桂杏水提取物调控TLR4/MyD88/NF-κB信号通路对脂多糖诱导的小鼠急性肺损伤的作用OA

Objective:To evaluate the efficacy and mechanism of Guixing(桂杏)Aqueous Extract(GX-AE)in a-meliorating acute lung injury induced by lipopolysaccharide(LPS)in mice.Methods:An acute lung injury model was established in C57BL/6J mice via tracheal instillation of LPS(20 mg/kg)and then treated with GX-AE at different do-ses through gavage.Histopathological changes in the lung tissue were assessed via hematoxylin-eosin staining.Enzyme-linked immunosorbent assays was performed to measure the levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-1β(IL-1β),and interleukin-10(IL-10)in the lung tissue.A fully automated hematology analyzer was used to count total cells(TC),white blood cells(WBC),and their subtypes in the bronchoalveolar lavage fluid(BALF).Colorimetric assays were employed to measure the serum levels of superoxide dismutase(SOD)and malondi-aldehyde(MDA).Immunohistochemistry was employed to detect the expression of nuclear factor κB p65(NF-κB p65),F4/80,and Toll-like receptor 4(TLR4).Western blotting was used to determine the protein levels of nuclear p65,phos-phorylated NF-κB p65(p-NF-κB p65),TLR4,zonula occludens-1(ZO-1),occludin,myeloid differentiation factor 88(MyD88),nuclear factor κB inhibitor alpha(IκBα),and its phosphorylated form(p-IκBα).Reverse transcription poly-merase chain reaction(RT-PCR)was employed to determine the mRNA levels of Il6,Iκbα,Tnfα,C-X-C chemokine lig-and 1(Cxcl1),hepatic nuclear factor 4α(Hnf4α),Snail,and zinc-finger E-box-binding homeobox 1(Zeb1).Results:Compared with the normal control group,the model control group showed increased lung tissue injury score(P<0.01),TC-BF,WBC-BF,and mono-nuclear lymphocyte count(MN#)in the BALF(P<0.05 or P<0.01),elevated levels of pro-inflammatory cytokines TNF-α,IL-6,and IL-1β(P<0.05 or P<0.01)and reduced level of the anti-inflammatory cytokine IL-10(P<0.01)in the lung tissue,increased level of MDA and decreased SOD activity in the serum(P<0.01),increased positive expression of NF-κB p65,F4/80,and TLR4 in the lung tissue(P<0.05 or P<0.01),upregu-lated protein levels of nuclear p65,p-NF-κB p65,TLR4,p-IκBα,and MyD88(P<0.05 or P<0.01),downregulated pro-tein levels of IκBα,ZO-1,and Occludin(P<0.05 or P<0.01),upregulated mRNA levels of Cxcl1,Hnf4α,Il6,Snail,Tnfα,and Zeb1(P<0.05 or P<0.01),and downregulated mRNA level of IκBα(P<0.05).Compared with the model control group,GX-AE(3 and 14 g/kg)groups showed decreased lung tissue injury scores(P<0.05).The GX-AE(7 and 14 g/kg)groups showed reduced TC-BF,WBC-BF,and polymorphonuclear neutrophil count(PMN#)in the BALF(P<0.05 or P<0.01),while the GX-AE(3 g/kg)group showed reduced MN#(P<0.01).GX-AE at all doses re-duced the levels of IL-6 and IL-1β and increased the level of IL-10 in the lung tissue(P<0.05 or P<0.01).The TNF-α level was reduced in the 14 g/kg group,and the MDA level was reduced in the 3 g/kg group(P<0.05).All the GX-AE groups showed enhanced SOD activity(P<0.01)and reduced TLR4-positive expression in the lung tissue(P<0.01).The positive expression of NF-κB p65 and F4/80 was reduced in the 7 and 14 g/kg groups(P<0.05).The 14 g/kg group showed downregulated protein levels of nuclear p65,p-NF-κB p65,MyD88,TLR4,and p-IκBα(P<0.05 or P<0.01),upregulated protein levels of IκBα,ZO-1,and Occludin(P<0.05 or P<0.01),downregulated mRNA lev-els of Il6,Tnfα,Cxcl1,Snail,and Zeb1,and upregulated mRNA levels of Hnf4α and Iκbα(P<0.05 or P<0.01).Conclusion:GX-AE may inhibit the TLR4/MyD88/NF-κB signaling pathway to attenuate inflammatory responses,oxida-tive stress,and epithelial barrier disruption,thereby exerting protective effects against LPS-induced acute lung injury.