基于TLR4/NF-κB/NLRP3信号通路研究三叶香茶菜含药血清对肝星状细胞的影响

Effect of Isodon ternifolius-medicated serum on hepatic stellate cells based on TLR4/NF-κB/NLRP3 signaling pathway

研究三叶香茶菜含药血清对脂多糖(lipopolysaccharide,LPS)诱导肝星状细胞(hepatic stellate cell,HSC)活化的抑制作用及机制。将LPS诱导的HSC分为空白对照组、LPS模型组、秋水仙碱含药血清组、LPS+空白血清对照组、三叶香茶菜含药血清组、Toll样受体4(Toll-like receptor 4,TLR4)阻断剂组、TLR4阻断剂+三叶香茶菜含药血清组。四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)法检测HSC增殖,酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法测定HSC中Ⅰ型胶原蛋白(typeⅠcollagen,COLⅠ)、Ⅲ型胶原蛋白(COLⅢ)、转化生长因子-β1(transforming growth factor-β1,TGF-β1)、细胞间黏附分子-1(intercellular adhesion molecule-1,ICAM-1)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、血管内皮细胞黏附分子-1(vascular cell adhesion molecule-1,VCAM-1)、含半胱氨酸的天冬氨酸蛋白水解酶-1(cysteinyl aspartate specific proteinase-1,caspase-1)、单核细胞趋化蛋白-1(monocyte chemotactic protein-1,MCP-1)。实时荧光定量PCR(real-time PCR,RT-PCR)检测HSC中TLR4、IκBα、NOD样受体蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)、核因子-κB(NF-κB)p65、gasdermin D(GSDMD)、凋亡相关微粒蛋白(apoptosis-associated speck-like protein containing CARD,ASC)mRNA表达;蛋白免疫印迹(Western blot,WB)检测HSC中TLR4、p-IκBα、NF-κB p65、NLRP3、ASC、GSDMD蛋白水平。结果显示三叶香茶菜含药血清可抑制HSC细胞增殖活性,并对HSC中COLⅠ、COLⅢ、α-SMA、TGF-β1、caspase-1、MCP-1、VCAM-1、ICAM-1的分泌具有抑制作用;与LPS模型组比,三叶香茶菜含药血清组、秋水仙碱含药血清组及TLR4阻断剂组p-IκBα、NLRP3、NF-κB p65、GSDMD、ASC表达显著下调,IκBα表达显著上调;与TLR4阻断剂组比,TLR4阻断剂+三叶香茶菜含药血清组TLR4、p-IκBα、NLRP3、NF-κB p65、GSDMD、ASC表达显著降低,IκBα表达显著升高。综上,三叶香茶菜含药血清通过抑制TLR4/NF-κB/NLRP3信号通路下调HSC的活化。

The present study aimed to investigate the inhibitory effect and mechanism of Isodon terricolous-medicated serum on lipopolysaccharide(LPS)-induced hepatic stellate cell(HSC)activation.LPS-induced HSCs were divided into a blank control group,an LPS model group,a colchicine-medicated serum group,an LPS+blank serum group,an I.terricolous-medicated serum group,a Toll-like receptor 4(TLR4)blocker group,and a TLR4 blocker+I.terricolous-medicated serum group.HSC proliferation was detected by methyl thiazolyl tetrazolium(MTT)assay.Enzyme-linked immunosorbent assay(ELISA)was used to measure typeⅠcollagen(COLⅠ),COLⅢ,transforming growth factor-β1(TGF-β1),intercellular adhesion molecule-1(ICAM-1),α-smooth muscle actin(α-SMA),vascular cell adhesion molecule-1(VCAM-1),cysteinyl aspartate-specific proteinase-1(caspase-1),and monocyte chemotactic protein-1(MCP-1).Real-time PCR(RT-PCR)was used to detect mRNA expression of TLR4,IκBα,and NOD-like receptor thermal protein domain associated protein 3(NLRP3),nuclear factor-κB(NF-κB)p65,gasdermin D(GSDMD),and apoptosis-associated speck-like protein containing a CARD(ASC)in HSCs.Western blot(WB)was used to detect the protein levels of TLR4,p-IκBα,NF-κB p65,NLRP3,ASC,and GSDMD in HSCs.The results showed that I.terricolous-medicated serum could inhibit the proliferation activity of HSCs and inhibit the secretion of COLⅠ,COLⅢ,α-SMA,TGF-β1,caspase-1,MCP-1,VCAM-1,and ICAM-1 in HSCs.Compared with the LPS model group,the I.terricolous-medicated serum group,the colchicine-medicated serum group,and the TLR4 blocker group showed down-regulated expression of p-IκBα,NLRP3,NF-κB p65,GSDMD,and ASC,and up-regulated expression of IκBα.Compared with the TLR4 blocker group,the TLR4 blocker+I.terricolous-medicated serum group showed decreased expression of TLR4,p-IκBα,NLRP3,NF-κB p65,GSDMD,and ASC,and increased expression of IκBα.In conclusion,I.terricolous-medicated serum down-regulates HSC activation by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway.