康复新液对脂多糖诱导炎症微环境中人食管成纤维细胞损伤的作用及机制OA

Objective To investigate the effects and underlying mechanism of Kangfuxin Liquid[an extract of Periplaneta americana(L.)]on human esophageal fibroblast(HEFs)injury within a lipopolysaccharide(LPS)-induced inflammatory microenvironment.Methods(1)Following intervention with varying concentrations of Kangfuxin Liquid(0%,0.5%,1%,2%,4%,8%,16%,32%)and LPS(0,2,4,6,8,10,20,40 μg·mL-1)for 24 hours,the HEFs proliferation rate was assessed via the CCK-8 assay to determine the optimal concentrations for LPS induction and Kangfuxin Liquid intervention.(2)HEFs in the logarithmic growth phase were divided into the following groups:Control,LPS,0.5%Kangfuxin Liquid,1%Kangfuxin Liquid,2%Kangfuxin Liquid,and Prednisolone Acetate(PDNN,50 μg·mL-1).An inflammatory microenvironment was induced by stimulation with LPS(10 μg·mL-1)for 1 hour.The levels of interleukin(IL)-1β,IL-6,tumor necrosis factor(TNF)-α,and inducible nitric oxide synthase(iNOS)in the cell supernatant were measured by ELISA.The expression levels of IL-6,nuclear factor(NF)-κB p-p65,p-Smad3,and α-smooth muscle actin(α-SMA)in the cells were detected by immunofluorescence.The mRNA expression levels of IL-1β,IL-6,TNF-α,NF-κB,transforming growth factor(TGF)-β1,Smad3,and α-SMA in the cells were determined using qRT-PCR.The protein expression levels of NF-κB p65,NF-κB p-p65,inhibitor of nuclear factor kappa-B alpha(IκBα),p-IκBα,TGF-β1,Smad3,p-Smad3,and α-SMA in the cells were assessed by Western Blot.Results(1)Subsequent experiments utilized Kangfuxin Liquid at concentrations of 0.5%,1%,and 2%,and an LPS concentration of 10 μ g·mL-1 to induce the inflammatory microenvironment in HEFs.(2)Compared with the control group,the LPS group exhibited significantly increased levels of IL-1β,IL-6,TNF-α,and iNOS in the HEF supernatant(P＜0.01);significantly upregulated mRNA expression of IL-1β,IL-6,TNF-α,and NF-κB(P＜0.01);significantly increased protein expression of NF-κB p65,NF-κB p-p65,IκBα,and p-IκBα(P＜0.01);significantly enhanced cytoplasmic red fluorescence signal for IL-6 and nuclear green fluorescence signal for NF-κB p-p65(P＜0.01);significantly upregulated mRNA expression of TGF-β1,Smad3,and α-SMA(P＜0.01);significantly increased protein expression of TGF-β1,Smad3,p-Smad3,and α-SMA(P＜0.01);and significantly intensified nuclear green fluorescence signal for p-Smad3 and cytoplasmic red fluorescence signal for α-SMA(P＜0.01).Compared with the LPS group,the 0.5%,1%,and 2%Kangfuxin Liquid groups and the PDNN group showed significantly reduced levels of IL-6,TNF-α,and iNOS in the cell supernatant(P＜0.01);significantly downregulated protein expression of NF-κB p65,NF-κB p-p65,IκBα,and p-IκBα(P＜0.01);significantly attenuated cytoplasmic red fluorescence signal for IL-6 and nuclear green fluorescence signal for NF-κB p-p65(P＜0.01);significantly decreased protein expression of TGF-β1 and p-Smad3(P＜0.01);and significantly weakened cytoplasmic red fluorescence signal for α-SMA(P＜0.01).Compared with the LPS group,the 2%Kangfuxin Liquid group and the PDNN group demonstrated significantly lower IL-1β content in the cell supernatant(P＜0.01);significantly downregulated mRNA expression of IL-1β,IL-6,TNF-α,and NF-κB(P＜0.05,P＜0.01);significantly downregulated mRNA expression of TGF-β1,Smad3,and α-SMA(P＜0.01);significantly decreased protein expression of Smad3 and α-SMA(P＜0.01);and a significantly weakened nuclear green fluorescence signal for p-Smad3(P＜0.01).Conclusion Kangfuxin Liquid can alleviate HEF injury and inhibit the phenotypic transition to myofibroblasts in an LPS-induced inflammatory microenvironment.The underlying mechanism may be associated with the suppression of the excessive activation of the TGF-β1/Smad3 signaling pathway.These findings provide experimental evidence supporting the potential use of Kangfuxin Liquid in treating esophageal strictures following endoscopic submucosal dissection.