The Effect And Mechanism Of Escherichia Coli-derived HtpG Regulating PRMT5 In Chronic Pancreatitis

As a permanent,recurring,and irreversible pancreatic-fibrosis-inflammatory disease,chronic pancreatitis(CP)is caused by a combination of genetic and environmental factors.CP is characterized by continuous pancreatic damage that eventually results in significant impairment of exocrine as well as endocrine functions of the gland.Common symptoms of CP include abdominal pain,pancreatic fibrosis,intestinal flora disorders,and complications such as diabetes,metabolic bone disease and pancreatic cancer.A large number of studies have shown that the intestinal flora of CP patients is different from that of healthy people,and pathogenic bacteria,especially Escherichia coli(E.coli),are abnormally enriched,besides,CP can be alleviated by antibiotics or probiotics.The virulence of E.coli is mainly dependent on high temperature protein G(Htp G).Heat shock protein 90,the ortholog of Htp G,plays an important role in the development of CP fibrosis,and upregulates the expression of protein arginine methyltransferase 5(PRMT5).As a post translational modification enzyme related to epigenetics and signal transduction,PRMT5 plays a significant role in tumor and inflammation through histone modification of key transcription factors,and participates in cell phenotypic transformation.Recent studies have shown that the polarization of macrophages can affect the pancreatic fibrosis-inflammatory microenvironment,thereby activating pancreatic stellate cells(PSCs)and participating in the development of CP.PRMT5 promotes the accumulation of KLF4 protein by binding to and methylation modifying the transcription factor Krüppel-like factor 4(KLF4)of macrophage polarization.Based on the above research background,this article speculates that the release of Htp G from E.coli upregulates the expression of PRMT5 during the development of CP,and methylation of KLF4 is involved in regulating the polarization of macrophages.Combining animal experiments and cell experiments,this article elucidates the role and mechanism of E.coli derived Htp G in regulating PRMT5 on CP from the perspective of flora.The main results are as follows:(1)In order to clarify the effect of E.coli on the expression of PRMT5 in pancreas during the development of CP,a mouse CP model was constructed by intraperitoneal injection of caerulein.The composition of intestinal flora in C57BL/6J mouse CP model was analyzed by using 16 S amplification sequencing method.The results showed that the abundance of E.coli was significantly changed,and elevated the expression of Htp G,increased CD91 receptor and NF-κB pathway was activated.In addition,it was found that the expression of PRMT5 was upregulated in pancreatic tissues.Immunofluorescence experiments confirmed that PRMT5 was mainly expressed in macrophages in CP patients and mouse CP models.To further demonstrate the regulatory effect of Htp G on the expression of PRMT5,we conducted intervention on bone marrow derived macrophages(BMDM)isolated from mice with a treatment of Htp G(E.coli)and 17 AAG,which is the inhibitor of heat shock protein 90.The results showed that the Htp G released by E.coli upregulated the expression of PRMT5 through activating NF-κB pathway by the CD91 receptor.(2)In order to explore the effect of pancreatic PRMT5 on CP,macrophage-specific knockout of PRMT5 mice and exogenous application of PRMT5 specific inhibitor EPZ015666 were used to detect CP phenotype,macrophage polarization,and fibrosis degree.The results showed that the PRMT5 knockout could alleviate damage and fibrosis of the pancreas during the development of CP,and reduce the expression of α-smooth muscle actin and type Ⅰ collagen alpha 1,and inhibit the phenotypic transformation of M2 macrophages.(3)In order to clarify the mechanism of PRMT5 exacerbating CP,the expression of KLF4 in pancreatic tissues was detected first.The results showed that the expression of KLF4 in the pancreas of CP mice was significantly lower than that of the control group,while the expression of KLF4 was decreased after PRMT5 downreglation.This study indicated that PRMT5 and KLF4 had a mutual binding relationship.It was further confirmed that PRMT5 mediated the methylation of KLF4,and inhibited its ubiquitination degradation,thereby stabilizing the expression of KLF4.Using a co-culture experiment of BMDM and PSCs in vitro,combined with small interference,overexpressed plasmid and mutant plasmid,to explore the activation effect of macrophage polarization on PSCs.The results showed that PRMT5 mediated KLF4 to regulate macrophage polarization into M2 type,and released transforming growth factor βto activate PSCs.Finally,Mφ-PRMT5-KO mice with CP model were constructed by injecting packaged KLF4 adeno-associated virus into the tail vein to verify the mechanism of PRMT5 on the development of CP.The results showed that the overexpression of KLF4 while knocking out PRMT5 reversed the effect of knocking out PRMT5 on alleviating pancreatic atrophy,injury and fibrosis in CP mice.This article revealed that the release of Htp G by E.coli in CP mice promoted the expression of pancreatic PRMT5,bound to and methylated KLF4,and then regulated M2 macrophage polarization,activated PSCs,and exacerbated CP.This study provides a theoretical basis for the gut microbiota-PRMT5 axis or the prevention and treatment of CP.