组织蛋白酶B通过TLR4非依赖途径调控小鼠肝脏Kupffer细胞内炎症的激活

Cathepsin B in hepatic Kupffer cells regulates activation of TLR4-independent inflammatory pathways in mice with lipopolysaccharide-induced sepsis

目的研究组织蛋白酶B在LPS引发小鼠脓毒血症中的作用。方法动物生存实验观察:采用腹腔注射致死剂量LPS(54 mg/kg)法,WT小鼠随机分为3组,TLR4-/-小鼠随机分为3组,两种小鼠的各组均依次分为生理盐水对照组、致死剂量组(LPS)及组织蛋白酶B抑制剂—CA-074预处理组(CA-074+LPS),观察小鼠生存时间及生存率;脓毒血症实验采:用大剂量LPS(20 mg/kg)腹腔注射法,其余处理及分组同前,各组小鼠造模结束后:(1)肝脏HE染色检测肝脏病理变化;(2)检测肝脏Kupffer细胞胞质内组织蛋白酶B的蛋白水平及活性;(3)检测血清中IL-1α、IL-1β、TNF-α、IL-18等炎症因子水平。结果经致死量LPS打击后,TLR4-/-小鼠生存时间延长至84 h,明显长于WT小鼠,但仍无法完全抵抗致死量LPS的打击,CA-074预处理可分别延长WT和TLR4-/-小鼠的生存时间至60和132 h。大剂量LPS刺激后,WT及TLR4-/-小鼠肝细胞变性坏死明显,Kupffer细胞胞质中组织蛋白酶B蛋白水平无明显变化,但其在胞质的活性显著增加(P?0.05);血清中IL-1α、IL-1β、TNF-α以及IL-18等炎症因子水平增高(P?0.05);各CA-074预处理组,肝细胞坏死减轻,Kupffer细胞中组织蛋白酶B蛋白水平无明显变化,胞质内活性明显降低(P?0.05);血清中IL-1α、IL-1β、TNF-α以及IL-18等炎症因子水平明显低于单纯大剂量LPS刺激组(P?0.05)。结论在大剂量LPS诱导的脓毒血症中,存在非依赖TLR4受体的炎症通路的激活过程,组织蛋白酶B在这个过程中具有重要作用。

Objective To investigate the role of cathepsin B in hepatic Kupffer cells (KCs) in activating Toll-like receptor 4 (TLR- 4)-independent inflammatory pathways in mice with lipopolysaccharide (LPS)-induced sepsis. Methods Eighteen wild-type (WT) mice and 18 TLR4-knockout (TLR4^-/-) mice were both divided into 3 groups for intraperitoneal injections of a lethal dose (54 mg/kg) of LPS, LPS and CA-074 (a cathepsin B inhibitor), or normal saline, and the survival of the mice were observed. Another 36 WT mice and 36 TLR4^-/- mice were also divided into 3 groups and subjected to intraperitoneal injections of normal saline, 20 mg/kg LPS, or LPS with CA-074 pretreatment. After the treatments, KCs were collected from the mice for assessing the protein level and activity of cathepsin B. The histopathological changes of the liver were observed with HE staining, and the serum levels of IL-1α, IL-1β, TNF-α and IL-18 were detected. Results Compared with the WT mice, TLR4^-/- mice receiving the lethal dose of LPS had significantly longer survival time (up to 84 h) after the injection, but were still unable to fully resist LPS challenge. CA-074 pretreatment prolonged the survival time of WT mice and TLR4^-/- mice to 60 h and 132 h, respectively. In the mouse models of sepsis, 20 mg/kg LPS induced significantly enhanced activity of cathepsin B without affecting its expression level in the KCs (P<0.05) and increased the serum levels of the inflammatory cytokines. CA-074 pretreatment of the mice obviously lessened the detrimental effects of LPS in TLR4^-/- mice by significantly lowering cathepsin B activity in the KCs, alleviating hepatocyte apoptosis and reducing the serum levels of inflammatory cytokines. Conclusion Cathepsin B plays an important role in activating TLR4-independent inflammatory pathways in mice with LPS-induced sepsis.