The Mechanism Of Qingnao Dropping Pill On Acute Cerebral Ischemia-reperfusion Injury Was Studied Based On The Regulation Of Apoptosis By Glial Cell HIF-1α Pathway

The 2010 Global Burden of Disease(GBD)showed that stroke was ranked second in the cause of death,and in 2004-2005,China’s third annual death review sample survey showed that cerebrovascular disease was the first Death,2010 disease burden report shows cerebrovascular disease is still the first death.Among them,the incidence of ischemic stroke age is much higher than the incidence of hemorrhagic stroke.In-depth study of the pathogenesis and treatment of ischemic stroke,has important medical value.Glial cells are a large group of cells in the nervous system,accounting for about 90%of the total number of central nervous system cells,in addition to support,to provide nutrition and help metabolism and other supporting role,while excitatory,involved in neuronal communication and promote contact formation and regeneration of nerve functions.In the event of acute cerebral ischemia,astrocytes are first damaged,damaged glial cells kill adjacent neurons,and damaged astrocytes will apoptosis,and then lead to neurons the secondary damage,and finally to expand the area of infarction.Under hypoxia conditions,hypoxia inducible factor-1(HIF-1)can directly or indirectly regulate the transcription of more than 100 downstream target genes into a hypoxic response to stabilize the intracellular environment of the regulatory center.HIF-1 is composed of stably expressed HIF-1β subunit and hypoxia-regulated HIF-1α subunit.HIF-lα is widely expressed in brain tissue,including neurons,glial cells and blood vessels Endothelial cells,etc.,which has a dual role in cerebral ischemia.When mild ischemia and hypoxia,HIF-1α can activate its downstream genes,by increasing angiogenesis,regulating glycolysis and other ways to reduce apoptosis.HIF-1α can induce apoptosis by binding to p53,or induce the expression of BNIP3(BCL2/adenovirus E1B 19kDa interacting protein 3)in the bcl2(B-cell lymphoma 2)family,Apoptosis,HIF-1 binds to the hypoxia-response element(HRE)sequence of the BNIP3 gene promoter under hypoxia to promote the expression of BNIP3.And BNIP3 can promote apoptosis by promoting H+ influx or cytochrome C release.Qingnao dripping pills(also known as Qingnao Xuanqiao dripping pills)is a long-term clinical practice summary of the prescription,the treatment of ischemic stroke in the early stage and early recovery have a good effect.This side by gardenia,Sanqi and borneol threeherbs composition,three drugs played a total of heat Xiehuo,Huoxue Xuanqiao effect,clinical research reports,Qingnao dripping pills to improve TCM syndrome,especially on the"Headache,dizziness,mouth pain throat,constipation,tongue coating yellow "hot,blood stasis symptoms have improved significantly,can improve the degree of neurological deficits in patients.Experimental study found that Qingnao dripping pills can reduce the acidosis,inhibit free radical production and adhesion molecules to regulate the expression of cerebral ischemia-reperfusion injury metabolic disorders,inhibition of JNK phosphorylated protein overexpression to reduce apoptosis,reduce the brain damage.[Research purposes]To investigate the effect of Qingnao dripping pills on cerebral ischemia-reperfusion injury and C6 glial cell oxygen glucose deprivation injury induced by HIF-la,and to elucidate the molecular mechanism of Qingnao dripping pill to protect cerebral ischemia-reperfusion injury.[Research methods]1.C6 rat glial cells were cultured in vitro,and the model of cell injury was established by oxygen glucose deprivation/reoxygenation.(0.625 g/L,QNDW 1.25 g/L),CCK8 method was used to determine the cell viability,and the best time and the best dose.was determined by the method.The expressions of HIF-1α,BNIP3 and Caspase-3 were observed by Western Blot.The expression of HIF-1α and Caspase-3 were observed by immunofluorescence staining.2.The model of cerebral ischemia-reperfusion injury was established by suture method.The degree of neurological deficit and the infarct volume were measured by Garcia GH and TTC staining.Western blot was used to detect the expression of HIF-1α,BNIP3 and Caspase-3 protein were detected by flow cytometry.The expression of HIF-1α and GFAP was detected by fluorescence staining.[Research result]1.Cell experiments①Compared with the corresponding normal group,the cell viability(P<0.01)was observed oxygen glucose deprivation 24h/reoxygenation 3h or 6h,and the cell viability was about 50%Point for the oxygen glucose deprivation 24h/reoxygenation 6h.② Compared with the model group(P<0.05),the cell viability of Qingnao dripping pills was significantly higher than that of the control group(P<0.05)The cell viability was the highest at 1.25 g/L concentration.③Western blotting showed that the expression of HIF-1α,BNIP3 and Caspase-3 protein in the 24h/reoxygenation group was significantly higher than that in the normal group(P<0.05).Compared with the normal group,the expression of HIF-1α,Compared with the 6h group,the expression of HIF-1α,BNIP3 and Caspase-3 were decreased(P<0.05)in the 1.25g/L concentration group.④Compared with the normal group,the expression of HIF-1α and Caspase-3 in the 24 h/h group was significantly higher than that in the normal group(P<0.05),and the expression of HIF-la and Caspase-3 was significantly decreased in 1.25 g/L group.2.Animal experiments①The neurological deficit score was decreased(P<0.01).Compared with the sham group,the cerebral infarct size was increased(P<0.05)(P<0.05),and the neurological deficit scores were increased(P<0.01),and the cerebral infarct size of the group was significantly lower than that of the control group(P<0.01).② Western blot analysis showed that the expression of HIF-1α,BNIP3 and Caspase-3 protein in the 24h group was significantly higher than that in the sham group(P<0.05)Compared with HIF-1α inhibitor group,the expression of HIF-1α,BNIP3 and Caspase-3 were decreased(P<0.05),and the levels of HIF-1α,BNIP3,Caspase-3 protein overexpression.③Immunofluorescence staining results:HIF-1α and GFAP can co-expression in cells,that is,HIF-1α can be expressed in astrocytes.(P<0.05).Compared with normal group,the expression of HIF-1α was significantly increased in 1.5 h reperfusion group(P<0.05).Compared with ischemia group(1.5 h),the levels of HIF-1α The expression of HIF-1α was significantly decreased(P<0.05).[Conclusion]From the level of cell molecules and the overall level found that Qingnao dripping pills can inhibit glial cell apoptosis to reduce acute cerebral ischemia and reperfusion injury,the mechanism may be by down-regulation of ischemia-reperfusion injury in the cerebral cortex of HIF-1α And to inhibit the expression of downstream gene BNIP3 to a certain extent,and ultimately reduce the expression of Caspase-3 protein in the promoter of the apoptotic promoter,and to reveal the pathogenesis of acute cerebral ischemia and reperfusion Scientific connotation.The innovation of this studyFrom the point of view of HIFla and apoptosis,the protective mechanism of brain drop pills on cerebral ischemia reperfusion injury was explained at the level of tissue,cell and molecule,which provided scientific basis for clinical medication.