| Background and objectiveThe incidence of lung cancer is currently a high incidence of malignant tumors,with the increase of environmental pollution,the incidence and mortality of lung cancer is still a clear upward trend,a serious threat to people's lives and health.Chinese medicine has made considerable achievements in the treatment of tumors,especially the anticancer active ingredients extracted from Chinese herbal medicines for plant development.It is the current research direction to analyze the chemical structure of various Chinese medicines and to extract anti-tumor active ingredients from them.There are also a large number of reports that gallic acid in the clinical application of the tumor has many pharmacological activities,such as gallic acid by reducing the content of free radicals in cells so as to enhance cell antioxidant capacity;while gallic acid can promote tumor cells The process of apoptosis and the intensive nourishment of neovascularization in the tumor,thereby inhibiting the formation and further metastasis of the tumor,exacerbation and the like,but the exact mechanism is not yet clear.Therefore,in this study,gallic acid on lung cancer cell proliferation and apoptosis of the activity and possible mechanism as a breakthrough point,so as to further the development of natural medicine for the treatment of lung cancer provide a theoretical basis and technical support.This part is divided into three parts: The first part is the effect of gallic acid on the proliferation and apoptosis of A549 and 95-D cells in lung cancer cells by cell experiment.The second part is to study the effect of gallic acid on tumor growth in vivo And detected the proliferation of A549 and 95-D in lung cancer cells in vivo.In the third part,NF-?? was detected by western blot in A549 and 95-D Protein,Caspase-3 protein,AKT total protein and AKT phosphorylation protein and P53 protein to understand the possible mechanism of gallic acid-induced lung cancer cell apoptosis.Part One Effect of Gallic Acid on the Proliferation and Apoptosis of A549 and 95-D Cells in Lung Cancer CellsMethods 1.Different concentrations of gallic acid solution,A549 and 95-D cell culture medium preparation.2.The proliferation of A549 and 95-D tumor cells was detected by CCK-8 assay.3.Flow cytometry was used to detect the cell cycle of A549 and 95-D tumor cells.4.Flow cytometry was used to detect the cells of A549 and 95-D tumor cells.5.Statistical methods: SPSS19.0 system analysis of all experimental data in the experiment,two sets of data between the application of t test,three sets and more data to compare ?2 test,while applying the chi-square test to detect the percentage of Counting data.Results1.There was no significant difference in the proliferation of A549 and 95-D in lung cancer cells when the concentration of gallic acid was 10?g / m L compared with the blank group(0?g / m L for gallic acid),p> 0.05.When the concentration of gallic acid was 20,30?g / m L,the proliferation of A549 and 95-D cells in lung cancer cells was inhibited,the inhibitory rate was significantly higher than that in blank group(0?g / m L for gallic acid),and the difference was significant at p <0.05;and when the concentration of gallic acid was> 10?g / With the increase of gallic acid concentration,the proliferation inhibition rates of lung cancer cells A549 and 95-D were significantly increased,p <0.05,the difference was significant.2.Compared with the blank group(gallic acid 0?g / m L),20?g / m L and 30?g / m L gallic acid can significantly reduce the number of A549 and 95-D lung cancer cells in the same field of vision,p <0.05 significant difference;and The difference between 10?g / m L gallic acid and blank group was not significant(p> 0.05).With the increase of gallic acid concentration,the number of apoptotic cells in lung cancer A549 and 95-D gradually increased.3.Compared with the blank group(gallic acid is 0?g / m L),20?g / m L and 30?g / m L of gallic acid can significantly reduce the number of A549 and 95-D lung cancer cells in the same field of vision,p <0.05 significant difference;10?g / m L gallate had no significant difference with that of blank group(p> 0.05).With the increase of gallic acid concentration,the number of apoptotic cells in lung cancer A549 and 95-D gradually increased.4.With the increase of the concentration of gallic acid,the cell apoptosis rate of lung cancer cell A549 was also significantly increased(p <0.05,the difference was significant,statistically significant);in the cell apoptosis rate of A549,There was no significant difference in the cell apoptosis rate of lung cancer cell A549 between each dose group of gallic acid(P> 0.05),but there was no statistical significance.Among the total cell apoptosis rate(early stage + late stage apoptosis rate)of lung cancer cell A549,As the concentration of gallic acid increased,the total cell apoptosis rate(early + late apoptotic rate)of lung cancer cell A549 also increased significantly(p <0.05,significant difference,statistically significant).There was no significant difference in the early apoptotic rate of 95-D cells in lung cancer cell line 95-D between 0?g / m L and 10?g / m L of gallic acid in 95-However,with the increase of gallic acid concentration,the early apoptotic rate of 95-D cells in lung cancer cells was also significantly increased when the concentration of gallic acid was> 10 ?g / m L(p <0.05,the difference was significant and statistically significant);With the increase of gallic acid concentration,the cell apoptosis rate of 95-D cells in lung cancer cells was significantly increased(p <0.05,the difference was significant,statistically significant);in the 95-D lung cancer cells,the total apoptosis rate(Early and late apoptotic rate)),with the increase of gallic acid concentration,the total cell apoptosis rate(early + late apoptotic rate)of 95-D lung cancer cells was also significantly increased(p <0.05,the difference was significant,there are Statistical significance).Part Two Effect of gallic acid on the tumorigenicity of A549 and 95-D in vivoMethods 1.The A549 and 95-D single cell turbid fluid was used to model animals in the underarm region of the SPF nude mice.2.After the success of modeling,they were divided into blank group and gallic acid administration group respectively to observe the change of tumor volume dynamically,and to make tumor growth curve of relative volume and to calculate the tumor suppression rate.3.Immunohistochemistry was used to detect the proliferation index of A549 and 95-D in the resected tumor specimens.4.Statistical methods: SPSS19.0 system analysis of all experimental data in the experiment,two sets of data between the application of t test,three sets and more data to compare ?2 test,while applying the chi-square test to detect the percentage of Counting data.Results 1.After 2 weeks of treatment with gallic acid,the relative tumor volume of nude mice + A549 + gallic acid group(30?g / m L)was significantly lower than that of nude mice + A549 + blank control group(p <0.05,statistically significant)The relative tumor volume of rats treated with 95 + D-gallic acid(30?g / m L)was significantly lower than that of control + 95-D + blank control group(p <0.05).By weighing the tumor weight of each group of mice,it can be concluded that the nude mice + A549 + blank control group,the nude mice + A549 + gallic acid administration group(30?g / m L),the nude mice + 95-D + blank control group,The tumor weights of the 95-D + Gallic acid group(30?g / m L)were 6936.5 ± 831.5mg,2135.9 ± 521.4mg,4556.7 ± 689.3mg and 862.3 ± 106.7mg,In vivo tumor inhibition rate was 69.21%,gallic acid(30?g / m L)on 95-D in vivo tumor inhibition rate was 81.08%.2.The number of tumor cells in nude mice + A549 + gallic acid group(30?g / m L)with proliferative ability was significantly less than that in nude mice + A549 + blank control group;The number of competent tumor cells was significantly less than that in nude mice + 95-D + blank control group(significant difference,p <0.05).3.The tumor proliferation index of nude mice + A549 + gallic acid group(30?g / m L)was 0.1835 ± 0.0186,which was significantly lower than that of nude mice + A549 + 0.2962 ± 0.0216(p <0.05)The tumor proliferation index of 0.0932 ±0.0109 in 95-D + gallic acid group(30?g / m L)was significantly less than that in nude mice + 95-D + blank control group(0.2268 ± 0.0207,p <0.05).4.Statistical methods: SPSS19.0 system analysis of all experimental data in the experiment,two sets of data between the application of t test,three sets and more data to compare ?2 test,while applying the chi-square test to detect the percentage of Counting data.Part Three Possible Mechanism of Gallic Acid Induced ApoptosisMethods1.Application of different concentrations of gallic acid in lung cancer cells A549 and 95-D culture medium mixed.2.Western blot was used to detect the expression of NF-k? in A549 and 95-D cells.3.The protein expression of Caspase-3 in A549 and 95-D cells was detected by Western blot.4.Detection of AKT total protein and AKT phosphorylation protein in A549 and 95-D cells by Western blot.5.Western blot was used to detect the expression of P53 protein in A549 and 95-D cells.6.The effect of gallic acid on the expression of Caspase-3 protein in A549 and 95-D cells was observed by si RNA transfection.7.Using SPSS19.0 statistical software for data analysis.Results 1.With the increase of the concentration of gallic acid,the expression of NF-?? protein in A549 and 95-D lung cancer cells was significantly decreased(p <0.05,significant difference,with statistical significance).2.With the increase of gallic acid concentration,the expression of Caspase-3 in lung cancer cells A549 and 95-D increased significantly(p <0.05,significant difference,statistically significant).3.With the increase of gallic acid concentration,the expression of AKT protein in lung cancer cells A549 had no significant change(p> 0.05,the difference was not significant,no statistical significance);AKT phosphorylation protein expression in lung cancer A549 cells was significantly decreased(p < 0.05,significant difference,statistically significant).Conclusion 1.Gallic acid can inhibit the proliferation of lung cancer cells and promote the apoptosis of lung cancer cells,and the concentration of gallic acid in a dose-dependent manner.2.Gallic acid inhibits the in vivo tumorigenicity of lung cancer cells A549 and 95-D.3.Gallic acid can affect the apoptosis of lung cancer cells by inhibiting the expression of NF-?? and AKT phosphorylation proteins in lung cancer cells and promoting the expression of Caspase-3 and P53 in lung cancer cells.4.Knockdown of p53 gene and found that the activation of gallic acid downstream protein caspase3 decreased,indicating that p53 gene may be gallic acid-induced apoptosis in lung cancer key proteins. |
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