基于氧化应激和缺氧探讨加味真武汤对HK-2细胞线粒体生物合成的影响  

Exploration on effects of Modified Zhenwu Decoction on mitochondrial biosynthesis in HK-2 cells based on the oxidative stress and hypoxia

作　　者：张圆圆 靳培培 宋婧雅 郭登洲[2] ZHANG Yuanyuan;JIN Peipei;SONG Jingya;GUO Dengzhou(Graduate School,Hebei University of Chinese Medicine,Shijiazhuang 050200,China;The First Affiliated Hospital of Hebei University of Chinese Medicine,Hebei Provincial Hospital of Traditional Chinese Medicine,Shijiazhuang 050011,China)

机构地区：[1]河北中医药大学研究生学院,石家庄050200 [2]河北中医药大学第一附属医院,河北省中医院,石家庄050011

出　　处：《中华中医药杂志》2024年第7期3663-3668,共6页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：河北省中医药管理局科研计划项目(No.2023021);河北中医药大学博士研究生创新资助项目(No.XCXZZBS2023009)。

摘　　要：目的:通过观察加味真武汤对低氧和脂多糖(LPS)共同处理的HK-2细胞中活性氧(ROS)、脯氨酰羟化酶2(PHD2)、缺氧诱导因子-1α(HIF-1α)、PPARγ辅助活化因子1α(PGC-1α)、核呼吸因子(NRF)1、NRF2、线粒体转录因子A(TFAM)表达的影响,探讨加味真武汤延缓慢性肾功能衰竭(CRF)进展的潜在机制。方法:将HK-2细胞分为正常组、模型组、空白血清组、含药血清组、FM19G11组、NAC组。干预72 h后,流式细胞仪检测ROS水平;免疫荧光检测PGC-1α表达;电镜观察线粒体形态变化;TUNEL法检测细胞凋亡;Western Blot检测HIF-1α、PHD2、PGC-1α、NRF1、NRF2、TFAM蛋白表达水平。结果:与正常组比较,模型组细胞中ROS含量显著升高(P<0.01);PGC-1α表达显著减少(P<0.01);PHD2、PGC-1α、NRF1、NRF2、TFAM蛋白表达显著降低(P<0.01),HIF-1α蛋白表达显著升高(P<0.01);线粒体数量减少,内室肿胀,嵴移向周围;细胞凋亡数量显著升高(P<0.01)。与模型组比较,经加味真武汤提前干预后,细胞中ROS含量显著降低(P<0.01),PGC-1α表达显著增加(P<0.01);PHD2、PGC-1α、NRF1、NRF2、TFAM蛋白表达显著升高(P<0.01),HIF-1α蛋白表达显著下降(P<0.01);线粒体数量增加,病理改变减轻;细胞凋亡数量显著下降(P<0.01)。结论:加味真武汤可能通过抑制近曲肾小管上皮细胞氧化应激和缺氧反应,改善线粒体生物合成,从而减少细胞凋亡,延缓CRF进展。Objective:To observe the effects of Modified Zhenwu Decoction on the expression of ROS,PHD2,HIF-1α,PGC-1α,NRF1,NRF2 and TFAM in HK-2 cells treated with hypoxia and LPS,the potential mechanism of Modified Zhenwu Decoction delaying the progression of chronic renal failure(CRF)was investigated.Methods:HK-2 cells were divided into normal group(A),model group(B),blank serum group(C),drug-containing serum group(D),FM19G11 group(E)and NAC group(F).After 72 h of intervention,the ROS levels were detected by flow cytometry.The expression of PGC-1αwas detected by immunofluorescence.The morphological changes of mitochondria were observed by electron microscope.Apoptosis was detected by TUNEL kit.The protein expression levels of HIF-1α,PHD2,PGC-1α,NRF1,NRF2 and TFAM were detected by Western Blot.Results:Compared with group A,ROS content in group B significantly increased(P<0.01),the expression of PGC-1αsignificantly decreased(P<0.01),the protein expressions of PHD2,PGC-1α,NRF1,NRF2 and TFAM significantly decreased(P<0.01),while the expressions of HIF-1αsignificantly increased(P<0.01),the number of mitochondria decreased,the inner chamber swelled,and the ridge shifted to the periphery,the number of apoptosis significantly increased(P<0.01).Compared with group B,ROS content in cells significantly decreased after advanced intervention with Modified Zhenwu Decoction(P<0.01),the expression of PGC-1αsignificantly increased(P<0.01),the protein expressions of PHD2,PGC-1α,NRF1,NRF2 and TFAM significantly increased(P<0.01),while the expressions of HIF-1αsignificantly decreased(P<0.01),the number of mitochondria increased and the pathological changes decreased,the number of apoptosis significantly decreased(P<0.01).Conclusion:Modified Zhenwu Decoction may reduce cell apoptosis and delay the progression of CRF by inhibiting oxidative stress and hypoxia of proximal curved renal tubular epithelial cells and improving mitochondrial biosynthesis.

关 键 词：加味真武汤 氧化应激 缺氧 线粒体生物合成 细胞凋亡 慢性肾功能衰竭 机制 

分 类 号：R285[医药卫生—中药学]