四氯化碳诱导大鼠肝纤维化过程中TLR4的作用机制研究

Expression of Toll-like Receptor 4 in Kupffer Cells During CCl_4-induced Hepatic Fibrosis in Rats

目的动态观察Kupffer细胞(KCs)Toll样受体4(TLR4)及其蛋白在四氯化碳(CCl4)致大鼠肝纤维化过程中的表达及作用。方法用40%四氯化碳花生油溶液皮下注射建立肝纤维化模型,于第0、4、6、8、10周收集血液和肝脏组织。检测血清内毒素、Ⅳ型胶原(collagenⅣ,CⅣ)、转化生长因子β1(TGF-β1)水平,并检测KCs TLR4 mRNA表达水平,免疫组化SP法检测肝脏TLR4蛋白、核因子κB(nuclear factor-κB,NF-κB)蛋白和组织金属蛋白酶组织抑制剂-1(TIMP-1)蛋白表达。结果大鼠4、6、8、10周组肝组织TLR4蛋白、NF-κB蛋白、纤维化积分和KCs TLR4 mRNA以及血清内毒素、TGF-β1、CⅣ型胶原水平明显升高,同0周组比较差异有统计学意义(P<0.01)。NF-κB蛋白、TLR4蛋白和KCs TLR4 mRNA在第10周时较第8周有轻度下降;肝KCsTLR4 mRNA的表达与血浆内毒素水平呈正相关(r=0.845,P<0.01),与血清TGF-β1水平成正相关(r=0.665,P<0.01)。结论在CCl4诱导的肝纤维化过程中,内毒素可上调Kupffer细胞TLR4的表达,其可能为通过TLR4信号途径在肝纤维化中起重要作用。

Objective To evaluate the role of TLR 4 in the pathogenesis of hepatic fibrosis in the rats with CCl4 - induced. Methods Male Wistar rats were randomly divided into the control group and model groups. The liver fibrosis was induced by 40% CCl4 hypodermic injection. Blood and liver samples were collected at the time of the 0^th, the 4 ^th, the 6 ^th, the 8 ^th and the 10 ^th week. The level of serum endotoxin, collagen Ⅳ （ C Ⅳ ） and transforming growth factor β1 （TGF - β1 ） were detected. The expression of tissue inhibitor of metalloproteinase - 1 （ TIMP - 1 ）, NF - κB and toll - like receptor 4 （TLR4） protein in hepatic tissue were assayed by immunohistochemistry. The expressions of TLR4 mRNA in Kupffer cells （KCs） were measured byreverse transcriptase polymerase chain reaction （RTPCR）. Results The expression of TLR4 mRNA in KCs, the level of TLR4, NF - κB protein in liver tissues and the level of endotoxin, C Ⅳ, TGF - β1 in serum, all in-creased significantly after4 - 10 weeks CCl4 administration compared with those of control group （P 〈 0. 001 ）. A positive correlations were found between the expression of TLR4 mRNA in KCs and serum endotoxin levels （r = 0. 845, P 〈 0. 001 ）, and serum TGF - β1 levels （r = 0. 665, P 〈 0. 001 ）. Conclusion The expression of TLR4 mRNA in KCs is up - regulated by endotoxin, the high expression of endotoxin and TLR4 may play an important role in the CCl4 -induced liver fibrosis.