| Purpose:In this study,rats cardiac microvascular endothelia cells(RCMECs)were used as experimental subjects and we established a model of myocardial microvascular endothelial cell injury by homocysteine(Hcy)induced myocardial microvascular endothelial cells(RCMECs)injury in rats at the level of cell culture in vitro.The purpose of this study is to observe the effect of Astragalus compound medicated serum on the expression of Bcl-2 and Bax protein and to explore the effect of Huangqi compound on homocysteine(Hcy)-induced injury of rats cardiac microvascular endothelia cells(RCMECs)from the perspective of cell apoptosis mechanism combined with the theory of simultaneous treatment of Qi and blood,and to further clarify the mechanism and methods of Huangqi compound in the prevention and treatment of coronary microcirculation dysfunction(CMD),it is helpful to provide theoretical basis and prevention targets for traditional Chinese medicine treatment of coronary microcirculation dysfunction(CMD),and also lay the foundation for wider clinical application and promotion of Huangqi compound.Material and method:(1)Preparation of Astragalus compound medicated serum :According to the need of the experiment,50 male SPF SD rats were randomly divided into 5groups according to the standard of 10 rats as a group.They were named as blank control group,model group,low-dose Huangqi compound group,medium-dose Huangqi compound group and high-dose Huangqi compound group,with 10 rats in each group.The dosage of rats was calculated according to the equivalent dose conversion formula between rats and human.The low-dose Huangqi compound group was given 1.8 g/kg Huangqi compound Chinese medicine concentrate,the medium-dose Huangqi compound group was given 3.6g/kg Huangqi compound Chinese medicine concentrate,the high-dose Huangqi compound group was given 7.2 g/kg Huangqi compound Chinese medicine concentrate,the blank control group and model group were given the same volume of distilled water.All rats were gavaged twice a day for 7 days.On the 7th day of gavage,1 hour after administration,the rats were anesthetized,and the serum was extracted from abdominal aorta,then static and centrifuged.The serum of each group was collected,heated and inactivated,filtered and sterilized,and stored in refrigerator at-80?.(2)Culture and identification of primary rats cardiac microvascular endothelia cells(RCMECs):the purchased rats cardiac microvascular endothelia cells(RCMECs)were subcultured,cryopreserved and resuscitated.The morphology of rats cardiac microvascular endothelia cells(RCMECs)was observed by inverted microscope,and the expression of CD31 protein on the cell membrane of rats cardiac microvascular endothelia cells(RCMECs)was detected by immunofluorescence.The identification results showed that the positive rate was more than 95%,which can be used for follow-up experiments.(3)Selection of homocysteine(Hcy)concentration: according to the gradient dilution method,Hcy was set to 7 concentrations : 8 mmol/L,4 mmol/L,2mmol/L,1 mmol/L,0.5 mmol/L,0.25 mmol/L and 0 mmol/L.Take 96 well culture plate,add 100?l complete culture medium containing rats cardiac microvascular endothelia cells(RCMECs)into each well,add ECM culture medium,and put it into CO2 incubator for 16 hours.After that,the 96 well culture plate was taken out,the ECM medium was discarded and seven different concentrations of homocysteine(Hcy)were added to each plate.Then they were put into the CO2 incubator for 4 hours,and the OD value of each group of rats cardiac microvascular endothelia cells(RCMECs)was detected by enzyme reader.(4)Grouping : normal cultured and identified rats cardiac microvascular endothelia cells(RCMECs)were randomly divided into blank control group(Blank),model group(Model),low-dose Huangqi compound group(HQFF-L),middle-dose Huangqi compound group(HQFF-M),high-dose Huangqi compound group(HQFF-H).The rats cardiac microvascular endothelia cells(RCMECs)in Model group,HQFF-L group,HQFF-M group and HQFF-H group were injured by homocysteine(Hcy).rats cardiac microvascular endothelia cells(RCMECs)of Blank group and Model group after homocysteine(Hcy)induced injury were co cultured with rats blank serum for 24 hours;rats cardiac microvascular endothelia cells(RCMECs)of HQFF-L group,HQFF-M group and HQFF-H group after homocysteine(Hcy)induced injury were co cultured with medicated serum of low-dose Huangqi compound group,middle-dose Huangqi compound group and high-dose Huangqi compound group respectively for 24 hours.After that,the rats cardiac microvascular endothelia cells(RCMECs)were collected and retained for later detection of related indicators.(5)Index detection:MTT method was used to detect the survival of the rats cardiac microvascular endothelia cells(RCMECs)in each group;TUNEL method was used to detect the apoptosis of the rats cardiac microvascular endothelia cells(RCMECs)in each group;The protein expression levels of Bcl-2 and Bax in the RCMECs were detected by Western blot method.Results:1.Morphological observation and immunofluorescence identification of rats cardiac microvascular endothelia cells(RCMECs):When the purchased rats cardiac microvascular endothelia cells were cultured to the second day,the single rats cardiac microvascular endothelia cells(RCMECs)were observed under the inverted microscope as single layer fusiform,star shaped or polygonal in the state of non fusion;when the tissue blocks were removed and cultured to the fifth day,the rats cardiac microvascular endothelia cells(RCMECs)showed typical paving stone.After identification by CD31 immunofluorescence,the purity of rats cardiac microvascular endothelia cells(RCMECs)was over 95%.2.Determination of homocysteine(Hcy)concentration:Seven different concentrations of homocysteine(Hcy)were added to the rats cardiac microvascular endothelia cells(RCMECs)in each plate.After incubation in CO2 incubator for 4 hours,the OD value of rats cardiac microvascular endothelia cells(RCMECs)in each group was detected by enzyme labeling instrument.Compared with normal rats cardiac microvascular endothelia cells(RCMECs),the OD value of rats cardiac microvascular endothelia cells(RCMECs)decreased gradually after adding different concentrations of homocysteine(Hcy),especially when the OD value of rats cardiac microvascular endothelia cells(RCMECs)was the lowest at 1 mmol/L,so 1 mmol/L was used as the concentration of homocysteine(Hcy).3.MTT assay detecting the effect of Huangqi compound medicated serum on the viability of rats cardiac microvascular endothelia cells(RCMECs)injured by homocysteine(Hcy)in each group:Compared with the blank control group,the OD value of the model group decreased significantly(P < 0.01);compared with the model group,the OD value of the low-dose,medium-dose and high-dose Haungqi compound groups increased significantly(P < 0.01,and there is a quantitative effect relationship between the dose groups of Haungqi compound.4.TUNEL method detecting the apoptosis of rats cardiac microvascular endothelia cells(RCMECs)injured by homocysteine(Hcy)in each group:The results showed that: there were only a small number of TUNEL positive cells in the blank control group;compared with the blank control group,the number of TUNEL positive cells in the model group was significantly increased(P < 0.01);compared with the model group,the number of TUNEL positive cells in the low,medium and high-dose Haungqi compound groups was significantly decreased(P < 0.01),and the decrease of TUNEL positive cells in the high-dose Haungqi compound group was the most significant(P < 0.01),and there is a quantitative effect relationship between the dose groups of Haungqi compound.5.Western blot detecting The protein expression levels of B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X(Bax)in the RCMECs:Compared with the blank control group,the protein expression level of Bcl-2 in the model group was significantly decreased(P < 0.01)and the protein expression level of Bax protein was significantly increased(P < 0.01);compared with the model group,the protein expression level of Bcl-2 in the low,medium and high-dose Huangqi compound groups was significantly decreased(P < 0.01)and the protein expression level of Bax was significantly decreased(P < 0.01),the protein expression of Bcl-2 and Bax in high-dose Huangqi compound group was the most significant(P < 0.01),and there is a quantitative effect relationship between the dose groups of Haungqi compound.Conclusion:1.Haungqi compound medicated serum can effectively inhibit the apoptosis of RCMECs induced by Hcy.2.Haungqi compound medicated serum can significantly up-regulate the protein expression level of B-cell lymphoma-2(Bcl-2),significantly down-regulate the protein expression level of Bcl-2 associated X(Bax)in the RCMECs.3.Haungqi compound medicated serum can up regulate the expression of Bcl-2 protein and down regulate the expression of Bax protein in RCMECs injured by Hcy,and protect the function of microcirculation endothelial cells from the perspective of inhibiting apoptosis may be one of the mechanisms of its treatment of CMD... |
PDF Full Text Request