Molecular Mechanism Study Of Acinar-to-ductal Metaplasia Promoted By The Imbalance Of Intestinal Flora In Pancreatitis

Pancreatitis is a high risk factor of pancreatic ductal adenocarcinoma(PDAC).It has been reported that acinar-to-ductal metaplasia(ADM)is the first response of acinar cells to pancreatitis injury.Under the stimulation of long-term inflammatory or carcinogenic signals,ADM lesion can cause pancreatic intraepithelial neoplasia(PanINs)and gradually worsen to PDAC.At the same time,some studies have shown that the imbalance of intestinal flora can aggravate the secondary infection and systemic inflammatory response of gastrointestinal diseases,especially pancreatic diseases.Therefore,it is of great significance to explore the role of intestinal flora in ADM lesion induced by pancreatitis for the study of molecular mechanisms in the transformation of pancreatitis to cancer and the early diagnosis and treatment of PDAC.In this study,the combination of microbiomics and metabonomics was used to analyze the intestinal flora and its metabolites of pancreatitis rats based on 16S rDNA sequencing technology and liquid chromatography-mass spectrometry assay,respectively.It was found that the fecal samples had the smallest individual difference and the highest species richness,which was the most suitable sample for the study of intestinal flora.Moreover,the flora in fecal samples is imbalance,such as,the relative abundances of Bacteroides and Escherichia are significantly increased,while the relative abundance of Lactobacillus decreased notably.Imbalance of intestinal flora leads to abnormal tryptophan metabolism and oxidative stress,and increased the generations of 5-hydroxytryptamine(5-HT)and reactive oxygen species(ROS).Then,primary pancreatic acinar cells and primary pancreatic stellate cells were extracted and pretreated with 5-HT.It was found that 5-HT could promote the occurrence of ADM lesion in acinar cells and the activation of stellate cells;meanwhile,the secretion level of 5-HT was significantly increased in pancreatic tissues with pancreatitis induced ADM lesion.Molecular mechanism studies have shown that 5-HT can increase RhoA/ROCK signaling pathway mediated nuclear translocation of nuclear factor-?B(NF-?B),and activate the downstream inflammatory response,which would promote the early lesions of PDAC,such as ADM and fibrosis.In addition,transmission electron microscopy and flow cytometry showed that pancreatitis induced mitochondrial damage in acinar cells,resulting in a large amount of ROS release.In order to explore the regulatory effect of ROS on ADM lesion,primary acinar cells and 266-6 acinar cell line were pretreated with H2O2 in vitro.It was found that H2O2 at a certain concentration could promote the occurrence of ADM lesion by activating S100A9 mediated inflammatory response.In addition,s100a9-/-mice were successfully constructed in this study,and it also found that knockout of S100A9 gene could significantly reduce the pancreatitis injury and the early pathological changes of ADM and even PanINs in mice.Molecular mechanism studies based on RNA-seq and qPCR assays showed that ROS could upregulate S100A9 expression and increase NF-?B mediated inflammatory response via its receptor TLR4,which can further activate MYC oncogene and promote ADM lesion and transformation of pancreatitis to cancer.Therefore,the imbalance of intestinal flora in pancreatitis leads to the abnormal metabolism of tryptophan and the increased level of oxidative stress,which activate the 5-HT/RhoA/NF-?B and ROS/S100A9/MYC signal axes,and finally promote the ADM lesion induced by pancreatitis.5-HT/RhoA/NF-?B and ROS/S100A9/MYC signal axes play important regulatory roles in the transformation of pancreatitis to cancer,which may be the early therapeutic targets of PDAC.