Effect And Mechanism Of Colorectal Injury Caused By Long-Term Low-Dose Exposure To Microcystin-LR

Objective:The purpose of this study is to use animal studies to gain insight into the function and mechanism of colorectal chronic inflammation,fibrosis,barrier disruption,structural alterations of gut bacteria,and their related metabolites.The mechanism of action was further verified by in vitro cell model and bacterial single colonization experiment.This study can provide theoretical basis and laboratory foundation for the investigation of the intestinal toxicity of MC-LR and the development of treatment approaches for intestinal inflammation.Methods:(1)A mouse model with long-term(12 m)exposure to low-dose MC-LR(0,1,60,120μg/L)was established in vivo,and the changes of inflammatory factors,fibrosis factors and tight junction factors in mouse colorectal tissues and cells were detected by q PCR and WB.Through transcriptomics and proteomics sequencing analysis techniques,differential genes and differential proteins were screened and analyzed jointly,key proteins and their downstream signaling pathway proteins were excavated and their expressions were verified.Different flora and metabolites were screened out for combined study using 16S r DNA high-throughput sequencing technology and metabolomics sequencing analysis technology,and potential important bacteria and metabolites connected to intestinal damage were discovered.(2)The in vitro model of low-dose MC-LR(0,60 nmol/L)cells exposed for a long time(3 m)was constructed to verify the mechanism of action excavated from the in vivo model of exposure to MC-LR.(3)An in vivo mouse model of MC-LR(20μg/kg)co-treated with C.perfringens/R.gnavus(10~8 CFU/m L)was established.In vitro models of cells co-treated by MC-LR(120μmol/L)and C.perfringens/R.gnavus(10~8CFU/m L)and MC-LR(120μmol/L)co-treated with D-Gluconolactone/3-indoxyl sulfate(100μmol/L)were constructed to validate the action mechanism discovered in the in vivo model of MC-LR exposure.Results:(1)No mortality and no significant change in body weight in all groups of mice in an in vivo model of chronic exposure to low doses of MC-LR.Compared with the control(CT)group,the colorectum of 60 and120μg/L MC-LR-treated mice was significantly shortened,and histopathology showed significant inflammatory responses and fibrosis development.The q PCR and WB data demonstrated that mice treated with60 and 120 g/L MC-LR had considerably higher expression of inflammatory markers and fibrosis factors in their colon tissues,while the expression of tight junction factors was significantly decreased compared with the CT group.Combined transcriptomic and proteomic analysis screened for the key factor CSF1R/Rap1b.Additionally,animals given 60and 120 g/L MC-LR showed significantly higher levels of CSF1R and Rap1b expression in their colon tissues compared to the CT group.In the in vivo model,combined genomic and metabolomic analyses showed that C.perfringens abundance was positively correlated with D-Gluconolactone abundance,and R.gnavus abundance was positively correlated with 3-indoxyl sulfate abundance.(2)The ROS production,MDA levels,CSF1R/Rap1b signaling pathway,inflammatory factors,and fibrosis factor expression were significantly increased in the NCM460 cell model after prolonged exposure to low doses of MC-LR,whereas SOD activity and tight junction factor expression were significantly decreased;while the alterations in these indices caused by MC-LR could be significantly inhibited after the intervention of oxidative stress response and CSF1R and Rap1b expression.(3)In an in vivo model of mice co-treated with MC-LR and Clostridium perfringens(C.perfringens)or Ruminococcus gnavus(R.gnavus),MC-LR and C.perfringens caused death and significant weight loss,with more pronounced weight loss in the MC-LR and C.perfringens co-treatment group,while R.gnavus protected mice from death and alleviated the weight loss caused by MC-LR.In the in vitro models of cells co-treated with MC-LR and C.perfringens/R.gnavus or MC-LR and D-Gluconolactone/3-indoxyl sulfate,compared with the CT group,the expression of CSF1R/Rap1b signaling pathway,inflammatory factors,and fibrosis factors were significantly increased,the levels of tight junction factors significantly decreased.The alterations of these indicators caused by MC-LR could be further aggravated after co-treatment with C.perfringens/D-Gluconolactone.The results of factorial analysis showed that the interaction of combined treatment on the damage phenomenon was synergistic.Whereas,after co-treatment with R.gnavus/3-indoxyl sulfate alleviated the altered expression of these indices caused by MC-LR.The results of factorial analysis showed that the interaction of combined treatment on the damage phenomenon was antagonistic.Conclusion(1)Long-term low-dose exposure to MC-LR can cause chronic inflammation,fibrosis and barrier disruption in the colorectal of mice,and the induced inflammatory response can affect the expression of antimicrobial peptides,and cause structural changes of intestinal microorganisms and their related metabolites,thus further aggravating intestinal injury.(2)Long-term low-dose exposure to MC-LR can activate oxidative stress,cause colorectal injury through CSF1R/Rap1b signaling pathway.(3)C.perfringens and its metabolite D-Gluconolactone affect CSF1R/Rap1b signaling pathway activation and promote MC-LR-induced colorectal inflammatory response,fibrosis and barrier disruption occurrence.R.gnavus and its metabolite 3-indoxyl sulfate affect CSF1R/Rap1b signaling pathway activation and alleviate MC-LR-induced colorectal inflammatory response,fibrosis and barrier disruption occurrence.