To Research The Mechanism Of Longzhi Decoction Serum Promoting Tube Formation Of Human Umbilical Vein Endothelial Cell On The Basis Of Autophagy

Objective:The human umbilical vein endothelial cells were induced by H₂O₂ and the autophagy model were constructed.To study the protective effect of Longzhi serum on the damaged HUVECs and its effect on the formation of tube,for further investigation on the mechanism of Longzhi serum in promoting tube formation of HUVECs.Methods:The serum of SD rats was prepared according to the serum pharmacology and pharmacokinetics of traditional Chinese medicine.Human umbilical vein endothelial cells were cultured and passaged,and HUVECs were incubated with drug-containing serum and related drugs during the logarithmic phase of growth.?1?CCK-8 method was used to detect the proliferation of HUVECs at different concentrations?gradient concentrations of 50umol/L,100umol/L,200umol/L,400umol/L,800umol/L and 1600umol/L?,and CCK-8method was used to detect the effect of 400umol/L H₂O₂ on the proliferation of HUVECs at different time?2h,4h,8h,12h,16h?.The optimal concentration of H2O2 and the appropriate action time were selected.The group was divided into normal group,blank serum group,H₂O₂ group,Longzhi decoction serum+H₂O₂ group,Buyang Huanwu decoction serum+H₂O₂ group.Morphological changes of the cells were observed by optical microscope,the migration of HUVECs was detected by scratch test,and the lumen formation capacity of the cells was detected by the artificial basement membrane?Matrigel?.?2?The optimal concentration of H₂O₂ and the appropriate time were obtained according to the experiment?1?,and the normal group,the blank serum group,the H₂O₂ group and the Longzhi decoction serum+H₂O₂ group were set.The HUVECs autophagy model was induced by H₂O₂ in vitro.Observation of morphological changes of cells by HE staining,meanwhile,detection of cell migration and lumen structure by scratching and matrigel.The ultrastructures and autophagy of HUVECs induced by H₂O₂ were detected by transmission electron microscopy.The expression of autophagy-related protein LC3 and Beclin-l was detected by western blot.To elucidate the possible mechanism of promoting H₂O₂-induced formation of HUVECs.Results:?1?Compared with the normal group and blank serum group,the cell morphology of H₂O₂ group changed greatly,irregular and accompanied with cell aging,and the cell activity of HUVECs was significantly inhibited?P<0.01?.Compared with H₂O₂ group,the proliferation ability of HUVECs in Buyang Huanwu Decoction group was increased?P<0.01?,migration distance was much higher than that in blank area?P<0.01?,and the number of vascular lumen formation increased?P<0.01?.Compared with the Buyang Huanwu Decoction group,the cells of the Longzhi decoction group had higher proliferative capacity,faster migration speed and migration distance,and more tubular cavities and small branches could be formed,the difference was significant?P<0.05?.in addition,aft 24 h,that cells of the Longzhi decoction could still maintain the complete lumen structure and morphology,while the Buyang Huanwu decoction group could not.?2?Compared with normal group and blank serum group,the migration distance of H₂O₂ group was significantly lower,and the number of intraluminal structures was small?P<0.01?.Compared with H₂O₂ group,Longzhi Decoction could decrease H₂O₂-induced cell damage,maintain cell morphology,enhance HUVECs migration and lumen formation,and the difference was significant?P<0.05?.In addition,after 24h,that cells of the Longzhi decoction could still maintain the complete lumen structure and morphology,while the Buyang Huanwu decoction group could not.?2?Compared with normal group and blank serum group,the migration distance of H₂O₂ group was significantly lower,and the number of intraluminal structures was small?P<0.01?.Compared with H₂O₂ group,Longzhi Decoction could decrease H₂O₂-induced cell damage,maintain cell morphology,enhance HUVECs migration and lumen formation,and the difference was significant?P<0.05?.Under transmission electron microscope,there were abundant mitochondria,endoplasmic reticulum and other organelles in HUVECs.There was a little autophagy in H₂O₂ group,the vacuolating structure in cytoplasm was increased,while the number of intracytoplasmic double-layer membrane-like structures in Longzhi decoction group was significantly increased,bending was extended,and some organelles were wrapped.From the expression of autophagy-related protein,the expression of LC3-II and Beclin-l protein in H₂O₂ group increased?P<0.05?.Compared with H₂O₂ group,the expression of LC3-II and Beclin-l increased significantly?P<0.01?.Conclusions:?1?Compared with Buyang Huanwu decoction,Longzhi decoction has better effect on the formation of HUVECs vascular lumen and the maintenance of the structure of the lumen,It is shown in quick effect,long time and so on.?2?Longzhi decoction has an up-regulation effect on the autophagy level by H2O2-induced and the increase of autophagy level may be one of the mechanisms for the tube formation of HUVECs.