| Purpose:Shenmai injection(Shen-mai Injection,SMI)is a commonly used traditional Chinese medicine injection,which is extracted from Red Ginseng and Ophiopogon japonicus.SMI is widely used in the treatment of cardiovascular diseases such as myocardial infarction,arrhythmia,myocardial fibrosis and so on.In addition,the combination of chemotherapy drugs in the treatment of lung cancer,breast cancer,pancreatic cancer and other malignant tumors can significantly improve the efficacy of chemotherapy and improve the quality of life of patients.At present,most of the studies on SMI are focused on clinical treatment,but its anti-tumor mechanism is still rare.In this study,parental human lung adenocarcinoma cell line A549 and cisplatin-resistant A549/DDP cells were used as model cells to study the molecular mechanism of SMI in improving cisplatin resistance in lung cancer from the point of view of mitochondrial dynamics,so as to provide scientific and theoretical basis for integrated traditional Chinese and western medicine in the treatment of malignant tumor.Material and method: 1.The correlation between the expression level of mitochondrial fusion protein-2(Mitofusin-2,Mfn2)and the survival time of patients with lung adenocarcinoma was analyzed by Kaplan Meier Plotter database.Immunohistochemical method was used to compare the expression level of Mfn2 in human lung adenocarcinoma and its adjacent tissues,and to further evaluate the correlation with its clinical indexes.2.Westernblot was used to compare the expression levels of mitochondrial fusion related protein-Mfn2,mitochondrial dynamics synthesis related protein-ATPase family AAA domain containing 3A(ATAD3A)in A549 and A549/DDP cells.3.SMI alone or in combination with cisplatin was used to interfere with A549/DDP cells.The inhibitory effect of SMI on the proliferation of A549/DDP cells and the sensitizing effect of SMI on cisplatin were detected by CCK-8.4.Detection of apoptosis related indexes: A549/DDP cells were intervened with(0 mg/m L SMI + 0 μg/m L cisplatin,0mg/m L SMI + 7 μg/m L cisplatin,10 mg/m L SMI + 7 μg/m L cisplatin,15 mg/m L SMI + 7 μg/m L cisplatin and 20 mg/Ml SMI + 7 μg/m L cisplatin)respectively.(1)Hoechst33234 staining was used to observe the changes of nuclei under fluorescence microscope.(2)Apoptosis was detected by Annexin V/PI double staining and flow cytometry.(3)The expression of apoptosis-related proteins(Bcl-2,Bax,Cleaved-caspase 3)was detected by Westernblotting.5.Detection of related indexes of mitochondrial dynamics in A549/DDP cells treated with SMI: A549/DDP cells were intervened with(0 mg/m L SMI + 0 μg/m L cisplatin,0 mg/m L SMI + 7 μg/m L cisplatin,10 mg/m L SMI + 7 μg/m L cisplatin,15 mg/m L SMI + 7 μg/m L cisplatin and 20 mg/m L SMI + 7 μg/m L cisplatin)respectively.(1)Mitochondrial fluorescence probe(Mito-Tracker Green)was labeled and the morphological changes of mitochondria in cells were observed by laser confocal microscope.(2)Rhodamine123 staining and flow cytometry were used to detect the changes of mitochondrial membrane potential.(3)The expression of mitochondrial protein molecules(Mfn2,ATAD3A)was detected by Western blotting.Results: 1.The correlation between Mfn2 expression level and lung adenocarcinoma was analyzed(1)According to the median Mfn2 expression level,504 patients with lung adenocarcinoma were divided into high Mfn2 expression group and low Mfn2 expression group.According to the analysis of survival curve,the prognosis of lung adenocarcinoma patients with low expression of Mfn2 was poor.The expression of Mfn2 in 75 cases of lung adenocarcinoma and paracancerous tissues was analyzed by immunohistochemistry.The results showed that the expression of Mfn2 in lung adenocarcinoma was lower than that in paracancerous tissues,but the expression level of Mfn2 was not related to sex,age,tumor size and TNM stage.(2)Western blot results showed that the expression of fusion-related protein-Mfn2 in mitochondria of cisplatin-resistant A549/DDP cells was lower than that of parent A549 cells;the expression of mitochondrial energy synthesis related proteins ATAD3 A increased.2.Effect of shenmai injection combined with cisplatin on drug resistance of lung adenocarcinoma cells(1)CCK8 showed that Shenmai injection could significantly inhibit the proliferation of A549/DDP cells,and Shenmai injection could enhance the cytotoxicity of cisplatin on A549/DDP.(2)Shenmai injection combined with cisplatin could induce mitochondrial apoptosis in A549/DDP cells.Compared with cisplatin alone,Shenmai injection combined with cisplatin could increase the number of apoptosis,activate apoptosis-related protein Caspase3,and up-regulate apoptosis-promoting protein Bax and down-regulate the expression of apoptosis-inhibiting protein Bcl-2.(3)Shenmai injection combined with cisplatin can induce mitochondrial apoptosis in A549/DDP cells,which is related to its effect on mitochondrial dynamics.The intervention of shenmai injection combined with cisplatin can change the mitochondrial dynamics and make it more prone to fusion,meanwhile,the mitochondrial quality and mitochondrial membrane potential are decreased,and the expression of mitochondrial fusion protein Mfn2 is up-regulated,and the expression of mitochondrial kinetics and cell death related protein ATAD3 A is down-regulated.Conclusion: 1.Compared with paracancerous tissues,the low expression of Mfn2 in lung adenocarcinoma was not related to sex,age,tumor size and stage,but the low expression of Mfn2 in lung adenocarcinoma was associated with poor prognosis,and the low expression of Mfn2 in lung adenocarcinoma was associated with cisplatin resistance.2.Shenmai injection combined with cisplatin can reverse the cisplatin resistance of lung adenocarcinoma cells by inducing mitochondrial apoptosis,and the mechanism may be related to the combined effect of drugs on mitochondrial dynamics of lung adenocarcinoma cells. |
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