TLR介导的Nrf2抗氧化通路在对乙酰氨基酚药物性肝损伤中的保护作用

TLR protects liver against acetaminophen-induced Hepatotoxicitin via activating the Nrf2 antioxidant signaling pathway

目的研究内毒素(LPS)及Toll样受体(TLR)在对乙酰氨基酚(APAP)药物性肝损伤中的保护作用及其相关机制。方法雄性C57BL/6小鼠40只,分为4组,每组10只。空白对照组腹腔注射0.9%氯化钠溶液,LPS组腹腔注射LPS 10μg/kg,APAP组腹腔注射APAP 300 mg/kg,LPS+APAP组在APAP造模前16 h给予LPS 10μg/kg预处理。通过比较各组血清ALT和AST水平,并通过HE染色评价肝组织损伤程度,观察LPS对小鼠肝损伤的保护作用。测定相应时间点的肝脏组织丙二醛(MDA)、还原型谷胱甘肽(GSH)的变化以及肝组织DHE染色,评价小鼠氧化应激水平。应用Western印迹及RT-PCR检测肝脏Nrf2,Gclc及HO-1的表达水平。结果 LPS预处理可明显减轻APAP所致的肝脏氧化应激反应及肝损伤程度。LPS预处理组的小鼠血清ALT(518.3±142.3对4542±498.4 U/L)、AST(643.3±105.6对5432.1±569.2 U/L)水平及肝组织MDA(78.0±14.5对141.7±26.4 mmoL/mg)水平与模型组相比明显降低,而GSH(6.2±1.7对3.5±1.0μmol/g)水平明显升高(P<0.05),肝组织病理损伤明显减轻。同时,LPS预处理可明显促进Nrf2及其下游抗氧化基因的表达。结论 LPS在小鼠APAP肝损伤中起到保护作用,作用机制与Nrf2抗氧化通路的激活相关,可能成为药物性肝损伤的新的治疗策略。

Objective To investigate the protective effect of Lipopolysaccharide （LPS）and toll-like receptors （TLRs） on acetaminophen （APAP）-induced liver injury and its potential mechanism.Methods Forty male mice were randomly divided into 4 groups.Mice in control group were intraperitneally （i.p.）injected with saline,in LPS group were i.p.given with 10 μg/kg LPS,and in APAP group were i.p.administrated with APAP （300 mg/kg）.LPS＋APAP group were i.p. pretreated with LPS （10 μg/kg）16 h before APAP （300 mg/kg）injection.Serum and liver tissue among 4 groups were collected for further analysis.Liver injury was assessed by detection of serum ALT and AST levels and HE staining of liver tissue.The oxidative stress was evaluated by measuring hepatic glutathione （GSH）and malondialdehyde （MDA）levels, and Dihydroethidium （DHE）staining.Expression of Nrf2,Gclc and HO-1 were measured by western blot and real time-polymerase chain reaction.Results Compared with APAP groups,LPS＋APAP group showed lower serum levels of ALT （518.3±142.3 vs.4542±498.4 U/L,P 〈0.05）and AST （643.3 ±105.6 vs.5432.1 ±569.2 U/L,P 〈0.05）,milder liver tissue damage,lower MDA levels （78.0±14.5 vs.141 .7±26.4 mmol/mg tissue,P 〈0.05 ）and higher GSH levels （6.2±1 .7 vs.3.5 ±1 .0 μmol/g tissue,P 〈0.05 ）,which revealed that APAP-induced liver injury and oxidative stress response were significantly attenuated by LPS pretreatment.Moreover,LPS pretreatment could enhance the expression of Nrf2 and other antioxidant genes significantly.Conclusion LPS plays an important role in protection against APAP-induced hepatotoxicity in mice via activating antioxidant signaling pathway of Nrf2,which might become a new strategy for APAP-induced liver injury therapy.