Sommerhalder Christian, Cummins Claire B, Wang Xiaofu, Ramdas Divya, Lopez Omar Nunez, Gu Yanping, Zhou Jia, Radhakrishnan Ravi S
Department of Surgery, University of Texas Medical Branch, Galveston, Texas.
School of Medicine, University of Texas Southwestern Medical Center, Dallas, Texas.
J Surg Res. 2021 May;261:334-342. doi: 10.1016/j.jss.2020.12.045. Epub 2021 Jan 21.
Hepatic fibrosis is wound-healing response that is the result of hepatic stellate cell (HSC) activation and subsequent excess extracellular matrix deposition. HSCs can be activated by a variety of inflammatory stimuli as well as through the signal transducer and activator of transcription 3 (STAT3) pathway. HJC0416 is a novel, orally bioavailable small-molecule inhibitor of STAT3 that was developed by our team using a fragment-based drug design approach. Previously, our team has shown that HJC0416 has antifibrogenic effects in activated HSCs. Recently, increasing evidence suggests that nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) plays an important role in the activation of HSCs. In the present study, we examined the role of NF-κB inhibition of HSC activation by HJC0416.
LX-2 (human) and HSC-T6 (rat) cell lines were used. Expression levels of extracellular proteins, NF-κB and STAT3 expression and DNA binding, and inflammatory cytokine levels were determined using western blot, ELISA, and immunofluorescence assay.
HJC0416 decreased cell viability in a dose-dependent manner in both cell lines and arrested the cell cycle at the S phase. Increased apoptosis was seen in LX-2 cells through Yo-Pro-1 and propidium iodide immunofluorescent stating. HJC0416 significantly decreased expression of fibronectin and collagen I as well as markedly decreased α-SMA and laminin. HJC0416 inhibited the STAT3 pathway by decreasing phosphorylation of STAT3, as well as signal transduction pathway activation. Notably, HJC0416 also inhibited the classic and alternative pathways of NF-κB activation. HJC0416 inhibited LPS-induced p65 nuclear translocation and DNA binding, as well as prevented phosphorylation and degradation of inhibitory protein IκBα. HJC0416 also prevented phosphorylation of serine residue 536 on p65.
HJC0416, an inhibitor of STAT3, was found to have antifibrogenic properties in activated hepatic stellate cell lines. In addition, HJC0416 was found to inhibit the NF-κB pathway. Owing to this double effect, HJC0416 demonstrates promise for in vivo experimentation as an antifibrosis treatment.
肝纤维化是一种伤口愈合反应，是肝星状细胞（HSC）激活及随后细胞外基质过度沉积的结果。HSC可被多种炎症刺激以及通过信号转导和转录激活因子3（STAT3）途径激活。HJC0416是一种新型的、口服生物可利用的STAT3小分子抑制剂，由我们团队采用基于片段的药物设计方法研发。此前，我们团队已表明HJC0416在活化的HSC中具有抗纤维化作用。最近，越来越多的证据表明，活化B细胞核因子κB（NF-κB）在HSC激活中起重要作用。在本研究中，我们研究了HJC0416抑制NF-κB对HSC激活的作用。
使用LX-2（人）和HSC-T6（大鼠）细胞系。采用蛋白质免疫印迹法、酶联免疫吸附测定法和免疫荧光测定法测定细胞外蛋白表达水平、NF-κB和STAT3表达及DNA结合情况以及炎性细胞因子水平。
HJC0416在两种细胞系中均以剂量依赖性方式降低细胞活力，并使细胞周期停滞于S期。通过Yo-Pro-1和碘化丙啶免疫荧光染色发现LX-2细胞凋亡增加。HJC0416显著降低纤连蛋白和I型胶原的表达，以及α-平滑肌肌动蛋白和层粘连蛋白的表达。HJC0416通过降低STAT3磷酸化以及信号转导途径激活来抑制STAT3途径。值得注意的是，HJC0416还抑制NF-κB激活的经典途径和替代途径。HJC0416抑制脂多糖诱导的p65核转位和DNA结合，并阻止抑制蛋白IκBα的磷酸化和降解。HJC0416还阻止p65上丝氨酸残基536的磷酸化。
发现STAT3抑制剂HJC0416在活化的肝星状细胞系中具有抗纤维化特性。此外，发现HJC0416可抑制NF-κB途径。由于这种双重作用，HJC0416作为一种抗纤维化治疗药物在体内实验中显示出前景。
