TLR4信号调节mfn2基因表达诱导线粒体融合障碍在非酒精性脂肪肝缺血再灌注损伤中的作用

Non-alcoholic fatty liver disease (NAFLD) has become the most common liver disease, which is susceptible to greater ischemia-reperfusion (I/R) injury and negatively impacts liver recovery from injury, and is associated with increased mortality of liver operation. Our recent studies have demonstrated that impair mitochondrial dynamics induced by mfn2 occurred in NAFLD and liver I/R injury separately. However, the mechanisms responsible for this are not fully understood. Our novel data demonstrate that Lipopolysaccharides (LPS), recognized by Toll-like receptor 4, induced impair mitochondrial function by suppressing the expression of mfn2. We also proved that TNF-a may decrease the expression of mfn2 through PGC-1a and leads to interruption of mitochondrial fusion process in I/R injury. However, it is still unknown whether TLR4 involved in the expression of mfn2. Our hypothesis is that saturated fatty acid depress the expression of mfn2 via activated the TLR4 on the hepatocyte (direct way) and the kupffer cells (indirect way) in earlier phase of NAFLD. ROS, produce by the impaired mitochondria, induced the recruitment of TLR4 into lipid rafts, which leading to susceptible to I/R and LPS. In this project, the alterations of mitochondrial dynamics will be extensively investigated in vivo and vitro model of liver I/R injury with NAFLD histopathology using a series of TLR4 signal knockout mice (Including TLR4KO, CD14KO, TLR4KO, MyD88 KO, TRIFKO, hepatocyte-TLR4 KO, and kupffer-TLR4KO). The project will help understand and develop novel effective strategies to protect NAFLD against I/R injury.

非酒精性脂肪肝病(NAFLD)已成为肝脏外科的“新常态”，其降低肝脏对缺血再灌注损伤(I/R)的耐受能力从而成为手术中的重要危险因素。申请者前期研究中首次发现mfn2基因表达抑制引起的线粒体融合障碍是NAFLD和I/R损伤共同的新机制，但抑制其表达的途径尚不清楚。进一步研究发现：细菌脂多糖（LPS）及TNF-α可调控肝细胞mfn2的表达，且不同结构脂肪酸会对mfn2表达产生与其对TLR4作用相同的反向作用。因此我们假设：饱和脂肪酸早期即可激活肝细胞表面（直接途径）以及Kupffer细胞表面（间接途径）的TLR4信号（LPS受体）抑制mfn2表达诱导线粒体融合障碍，受损线粒体产生的ROS通过诱导TLR4向脂筏募集放大的TLR4信号，导致NAFLD的进展与形成，同时也使肝细胞对I/R和感染损伤更为敏感。本项目将以TLR4/mfn2为靶点，为NAFLD肝脏I/R损伤的早期预测和治疗提供新的策略。

非酒精性脂肪肝病(NAFLD)已成为肝脏外科的“新常态”，其降低肝脏对缺血再灌注损伤(I/R)的耐受能力从而成为手术中的重要危险因素。前期研究中首次发现mfn2基因表达抑制引起的线粒体融合障碍是NAFLD和I/R损伤共同的新机制。本研究主要以TLR4/mfn2为靶点，进一步探索NAFLD肝细胞对I/R敏感的机制。野生型小鼠和基因敲除小鼠（TLR4 KO，MYD88 KO，TIRF KO）同时给予喂食正常饮食和高脂饮食，进食12周后，每组随机选取部分小鼠进行肝缺血-再灌注模型（缺血1小时，再灌注6小时）。实验结果发现:与野生小鼠相比较，敲除小鼠显著降低肝脏脂肪变性程度，且提高脂肪肝小鼠耐受肝脏缺血再灌注损伤。结论：TLR4 KO、MYD88 KO、TIRF KO小鼠显著改变PGC-1α/mfn2表达，进而调控线粒体功能，影响肝细胞凋亡过程。另一方面，敲除小鼠均减轻炎症因子（如IL-6、TNF-α）释放，促进抑炎因子（IL-10）释放，促进细胞存活。可以推测TLR4 -MYD88/TIRF- NF-κB -PGC-1α/ mfn2信号通路参与线粒体融合调控和细胞凋亡途径；炎症因子（TNF-α 和IL-6）可能成为预测 NAFLD 耐受 I/R 损伤程度的指标；促进线粒体融合可能成为预防或治疗非酒精性脂肪肝缺血再灌注损伤的新途径，靶向干预TLR4受体可能会成为临床上提高肝脏对缺血再灌注损伤(I/R)的耐受能力的潜在治疗方案。