Effect Of Serum Containing Huoxue Dingxuan Capsule On Autophagy And Expression Of Related Proteins In BEnd.3 Cells In ODG Environment

Objective:In this study,bEnd.3 cells that were used to establish an oxygen glucose deprivation model was used to simulate the hypoxic state of vertebrobasilar ischemia and hypoxia.The serum of Huoxue Dingxuan Capsule was used to investigate the autophagy and related protein expression of bEnd.3 cells in ischemic hypoxic environment.The influence provided a scientific basis for the clinical application and further promotion of Huoxue Dingxuan Capsule,and finally provided research ideas for the prevention and treatment of vertebral artery type cervical spondylosis by Chinese medicine at the cell and protein level.Methods:Preparation of Huoxue Dingxuan Capsule-containing serum,cultured bEnd.3 cells and passaged them,establishment of OGD model.Set grouping:blank group,OGD model group,Huoxue Dingxuan capsule high,medium and low dose group(15%,10%,5%),western medicine group,according to the corresponding conditions for drug administration and intervention for 6h.Observing normal cell morphology under a microscope.Single-dansyl cadaverine staining for bEnd.3 autophagosomes.the content of nitric oxide was detected by nitrate reductase method;the release of lactate dehydrogenase was detected by micro-enzyme method;The expression levels of p62,Bcline-1 and LC3 in bEnd.3 cells were determined by Western blot.Finally,the effect of serum containing Huoxue Dingxuan Capsule on autophagy of bEnd.3 cells was observed.The results:1 Resuscitation of P1 bEnd.3 cell.On the second day,bEnd.3 cell grow along the wall of the test tube.The growth morphology is long,fusiform and polygonal,and the cytoplasm of bEnd.3 cell is rich and full.On the third day,bEnd.3 cell proliferation and division accelerated and overgrown the bottle,showing the posture of paving stones.2 By observing autophagosomes under a fluorescence microscope,autophagosomes were found to be fluorescent particles of different sizes,shapes,and dense staining.3 Effect of serum containing Huoxue Dingxuan Capsule on NO content in bEnd.3 cells:Compared with the blank group,the OGD model group was hypoxic starved,cell activity was reduced,cells were damaged,NO secretion was reduced,and the difference between the two groups was statistically significant.(P<0.01).Compared with the model group,the content of NO in the serum-containing serum group and the western medicine group was higher(P<0.05).There was no significant difference between the dose group and the western medicine group.The efficacy of the two groups was equivalent,and the medium-dose group containing the drug was higher than the high-dose and low-dose group containing the serum of Huoxue Dingxuan Capsule;4 The effect of serum containing Huoxue Dingxuan Capsule on the release of LDH in bEnd.3 cells:Compared with the blank group,the release of LDH in the model group was increased significantly,and the difference was significant(P<0.01).Compared with the model group,the serum containing huoxue dingxuan capsules in the high and low dose groups and the western medicine group had statistical significance(P<0.05).5 Effect of serum containing Huoxue Dingxuan Capsule on autophagy-associated proteins Bcline-1,LC3,p62:compared with the black group,the expression levels of p62,Becline-1,LC3II/LC3 I in the OGD model group were increased,and the expressions of Becline-1 and LC3II/LC3 I were statistical different(P<0.01).The expression of p62 protein were not significantly statistical different(P>0.05).Compared with the model group,the expression of p62 in the serum-dose group and the western medicine group was significantly decreased,and the expression of Becline-1 and LC3II/LC3 I was increased(P<0.01).There was no statistically significant difference between the middle dose group of Huoxue Dingxuan Capsule and the western medicine group(P>0.05).There was a negative correlation between p62 and Becline-1,LC3II/LC3 I.Conclusion:1 Huoxue Dingxuan Capsule can significantly improve the damage caused by hypoxic starvation conditions to bEnd.3 cells,increase the secretion of NO and inhibit the release of LDH,and improve the activity of vascular endothelial cells.2 The serum containing Huoxue Dingxuan Capsule can regulate the autophagy ofbEnd.3 cells in hypoxic starvation environment by affecting the expression levels of p62,Bcline-1 and LC3 related autophagy proteins,further improve blood flow,regulate endothelial function and promote angiogenesis.To provide new ideas for the prevention and treatment of CSA by Huoxue Dingxuan Capsule.