| Objective: To observe the effects of Qingre Jiedu Formula and Ruanjian Sanjie Formula on the growth,proliferation,apoptosis of human lung adenocarcinoma A549 cells,and then compare the effects of the two traditional Chinese medicine formulas on the apoptosis of A549 cells,and observe the expression of Bcl-2 mRNA in A549 cells.Methods: The human lung adenocarcinoma A549 cells were cultured in vitro,and A549 cells with good growth state in logarithmic phase were randomly divided into three groups: two Chinese medicine formula groups with different efficacies(Qingre Jiedu Formula and Ruanjian Sanjie Formula)and a negative control group with DMEM medium.After the cells in each group were cultured for 48 h,the following tests were performed:(1)the proliferation inhibition and growth inhibition rate changes of two Chinese medicine formulas with different concentrations and different efficacies on human lung adenocarcinoma A549 cells were detected by CCK8 method,and the IC50 values of Qingre Jiedu Formula and Ruanjian Sanjie Formula were calculated respectively;(2)The growth status and morphological changes of human lung adenocarcinoma A549 cells induced by two Chinese medicinal formulas with different efficacies were observed under an inverted phase contrast microscope;(3)Hoechst33258staining was used to observe the morphological changes of A549 cell apoptosis in each group;(4)The changes of apoptotic rate of human lung adenocarcinoma A549 cells detected by Annexin V-FITC/PI double-staining flow cytometry;(5)Real-time fluorescent quantitative(RT-PCR)detection was performed to examine the effects of the two Chinese medicinal formulas with different efficacies on the expression of apoptotic gene Bcl-2 mRNA in human lung adenocarcinoma A549 cells.Results:(1)The results of CCK8 method showed that different concentrations of Qingre Jiedu Formula and Ruanjian Sanjie Formula inhibited the proliferation of human lung adenocarcinoma A549 cells in a dose-dependent manner after 48 h.Compared with the negative control group,the absorbance of A549 cells decreased after treatment with2.5mg/ml Ruanjian Sanjie Formula,without statistical significance(P > 0.05).The absorbance of A549 cells decreased after treatment with the other two groups of Chinese medicine formulas with different concentrations,with statistical significance(P < 0.05,P< 0.01).The IC50 values of Qingre Jiedu Formula and Ruanjian Sanjie Formula after 48 h of exposure to A549 cells were 11.313 mg/ml and 23.874 mg/ml respectively.(2)The observation under an inverted phase contrast microscope shows that the cells of the negative control group: The cells grew vigorously,distributed intensively,and tended to be consistent in trend.The cells were extended,flat,and grew in polygonal adherence,with two to three flat and long protrusions.The cell body of the cells was full,and the cells were homogeneous and transparent.The cells generally had two to four nucleoli.The nuclear membrane and nucleoli had obvious outlines,and they were oval and mostly in the middle.The cells in the two groups with different efficacies of traditional Chinese medicine formula groups: cell proliferation was slowed down,the density was reduced,the intercellular space was increased,the protrusions were shortened,the cell body was reduced and in a suspended state,the cytoplasm was decreased,and black particles in the cytoplasm were increased.(3)The results of Hoechst33258 staining showed that after acting on A549 cells for 48 h with Qingre Jiedu Formula and Ruanjian Sanjie Formula,respectively,they could both observe dense and whitish apoptotic cells,with obvious karyopycnosis and fragmentation,The cell density was lower than that of the negative control group.(4)The results of Annexin V-FITC/PI double-staining flow cytometry showed that after acting on A549 cells for 48 h,the apoptotic rates(early apoptosis+late apoptosis)of Qingre Jiedu Formula and Ruanjian Sanjie Formula were 8.66% and11.17%,respectively.Compared with the negative control group(3.53%),the apoptotic rates in the groups with different efficacies of traditional Chinese medicine prescription were increased,and the difference was statistically significant(P < 0.01).The increase in the apoptotic rate of Ruanjian Sanjie Formula was the most significant.(5)Real-time fluorescent quantitative(RT-PCR)results showed that compared with the negative control group,both Qingre Jiedu Formula and Ruanjian Sanjie Formula could down-regulate Bcl-2 mRNA expression in different degrees after 48 h of exposure to A549 cells,and the difference was statistically significant(P < 0.05,P < 0.01).Conclusion: Both Qingre Jiedu Formula and Ruanjian Sanjie Formula can inhibit the growth and proliferation of human lung adenocarcinoma A549 cells to different degrees,and promote the apoptosis of human lung adenocarcinoma A549 cells,especially Ruanjian Sanjie Formula.In addition,Qingre Jiedu Formula and Ruanjian Sanjie Formula can down-regulate the expression of Bcl-2 mRNA in human lung adenocarcinoma A549 cells. |
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