菊花总黄酮含药血清对干眼细胞模型AR、NF-κB磷酸化蛋白及TGF-β1表达的影响

Effects of Chrysanthemum Total Flavonoids-containing Serum on AR,NF-κB Phosphorylated Protein and TGF-β1 in Dry Eye Cell

目的观察菊花总黄酮在体外条件下对泪腺上皮细胞中雄激素受体(Androgen Receptor,AR)的调控作用及对下游的核因子κB(Nuclear Factor kappa B,NF-κB)、转化生长因子-β1(transforming growth factor-beta1,TGF-β1)的表达影响。方法以过氧化氢(H2O2)诱导泪腺上皮细胞凋亡,建立细胞模型,设立无雄激素培养空白对照组、含雄激素培养对照组、无雄激素培养黄酮治疗组。以MTT法摸索含药血清最佳干预加入量;免疫印迹实验(Western Blot)以及实时荧光定量PCR法(Quantitative Real-Time PCR,qPCR)检测泪腺上皮细胞中AR、NF-κB及TGF-β1的表达情况;并应用雄激素受体阻断剂氟他胺进行AR阻断后再次检测上述指标。结果MTT法检测后计算得出含药血清干预细胞的最终浓度为13.2%;无雄激素培养黄酮治疗组及含雄激素培养对照组中AR、NF-κB及TGF-β1的表达增强,与空白对照组对比有显著差异(P<0.01);无雄激素培养黄酮治疗组中NF-κB表达量比含雄激素培养对照组表达量低,两者间的差异有统计学意义。氟他胺及含药血清干预后,无雄激素培养黄酮治疗组及含雄激素培养对照组中AR及NF-κB的表达未见提高,无统计学意义。结论菊花总黄酮对泪腺上皮细胞可能存在拟雄激素效应,从而使AR、NF-κB表达增加,并通过上调TGF-β1的表达,可能起到在泪腺局部的抗炎作用。

Objective To observe the regulation effect of chrysanthemum total flavonoids on Androgen Receptor(AR)in lacrimal epithelial cells under in vitro conditions and its influence on downstream nuclear factor-κB(NF-κB)and transforming growth factor-β(TGF-β1)expression.Methods H2O2was used to induce the apoptosis of lacrimal epithelial cells,establish cell model,and androgen-free culture blank control group,androgen-containing culture control group,and androgen-free culture flavonoid treatment group.MTT method was used to find out the optimal intervention dosage of drug-containing plasma.Western Blot and Quantitative Real-time PCR(qPCR)were used to detect the expression of AR mRNA,NF-κB phosphorylated protein and TGF-β1 in lacrimal epithelial cells,Use the androgen receptor blocker flutamide to block AR,and then test the above indicators again.Results The final concentration of the interfering cells was calculated by MTT method to be 13.2%.The expression of AR protein,NF-κB and TGF-β1 in the chrysanthemum flavonoid plasma intervention group and testosterone propionate intervention group were enhanced,and there were significant differences from the blank control group(P<0.01),The expression level of NF-κB in the total flavonoid-containing plasma intervention group was lower than that in the testosterone propionate intervention group,the difference between the two was significant.The expression of AR and NF-κB phosphorylated protein did not increase after flutamide intervention in lacrimal epithelial cells,and there was no statistical significance.Conclusions Chrysanthemum flavonoids may have an androgen-like effect on lacrimal epithelial cells,thereby increasing the expression of AR and NF-κB,and can play an anti-inflammatory effect by up-regulating the expression of TGF-β1.