The Molecular Mechanism Research Of Acupuncture Intervention For ERS Regulating Cell Autophagy In Cerebral Ischemia Reperfusion Rats

Research BackgroundStroke has become a main cause of death in urban and rural residents'disease.Cerebral ischemia-reperfusion injury directly affects the prognosis of cerebral vascular disease.How to reduce cerebral ischemia reperfusion injury is the key to the prevention and treatment of cerebral ischemic disease.As a new way of programmed cell death,autophagy has great significance in the switch of"live"and"destruction"in cells.Under the condition of cerebral ischemia hypoxia stress,autophagy can remove aging organelles and misfolded proteins in the nerve cells,maintain cell survival and energy.It is an important metabolic activity.If excessive rised,it would lead to cell death.Therefore,the study provides the opportunity to further explore the new cells"live"and"death"regulation of nerve cells autophagy and its control mechanism.Recent studies have shown that un-folded protein response(UPR)in endoplasmic reticulum stress is an important signaling pathway inducing autophagy.After UPR signaling pathways are activated,unfolded proteins in the endoplasmic reticulum make GRP78 release PERK/elF2??Ire1/TRAF2?ATF6?,regulate downstream CHOP,and inhibit antiapoptotic proteins BCL-2,and ultimately affect autophagy by inducing autophagy related protein expression Beclin.Based on this new scientific discoveries,we suspect that acupuncture may relieve cerebral ischemia reperfusion injury is closely related to the process of UPR three signal transduction pathways to induce cell autophagy.The development of this research is to help further reveal the pathogenesis and prevention of cerebral ischemia reperfusion injury,which can provide objective experimental basis with important scientific significance and good prospect.The effect of UPR signaling pathways regulating autophagy by needling Shuigou(DU26)after cerebral ischemia and reperfusion injury would be dynamically observed.From the molecule function way of three signal transduction pathways PERK/elF2?,Ire1/TRAF2,ATF6?of UPR,the mechanism of autophagy,material base and effective acting targets by needling Shuigou(DU 26)after cerebral ischemia reperfusion injury would be further explained.It can provide experimental and theoretical basis of treating cerebral ischemia by acupuncture for the clinical application.Research MethodsThis research will be divided into four parts:Part One:Dynamic observation of alleviate ischemia reperfusion injury by acupunctureReference to improved Longa method,rat middle cerebral artery embolism focal ischemia reperfusion(MCAO/R)model was produced.According to experimental random number table method,rats were randomly divided into 3 groups,respectively control group,model group,acupuncture treatment group(n=15).The model groups and acupuncture treatment groups is further divided into 6 h,24 h,72 h subgroups.Nerve defect signs was observed by Bederson neural function defect score evaluation method,change of the cerebral infarction area and tissue morphological changes by TTC staining and light microscope.Part Two:Dynamic observation of autophagy change in ischemic reperfusion rats by acupunctureMCAO/R model was produced similar to A.According to experimental random number table method,rats were randomly divided into 3 groups,respectively control group,model group,acupuncture treatment group(n=4).The model groups and acupuncture treatment groups were further divided into 6h,24h,72h subgroups.Autophagy body in neural cell was observed by transmission electron microscope;the expression of autophagy related protein Beclin 1 and LC3 were detected by Western blot.Part Three:Dynamic observation of endoplasmic reticulum stress in ischemic reperfusion rats by acupunctureMCAO/R model was produced similar to A.According to experimental random number table method,rats were randomly divided into 3 groups,respectively control group,model group,acupuncture treatment group(n=4).The model groups and acupuncture treatment groups were further divided into 6h,24h,and 72h subgroups.Autophagy body in neural cell was observed by transmission electron microscope;the expression of GRP78,CHOP,PERK?p-eIF2 and IRE1 were detected by Western blot.Part Four:The mechanism of endoplasmic reticulum stress(ERS)regulating autophagy and acupuncture intervention effectAccording to the GRP78 gene sequences,we designed three kinds of jamming shRNA-GRP78 gene fragments,and on this basis,three different interferenced lentivirus were constructed.At the same time,a lentiviral vector LV-CHOP to over-express CHOP genes was framed.After packing lentiviral vector successfully with normal virus titer,LV-shRNA-GRP78 and LV-CHOP were detected in vitro.Identification method was infecting BRL cells respectively by LV-shRNA GRP78,LV-CHOP,detecting the expression of GRP78and CHOP genes with qPCR.1 days later,separate rat cortex,assess rat cortex mRNA change of GRP78 and CHOP with qPCR analysis.Finally,use Western blot to detect protein expression of Beclin-1after lentivirus combined acupuncture interfered in MCAO/R rats.ResultsPart One:Acupuncture on cerebral ischemia reperfusion(MCAO/R)in rats.A.Infarction area changes of MCAO/R model rat:TTC staining results showed that rose-red brain tissue in all sham groups and obvious white infarction areas in each phase of model and treatment groups.The volume of white infarction area increases over time.Compared with model group,white infarction area was decreased in treatment groups,especially in 24 h and 72 h.B.Nerve functional recovery in cerebral ischemia reperfusion(MCAO/R)model rats:the sham groups rats did not appear nervous function defect signs,while model group rats were obvious lack of nerve function.There is obvious difference between sham and model group at the same phase(p<0.01);nerve function defect score in acupuncture group rats were significantly lower than model groups,there is no statistical difference in 6 h after reperfusion(p>0.05),while significant statistical differences in 24 h and 72 h(p<0.05).C.The histomorphology changes in cerebral ischemia reperfusion brain:even dyeing nerve cells in sham groups,clear outline without edema.Depletion in numbers of nerve cells in model group,less clear of cellular level,swelling and disordered cells with pale cytoplasm.Acupuncture groups:swelling nerve cells,decreased cell numbers,while neuron structure is much recovered compared with 24 h,72 h model groups.Part Two:Dynamic observation of acupuncture on autophagy in ischemic reperfusion ratsA.Ultrastructure changes under electron microscope:nerve myelin damage in M6h group with normal chromatin distribution,swelling mitochondrial and mild expanding endoplasmic reticulum,autophagy bubble is forming.Multiple visible autophagy bubble in M24h group;rarely observed phage or lysosomes in M72h.Visible autophagy bubble in each acupuncture group were less than that in each phase of model group.B.The expression changes of Beclin-1,LC3 in MCAO/R rats:compared with sham group,relative expression of Beclin 1 protein in model groups increased,the difference was statistically significant(p<0.05);compared with model group,relative expression of Beclin1 protein in each point of acupuncture groups were reduced,there is statistically significance in 24 h and 72 h(p<0.05).LC3-II/LC3-I ratio was increased significantly in each model groups compared with sham groups(p<0.05).LC3-II/LC3-I ratio in 6 h acupuncture group reduce significantly compared with the same phase model group(p<0.05).Part Three:Acupuncture effect on endoplasmic reticulum stress in ischemic reperfusion ratsA.Western blot showed that the expression level of GRP78 protein in MCAO/R model rats up-regulated in each phase,peak 6h after MCAO/R.There is significant statistical significance compared with sham group(p<0.01).After acupuncture treatment,the expression decreased at 6h(p<0.05)but increased after 24h,72h.There is statistically significant difference between 24h groups(p<0.05).The expression of CHOP protein in model groups were obviously up-regulated,24h was the peak(p<0.01).CHOP expression decreased in acupuncture groups,there is statistically significant difference in 24h,72h groups(p<0.01).B.The expression of PERK?p-eIF2?both up regulated after 24 h ischemia-reperfusion in model group(p<0.01),while both decreased significantly in acupuncture group(p<0.01,p<0.01).Ire1 protein in 24h model group increased,and down-regulated after acupuncture treatment.There is statistically significant difference among three groups(p<0.05).Part Four:The mechanism of endoplasmic reticulum stress(ERS)regulating autophagy and acupuncture intervention effectA.We designed three kinds of jamming shRNA-GRP78 gene fragments,and on this basis,three different kinds of interfering lentivirus were constructed,LV-shRNA-GRP78-581,LV-shRNA-GRP78-1098,LV-shRNA-GRP78-1852.At the same time,we framed LV–CHOPandcalculatedthevirustiterofLV-shRNA-GRP78-581?LV-shRNA-GRP78-1098?LV-shRNA-GRP78-1852 and LV-CHOP,2.0×10~8?1.8×10~8?1.6×10~8 and 2.0×10~8TU/ml.B.After packing lentiviral vector successfully with normal virus titer,LV-shRNA-GRP78 and LV-CHOP were detected in vitro.(a)LV-shRNA-GRP78-581?LV-shRNA-GRP78-1098?LV-shRNA-GRP78-1852were built to infect BRL cells.The results of detecting the expression of GRP78mRNA with qPCR showed GRP78 mRNA levels were significantly lower than average irrelevant sequence of virus group(p<0.01);GRP78 mRNA level in LV-shRNA-GRP78-581 BRL cells was lower than that of LV-shRNA-GRP78-1098(p<0.01)?LV-shRNA-GRP78-1852(p<0.01)?(b)LV-CHOP and blank LV were built to infect BRL cells.The results of detecting the expression of CHOP mRNA with qPCR showed CHOP mRNA levels in LV-CHOP infected cells were obviously higher than that in blank sequence virus group(p<0.01).(c)Determine the best interference effect of LV-shRNA-GRP78-581 and LV-CHOP,inject them into rat's cortex.qPCR results showed both LV-shRNA-GRP78 group and LV-shRNA GRP78 combined acupuncture group have no obvious difference with normal group(p>0.05).In model group,the expression of GRP78 protein in LV-shRNA-GRP78 group and LV-shRNA GRP78 combined acupuncture group have no obvious difference(p>0.05).Therefore,transfection effect of LV-shRNA-GRP78-581 in vivo failed.After acupuncture on the model rats,CHOP mRNA decreased significantly(p<0.01),while CHOP mRNA increase obviously in LV-CHOP group(p<0.01);After acupuncture,CHOP mRNA significantly decreased(p<0.05),but still higher than model group(p<0.05).(d)Detect related expression of Beclin-1 in LV-CHOP combined acupuncture group with Western blot.Results showed that Beclin 1 protein in acupuncture group reduced with statistically significant difference(p<0.01);Beclin 1 protein in LV-CHOP group increased obviously(p<0.05);Compared with LV-CHOP group,Beclin-1 in LV-CHOP combined acupuncture group decreased,but much more than acupuncture group,there are significant differences(p<0.01).Conclusion1.Electroacupuncture at Shuigou has a definite therapeutic effect,which could promote recovery of cerebral ischemia reperfusion injury.2.The mechanism of electroacupuncture at Shuigou on cerebral ischemia reperfusion injury rats is closely related to inhibiting Beclin-l,LC3 expression,and down-regulating autophagy in the nerve cells.3.Electroacupuncture at Shuigou may regulate the expression level of GRP78,CHOP,UPR PERK/eIF2?and IRE1 pathway to influence the endoplasmic reticulum stress.4.Electroacupuncture at Shuigou to reduce cerebral ischemic reperfusion injury,maintain the cell survival has certain relevance with CHOP pathway regulating cell autophagy in endoplasmic reticulum stress.