摘要
目的:观察芪参汤对肝窦内皮细胞去分化及自噬的影响。方法:大鼠肝窦内皮细胞(LSEC)随机分为空白组、模型组、芪参汤低、中、高和抑制剂组,通过100μg/mL的氧化型低密度脂蛋白(oxLDL)诱导24 h建模并给予相应的药物或血清进行干预。分别采用RT-qPCR及免疫荧光染色法检测VEGFR2及ET1的表达水平,透射电镜检测LSEC超微结构,ELISA法检测NO含量,Western blot法检测自噬相关蛋白(LC3BI、LC3BII和p62)及内皮细胞功能相关蛋白(eNOS及其磷酸化水平)的表达水平。结果:透射电镜结果显示,芪参汤含药血清能增加LSEC细胞中窗孔、自噬体的数量,抑制内皮下基底膜形成。与模型组相比,芪参汤含药血清能够明显上调LSEC中VEGFR2 mRNA及蛋白的表达水平,下调ET1 mRNA及蛋白的表达水平,差异均具有统计学意义(P<0.05)。此外,芪参汤还能明显增加LSEC中LC3BⅡ、p-eNOS、eNOS蛋白的表达和LC3BⅡ/LC3BI、p-eNOS/eNOS比值,降低LC3BI和p62蛋白的表达,与模型组比较,差异均具有统计学意义(P<0.05)。结论:芪参汤能够通过促进LSEC自噬水平抑制其去分化水平,进而发挥抗肝纤维化的作用。
Objective:To observe the effect of Qishen decoction on dedifferentiation and autophagy of liver sinusoid endothelial cells(LSEC).Methods:LSEC were randomly divided into the control group,the model group,the low,medium and high dose groups of Qishen decoction,and the inhibitor group.The model was induced by 100μg/mL oxidized low-density lipoprotein(oxLDL)for 24 h,and the corresponding drugs or medicated serum were given for intervention.The expression levels of VEGFR2 and ET1 were detected by RT-qPCR and immunofluorescence staining,the ultrastructure of LSEC was detected by transmission electron microscopy,the content of NO was detected by ELISA,and the expression levels of autophagy related proteins(LC3BI,LC3BⅡand p62)and endothelial function related proteins(eNOS and p-eNOS)were detected by Western blot.Results:The results of transmission electron microscopy showed that Qishen decoction medicated serum could increase the number of fenestra and autophagy in LSEC cells,and inhibit the formation of basement membrane under endothelium.Compared with the model group,Qishen decoction medicated serum could significantly up-regulate the expression level of VEGFR2 mRNA and protein in LSEC,and down-regulate the expression level of ET1 mRNA and protein,with statistically significant differences(P<0.05).In addition,Qishen decoction medicated serum could significantly increase the expression of LC3BⅡ,p-eNOS,eNOS protein and the ratio of LC3BⅡ/LC3BI,p-eNOS/eNOS and reduce the expression of LC3BI and p62 protein in LSEC,which was statistically significant compared with the model group(all P<0.05).Conclusion:Qishen decoction can inhibit the dedifferentiation of LSEC by promoting the autophagy level of LSEC and play an anti-fibrosis role.
作者
毛旭
袁星星
杨磊
李丹丹
王炳予
MAO Xu;YUAN Xing-xing;YANG Lei;LI Dan-dan;WANG Bing-yu(Heilongjiang University of Traditional Chinese Medicine, Jiamusi 154000, China;Nangang Branch of Heilongjiang Academy of Chinese Medicine Sciences, Harbin 1500001, China)
出处
《海南医学院学报》
CAS
2020年第16期1222-1227,共6页
Journal of Hainan Medical University
基金
黑龙江省中医药科研项目(ZHY18-029,ZHY19-061,ZHY19-062)
黑龙江省自然科学基金联合引导项目(LH2019H095)
国家中医药管理局科研项目(2016ZX05)。
关键词
芪参汤
肝窦内皮细胞
自噬
去分化
含药血清
Qishen Decoction
Liver sinusoidal endothelial cells
Autophagy
Dedifferentiation
Medicated serum
