Experimental Study On The Mechanism Of Anti-Artherosclerosis By Homocysteine Of GANXINKANG

Methods:(1) In the strict aseptic conditions, isolate human umbilical vein endothelial cells with Trypsin from newborn umbilical cord,then culture the cell with DMEM medium containing 20% fetal bovine serum.When the cells reach 80% confluence, digeste passaged,identification and cryopreservation;Application of Chinese medicine serum pharmacology, Prepare drug serum containing different concentrations of medicine drugs. (2) Divide the HUVEC into 6 groups randomly, each group of cells separately using 0.0mmol/L (i.e. for blank control group), 0.01 mmol / L, 0.5mmol / L, 1.0mmol / L, 2.0mmol / L, 3.0mmol / L Hcy role in HUVEC, and then at 3 h, 6h, 12h, 24h rate of cell apoptosis percentage(PI method), mitochondrial membrane potential (JC-1) and protein expression of the endoplasmic reticulum chaperone protein--Grp78 changes at 24h.(3) Divide the HUVEC into 5 groups randomly: control group,model control group,Guanxinkang low concentrations group,Guanxinkang middle concentration group,high concentration groups. Actting for 2h, added 3.0 mmol / L Hcy in every group of cells.At 6 h and 12h and 24h observe vascular endothelial cell apoptosis percentage , mitochondrial membrane potential, Grp78 protein expression as well as to different levels of ROS and LOX-1 changes.Results:(1) With the identification, the cells have proofed it is HUVEC. The condition of the cell growth is good, and save the recovery after frozen the cell ,there has no difference from the cell which has recovered with the cell which has not saved frozen frozen. And we Have prepared drug serum with different concentrations of Chinese medicine.(2) after 0.1mmol/L Hcy affects 6h, apoptosis was significantly increased , mitochondrial membrane potential decreased significantly. The result of increasing concentrations of Hcy, there are gaps in the control group significant difference (P <0.05)as well as the extension of time.when Hcy reached 5.0mmol / L at 24h it reach a peak; Grp78 protein expression in 0.5 mmol / L increase the expression, and a concentration -dependent manner, at 3.0 mmol / L reached a peak.(3) With model group compared with control group, has significantly increased , mitochondrial membrane potential has decreased significantly and Grp78 protein expression has significantly increased (P <0.05). ROS and LOX-1 in endothelial cells has both increased.GuanxinKang high concentration of coronary intervention, the apoptosis percentage significantly decreased, mitochondrial membrane potential declined the reduce and the protein expression of Grp78 reduced.ROS and LOX-1 production decreased.Compared with the model group, there were significantly different ( P <0.01). And with the extension of time, the more significant roled.Conclusions: (1) We can obtained HUVEC with the Tryption digestion,and can be carried out through technical cryopreservation and recovery.Application of the principle of serum pharmacology,we can prepare drug serum of traditional Chinese medicine containing different concentrat -ions.These builds the foundation for further study. (2) Hcy can induce apoptosis in vascular endothelial cells and upward the level of protein expression of Grp78.And its result assumes the density or the response time dependence.The mechanism of that may be that Hcy can cause endoplasmic reticulum stress of VEC, and promote endoplasmic reticulum, so the unfolding protein accumulated, and the endoplasmic reticulum protein expression increased by the partner. (3) Guanxinkang can antag -onistic the injury induced by Hcy of the vascular endothelial cell. And its result assumes the density or the response time dependence of the positive relationship.The mechanism may be that Guanxinkang can inhibite the endoplasmic reticulum stress induced by homocysteine, thereby decrease endothelial cell apoptosis percentage, reduce the oxidative stress induced homocysteine ,and be achievable finally.