火炭母缓解鼠伤寒沙门菌致小鼠肝损伤的机制研究

Study on the Mechanism of Polygonum chinense L.Alleviating Liver Injury Induced by Salmonella Typhimurium in Mice

【目的】探讨火炭母口服液对鼠伤寒沙门菌感染小鼠肝脏的保护作用及其机制。【方法】将48只雄性BALB/c小鼠随机分为6组:空白对照组、模型组、阳性药物组及火炭母高、中、低剂量组,每组8只。阳性药物组小鼠灌胃庆大霉素(20 mg/kg),火炭母高、中、低剂量组小鼠分别灌胃16、8和4 g/kg火炭母,空白对照组和模型组小鼠分别给予等体积的PBS,连续给药8 d。预防性给药2 d后,空白对照组小鼠灌胃0.2 mL PBS,其他组小鼠一次性灌胃0.2 mL 5×10^(4) CFU/mL鼠伤寒沙门菌溶液。给药结束后处死小鼠,解剖取肝脏,制作组织切片并经HE染色观察肝脏病理变化,平板培养基培养肝脏组织悬液检测小鼠肝脏载菌量,Western blotting检测α干扰素(IFN-α)、IFN-β、IFN-γ、肿瘤坏死因子α(TNF-α)、白介素1β(IL-1β)、TANK结合激酶1(TBK1)、干扰素调节因子3(IRF3)蛋白表达水平。【结果】肝脏病理观察结果显示,模型组小鼠肝脏有淤血,大量炎性细胞浸润,肝细胞明显肿胀、排列紊乱,胞质染色加深,肝细胞坏死和凋亡;经火炭母处理后,高剂量组小鼠肝脏组织中少量炎症细胞,淤血及肝细胞坏死和凋亡的情况得到改善,肝细胞染色深、轻微肿胀、排列紊乱,而中、低剂量组除肝细胞胞质染色深、轻微肿胀、排列紊乱外,其他病变不明显。与空白对照组相比,模型组小鼠肝脏中鼠伤寒沙门菌负荷显著增加(P<0.05),IRF3、P-IRF3、IFN-α、IFN-β和IFN-γ表达显著下降(P<0.05),TNF-α和IL-1β表达水平则显著上升(P<0.05);与模型组相比,火炭母各剂量组小鼠肝脏中鼠伤寒沙门菌负荷均显著降低(P<0.05),中剂量火炭母能明显增强TBK1蛋白的磷酸化和IRF3蛋白的表达及其磷酸化水平(P<0.05),增加IFN-α、IFN-β、IFN-γ的蛋白水平(P<0.05),且能显著降低TNF-α和IL-1β的分泌(P<0.05)。【结论】火炭母可能通过上调TBK1-IRF3途径诱导IFN产生,缓解鼠伤寒沙门菌感染致小鼠肝损伤,以8 g/kg给药剂量效果较好。

【Objective】This study was aimed to investigate the protective effects of Polygonum chinense L.(P.chinense,PCL)on the liver of mice infected with Salmonella Typhimurium(S.Typhimurium)and its underlying mechanism.【Method】48 male BALB/c mice were randomly divided into 6 groups:Normal,model,positive drug,PCL-H,PCL-M and PCL-L groups.Mice in the positive drug group were given gentamicin(20 mg/kg)orally.Mice in the PCL-H,PCL-M and PCL-L groups were orally administered with PCL at 16,8 and 4 g/kg,respectively.The normal and model groups mice were given with the same volume of PBS.The drugs were administered for 8 consecutive days.After 2 days of prophylactic administration,the mice in the normal group were given 0.2 mL PBS,and the mice in the other groups were given 0.2 mL S.Typhimurium solution(5×10^(4) CFU/mL)at one time.After the administration,the mice were killed,and the liver was dissected to make tissue sections and stained with HE to observe the pathological changes of the liver.The liver tissue suspension was cultured in the plate medium to detect the bacterial load in the liver of the mice.Western blotting was used to detect the expression levels interferon alpha(IFN-α),interferon beta(IFN-β),interferon gamma(IFN-γ),tumor necrosis factorα(TNF-α),interleukin 1β(IL-1β),TANK-binding kinase 1(TBK1)and interferon regulatory factor 3(IRF3)protein.【Result】The pathological observation of liver showed that the liver of mice in the model group had congestion and a large number of inflammatory cells infiltrated.The hepatocytes were obviously swollen and disordered,the cytoplasmic staining was deepened,and the hepatocytes were necrotic and apoptotic.In PCL-H mice,there were a few inflammatory cells in liver,congestion and necrosis and apoptosis of liver cells were improved.In PCL-M and PCL-L,except for hepatocytes with dark cytoplasmic staining,slight swelling,and disordered arrangement,other lesions were not obvious.Compared with the normal group,the load of S.Typhimurium in liver of the model group mice was significantly increased(P<0.05),and the expressions of IRF3,P-IRF3,IFN-α,IFN-βand IFN-γwere significantly decreased(P<0.05),the expressions of TNF-αand IL-1βwere significantly increased(P<0.05).Compared with the model group,the load of S.Typhimurium in liver of mice in PCL-H,PCL-M and PCL-L groups was significantly decreased(P<0.05).Among them,PCL-M could significantly enhance the phosphorylation of TBK1 and IRF3 proteins and the expression of IRF3 protein(P<0.05).The protein levels of IFN-α,IFN-βand IFN-γwere significantly increased(P<0.05),and the secretions of TNF-αand IL-1βwere significantly decreased(P<0.05).【Conclusion】PCL could induce the production of interferon through enhancing TBK1-IRF3 pathway to alleviate the liver damage of mice infected by S.Typhimurium,and the dosage of 8 g/kg was better.