 group had obvious chromatin coagulation,and the number of neurons was significantly reduced,and with the passage of time,chromatin coagulation was more obvious,and the number of neurons continued reduce.When AG490 was injected into the cerebral ventricle of rats,the effect of EA on improving chromatin coagulation and reducing neuronal apoptosis was significantly reduced,and the repairing effect of EA on brain neurons was significantly weakened.(3)The effects of EA on neuronal apoptosis in rats with focal cerebral ischemia.One-way analysis of variance between groups showed:there are fewer TUNEL stained cells at each time point in the sham operation group,and there was a very small number of cells undergo apoptosis.With the prolongation of ischemia time(2 h-24 h),the apoptosis in the cerebral ischemia model rats continued to increase.After 24 h(24 h-72 h)ischemia,the apoptosis in the model group was basically unchanged.After EA treatment for 2 h and 12 h,the apoptosis gradually increased with the prolongation of ischemia time,and there was no significant difference compared with the model group;after EA treatment for 24 h,the EA group had the most serious apoptosis.After treatment for 48 h and 72 h,compared with the model group,the number of apoptosis in the EA group was significantly reduced,meanwhile TUNEL index was significantly decreased.When AG490 was injected into the rat’s lateral ventricle,the TUNEL index continued to increase with the prolongation of AG490’s action time.After AG490 was injected into the lateral ventricle of rats and given EA at the same time,the TUNEL index continued to increase within 24 h of ischemia;after 24 h of ischemia,the TUNEL index showed a continuous downward trend.One-way analysis of variance at different time periods showed:there was no significant difference in the TUNEL index of rats in the sham group at different time periods(P>0.05).Compared with 2 h,the TUNEL index of rats in the model group increased significantly at 12 h,24 h,48 h and 72 h,and compared with the previous period,there were statistical differences(all P<0.05).The TUNEL index reached the highest value at 24 h after EA treatment,and then the TUNEL index began to decrease(P<0.05);after AG490 lateral ventricle injection,compared with 2 h,the TUNEL index at 12 h,24 h,48 h and 72 h significant increase,compared with the previous period,there were statistical differences(P<0.05).The change trend of the TUNEL index in the EA combined with AG490 group was similar to that in the AG490 group,and the TUNEL index showed a downward trend at 48 h(including 72 h)after EA treatment(P<0.05).(4)Effects of EA on EPO-JAK2-STAT5 pathway related genes/proteins in focal cerebral ischemia model rats.One-way analysis of variance between groups showed:Compared with the sham group,the levels of HIF-1α/EPO mRNA/proteins in the model group increased significantly(P<0.05),and with the prolongation of ischemia time,their expression levels continued to increase.After EA treatment for 12 h,24 h,48 h and 72 h,the levels of HIF-1α/EPO mRNA/proteins were significantly higher than that of the model group(P<0.05).After injection of AG490 into the lateral ventricle,there was no significant difference in the levels of HIF-1α/EPO mRNA/proteins in the AG490 group,compared with the model(P>0.05).After combination of AG490 and EA treatment,the levels of HIF-1α/EPO mRNA/proteins was significantly higher than that of the model group/AG490 group(P<0.05),but not significantly difference compared with the EA group(P>0.05).JAK2 and STAT5 mRNA levels were not significantly different among the groups and different time periods(P>0.05).Compared with the sham group,the levels of p-JAK2/p-STAT5 proteins in the cerebral ischemic focus of the model group were significantly increased(P<0.05).After EA treatment for 24 h,48 h and 72 h,the protein levels of p-JAK2/p-STAT5 were significantly higher than that of the model group(P<0.05).2 h after the injection of AG490 into the lateral ventricle,the levels of p-JAK2/p-STAT5 proteins in the AG490 group was significantly lower than that of the model group/EA group(P<0.05).After combination of AG490 and EA treatment,the protein levels of p-JAK2/p-STAT5 was significantly lower than that of the model group/EA group(P<0.05),but there was no significant difference compared with AG490 group(P>0.05).One-way analysis of variance at different time periods showed that there was no significant difference in HIF-1α/EPO mRNA/protein levels in the sham group at different time periods(P>0.05).Compared with 2 h,the HIF-1α/EPO mRNA/protein levels in the model group increased continuously at 12h,24 h,48 h and 72 h,which were statistically different from the previous period(all P<0.05).Compared with the 2 h of the corresponding group,the levels of HIF-1α/EPO mRNA/protein levels at 12 h,24 h,48 h and 72 h in EA group,AG490 group and EA+AG490 group increased,and compared with the previous period,there were statistical differences(all P<0.05).Compared with the corresponding group at 2 h,there was no significant change in the relative expression of JAK2/STAT5 mRNA in the model group,EA group,AG490 group and EA+AG490 group at each time period(P>0.05).There was no significant difference in p-JAK2/p-STAT5 protein levels in the sham group at different time periods(P>0.05).Compared with 2 h,the protein levels of p-JAK2/p-STAT5 in the model group was at a stable high level at 12 h and 24 h.After 48 h(including 72 h),the levels began to decrease,significantly lower than 2 h(all P<0.05).After 24 h,the p-JAK2/p-STAT5 protein level in the EA group increased significantly,and compared with the previous period,the difference was statistically significant(P<0.05).After the intervention of AG490,the p-JAK2/p-STAT5 protein levels were at a reduced level at each time period,and there was no significant change(P>0.05).(5)The effects of EA on the apoptosis-related genes/proteins(HSP70s,Bax and Bcl-2)of focal cerebral ischemia in rats with focal cerebral ischemia.One-way analysis of variance between groups showed:compared with the sham group,the levels of HSP70/Bcl-2 mRNA/proteins in the model group were significantly increased(P<0.05).After EA treatment for 2 h,12 h,24 h,48 h and 72 h,the levels of HSP70/Bcl-2 mRNA/proteins gradually increased,and after 48 h and 72 h,the levels of HSP70/Bcl-2 mRNA/proteins increased significantly(P<0.05).After injection of AG490 into the lateral ventricle,there was no significant change in the levels of HSP70/Bcl-2 mRNA/proteins(P>0.05).After combination of AG490 and EA treatment,the levels of HSP70/Bcl-2 mRNA/proteins were significantly higher than that in the AG490 group(P<0.05).Compared with the sham group,the levels of Bax mRNA/proteins in the model group increased significantly(P<0.05).After EA treatment for 12 h,24 h,48 h and 72 h,the Bax mRNA/proteina levels were significantly lower than that of the model group(P<0.05).After intracerebroventricular injection of AG490,the levels of Bax mRNA/protein in the AG490 group and the EA+AG490 group had no significant changes compared with the model group(P>0.05).One-way analysis of variance at different time periods showed:compared with the 2 h of the corresponding group,the levels of HSP70/Bcl-2 mRNA in the EA group and EA+AG490 group increased significantly at 24 h,48 h,and 72 h,and the levels at 24 h,48 h and 72 h were all significant differences compared with that of the previous period(all P<0.05).Compared with the 2 h of the corresponding group,the HSP70 protein levels in the EA group and the EA+AG490 group increased significantly at,48 h and 72 h,and the levels at 48 h and 72 h were all significant differences compared with that of the previous period(all P<0.05).Compared with 2 h,the Bcl-2 protein level in the model group increased significantly at 12 h,24 h,48 h and 72 h(P<0.05),reached the highest level at 12 h,and then gradually decreased.Compared with 2 h,the Bcl-2 protein level in the EA group increased significantly at 12 h,24 h,48 h and 72 h(P<0.05),and reached the highest level at 24 h,and then the level gradually increased,but higher than that of the model group in the corresponding period.Compared with 2 h,the Bcl-2 protein level in the AG490 group reached the highest level at 24 h,and then the level gradually decreased,which was lower than the model group in the corresponding period.Compared with 2 h,the Bcl-2 protein level in the EA+AG490 group increased significantly at 12 h,24 h,48 h,and 72 h(P<0.05),and reached the highest level at 48 h.There was no significant difference in Bax mRNA in the sham group at different time periods(all P>0.05).Compared with the corresponding group at 2 h,Bax mRNA in the model group,AG490 group,and EA+AG490 group increased significantly at 12 h,24 h,48 h,and 72 h,and the levels at 24 h,48 h and 72 h were all significant differences compared with that of the previous period(all P<0.05).Compared with 2 h,Bax mRNA in the EA group increased significantly at 12 h,24 h,48 h and 72 h,reached the highest level at 48 h,and began to decrease significantly at 72 h(all P<0.05).Compared with the 2 h of the corresponding group,the Bax protein levels in the model group,EA group,AG490 group and EA+AG490 group increased significantly at 24 h,48 h and 72 h,and the levels at 24 h,48 h and 72 h were all significant differences compared with that of the previous period(all P<0.05).Conclusion(1)After the MCAO rat model is successfully constructed by the electrocoagulation method,EA at Baihui and Dazhui acupoints can improve neurological deficits in rats with cerebral ischemia,reduce neurons apoptosis,and promote brain tissue repair.(2)The early intervention of EA after cerebral ischemia can improve the neurological symptoms and inhibit the expression of apoptosis-related proteins,which effect is related to the EA time(number of times),as the numbers of EA treatment increases,its role in promoting brain tissue repair is more obvious.(3)The effects of EA in promoting brain tissue repair and anti-apoptosis of brain tissue may be mainly through activating the HIF-1α/EPO/p-JAK2/p-STAT5 signal transduction pathway and promoting the expression of anti-apoptosis-related proteins(HSP70 and Bcl-2)and inhibition of Bax expression are achieved.However,HIF-1α/EPO/p-JAK2/p-STAT5 pathway is not the only pathway,EA may activate other pathways to inhibit cell apoptosis.(4)The application of EA in the treatment of cerebral ischemic diseases in this study may be through acupoint stimulation to increase the content of protein factors such as HIF,and activate the EPO-JAK2-STAT5 signal transduction pathway,thereby inhibiting cell apoptosis and promoting the repair of damaged brain tissue cell function.This study further verified the scientific hypothesis that "acupuncture stimulation-signal regulation-homeostasis regulation initiation is one of the key mechanisms of cerebral ischemic local self-repair",and perfected the molecular mechanism of acupuncture treatment of ischemic cerebrovascular disease. |
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