火炭母通过调控TLR4-TBK1信号通路改善鼠伤寒沙门菌感染小鼠空肠的炎症反应

Polygonum chinense L.Alleviated Jejunum Inflammatory Response in Mice Infected by Salmonella typhimurium through Regulating TLR4-TBK1 Signaling Pathway

为了探讨火炭母对鼠伤寒沙门菌感染小鼠空肠的免疫保护作用及其潜在机制。本试验将48只雌性BALB/c小鼠随机分为6个组:空白对照组、模型组、阳性药物组和火炭母高、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·bw)],火炭母高、中、低剂量组小鼠分别按16、8和4 g/(kg·bw)剂量灌胃火炭母,空白对照组和模型组小鼠给予等体积的灭菌生理盐水,共连续给药8 d。预防性给药2 d后,每只小鼠(空白对照组除外)灌胃0.2 mL半数致死量(LD 50)鼠伤寒沙门菌(5×10^(4) CFU/mL)致病。给药结束后,处死小鼠取空肠用于组织病理学检查和Western blot。结果显示,鼠伤寒沙门菌感染后,小鼠空肠杯状细胞和肠腺红细胞增多、肠绒毛结构松散,火炭母各剂量组小鼠空肠杯状细胞和肠腺红细胞明显减少,火炭母减轻了小鼠空肠组织损伤。Western blot结果显示,与空白对照组相比,模型组小鼠空肠中免疫因子β干扰素(IFN-β)蛋白表达显著上升(P<0.05),γ干扰素(IFN-γ)蛋白表达显著下降(P<0.05),干扰素调节因子3(IRF3)、磷酸化TANK结合激酶1(p-TBK1)和Toll样受体4(TLR4)蛋白表达显著上调(P<0.05),炎性因子白介素-6(IL-6)、白介素-1β(IL-1β)和核因子κB p65(NF-κB p65)蛋白表达显著上升(P<0.05);与模型组相比,不同剂量火炭母可以不同程度降低炎症因子IL-6(P<0.05或P>0.05)、IL-1β(P<0.05)和NF-κB p65(P<0.05)蛋白表达,显著提高TLR4、β干扰素TIR结构域衔接蛋白(TRIF)蛋白的表达和TANK结合激酶1(TBK1)蛋白表达及其磷酸化水平(P<0.05),提高下游信号IRF3蛋白的表达(P<0.05)及其磷酸化水平(P<0.05或P>0.05),进而提高空肠中IFN-β(P<0.05或P>0.05)和IFN-γ(P<0.05)蛋白的表达。结果表明,火炭母通过调控TLR4-TBK1信号通路改善鼠伤寒沙门菌感染小鼠空肠的炎症反应。

In order to investigate the immunoprotective effects of Polygonum chinense L.(PCL)on the jejunum of mice infected with Salmonella typhimurium and its potential mechanisms,48 female BALB/c mice were randomly divided into six groups:Blank control group,Model group,Positive drug group,and High-,Medium-,and Low-dose PLC groups,with 8 mice in each group.Mice in the Positive drug group were orally administered with gentamicin[20 mg/(kg·bw)],and mice in the High-,Medium-,and Low-dose PLC groups were orally administered with PLC at doses of 16,8,and 4 g/(kg·bw),respectively.Mice in the Blank control group and Model group were given an equal volume of sterile saline,with a total of 8 days of continuous administration.After 2 days of prophylactic treatment,each mouse(excluding the Blank control group)was orally challenged with 0.2 mL of half-lethal dose(LD 50)of Salmonella typhimurium(5×10^(4) CFU/mL)to induce infection.After completion of the treatment,the mice were euthanized,and the jejunum was collected for histopathological examination and Western blot.The results showed that following infection with Salmonella typhimurium,the mice in the Model group exhibited increased goblet cells in jejunum and erythrocytes in intestinal gland,and loose structure of intestinal villi.Mice in the PLC groups showed significant reduction of goblet cells in jejunum and erythrocytes in intestinal gland,indicating that PLC alleviated intestinal tissue damage in mice.Western blot results demonstrated that compared with the Blank control group,the Model group exhibited significant increase in interferon-β(IFN-β)protein expression(P<0.05),significant decrease in interferon-γ(IFN-γ)protein expression(P<0.05),and significant upregulation of interferon regulatory factor 3(IRF3),phosphorylated TANK-binding kinase 1(p-TBK1),and Toll-like receptor 4(TLR4)protein expression(P<0.05).Additionally,inflammatory factors interleukin-6(IL-6),interleukin-1β(IL-1β),and nuclear factor kappa B p65(NF-κB p65)showed significant increase in expression(P<0.05).In comparison to the Model group,different doses of PLC reduced the expression of inflammatory factors IL-6(P<0.05 or P>0.05),IL-1β(P<0.05),and NF-κB p65(P<0.05),increased the expression of TLR4,interferon-βTIR domain-containing adaptor protein(TRIF),TANK-binding kinase 1(TBK1),and its phosphorylation levels(P<0.05),as well as the downstream signaling protein IRF3(P<0.05)and its phosphorylation levels(P<0.05 or P>0.05).This led to increase in IFN-β(P<0.05 or P>0.05)and IFN-γ(P<0.05)protein expression in the jejunum.The results suggested that PCL alleviated jejunum inflammatory response in mice infected by Salmonella typhimurium through regulating TLR4-TBK1 signaling pathway.