| The incidence of ischemic stroke is increasing year by year,and the population has become younger,which seriously affects the health and quality of life of Chinese residents.At present,thrombolytic therapy is the first choice for the treatment of ischemic stroke.Because of the blood flow reperfusion of ischemic blood vessels after thrombolysis,the degree of brain tissue damage is further aggravated,that is,the damage of cerebral ischemia-reperfusion(I/R).The mode of neuron death caused by brain I/R injury is mainly apoptosis,which mainly occurs in the ischemic penumbra area around the ischemic central area,and the severity of brain ischemic injury is directly related to apoptosis.Mitochondrion apoptotic pathway is one of the main ways of cell apoptosis.Cyt C/Apaf-1 is the classic pathway of mitochondrion apoptotic pathway,which plays a key role in regulating cell apoptosis and maintaining the stability of mitochondrial function.Astragalus membranaceus has the effect of invigorating Qi and promoting blood circulation.It has been used by doctors of all ages to treat ischemic stroke and has achieved good results.Calycosin is an effective monomer component of astragalus flavonoids.It has been found that calycosin has biological effects of antioxidation,anti-inflammatory,anti-virus,liver protection and anti-aging.But does it have the effect of inhibiting brain I/R injury? Can it play a neuroprotective role by inhibiting cell apoptosis,or even regulating Cyt C/Apaf-1 mitochondrial apoptosis pathway,thus antagonizing brain I/R injury? There is a lack of research.Therefore,the purpose of this study is to establish a model of oxygen and glucose deprivation/reoxygenation PC12 cells,to simulate the I/R injury environment of neurons in vitro,and to explore whether calycosin can inhibit cell apoptosis through Cyt C/Apaf-1 mitochondrial apoptosis pathway,so as to play a protective role in oxygen and glucose deprivation/reoxygenation PC12 cells.In this study,PC12 cells were used to simulate the process of brain I/R injury in vitro.The research is divided into three parts: 1.The effects of calycosin on the apoptosis of oxygen and glucose deprivation/reoxygenation PC12 cells;2.The effects of calycosin on the apoptosis of oxygen and glucose deprivation/reoxygenation PC12 cells;3.The effects of calycosin on the apoptosis pathway of Cyt C/Apaf-1mitochondria of oxygen and glucose deprivation/reoxygenation PC12 cells.Part one Effects of calycosin on oxygen and glucose deprivation/reoxygenation PC12 cell damageObjective: To observe the effect of calycosin on PC12 cells injured by oxygen and glucose deprivation/reoxygenation.Methods: PC12 cells were randomly divided into 5 groups: normal control group(control),OGD/R group(model),calycosin(0.140μmol/L,0.070 μmol/L,0.035 μmol/L)dose group.In addition to the control group,the other groups were treated with oxygen and glucose deprivation for 2 hours,reoxygenation and glucose recovery for 24 hours.At the same time of reoxygenation,different doses of calycosin were treated with mediums containing calycosin(0.140 μmol/L,0.070μmol/L,0.035 μmol/L).Cell morphology was observed under inverted microscope;cell viability was detected by CCK-8;LDH was used to detect LDH leakage rate;PI fluorescence staining was used to detect cell membrane integrity.Results: Compared with the control group,the number of cells in the model group decreased,the refractive index of cells decreased,the synapses decreased,the cell morphology shrunk,the cell membrane was damaged,and the cell activity decreased significantly(P<0.05),the leakage rate of LDH increased significantly(P<0.05),and the number of PI red staining cells increased(P<0.05);Compared with the model group,the number of cells,synapses and cell bodies in the middle and low dose groups of calycosin were increased,rounder,more regular,more refractive and more active(P<0.05),The leakage rate of LDH decreased significantly(P<0.05),the number of PI red cells decreased(P<0.05),and the effect of the above indexes was more significant in the middle dose group,while the difference of the above indexes between the high dose group and the model group was not significant(P>0.05).Summary: Calycosin can optimize the growth state of PC12 cells,improve cell vitality,inhibit LDH leakage,effectively reduce cell membrane damage,and play a protective role on PC12 cells.In addition,the middle dose group(0.070 μmol/L)has the most significant effect,which will be applied to the second and third part of the experiment.Part two Effects of calycosin on apoptosis of oxygen and glucose deprivation/reoxygenation PC12 cellsObjective: To investigate the effect of calycosin on apoptosis of oxygen and glucose deprivation/reoxygenation PC12 cellsMethods: PC12 cells were randomly divided into four groups:control group,model group,calycosin group(0.07 μmol/L)and nimodipine group(5.00 μmol/L,positive control group).The method of modeling is the same as that in the first part.The calycosin group and nimodipine group were treated with the medium containing calycosin(0.07 μmol/L)and nimodipine(5.00 μmol/L)at the same time of reoxygenation.Oxygen and glucose deprivation for 2 hours,reoxygenation and glucose recovery for 24 hours,flow cytometry and TUNEL staining were used to detect apoptosis rate and apoptosis index;Bax/Bcl-2 ratio was detected by immunofluorescence staining;caspase-3expression was detected by immunohistochemistry.Results: Compared with control group,the apoptosis rate and apoptosis index of model group were significantly increased(P<0.05),Bax/Bcl-2 ratio was up-regulated(P<0.05),and caspase-3 protein expression was significantly increased(P<0.05);Compared with the model group,the apoptosis rate and apoptosis index of calycosin group and nimodipine group decreased significantly(P<0.05),Bax/Bcl-2 ratio decreased significantly(P<0.05),caspase-3 protein expression decreased significantly(P<0.05),the difference was statistically significant.Summary: Calycosin can significantly reduce the apoptosis rate and apoptosis index of oxygen and glucose deprivation/reoxygenation PC12 cells,down regulate the Bax/Bcl-2 ratio,reduce the expression of apoptosis protein caspase-3,and play a protective role on oxygen and glucose deprivation/reoxygenation PC12 cells by inhibiting apoptosis.Part three Effects of calycosin on apoptosis pathway of Cyt C/Apaf-1 mitochondria on oxygen and glucose deprivation/reoxygenation PC12 cellsObjective: To study the effect of calycosin on the mitochondrial apoptosis pathway of Cyt C/Apaf-1 on oxygen and glucose deprivation/reoxygenation PC12 cells.Methods:(1)Firstly,gene silencing method was used to clarify the key role of mitochondrial apoptotic pathway core protein Apaf-1 in regulating apoptosis in a special model of PC12 cells with oxygen and glucose deprivation/reoxygenation.The experiment was divided into normal control group,model group and Apaf-1-si RNA group.The molding method is the same as the second part.In the Apaf-1-sirna group,the chemically synthesized si RNA was transfected into PC12 cells by liposome before modeling,targeting to silence Apaf-1 gene.The transfection efficiency of Apaf-1 was detected by fluorescence labeled si RNA and Western blot respectively,then the apoptosis rate of each group was detected by flow cytometry,and the expression of Apaf-1protein in each group was detected by Western blot.(2)To explore the effect of calycosin on the mitochondrial apoptosis pathway of Cyt C/Apaf-1 in oxygen and glucose deprivation/reoxygenation PC12 cells.The experiment was divided into normal control group,model group and calycosin group(0.07 μmol/L).Western blot was used to detect the expression of Cyt C,Apaf-1,caspase-9 and caspase-3,the key proteins of mitochondrial apoptosis pathway.Results:(1)Apaf-1-si RNA can effectively silence the expression of Apaf-1 protein in PC12 cells(P<0.05).Compared with the model group,Apaf-1-si RNA can significantly reduce the apoptosis rate(P<0.05),and the expression of Apaf-1 protein can significantly reduce(P<0.05),indicating that targeted silencing of Apaf-1 gene can effectively inhibit the apoptosis of oxygen and glucose deprivation/reoxygenation PC12 cells.It is clear that Apaf-1 plays a key role in regulating apoptosis in a special model of oxygen and glucose deprivation/reoxygenation PC12 cells.(2)To observe the effect of calycosin on Cyt C/Apaf-1mitochondrial apoptosis pathway,compared with the control group,the expression of Cyt C,Apaf-1,caspase-9 and caspase-3 in the model group was significantly increased(P<0.05).Compared with the model group,the expression of Cyt C,Apaf-1,caspase-9 and caspase-3 in the calycosin group was significantly decreased(P<0.05),difference has statistical significance.Summary: The inhibition mechanism of calycosin on apoptosis of oxygen and glucose deprivation/reoxygenation PC12 cells is closely related to its regulation of the mitochondrial apoptosis pathway of Cyt C/Apaf-1 and its inhibition of the expression of key proteins Cyt C,Apaf-1,caspase-9 and caspase-3 in the mitochondrial apoptosis pathway.Conclusions:1.Calycosin can improve the growth state of PC12 cells,enhance the cell vitality,inhibit the leakage of LDH,protect the integrity of cell membrane,effectively reduce the damage of oxygen and glucose deprivation/reoxygenation PC12 cells and play a protective role.2.Calycosin can significantly reduce the apoptosis rate and apoptosis index of oxygen and glucose deprivation/reoxygenation PC12 cells,decrease the cell Bax/Bcl-2 ratio,and reduce the expression of caspase-3 protein,and play a protective role on oxygen and glucose deprivation/reoxygenation PC12 cells by inhibiting apoptosis.3.The inhibition mechanism of calycosin on apoptosis of oxygen and glucose deprivation/reoxygenation PC12 cells is closely related to its regulation of the mitochondrial apoptosis pathway of Cyt C/Apaf-1 and its inhibition of the expression of key proteins Cyt C,Apaf-1,caspase-9 and caspase-3 in the mitochondrial apoptosis pathway. |
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