基于肠细胞TRPV1/Caspase3信号探讨辣椒素抑制酒精性脂肪肝作用机制

Intestinal leakage plays a major role in the alcoholic liver disease. Capsaicin can inhibit intestinal leakage, but the mechanism needs to be further clarified. The expression of transient receptor potential vanilloid type-1 (TRPV1) is closely related to intestinal disease. Our previous study found that TRPV1 was down-regulated and Caspase3 was up-regulated in alcohol-induced liver injury WT mice, on which capsaicin has significantly regulatory effects. However, it cannot restrain the over-expression of Caspase3 when TRPV1 were knock-down. Based on these new findings, we could hypothesize that Capsaicin inhibit alcoholic fatty liver disease by regulating TRPV1/caspase3 signal transduction in enterocyte. We would utilize real-time PCR analysis and patch clamp technique to elucidate the effect of alcohol on the expression and regulation of TRPV1 in Caco-2 cells. Simultaneously, we would study the effect of TRPV1 on Casase3 activity and the intervention of capsaicin using RNA interference and flow cytometry. Furthermore, we would use the Lieber-DeCarli plus Binge alcohol liver disease model to explore the intervention of capsaicin on intestinal cell apoptosis and ALD progression in TRPV1-/- mice. This project will help us find the molecular mechanism of capsaicin inhibiting ALD, which provides a new therapeutic strategy for clinical treatment.

肠渗漏是酒精性肝病（ALD）的主要原因之一，辣椒素具有抑制肠渗漏的作用，但机制有待进一步阐明。辣椒素受体（TRPV1）表达与肠病变关系密切，我们前期发现：辣椒素对野生型酒精肝小鼠肠细胞Caspase3表达增高和TRPV1表达降低有显著调控作用，而对TRPV1-/-酒精肝Caspase3升高无抑制作用。因此，提出“辣椒素调控肠细胞TRPV1/caspase3信号抑制酒精性脂肪肝”的科学假说；拟采用实时定量PCR和膜片钳等技术，研究酒精对Caco-2细胞TRPV1基因表达、调控作用，运用RNA干扰和流式细胞仪等技术，明确TRPV1对Caspase3活性和紧密连接的影响及辣椒素的干预作用；进而运用Lieber-DeCarli复合Binge酒精肝模型，研究TRPV1-/-小鼠肠细胞凋亡与ALD进展及辣椒素的干预作用。本项目的完成有助于发现辣椒素抑制ALD的分子机制，为其治疗开辟新途径。

酒精性肝病（ALD）在全世界的患病率和死亡率均高，肠道屏障功能障碍是导致ALD的重要因素。辣椒素是辣椒的活性成分，被证实具有抑制肠渗漏的作用。本项目通过建立酒精性脂肪肝小鼠模型及TRPV1基因敲除小鼠酒精性脂肪肝模型，检测肝脏炎症、脂肪变、血清内毒素水平及肠粘膜炎症、紧密连接、凋亡指标，证实酒精性肝病存在肠渗漏，并检测辣椒素对酒精性脂肪肝小鼠肝脏及肠细胞作用；采用Caco-2细胞培养，研究酒精对Caco-2细胞TRPV1基因表达调控作用及辣椒素对TRPV1/Caspase3的干预作用。负责人在项目研究中发现：1.辣椒素能减轻酒精性脂肪肝小鼠的血清ALT及TG含量，并改善肝脏炎症及脂肪变。2.辣椒素能改善酒精性脂肪肝小鼠小肠黏膜炎症、黏膜屏障及减少肠细胞凋亡。3.酒精性脂肪肝小鼠肠TRPV1表达下调，Caspase3显著上调，辣椒素能降低酒精性脂肪肝小鼠的肠Caspase3水平。4.TRPV基因敲除后，酒精性脂肪肝小鼠肝脏炎症、脂肪变及肠细胞炎症、凋亡加重，而辣椒素对TRPV1基因敲除酒精性脂肪肝小鼠肝损害及肠细胞凋亡无明显改善。5.Caco-2细胞实验表明：酒精刺激可减低TRPV1表达，而增加细胞Caspase3表达；辣椒素处理后上调TRPV1，减少细胞凋亡。敲减TRPV1后，辣椒素不能有效抑制酒精引起的Caco-2细胞凋亡。从而证实辣椒素通过调控肠细胞TRPV1/caspase3抑制酒精性脂肪肝的科学假说，为临床应用提供理论基础。

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