基于TLR4/NF-κB/NLRP3信号通路探讨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].
作者信息
Huang Gui-Dong, Zhou Zhi-Pin, Pang Zhi, Qin Le, Wu Rui-Sheng, Chen Yong, Ye Xiao-Xue
机构信息
School of Pharmacy,Guangxi University of Chinese Medicine Nanning 530001,China.
Liuzhou People's Hospital Affiliated to Guangxi Medical University,Guangxi Key Laboratory of Biotechnology Research for Clinical Diseases,Health Commission of Guangxi Zhuang Autonomous Region,Liuzhou Engineering Technology Research Center of Gastrointestinal Chinese Patent Medicine Liuzhou 545006,China.
出版信息
Zhongguo Zhong Yao Za Zhi. 2023 Jul;48(14):3913-3921. doi: 10.19540/j.cnki.cjcmm.20230306.704.
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 type Ⅰ collagen(COL Ⅰ), COL Ⅲ, 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 COL Ⅰ, COL Ⅲ, 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.
本研究旨在探讨地生香茶菜含药血清对脂多糖（LPS）诱导的肝星状细胞（HSC）活化的抑制作用及其机制。将LPS诱导的HSCs分为空白对照组、LPS模型组、秋水仙碱含药血清组、LPS+空白血清组、地生香茶菜含药血清组、Toll样受体4（TLR4）阻断剂组和TLR4阻断剂+地生香茶菜含药血清组。采用甲基噻唑基四氮唑（MTT）法检测HSC增殖。采用酶联免疫吸附测定（ELISA）法检测Ⅰ型胶原（COL Ⅰ）、COL Ⅲ、转化生长因子-β1（TGF-β1）、细胞间黏附分子-1（ICAM-1）、α-平滑肌肌动蛋白（α-SMA）、血管细胞黏附分子-1（VCAM-1）、半胱天冬酶-1（caspase-1）和单核细胞趋化蛋白-1（MCP-1）。采用实时荧光定量聚合酶链反应（RT-PCR）检测HSCs中TLR4、IκBα、NOD样受体热蛋白结构域相关蛋白3（NLRP3）、核因子-κB（NF-κB）p65、gasdermin D（GSDMD）和含半胱天冬酶激活和招募结构域的凋亡相关斑点样蛋白（ASC）的mRNA表达。采用蛋白质免疫印迹法（WB）检测HSCs中TLR4、p-IκBα、NF-κB p65、NLRP3、ASC和GSDMD的蛋白水平。结果表明，地生香茶菜含药血清可抑制HSCs的增殖活性，并抑制HSCs中COL Ⅰ、COL Ⅲ、α-SMA、TGF-β1、caspase-1、MCP-1、VCAM-1和ICAM-1的分泌。与LPS模型组相比，地生香茶菜含药血清组、秋水仙碱含药血清组和TLR4阻断剂组p-IκBα、NLRP3、NF-κB p65、GSDMD和ASC的表达下调，IκBα的表达上调。与TLR4阻断剂组相比，TLR4阻断剂+地生香茶菜含药血清组TLR4、p-IκBα、NLRP3、NF-κB p65、GSDMD和ASC的表达降低，IκBα的表达增加。综上所述，地生香茶菜含药血清通过抑制TLR4/NF-κB/NLRP3信号通路下调HSC活化。
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