| Research Background:Nonalcoholic steatohepatitis(NASH)refers to the destruction of liver cells by a variety of pathogenic factors such as bacterial and virus infection,autoimmune factors,obesity,metabolism and so on,which damages the function of the liver and causes a series of unwell symptoms.The pathology is characterized by varying degrees of fatty infiltration and inflammatory response,followed by liver fibrosis,and eventually cirrhosis and even liver cancer.Porphyromonas gingivalis(P.g),an obligate anaerobic gram-negative bacterium,is currently recognized as one of the most important pathogens causing periodontitis.P.g can cause the disturbance of oral microenvironment and a series of periodontal diseases.P.g can also cause intestinal microenvironment disturbance to induce intestinal inflammation and damage the intestinal barrier,allowing autovirulence factors to enter the blood circulation,thus contributing to the occurrence of NASH and the worsening of digestive system diseases.Meanwhile,in NASH induced by P.g,P.g leads to the activation of NF-κB signaling pathway in hepatocytes,leading to an inflammatory response in hepatocytes.However,increased liver inflammation can lead to increased ROS and increased oxidative stress,leading to ferroptosis of liver cells.Therefore,it is of research value to explore the role and mechanism of P.g in regulating ferroptosis of hepatocytes through NF-κB signaling pathway in non-alcoholic steatohepatitis,clarify the relationship between periodontal disease bacteria and NASH,and provide theoretical basis for alleviating or preventing NASH through treatment of periodontitis in the later stage,and provide new ideas and strategies for NASH treatment.Research objectives:The role and molecular mechanism of P.g in regulating ferroptosis of hepatocytes through activation of NF-κB signaling pathway in non-alcoholic steatohepatitis were analyzed in vitro and in vivo.Research methods:Modeling method of animal model:BALB/C mice in the experimental group were fed with about 1×109 P.g,while the control group was given the same amount of BHI bacterial medium.During the experiment,each group of mice was weighed weekly and their survival rates monitored.At the end of the third and fifth week,200μL of blood was collected through the posterior orbital venous plexus.Blood,feces,liver,spleen,maxilla,and intestinal organs were collected after gavage at the seventh week.In vivo periodontal,intestinal and liver injury tests:The collected blood was used to determine the biochemical indexes of liver function,ALT and AST.Fecal 16 Sr RNA sequencing was used to analyze the degree of intestinal flora disorder.H&E staining and immunohistochemistry were used to evaluate the degree of hepatic and intestinal inflammatory infiltration,periodontal inflammatory infiltration and alveolar bone resorption.The changes of ferroptosis related regulatory proteins(GPX4,ACSL4,SLC7A11)in liver and intestine were analyzed by immunohistochemistry.The m RNA expression changes of ferroptosis related genes(Gpx4,Ptgs2,Ncoa4)in liver were detected by RT-PCR.The changes of ferroptosis related regulatory proteins in liver tissue protein were analyzed by Western blot analysis.The m RNA expression of P.g bacteria-specific protease(Kgp,Rgp A,Rgp B)in liver was detected by RT-PCR.The expression level of key protein of NF-κB signaling pathway in liver tissue protein was analyzed by Western blot.In vitro experiment of hepatocyte injury model:The L-02 cell line was co-cultured with 26.7% P.g supernatant for 24 h,and the effects of P.g on hepatocyte viability were analyzed by CCK-8 and cell death staining.After collecting RNA and protein from liver cells,RT-PCR was used to detect m RNA expression changes of ferroptosis related genes(GPX4,PTGS2,NCOA4)in liver cells,and Western blot was used to analyze the changes of iron death related regulatory proteins(GPX4,ACSL4,SLC7A11)in liver cell proteins.The activation of NF-κB signaling pathway(NF-κB-p65,p-NF-κB-p65,IKBα)and the expression of inflammatory cytokines in hepatocytes(IL-17,IL-6,IL-10)were analyzed.The nuclear entry of NF-κB was analyzed by Western blot.The liver cells were treated with Fer-1at 5μmol/m L for 2h,and then co-cultured with 26.7%-P.g supernatant for 24 h.Cell viability was assessed.RNA and protein were collected from liver cells.The changes of m RNA expression of GPX4,PTGS2,NCOA4,and the changes of ferroptosis related regulatory proteins(GPX4,ACSL4,SLC7A11)in hepatocyte proteins were analyzed by Western blot analysis.NF-κB signaling pathway predominate inhibitors(QNZ) concentration of 30 nmol/m L treatment liver cells after 2 h,then co-cultured with26.7%-P.g supernatant for 24 h.Tnen,collecting L-02 cell RNA and protein,RT-PCR detection of liver ferroptosis related gene(GPX4 PTGS2,NCOA4)m RNA expression,Western blot analysis of ferroptosis related regulatory proteins(GPX4,ACSL4,SLC7A11),the expression of NF-κB signaling pathway(NF-κB-p65,p-NF-κB-p65,IKBα),and the expression of hepatocyte inflammatory cytokines(IL-17,IL-6,IL-10).Research results:1.Immunohistochemistry and H&E staining showed that the alveolar bone resorption and the expression of IL-17 and IL-6 were increased,suggesting that P.g bacteria feeding could cause periodontal inflammation in mice.16 Sr RNA sequencing results showed that the intestinal microenvironment disorder of mice after P.g irrigation showed an increase of bacteroides and a decrease of firmicutes.Intestinal inflammation increased and villous interstitial edema complicated with ferroptosis;The expression of P.g bacteria and its specific proteins Kgp,Rgp A and Rgp B in liver after intestinal barrier changes suggested that P.g bacteria might colonize in liver.2.After 7 weeks of feeding P.g bacteria,serological analysis showed that ALT and AST levels increased;The results of H&E staining showed that 33.33% of mice liver had fatty lesions,and 100% mice liver had a large number of inflammatory cell infiltration,suggesting that P.g bacteria feeding could cause NASH.3.P.g leads to the inflammatory response in hepatocytes and activates the NF-κB signaling pathway: Immunohistochemistry confirmed the increased expression of inflammatory factors in liver and spleen;Western blot and RT-PCR showed increased expression of inflammatory cytokines and activation of NF-κB signaling pathway in liver tissues.The expression of NF-κB in liver was enhanced by immunofluorescence staining.In vitro experiments demonstrated that inhibitor QNZ of NF-κB signaling pathway can inhibit the activation of NF-κB signaling pathway and relieve the hepatocyte inflammation induced by P.g.4.P.g induced ferroptosis in hepatocytes: Immunohistochemistry confirmed that the expressions of ferroptosis regulatory proteins GPX4 and SLC7A11 were decreased and the expressions of ACSL4 were increased in liver tissue.Western blot results showed that the expressions of ferroptosis regulation proteins GPX4 and SLC7A11 were decreased,while the expressions of ACSL4 were increased.RT-PCR showed that the m RNA expression level of GPX4,the regulator of ferroptosis,was decreased in liver tissue,and the m RNA expression levels of PTGS2 and NCOA4 were increased,suggesting that P.g induced ferroptosis in liver cells after gavage.In vitro experiments,Fer-1,an ferroptosis inhibitor,alleviated liver cell activity,ferroptosis and inflammation induced by P.g supernatant.5.The NF-κB signaling pathway inhibitor QNZ can inhibit the ferroptosis and inflammatory response of hepatocytes induced by P.g supernatant.Western blot results showed that the expression of ferroptosis protein ACSL4 was inhibited by QNZ,and QNZ effectively alleviated the expression levels of GPX4 and SLC7A11.RT-PCR results showed that GPX4 m RNA levels were alleviated by QNZ,while PTGS2 and NCOA4 m RNA levels were inhibited by QNZ.Western blot results showed that QNZ inhibited the expression of inflammatory cytokines IL-17 and IL-6,and increased the expression of anti-inflammatory cytokines IL-10.Research conclusion:1.In vivo studies have confirmed that oral feeding of P.g can lead to periodontal inflammation,intestinal inflammation,ferroptosis and intestinal microenvironment disorders,and found that P.g bacteria may colonize in the liver.2.In vivo studies have found that P.g can cause liver function injury,liver inflammation and fatty lesions by gavage feeding;In vivo studies confirmed that P.g activated NF-κB signaling pathway in mouse liver,and in vitro studies confirmed that P.g supernatant could activate NF-κB signaling pathway.Further in vitro studies showed that P.g supernatant could affect the activity and inflammatory response of liver cells and cause ferroptosis in liver cells,and ferroptosis inhibitor could alleviate the occurrence of ferroptosis in vitro.3.In vitro studies have found that the use of NF-κB signaling pathway inhibitor QNZ can inhibit the ferroptosis of hepatocytes caused by P.g supernatant,relieve the inflammatory response of hepatocytes,and inhibit the expression of pro-inflammatory factors.This indicated that P.g bacteria could further promote ferroptosis by activating the NF-κB signaling pathway,causing inflammation in hepatocytes.Figure 17 Table 14 Reference 65... |
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