組織蛋白酶B通過TLR4非依賴途徑調(diào)控小鼠肝臟Kupffer細胞內(nèi)炎癥的激活

摘要： 目的研究組織蛋白酶B在LPS引發(fā)小鼠膿毒血癥中的作用。方法動物生存實驗觀察:采用腹腔注射致死劑量LPS(54 mg/kg)法,WT小鼠隨機分為3組,TLR4-/-小鼠隨機分為3組,兩種小鼠的各組均依次分為生理鹽水對照組、致死劑量組(LPS)及組織蛋白酶B抑制劑—CA-074預處理組(CA-074+LPS),觀察小鼠生存時間及生存率;膿毒血癥實驗采:用大劑量LPS(20 mg/kg)腹腔注射法,其余處理及分組同前,各組小鼠造模結(jié)束后:(1)肝臟HE染色檢測肝臟病理變化;(2)檢測肝臟Kupffer細胞胞質(zhì)內(nèi)組織蛋白酶B的蛋白水平及活性;(3)檢測血清中IL-1α、IL-1β、TNF-α、IL-18等炎癥因子水平。結(jié)果經(jīng)致死量LPS打擊后,TLR4-/-小鼠生存時間延長至84 h,明顯長于WT小鼠,但仍無法完全抵抗致死量LPS的打擊,CA-074預處理可分別延長WT和TLR4-/-小鼠的生存時間至60和132 h。大劑量LPS刺激后,WT及TLR4-/-小鼠肝細胞變性壞死明顯,Kupffer細胞胞質(zhì)中組織蛋白酶B蛋白水平無明顯變化,但其在胞質(zhì)的活性顯著增加(P?0.05);血清中IL-1α、IL-1β、TNF-α以及IL-18等炎癥因子水平增高(P?0.05);各CA-074預處理組,肝細胞壞死減輕,Kupffer細胞中組織蛋白酶B蛋白水平無明顯變化,胞質(zhì)內(nèi)活性明顯降低(P?0.05);血清中IL-1α、IL-1β、TNF-α以及IL-18等炎癥因子水平明顯低于單純大劑量LPS刺激組(P?0.05)。結(jié)論在大劑量LPS誘導的膿毒血癥中,存在非依賴TLR4受體的炎癥通路的激活過程,組織蛋白酶B在這個過程中具有重要作用。
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.