Experimental Research On The Mechanism Of The Effect Of Ai Kang Fang On MTOR And Related Apoptotic Proteins In Human Lung Squamous Cell Carcinoma SK-MES-1

Objective Previous research by the research group has fully demonstrated that Ai Kang Fang medicated serum can induce SK-MES-1 cell apoptosis.Based on these experiments,20% of the optimal concentration of drug-containing serum was used to intervene the cells,and to observe the effect of Ai Kang Fang drug-containing serum on Caspase-9,Bad,Survivin,and mTOR in human lung squamous cell carcinoma SK-MES-1 cells for protein expressions and mRNA levels,further explore the possible molecular mechanism of this formula in promoting lung cancer cell apoptosis.Method Sixty male SPF-grade SD rats weighed 220±20 grams were randomly divided into normal saline control group,cyclophosphamide group,Ai Kang Fang group,and Ai Kang Fang combined cyclophosphamide group,each group 15 only.According to the method of preparing the medicated serum by the previous research group,the rats in the above four groups were administered with saline,intraperitoneal injection of cyclophosphamide,intragastric administration of Ai Kang Fang,intragastric injection of Ai Kang Fang and intraperitoneal injection of cyclophosphamide,then prepared for drug containing serum.After SK-MES-1 cells were cultured,according to the different groups of drug-containing serum added,the cells were divided int o: blank control group(only add DMEM complete medium),control group(normal saline group),and experimental groups(Ai Kang Fang group?cyclop-hosphamide group?Ai Kang Fang combined cyclophosphamide group).After the cells were cultured for 24 hours,using the immunocytochemistry and W-estern blot methods to detect the protein expressions of Caspase-9,Bad,Survivin,and mTOR,using the Real-time PCR method to detect the Caspase-9,Bad,Survivin,and mTOR mRNA levels.Results1.Effect of Ai Kang Fang on Caspase-9,Bad protein expression and mRNA level in SK-MES-1 Cells.Immunocytochemistry and the Western blot methods detected the protein expression levels of Caspase-9 and Bad: The protein expression in the experimental groups is higher than that in the saline control group(P<0.05),compared with the experimental groups,the cyclophosphamide group's protein expression was higher than that of the Ai Kang Fang group(P<0.05),and the Ai Kang Fang combined with the cyclophosphamide group's protein expression was higher than the cyclophosphamide group,the difference was statistical significance(P<0.05).Real-time PCR detection of Caspase-9 and Bad mRNA levels: The experimental results show that the mRNA levels of each experimental group in Caspase-9 and Bad are higher than those in the normal saline control group(P<0.05),compared with the experimental groups,cyclophosphamide group was higher than that of Ai Kang Fang group(P<0.05),the mRNA level of Ai Kang Fang combined with cyclophosphamide group was higher than that of cyclophosphamide group,and the difference was statistically significant(P <0.05).2.Effect of Ai Kang Fang on Survivin,mTOR protein expression and mRNA level in SK-MES-1 cells.Immunocytochemistry and Western blot methods detected the Survivin and mTOR protein expression levels: The protein expression in the experimental group was lower than that in the normal saline control group(P<0.05),comparing the experimental groups,the cyclophosphamide histone expression was lower than that of the Ai Kang Fang group(P<0.05),and the Ai Kang Fang combined with the cyclophosphamide grouo protein expression was lower than the cyclophosphamide group,the difference was statistically significant(P<0.05).Real-time PCR detection of Survivin and mTOR levels: The experimental results show that the mRNA levels of each experimental group in Survivin and mTOR are lower than those in the normal saline control group(P<0.05),compared with the experimental groups,cyclophosphamide group was lower than that of Ai Kang Fang group(P<0.05),the mRNA level of Ai Kang Fang combined with cyclophosphamide group was lower than that of cyclophosphamide group,and the difference was statistically significant(P <0.05).Conclusion1.Ai Kang Fang and Ai Kang Fang combined with Cyclophosphamide can increase Caspase-9 and Bad protein expression and mRNA levels in human lung squamous cell carcinoma SK-MES-1 cells.2.Ai Kang Fang and Ai Kang Fang combined with cyclophosphamide can reduce Survivin and mTOR protein expression and mRNA levels in human lung squamous cell carcinoma SK-MES-1 Cells.3.Ai Kang Fang may promote the expression of human lung squamous cell carcinoma SK-MES-1 cells by regulating the expression of apoptosis-related proteins Caspase-9,Bad,and Survivin and by regulating the expression of mTOR to inhibit the PI3K/AKT/mTOR signaling pathway apoptosis and synergistic effect with cyclophosphamide.