Effect Of Drug-containing Serum Of Huo Xue Ding Xuan Capsule On Hypoxia Injury Of BEnd.3 Cell Based On Autophagy

Objective:1.By culturing mouse brain microvascular endothelial cells(bEnd.3 cells),the proliferation of bEnd.3 cells and the optimal OGD time were studied under different Oxygen Glucose Deprivation(OGD)model time.2.Under the optimal OGD time,explore the protective effect of different concentrations of Huoxue Dingxuan Capsule on bEnd.3 cells and the relationship between autophagy,and improve the mechanism of Huoxue Dingxuan Capsule in treating vertebral artery type cervical spondylosis.To lay the foundation for further clinical application of Huoxue Dingxuan CapsuleMethods:1.The bEnd.3 cells with good growth status were divided into 5 groups,which were normal control group,OGD 3h group,OGD 6h group,OGD 9h group and OGD 12h group.The cells were first cultured in a normal carbon dioxide cell incubator for 36 hours,and then the cells were subjected to OGD treatment according to the above grouping.The OGD treatment method was as follows.First,the sugar-free DMEM medium was placed in a three-gas cell incubator at 37�C,5%CO 2,94%N 2,1%02 and saturated humidity for 30min.The old medium of each OGD group was discarded and replaced with a hypoxic pre-treated,sugar-free DMEM medium,which were then placed in the above three-gas cell incubator for 3 h,6 h,9 h,and 12 h,respectively.The normal control group was OGD 0h group,so after 36 hours of co-culture,the culture medium was replaced with sugar-free DMEM and directly tested.The cell morphology of bEnd.3 was observed by ordinary light microscope and photographed.The cell morphology of the control group and OGD treatment group were compared and the cell viability of each group was detected by CCK-8.2.Serum serum and blank serum of Huoxue Dingxuan Capsule were prepared by serum pharmacological theory.The bEnd.3 cells with good growth status were divided into 7 groups under the same generation,which were model control group,different concentration of Huoxue Dingxuan capsule containing serum OGD group(5%,10%,15%),different concentration blanks.The serum OGD control group(5%,10%,15%)was first cultured in a normal carbon dioxide cell incubator.When the cells proliferated to 80%-90%density,the basal medium of each group was replaced.For the sugar-free DMEM medium,the corresponding serum was added according to the different groups.In the model control group,no serum was placed,and according to the results of the previous part,the optimal time of OGD was obtained,and each group was placed in three gas.The optimal time for OGD co-culture in the cell culture incubator was taken out,and the supernatant of the cell culture medium was aspirated.The activity of lactate dehydrogenase(LDH)released by cytotoxicity was detected by colorimetry,and the nitric oxide was detected by nitrate reductase method.NO);The apoptosis rate was detected by flow cytometry.The expressions of autophagy markers LC3II/LC3I,P62 and Beclin 1 were detected by Western Blot.The expressions of P62 and Beclin 1 mRNA were detected by qPCR.to sum it up If the relationship analysis Huoxue Dingxuan capsules containing Serum protective effect on cells and with bEnd.3autophagy.Result:1.Under the light microscope,the bEnd.3 cells in the normal control group were long,shuttle-shaped and distributed,connected to each other and arranged in a wire mesh order,and in the OGD model group(3h,6h,9h and In 12h),with the prolongation of time,bEnd.3 cells gradually evolved from the original shuttle type to elliptic type,and at 12h,bEnd.3 cells died in large numbers;by CCK-8 detection,compared with the normal group,OGD was compared with the normal group.The OD values of the model group showed a downward trend at 4time points(P<0.05),indicating that the activity of bEnd.3 cells gradually decreased with the prolongation of time,and combined with optical microscopy results,bEnd.3 with time.The apoptotic rate gradually increased,and at 12 h of OGD,the cells died in large numbers.According to the linear regression equation:y=-0.1186x+1.7375(R~2=0.9881),when the percentage of living cells is 50%,the OGD induction time takes about 7.3 hours.According to the results of this experiment,the literature can be obtained.The optimal time for OGD induction in the experiment was 6 hours,and 6 hours was determined as the optimal time for OGD.2.After 6 hours of OGD induction,compared with the model control group,different concentrations of drug-containing serum group and different concentration of blank serum group can reduce the release of LDH and increase the secretion of NO in the cell to reduce bEnd caused by OGD model.indicating that rat serum can alleviate cell damage and reduce cell membrane permeability;different concentrations of blank serum group and different concentrations of drug-containing serum group,at the same dose,Huoxue Dingxuan capsule drug-containing serum It can significantly inhibit the increase of LDH in the cell supernatant and enhance the amount of NO to protect the cells.The 10%and 15%Huoxue Dingxuan capsules have significant differences(P<0.05),of which 10%contain serum.The NO content was the highest in the other groups,and the difference was significant(P<0.05).In the apoptosis assay,we found that the apoptosis of bEnd.3 cells in the model control group was 35.5%.The apoptosis rates of bEnd.3 cells in the 5%,10%,and 15%drug-containing sera groups were 18.4%,5.7%,and 11.2%,respectively,while in the 5%,10%,and 15%blank sera groups,bEnd.3 cells were apoptotic.The rates were 25.7%,20.7%,and 15.3%,respectively.Compared with the blank sera group,the drug-containing serum of the same dose can better inhibit the apoptosis rate of BAD.3 cells induced by OGD,and compared with other groups,10%drug-containing serum group can Significant inhibition of the progression of bEnd.3 cell apoptosis.The results of Western Blot showed that:(1)Compared with the model control group,the relative expression levels of P62/?-Actin in the 5%,10%,and 15%drug-containing serum groups and 5%and 10%blank serum groups were significantly decreased.(P<0.05),while the relative content of P62/?-Actin in the 15%blank serum group was higher than that in the model control group(P<0.05),while the relative expression of P62/?-Actin in the 10%drug-containing serum group was higher than that in the other groups.The group was low(P<0.05);(2)in the LC3-II/LC3-I data,we found that the 10%drug-containing serum group was significantly higher than the other groups(P<0.05);5%,The ratio of LC3-II/LC3-I in the 15%drug-containing serum group was higher than that in the 5%and 15%blank serum groups(P<0.05);(3)in Beclin 1/?-actin,10%drug-containing The relative expression of serum protein was significantly higher than that of other groups(P<0.05).The relative content of Beclin 1/?-actin in the 5%drug serum group was less than 5%blank serum group(P<0.05),while at 15 In the%drug-containing serum group,the content was higher than the 15%blank serum group(P<0.05).The results of qPCR showed that:(1)The content of Beclin 1 mRNA(BECN-1)in the drug-containing serum group was higher than that in the same dose of blank serum group;in P62 mRNA,the expression level of blank serum group was significantly higher than that of the same dose of drug-containing serum.In the group,(2)the expression of P62 mRNA was decreased and the expression of BECN-1 was increased in the model control group?Conclusion:1.The optimal induction time of bEnd.3 cells at 6 hours under different OGD time induction2.The Notch pathway mainly plays the role of inhibiting the osteogenic differentiation of BMSCs in this experiment.2.Huoxue Dingxuan Capsule containing serum can significantly inhibit OGD-induced bEnd.3 cell injury,significantly increase NO content,reduce LDH release,reduce the number of bEnd.3 cell apoptosis,and promote bEnd.3 cell proliferation.3.Huoxue Dingxuan Capsule-containing serum can reduce the damage of OGD-induced bEnd.3 cells by up-regulating the expression of autophagy-related proteins Beclin 1,LC3 and down-regulating P62 protein,thereby regulating the function of vascular endothelial cells.