Effect Of Yanghe Decoction On Proliferation Of Human Triple-Negative Breast Cancer Cells Based On EZH2/NF-κB Signaling Pathway

Objective: To investigate the proliferation of Yanghe Decoction on human breast cancer cell MDA-MB-231 and its mechanism by intervention of Yanghe Decoction containing serum on human triple-negative breast cancer cells.The effect of Yanghe Decoction on EZH2/NF-κB signaling pathway in MDA-MB-231 are explored.To provide theoretical basis for Yanghe Decoction in vivo experiment and clinical application.Methods: According to the composition of Yanghe Decoction and the preparation method,the drugs are decocted to prepare a liquid medicine of 0.7 g crude drug amount/m L.Thirty SD female rats are randomly divided into 5 groups: blank control group,low,medium and high dose groups of Yanghe Decoction and positive drug group(6 in each group).The rats in blank control group are treated with distilled water.The rats in low,middle and high doses of Yanghe Decoction are treated at a dose of 4.5 g/kg,9 g/kg,18 g/kg(equivalent to 5 times,10 times,20 times the clinical dose).At the same time,the rats in blank control group,low,middle and high dose group of Yanghe Decoction were injected with 5-Fluorouracil solution through tail vein at the dose of 15 mg/kg.The rats in positive drug group are injected with 5-fluorouracil via the tail vein at a dose of 15 mg/kg(equivalent to clinical human body dosage).At the same time,the rats in the positive group are treated with distilled water.The administration volume of rats in each group is calculated as 10 m L/kg.The rats in each group are given continuous treat for 3 days,blood is taken from abdominal aorta,centrifuged and serum is separated.In the experiment,each group by 10% drug serum intervention.Treated with low,medium and high dose of Yanghe Decoction and positive drug drug-containing serum for 24,48,72 h,the OD value of each group was detected by CCK-8 assay.Treated with low,medium and high dose of Yanghe Decoction and positive drug drug-containing serum for 48 h,the cell cycle distribution and apoptosis are detected by flow cytometry,the secretion of TNF-IL-6 and IL-8 were measured.The expression of TNF-α,IL-6 and IL-8 in the cells are detected by Western blot.The expression of EZH2 and NF-κB are detected by Western blot in the nucleus.Result: 1.The cell growth inhibition rates of MDA-MB-231 cells treated with low,medium and high dose of Yanghe Decoction and positive drug drug-containing serum for 48 h were 7.75%,23.08%,29.46% and 49.31% respectively.For 48 h,the cell growth inhibition rates were 11.95%,28.80%,37.80% and 53.78%,respectively.For 72 h,the cell growth inhibition rates were 19.29%,36.36%,46.86% and 63.63%,respectively.After treated with low,medium and high dose of Yanghe Decoction and positive drug drug-containing serum for 24,48 and 72 h,compared with the blank control group,the growth inhibition rate of each group of low,medium and high dose of Yanghe Decoction and positive drug drug-containing serum groups increased(P <0.05 or 0.01).After treated with low,medium and high dose of Yanghe Decoction and positive drug drug-containing serum for 24,48 and 72 h,the inhibitory rate of each group of MDA-MB-231 cells increased with the increase of time(P <0.01).2.After treated with low,medium and high dose of Yanghe Decoction and positive drug drug-containing serum for 48 h,the proportion of each group of G0/G1 phase were 74.51%,67.93%,60.43%,52.12% and 54.24% respectively.the proportion of each group of S phase were 14.12%,13.53%,16.06%,14.94% and 10.67% respectively,the proportions of each group of G2/M phase were 11.36%,18.53%,23.51%,32.97% and 35.09% respectively.Compared with the blank control group,the proportion of each group of G0/G1 phase cells in the middle and high dose groups of Yanghe Decoction decreased(P <0.05),the proportion of each group of G2/M phase increased(P <0.05 or 0.01).3.After treated with low,medium and high dose of Yanghe Decoction and positive drug drug-containing serum for 48 h,the apoptotic rate of each group of them was 4.65%,8.16%,11.98%,15.65%,23.65% respectively.The apoptosis rates of the blank control group,low,medium and high dose of Yanghe Decoction and positive drug group were compared between groups,the apoptosis rates of low,medium and high dose of Yanghe Decoction and positive drug groups gradually increased(P <0.01).4.The serum levels of TNF-α,IL-6 and IL-8 in the corresponding serum of each group were statistically analyzed.The results showed that there was no significant difference in the concentrations of TNF-α,IL-6 and IL-8(P >0.05).After treated with low,medium and high dose of Yanghe Decoction and positive drug drug-containing serum for 48 h,compared with the blank control group,the level of each group of TNF-α to extracellular secretion of low,medium and high dose of Yanghe Decoction and positive drug drug-containing serum decreased(P <0.05 or 0.01).Compared with the blank control group,the level of each group of IL-6 to extracellular secretion of low,medium and high dose of Yanghe Decoction and positive drug drug-containing serum decreased(P <0.05 or 0.01).Compared with the blank control group,the level of each group of IL-8 to extracellular secretion of low,medium and high dose of Yanghe Decoction and positive drug drug-containing serum decreased(P <0.05 or 0.01).5.After treated with low,medium and high dose drug-containing serum of Yanghe Decoction for 48 h,compared with the blank control group,the expression of each group of TNF-α in MDA-MB-231 cells of low,middle and high dose group of Yanghe Decoction decreased(P <0.05 or 0.01).Compared with the blank control group,the expression of each group of IL-6 in MDA-MB-231 cells of middle and high dose group of Yanghe Decoction decreased(P <0.05).Compared with the blank control group,the expression of each group of IL-8 in MDA-MB-231 cells of middle and high dose group of Yanghe Decoction decreased(P <0.05).6.After treated with low,medium and high dose drug-containing serum of Yanghe Decoction for 48 h,compared with the blank control group,the level of each group of EZH2 in the nucleus of low,middle and high dose group of Yanghe Decoction decreased(P <0.05 or 0.01).Compared with the blank control group,the level of each group of p65 in the nucleus of middle and high dose group of Yanghe Decoction decreased(P <0.01).Conclusion: Yanghe Decoction can reduce the expression of EZH2,reduce the nuclear entry of NF-κB(p65)and inhibit the extracellular secretion of TNF-α,IL-6 and IL-8,thereby inhibit the proliferation of triple-negative breast cancer,arrest cell division in G2/M phase,induce cell apoptosis.Yanghe Decoction plays an important role in anti-tumor.