The Protective Effect And Molecular Mechanism Of Shen Fu Preparation On Inflammatory Diseases Of Digestive System

Part 1 Shen Fu preparation protects AML12 cells against palmitic acid-induced injury through inhibition of both JNK/Nox4 and JNK/NF?B pathwaysObjective:Nonalcoholic steatohepatitis(NASH)includes steatosis along with liver inflammation,hepatocyte injury and fibrosis.In this study,we investigated the protective role and the potential mechanisms of a traditional Chinese medicine Shen Fu(SF)preparation in an in vitro hepatic steatosis model.Methods:In palmitic acid(PA)-induced murine hepatic AML12 cell injury,effects of SF preparation on cellular apoptosis and intracellular triglyceride(i TG)level were assessed using TUNEL and TG Colorimetric Assay.Reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)levels were measured using DCF and JC-1 assay.Cytokine levels were evaluated using ELISA assay.Immunoblot was used to compare the activation level of c-Jun N terminal kinase(JNK),NADPH oxidase(Nox4),and NF?B pathways.Results:Addition of SF preparation prevented PA-mediated increase of apoptosis and i TG as well as IL-8 and IL-6.In PA-treated cell,SF preparation reduced the level of Nox4 and ROS,while increasing the level of MMP and the expression of manganese superoxide dismutase(Mn SOD)and catalase,indicating emendation of mitochondrial dysfunction.Nox4 inhibitor GKT137381 prevented PA-induced increase of ROS and apoptosis,while decreasing i TG slightly and not influencing the level of IL-8 and IL-6.SF preparation prevented PA-induced upregulation of phospho-JNK.JNK inhibitor SP600125 prevented PA-mediated increase of Nox4,IL-8,IL-6 and i TG.Nuclear translocation of NF?B/p65 was detected in PA-treated cells,which was prevented by SF preparation.An I?B degradation inhibitor,BAY11-7082,prevented PA-induced increase of IL-8 and IL-6 as well as i TG,whereas it only decreased ROS levels slightly and showed no influence on cellular apoptosis.Conclusion:SF preparation shows a beneficial role in prevention of hepatocyte injury by attenuating oxidative stress and cytokines production at least partially through inhibition of JNK/Nox4 and JNK/NF?B pathway,respectively.Part 2 Shen Fu preparation ameliorates lipopolysaccharide-induced intestinal epithelial cell injuryObjective:Intestinal epithelial cells play a crucial role inmaintenance of the gut barrier function,whereas the underlying injury mechanisms are still not fully clarified.Here,we investigated the protective effects and molecular mechanisms of a traditional Chinese medicine-Shen Fu(SF)preparation on lipopolysaccharide(LPS)-induced intestinal epithelial cell injury.Methods:Human intestinal epithelial cell line HIEC-6 was treated with LPS alone or combination of LPS and SF.Apoptosis was assessed using TUNEL assay.Reactive oxygen species(ROS)level was measured using DCF assay.Monolayer HIEC-6 cells cultured in Transwell plates were used for analyzing the permeability by FITC-albumin influx assay.Real time RT-PCR was used for quantification of cytokines IL-1?,IL-6 and TNF-?.Western blot was performed for the abundance of cytosolic I?B? and nuclear NF?B/p65 subunit.Results : Increased apoptosis,ROS and cytokines production as well as NF?B/p65 nuclear translocation were detected in LPS-treated cells.The application of Diphenylene iodonium(DPI),a specific ROS inhibitor,prevented increase of apoptosis,cytokines productionand nuclear translocation of NF?B/p65 in LPS-treated cells.In addition,LPS-induced overproduction of cytokines was abolished by an I?B degradation inhibitor BAY11-7082.Most interestingly,SFE prevented degradation of cytosolic I?B?,resulting in reduction of NF?B/p65 in nuclear fraction in LPS-treated cells.SF dose-dependently inhibited increase of apoptosis,ROS production,and permeability in LPS-treated cells.Moreover,LPS-mediated overproduction of TNF-? and IL-6 was prevented by SF.Conclusion:These data demonstrate that SF has the ability to protect LPS-induced intestinal epithelial injury by inhibition of ROS production and NF?B/p65 nuclear translocation,thus leading to reduction of inflammatory cytokines.