Research On The Intervention Effect Of Soufeng Qutan Chinese Medicine On Hcy-induced Damage Of RCMECs And The Mechanism Of Oxidative Stress

Purpose:In this experiment,at the level of in vitro cell culture,to observe the eff ect of Soufeng expectorant Chinese medicine on homocysteine-induced myocardial micr ovascular endothelial cell injury in rats and the mechanism of oxidative stress.The m echanism of pulse microcirculation disorder provides a theoretical basis for the clinical application of Soufeng qutan Chinese medicine in the treatment of coronary microcirc ulation disorder.Material and method:40 male SPF-grade SD rats were randomly divided into blank control group,low-dose Soufengqutan Chinese medicine group,medium-dose Soufengqutan Chinese medicine group,high-dose Soufengqutan Chinese medicine group,each group of 10 only.Soufengqutan Chinese medicine low-dose group,Soufengqutan Chinese medicine middle-dose group,Soufengqutan Chinese medicine high-dose group were given Soufengqutan Chinese medicine low-dose 4.05 g / kg,Soufengqutan Chinese medicine-dose 8.1g / kg,Soufengqutan Chinese medicine high-dose 16.2g / kg and physiological saline for intragastric administration,followed by intragastric administration for seven consecutive days.On the seventh day after gavage,the serum was extracted one hour after the administration.The method of extracting the serum was to anaesthetize the rats to take blood through the abdominal aorta,and the obtained serum was separated in a sterile environment and inactivated by heating.The purchased rat myocardial microvascular endothelial cells were subcultured,frozen and resuscitated.They were divided into blank control group,model group and Soufengqutan Chinese medicine low,medium and high dose groups.The myocardial microvascular endothelial cells of each group were isotyped.Cysteine intervention(except for the blank control group).After homocysteine intervention,the cells of the blank control group,model group and rat blank serum were co-cultured for 24 hours;Soufengqutan Chinese medicine low-dose group,Soufengqutan Chinese medicine The medium-dose group and Soufengqutan Chinese medicine high-dose group were cultured with Soufengqutan Chinese medicine low-dose group,Soufengqutan Chinese medicine-dose group,and Soufengqutan Chinesemedicine-high-dose group together with the drug-containing serum and cells for 24 hours.After the cells are collected,they are kept for future use for subsequent indicatordetection.Cell morphology was observed using an inverted microscope,and rat myocardial microvascular endothelial cells were identified and identified by CD31 immunofluorescence.The positive rate was more than 95% for subsequent experiments.Normally cultured rat myocardial microvascular endothelial cells were randomly divided into a blank control group(Control),a model group(Model),a Soufengqutan Chinese medicine low-dose group(SFQTZY-L),and a Soufengqutan Chinese medicine-dose group(SFQTZY)-M),Soufengqutan Chinese medicine high-dose group(SFQTZY-H).MTT method was used to detect the cell survival rate of each group,TBA method was used to detect the malondialdehyde(MDA)content in the cell supernatant,hydroxylamine method was used to detect the superoxide dismutase(SOD)activity in the cell supernatant,and RT-PCR was used to detect the endothelin-1(ET-1)level and Elisa method were used to detect the changes of nitric oxide(NO)expression level.Results:1.Morphology and identification of rat myocardial microvascular endothelial cells:When the purchased RCMECs were cultured to 48 h and observed with an inverted microscope,it was seen that the single layer of single microvascular endothelial cells arranged in a fusiform,star or polygonal shape in the unfused state,and cultured to the 5th day,typical paving stones were seen.CD31 immunofluorescence method was used to identify the cells.The results showed that the purity of the rat myocardial microvascular endothelial cells was greater than 95%.2.Determination of homocysteine concentration:Different concentrations of Hcy were added to the cells,and after 24 hours of intervention,the cell OD values were measured.Compared with normal rat myocardial microvascular endothelial cells,the cell OD value gradually decreased after adding different concentrations of Hcy,especially at 1mmol / L,so the cell OD value was determined to be 1mmol / L3.MTT assay for cell viability: Compared with the blank control group,the OD value of the model group was significantly reduced(P <0.01);compared with the model group,the OD value of Soufengqutan Chinese medicine groups were significantly increased(P <0.01).4.MDA content of cell supernatant measured by TBA method: Compared with the blank control group,the MDA content of the cell supernatant of the model group was significantly increased(P <0.01);compared with the model group,the cell supernatant of each dose group of Soufengqutan Chinese medicine The MDA content in the liquid was significantly reduced(P <0.01)5.SOD activity in the cell supernatant measured by hydroxylamine method: Compared with the blank control group,the SOD activity in the cell supernatant of the model group was significantly reduced(P <0.01),compared with the model group,the cell supernatant of each dose of Soufengqutan Chinese medicine group SOD activity was significantly increased(P<0.01).6.RT-PCR detection of ET-1 m RNA level in rat myocardial microvascular endothelial cells:Compared with the blank control group,the expression of ET-1 m RNA in the model group was significantly increased(P <0.01);compared with the model group,Soufengqutan The expression of ET-1 m RNA was significantly reduced in all dose groups of Chinese medicine(P <0.01).7.Elisa method to detect NO expression in myocardial microvascular endothelial cells of homocysteine-injured rats: Compared with the blank control group,the expression of NO in the model group was significantly reduced(P <0.01);The expression of NO in the dose group was significantly increased(P <0.01).Conclusion:1.Soufengqutan Chinese medicine-containing serum has protective effect on Hcy-induced myocardial microvascular endothelial cell injury in rats.2.Soufengqutan Chinese medicine-containing serum can increase the cell supernatant SOD activity and down-regulate the cell supernatant MDA content;indicating that Soufengqutan Chinese medicine can alleviate coronary microcirculation disturbance by improving oxidative stress response.3.Soufengqutan Chinese medicine-containing serum can down-regulate the expression of ET-1 m RNA and up-regulate the expression of NO;it indicates that Soufengqutan Chinese medicine may effectively improve endothelial dysfunction and then improve coronarymicrocirculation disorder.