Toll 样受体4（TLR4）在华支睾吸虫感染致肝纤维化中的作用及其分子机制

Infection by Clonorchis sinensis can lead to liver fibrosis/cirrhosis, which poses great threatens to both humans and animals health. Accumulated lines of evidence indicate that Toll-like receptor 4 (TLR4) activated by pathogen-associated molecular patterns (PAMPs) plays a critical role in activation of hepatic stellate cells (HSCs), which causes hepatic fibrosis/cirrhosis ultimately. Our previous study showed that a significant reduction in hepatic fibrosis of TLR4 mutant mice was observed, compared with TLR4 wild mice with overt hepatic fibrosis in a mouse model of clonorchiasis. However，the mechanisms by which TLR4 signaling enhances HSC activation and liver fibrosis caused by C. sinensis remains unknown. The objective of the present project is to explore the underlying mechanisms of TLR4 signaling in promotion of liver fibrosis caused by C. sinensis. To achieve this goal, we established a mouse model of clonorchiasis; Based this mouse model, the roles of TLR4 signaling in the activation of HSC and promotion of liver fibrosis are to be analyzed by real-time PCR, Western-blot and Flow cytometry in TLR4 mutation and TLR4 wild mice, as well as in mice with Kupffer cell depletion. Furthermore, The roles of TLR4 activated by PAMPs from C. sinensis in TGF-β1- and Kupffer cell-mediated HSC activation are also to be explored in vivo. Our foundlings will dramatically advance our understanding of the interactions between Toll-like receptors of host cells and PAMPs sourced from C. sinensis, as well as provide novel therapeutic targets to treat this disease.

华支睾吸虫感染导致的肝纤维化或肝硬化严重威胁人类和动物的健康。研究表明TLR4 能促进肝星状细胞的活化，从而导致肝纤维化的发生。本课题组前期研究发现TLR4突变型小鼠能明显减轻华支睾吸虫感染所导致的肝纤维化，但其作用机制还未知。因此，本项目拟以TLR4识别华支睾吸虫有效虫体抗原并促进肝纤维化发生为切入点，利用相关基因缺陷型小鼠以及清除体内枯否细胞的小鼠建立华支睾吸虫感染模型，采用实时定量PCR、Western blot及流式细胞术等手段分析TLR4及其介导的信号通路在促肝纤维化中的作用机制。以此为基础，在细胞水平深入研究经华支睾吸虫有效抗原激活的TLR4及其介导的信号通路直接或间接促进TGF-β1/Smads信号通路活化肝星状细胞的作用机制。拟通过上述研究，揭示宿主细胞模式识别受体-TLR4促进华支睾吸虫致肝纤维化发生的分子机制，为防治华支睾吸虫致肝纤维化或肝硬化提供新的有效干预靶点。

华支睾吸虫感染导致的肝纤维化或肝硬化严重威胁人类和动物的健康。为此，本项目组以TLR4为切入点，在细胞水平和动物水平深入探讨了TLR4及其介导的信号通路在华支睾吸虫致肝纤维化中的作用及分子机制；在此基础上，我们深入地探讨了小分子核酸（miRNA）在调控TGF-β1/Smads信号通路影响华支睾吸虫致肝纤维化的分子机制，研究进展总结如下：.（1）在国内率先建立华支睾吸虫感染C3H小鼠致肝纤维化动物模型，并发现华支睾吸虫感染该品系小鼠TLR4表达升高，其介导的下游信号通路蛋白活化，提示TLR4在华支睾吸虫感染致肝纤维化中发挥一定作用。.（2）成功建立了华支睾吸虫感染TLR4野生型和TLR4缺陷型小鼠模型，发现 TLR4功能缺陷后， TLR4介导的下游蛋白分子如MyD88、pNF-κB的表达量明显减低， TGFβ/smads 信号通路关键蛋白（如pSmad2/3）表达量变弱，TLR4缺陷型小鼠胆管周围炎症变轻，肝细胞结构破坏较轻，肝纤维化程度明显减轻，表明TLR4促进华支睾吸虫致小鼠肝纤维化的发生、发展。.（3） 体外水平我们发现华支睾吸虫排泄/分泌产物（ESPs）能够诱导人肝星状细胞（HSCs）的活化，体外阻断TLR4的活化后，TGFβ/smads 信号通路关键蛋白（如pSmad2/3）表达量变弱，HSCs的活化程度明显减弱，表明宿主肝星状细胞（HSC）Toll 样受体 4（TLR4）在 TGF-β/Smads 信号通路中活化 HSC中发挥重要作用。.（4）成功分离了小鼠肝内胆管上皮细胞（MIBECs），并证实TLR4 在华支睾吸虫排泄/分泌产物能够诱导MIBECs NF-κB 的活化以及相关细胞因子/趋化因子的释放中发挥重要作用。.（5）利用miRNA芯片技术结合生物信息学技术成功筛选出了若干个可能调控华支睾吸虫致肝纤维化发生、发展的小分子核酸，并进一步分析证实miRNA-32和miRNA-497可能作用于smad7从而影响TGF-β1/Smads信号通路的活化来调控肝纤维化的发生、发展。.上述研究首次阐明TLR4促进华支睾吸虫感染致肝纤维化中的详尽机制，为阐明华支睾吸虫致病机制提供更为详尽的实验依据，也为有效防治华支睾吸虫及其相关因素引起的肝纤维化或肝硬化提供有效的干预靶点。