基于TNF-α/P38MAPK/P53通路研究养阴润目丸对TNF-α诱导的兔泪腺细胞损伤保护作用

Protective effect of Yangyin Runmu Pill on rabbit lacrimal gland cell injury induced by TNF-αbased on TNF-α/P38MAPK/P53 pathway

目的:通过TNF-α诱导兔泪腺损伤建立干眼细胞模型,观察养阴润目丸对干眼兔泪腺细胞存活、凋亡和炎症因子分泌的影响,以及调控TNF-α/P38MAPK/P53通路的作用,探讨养阴润目丸对干眼兔泪腺细胞的保护作用及其机制。方法:原代分离培养泪腺细胞,并分为Con组、TNF-α组、Positive组和YYRM组。制备杞菊地黄丸、养阴润目丸的含药血清和无含药血清,给予相应干预后,采用MTT法检测各组细胞存活率,流式细胞术检测细胞凋亡率,ELISA法检测炎症因子(IL-1β、IL-6)的分泌水平,Western Blot法检测细胞中TNF-α、P38MAPK、P53蛋白的表达情况。结果:与Con组比较,TNF-α组泪腺细胞存活率显著下降(P<0.05),细胞凋亡率显著升高(P<0.05),炎症因子IL-1β和IL-6分泌显著增多(P<0.05);与TNF-α组比较,Positive组和YYRM组细胞存活率显著升高(P<0.05),细胞凋亡率降低(P<0.05),炎症因子IL-1β和IL-6分泌显著减少(P<0.05)。TNF-α诱导后激活了TNF-α/P38MAPK/P53通路的关键蛋白表达,而养阴润目丸能抑制该系列的关键蛋白表达,且与杞菊地黄丸效果相近。结论:干眼的发病可能与激活TNF-α/P38MAPK/P53通路有关,养阴润目丸可下调TNF-α、P38MAPK和P53蛋白表达,从而减少炎症因子分泌,抑制其凋亡率,改善其存活率,提示养阴润目丸可能通过抑制TNF-α激活的TNF-α/P38MAPK/P53信号通路治疗干眼。

Objective:To observe the effects of Yangyin Runmu Pill on the survival,apoptosis and secretion of inflammatory factors of lacrimal gland cells in rabbits with dry eye induced by TNF-α,and to explore the protective effect and mechanism of Yangyin Runmu Pill on lacrimal gland cells of dry eye rabbits.Methods:Lacrimal gland cells were isolated and cultured and divided into Con group,TNF-αgroup,Positive group and YYAM group.After the intervention,the cell survival rate was detected by MTT method,the apoptosis rate was detected by flow cytometry,the secretion level of inflammatory factors(IL-1β,IL-6)was detected by ELISA,and the expression of TNF-α/P38MAPK/P53 pathway protein was detected by Western Blot.Results:Compared with Con group,TNF-αinduced lacrimal gland cells in TNF-αgroup significantly decreased cell survival rate(P<0.05),increased apoptosis rate(P<0.05),and increased secretion of inflammatory cytokines IL-βand IL-6(P<0.05).After drug intervention,compared with TNF-αgroup,Positive group and YYRM group showed different degrees of increase in cell activity(P<0.05),decreased apoptosis rate(P<0.05)and secretion of inflammatory factors IL-1βand IL-6(P<0.05).Further studies showed that TNF-αcould activate the key protein expression of TNF-α/P38MAPK/P53 pathway,while Yangyin Runmu Pill could inhibit the expression of key proteins,and the effect was similar to that of Qiju Dihuang Pill.Conclusion:The pathogenesis of dry eye may be related to the activation of TNF-α/P38MAPK/P53 pathway.Yangyin Runmu Pill can down regulate the protein expression of TNF-α,P38MAPK and P53,thus reducing the secretion of inflammatory factors,inhibiting its apoptosis rate and improving its survival rate.It is suggested that Yangyin Runmu Pill may treat dry eye by inhibiting TNF-α/P38MAPK/P53 signal pathway activated by TNF-α.