To Investigate The Effects Of Different Concentrations Of IL-33 On Invasion And Apoptosis Of Huh-7 Cells Based On NF-κB Pathway

Objective:to investigate the effect of Nuclear factor-kappa B（NF-κB）on the invasion and apoptosis of human hepatocellular carcinoma Huh-7 cells after different concentrations of interleukin-33（IL-33）.Methods:（1）Human hepatoma Huh-7 cells were cultured in vitro,and the Huh-7 cells in logarithmic phase were randomly divided into 5 groups:（1）blank control group（cultured in5%CO₂incubator at 37℃）,（2）20ng/L IL-33 group（IL-33 was added,and the final concentration was 20ng/L）,（3）40ng/L IL-33 group（IL-33 was added,and the final concentration was 40ng/L）,（4）60ng/L IL-33 group（IL-33 was added,and the final concentration was 60ng/L）,（5）ant-ST2L group（adding ST2L antagonist,final concentration was 10ng/m L）.（2）Transwell invasion assay was used to detect the effects of different concentrations of IL-33 and ST2L antagonists on the invasion of human hepatocellular carcinoma Huh-7 cells for 24 hours,and the cells entering the subventricular membrane of each group were detected.（3）Flow cytometry was used to detect the effects of different concentrations of IL-33 and ST2L antagonists on the apoptosis of human hepatocellular carcinoma Huh-7 cells treated for 24 hours.（4）q RT-PCR was used to detect the effects of different concentrations of IL-33 and ST2L antagonists on the expression of NF-κB m RNA in human hepatoma Huh-7 cells.（5）Westen blot was used to detect the effect of different concentrations of IL-33 and ST2L antagonist on the expression of NF-kappa B protein in human hepatocellular carcinoma Huh-7 cells for 24 hours,and the expression of NF-kappa B protein in each group was detected.（6）SPSS26.0 statistical software was used to analyze the experimental data,The measurement data were expressed by mean±standard deviation.Independent sample t-test was used for comparison between the two groups,and one-way ANOVA was used for multiple sample means,correlation analysis was performed with Spearman rank correlation analysis,indicating that the difference was statistically significant in P<0.05.Results:（1）Transwell invasion assay showed that the invasion number of cells treated with IL-33（20ng/L,40ng/L,60ng/L）was significantly lower than that of the control group,and decreased with the increase of IL-33 concentration,while the invasion number of cells treated with ST2L antagonist was significantly higher than that of the control group（P<0.01）.（2）Apoptosis experiment showed that the apoptosis rate decreased significantly after intervention with different concentrations of IL-33,and the apoptosis rate decreased with the increase of IL-33 concentration,and the apoptosis rate increased significantly after intervention with ST2L antagonist group（P<0.01）.（3）q RT-PCR analysis showed that after treatment with IL-33（20ng/L,40ng/L,60ng/L）,the m RNA expression of NF-κBp65 was significantly up-regulated compared with the control group,and the m RNA expression of NF-κBp65 was significantly down-regulated compared with the control group after treatment with ST2L antagonist.（4）The results of Westen blot assay showed that the protein expression of NF-κBp65 was significantly increased after treatment with IL-33（20ng/L,40ng/L,60ng/L）compared with the control group,while the protein expression of NF-κBp65 was significantly decreased after treatment with ST2L antagonist compared with the control group.Conclusion:（1）IL-33 can significantly reduce the invasive ability and inhibit the apoptosis of human hepatocellular carcinoma Huh-7 cells in a dose-dependent manner.（2）IL-33 can increase the m RNA and protein expression of NF-κBp65 in human hepatoma Huh-7 cells.（3）IL-33 may inhibit the invasion and apoptosis of human hepatoma Huh-7 cells by activating NF-κB pathway in combination with ST2L.