杞精明目汤颗粒剂对肿瘤坏死因子-α刺激下结膜松弛症成纤维细胞MAPK信号通路的影响

Effect of Qijingmingmu decoction granule on MAPK signal pathway in fibroblasts of conjunctivochalasis stimulated by TNF-α

目的观察肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)刺激下,杞精明目汤颗粒剂对结膜松弛症(conjunctivochalasis,CCH)结膜成纤维细胞表达丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)的影响,探讨CCH的发病机制及有效治疗策略。方法体外培养CCH结膜成纤维细胞,分为CCH组、CCH+TNF-α组及CCH+TNF-α+杞精明目汤组。CCK-8确定杞精明目汤颗粒剂有效浓度,用含10^(-2)mg·L^(-1)的TNF-α刺激培养的成纤维细胞,加入杞精明目汤颗粒剂干预48 h,分别采用ELISA、Western Blot和RT-PCR检测MAPK信号通路相关蛋白及mRNA的表达,并行统计学分析。结果 CCK-8确定杞精明目汤颗粒剂有效浓度为1.61 g·L^(-1)。三组MAPK信号通路相关分子细胞外调节蛋白激酶(extracellular regulated protein kinase,ERK)、c-jun氨基末端激酶(c-jun N-terminal kinase JNK)、p38 MAPK及其磷酸化水平的吸光度(A_(450))值总体差异均有统计学意义(均为P<0.05),且TNF-α可明显上调CCH表达ERK1/2、JNK1/2、p-JNK1/2、p38 MAPK、p-p38 MAPK(均为P<0.05),而杞精明目汤可显著下调TNF-α刺激后的CCH成纤维细胞表达ERK1/2、p-ERK1/2、JNK1/2、p38 MAPK、p-p38 MAPK(均为P<0.05)。三组p-ERK1/2、p38 MAPK、p-p38 MAPK蛋白总体差异均有统计学意义(均为P<0.05),且TNF-α可明显上调CCH成纤维细胞p-ERK1/2、p-JNK1/2、p38 MAPK、p-p38 MAPK蛋白的表达(均为P<0.05),杞精明目汤可显著下调TNF-α刺激后的CCH成纤维细胞表达p-ERK1/2、p38 MAPK(均为P<0.05)。三组ERK1/2、p38 MAPK mRNA表达总体差异均有统计学意义(均为P<0.05),且TNF-α可明显上调CCH成纤维细胞ERK1/2、p38 MAPK mRNA的表达(均为P<0.05),杞精明目汤可显著下调TNF-α刺激后的CCH成纤维细胞p38 MAPK mRNA表达(P<0.05)。结论 TNF-α可上调CCH成纤维细胞MAPK信号通路的表达,导致CCH的发生发展,而杞精明目汤颗粒剂在一定程度上可下调MAPK信号通路的表达从而发挥治疗作用。

Objective To observe the effects of Qijingmingmu decoction granule on the expression of mitogen-activated protein kinase(MAPK)signal pathway in fibroblasts of conjunctivochalasis(CCH)under the stimulation of tumor necrosis factor(TNF-α),and to explore the pathogenesis and effective treatment method of CCH.Methods CCH conjunctival fibroblasts were cultured in vitro and divided into CCH group,CCH+TNF-αgroup and CCH+TNF-α+Qijingmingmu decoction group.CCK-8 assay was used to determine the effective concentration of Qijingmingmu decoction granule,and 10-2 mg·L-1 TNF-αwas added in the cultured fibroblasts of the latter two groups,followed by interference with Qijingmingmu decoction granule for 48h.The expression of MAPK signal pathway related protein and mRNA were detected by ELISA,Western Blot and RT-PCR.Then the results were statistically analyzed.Results CCK-8 assay showed the effective concentration of Qijingmingmu decoction granule was 1.61 g·L-1.And there were significant differences in the total A values(A450)of extracellular regulated protein kinase(ERK),c-jun N-terminal kinase(JNK),p38 mitogen activated protein kinase(p38 MAPK)and their phosphorylation levels among the three groups(all P<0.05).Moreover,TNF-αcould significantly up-regulate the expression of ERK1/2,JNK1/2,p-JNK1/2,p38 MAPK and p-p38 MAPK in CCH fibroblasts(all P<0.05),while Qijingmingmu decoction down-regulated their expressions in CCK fibroblasts after TNF-αstimulation(all P<0.05).Furthermore,the total differences in p-ERK1/2,p38 MAPK and p-p38 MAPK protein were significant in the three groups(all P<0.05);and TNF-αcould significantly up-regulate the expression of p-ERK1/2,p-JNK1/2,p38 MAPK and p-p38 MAPK protein in CCH fibroblasts(all P<0.05),while Qijingmingmu decoction down-regulated p-ERK1/2 and p38 MAPK expressions in CCK fibroblasts after TNF-αstimulation(both P<0.05).In addition,the total differences in ERK1/2 and p38 MAPK mRNA were significant in the three groups(both P<0.05);and TNF-αcould significantly up-regulate the expression of ERK1/2 and p38 MAPK mRNA in CCH fibroblasts(both P<0.05),while Qijingmingmu decoction down-regulated p38 MAPK mRNA expressions in CCK fibroblasts after TNF-αstimulation(P<0.05).Conclusion The inflammatory factor TNF-αcan up-regulate the expressions of MAPK signal pathway related protein and mRNA in CCH fibroblasts,resulting in the occurrence and development of CCH,and meanwhile Qijingmingmu decoction granule can down-regulate the expression of MAPK signal pathway to play the therapeutic role in CCH to some extent.