HMGB1在肾脏纤维化形成中的作用和机制研究

Renal injury triggers inflammatory responses induced by HMGB1 that promote renal fibrosis; however the mechanisms that couple tissue injury, inflammation and renal fibroblast activation are not known. HMGB1 is released outside by damaged cells that triggers tissue inflammation and involves tissue repair and regeneration. Renal fibroblasts are essential for renal fibrosis that determine the accumulation rate and composition of the extracellular matrix proteins. Given our findings that crosstalk exists between inflammatory cells and fibroblasts; and HMGB1 provoked a significant increase in NIH/3T3 fibroblast proliferation, we postulated that dying or injured renal tubular epithelial cells and macrophages release HMGB1, which exerts its effects on renal fibroblasts through TLR4 and/or RAGE receptors directly, and/or increases the production of profibrotic cytokines indirectly; the involving PI3K/Akt, TGF-β signaling pathways contribute to activation of renal fibroblasts to produce excess collagen. To confirm our proposal, we will utilize cell co-culture system and animal model to perform the experiments that HMGB1 is sufficient to activate renal fibroblasts in vitro and to provoke renal inflammation/fibrosis in vivo; siRNA transfection, neutralizing antibody technology and TLR4, RAGE deficient mice to prove that HMGB1 exerts its effects through TLR4 and/or RAGE receptors in vitro and in vivo; reverse blocking the downstream signaling pathways to explore the effects of different pathways on renal fibroblast activation; which provide us better understand and identify novel therapeutic targets to renal fibrosis.

肾脏损伤时受损细胞释放HMGB1到局部或循环引起炎症反应，促进肾脏纤维化形成，其确切发病机制尚未明确。释放到细胞外的HMGB1与TLR4或/和RAGE受体结合激活免疫细胞，促进组织修复和再生。我们发现炎症细胞和成纤维细胞相互调控；HMGB1由坏死肾小管上皮细胞释出激活NIH/3T3成纤维细胞；因此推测HMGB1由受损肾小管上皮细胞或巨噬细胞释出，与肾成纤维细胞受体结合，或促进促纤维化细胞因子分泌，通过PI3K/Akt、TGF-β等通路激活成纤维细胞，过度合成胶原蛋白。为证实假设，拟采用细胞共培养和动物模型，体内外观察HMGB1对肾成纤维细胞直接或间接激活及对肾组织炎症和纤维化的作用；应用siRNA和抗体中和受体，及TLR4、RAGE缺失小鼠模型，体内外明确HMGB1通过TLR4或/和RAGE发挥其作用；反向阻断下游信号通路，探讨不同通路对细胞功能的影响，为抗肾纤维化治疗提供新颖的理论依据。

肾脏损伤时受损细胞释放HMGB1到局部或循环引起炎症反应，促进肾脏纤维化形成，其确切发病机制尚未明确。释放到细胞外的HMGB1与TLR4受体结合激活免疫细胞，促进组织修复和再生。我们发现炎症细胞和成纤维细胞相互调控；HMGB1由坏死肾小管上皮细胞释出激活NIH/3T3成纤维细胞；因此推测HMGB1由受损肾小管上皮细胞释出，与肾成纤维细胞受体结合，或促进促纤维化细胞因子分泌，通过PI3K/Akt、TGF-β等通路激活成纤维细胞，过度合成胶原蛋白。为证实假设，采用细胞共培养系统和动物模型，体内外观察HMGB1对肾成纤维细胞激活及对肾组织炎症和纤维化的作用；应用特异性抗体中和受体，及TLR4缺失小鼠模型，通过肾脏内注射HMGB1和单侧输尿管阻断肾脏损伤2种小鼠模型，明确HMGB1通过TLR4发挥其作用；并反向阻断下游信号通路，探讨不同通路对细胞功能的影响。实验结果表明：HMGB1可激活肾成纤维细胞，促进成纤维细胞增殖、α-SMA表达和胶原蛋白的合成增加，以及细胞迁移。通过免疫缺失肾成纤维细胞TLR4受体，发现HMGB1对肾成纤维细胞的增殖作用通过TLR4受体，TLR4受体缺失细胞划痕实验延缓了HMGB1所致的细胞迁移。Transwell共培养和条件培养基可促进肾成纤维细胞的增殖，并促进胶原基因表达。HMGB1可促进肾成纤维细胞Akt，c-Jun，RAS，p-c-Jun，TLR4等蛋白表达上调。Akt、ERK和PI3K抑制剂不同程度地抑制HMGB1所致细胞内相应蛋白及胶原蛋白的表达，并减弱HMGB1所致的细胞增殖及α-SMA表达，并延缓细胞迁移。肾小管上皮细胞划痕损伤后24、48小时上清中AngII表达水平逐渐升高。将HMGB1注射到TLR4缺失小鼠和野生型小鼠肾脏内，相比于野生型小鼠，TLR4缺失小鼠TNF-α含量在给予HMGB1干预后3天含量有所下降。本项目首次探讨了肾脏损伤，炎症反应和肾脏成纤维细胞激活共同作用导致肾脏纤维化的机制。首次利用细胞共培养系统，探讨肾脏不同细胞间的相互调控关系，及HMGB1通过TLR4受体和信号通路激活肾脏间质成纤维细胞参与肾脏纤维化的发生。本项目的成功实施为抗肾纤维化治疗提供新颖的理论依据。