A Proteomic Study On Gastric Impairment In Rats Induced By Microcystin-LR Exposure

Objective:A growing body of research confirms that Microcystins are a significant risk to human health,and that Microcystins exposure can lead to gastrointestinal disease.The aim of this study was to investigate the mechanism of gastric tissue injury induced by Microcystin-LR exposure in rats by means of histopathology,molecular biology and proteomics,this study provides a theoretical basis for further research and prevention of gastric tissue injury induced by Microcystins exposure.Methods:MC-LR（purity≥95%）was purchased from Beijing Puhuaren Technology Development Co.,Ltd.,and the LD₅₀ of MC-LR was determined after intravenous injection（i.v.）in Wistar Rats.The SPF level 6-week-old male Wistar rats were fed for 5 days and randomly divided into 4 groups:blank control group（normal saline group）,low-dose MC-LR exposure group(0.5LD₅₀MC-LR),medium-dose MC-LR exposure group(075 LD₅₀MC-LR),and high-dose MC-LR exposure group(1.0 LD₅₀MC-LR).Rats in each group were intravenously injected with MC-LR once.After 24 hours of exposure,the gastric tissue of rats was dissected.The Pathological changes of gastric tissue were observed by HE staining in rats.The activities of CAT,SOD and GPx,and the contents of GSH and MDA in gastric tissue were determined.TMT-based proteomics analysis was used to identify the differentially expressed proteins in the study.The differentially expressed proteins were screened in accordance with the criteria of 1.2-fold expression and P<0.05,and the differentially expressed proteins were analyzed by functional classification,enrichment analysis and protein-protein interaction network analysis.Results:The LD₅₀ of male Wistar rats intravenously injected with MC-LR was 100μg/kg,and the 95%confidence interval was 70.68-131μg/kg.Compared with the control group,the different exposure dose of MC-LR had different pathological damage to gastric fundus,gastric body and gastric antrum of rats.The main manifestations were different degrees of bleeding in the gastric fundus of the medium-dose and high-dose MC-LR exposure group and the gastric body of the high-dose MC-LR exposure group,necrosis of gastric body parietal cells in the medium-dose and high-dose MC-LR exposure group and abscission of gastric antrum mucosa epithelial cells in the high-dose MC-LR exposure group.Compared with the control group,SOD activity in gastric tissue increased with the increase of MC-LR exposure dose（P<0.01）,CAT activity in low-dose MC-LR exposure group significantly decreased（P<0.01）,there was no significant change in other exposure groups,and there was no significant change in GPx activity in all dose groups（P>0.05）.The content of GSH in high-dose MC-LR exposed group was significantly higher than that in control group and low-dose MC-LR exposed group（P<0.05）.Compared with the control group,MDA content in all exposure groups was significantly increased（P<0.05）.Compared with the control group,37360peptides were identified by proteomics in the stomach tissue of the medium dose MC-LR exposure group,among which 34922 peptides were unique peptides.A total of 5827 proteins were identified,including 5160 quantifiable proteins.The total number of differentially expressed proteins was 277,including 187 up-regulated proteins and 90 down-regulated proteins.Proteomics results showed that the differentially expressed proteins were mainly involved in metabolism,immune response,inflammatory response,oxidative stress,procoagulant and anticoagulant,ion binding and gene expression.Functional enrichment analysis showed that MC-LR may cause gastric bleeding,oxidative stress,inflammation and ferroptosis by affecting domains such as albumin N-terminal,fibrinogen,alpha/beta/gamma chain,coiled coil domain and ferritin/DPS protein domain,as well as cellular component such as spectrin-associated cytoskeleton,fibrinogen complex and hemoglobin complex.The enrichment analysis of differential protein KEGG pathway showed that there were 11 pathways with statistical significance（P<0.05）,of which Complement and coagulation cascades（Map04610）were the most significant difference（P=0.0000002）,and contains the most number of differential proteins.Protein-protein interaction network analysis showed that MC-LR induced gastric tissue injury in rats is a multi-channel interaction.The interaction of Apolipoprotein B-100（Apob）,Serum albumin（Alb）,Kininogen-1（Kng1）,Plasminogen（Plg）,Fibrinogen alpha chain（Fga）,Fibrinogen beta chain（Fgb）,Fibrinogen gamma chain（Fgg）,Serine（or cysteine）peptidase inhibitor（Serpinc）and Complement C3（C3）affects the coagulation regulatory systems and the complement regulatory systems.Conclusion:1.MC-LR exposure can cause pathological damage of gastric tissue in rats,destroy the balance of antioxidant system of gastric tissue,induce oxidative stress,and resulting in oxidative damage of gastric tissue.2.Compared with the control group,a total of 277 differentially expressed proteins were identified by proteomics in the gastric tissue of the medium dose MC-LR exposure group.They are mainly involved in metabolism,immune response,inflammatory response,oxidative stress,procoagulant and anticoagulant,ion binding and gene expression.3.KEGG pathway enrichment analysis and protein-protein interaction network analysis showed that MC-LR induced acute gastric tissue injury in rats was the result of multi-center and multi-pathway interaction,and the key pathways of MC-LR induced gastric injury in rats may be Complement and coagulation cascades and Ferroptosis,which needs to be further verified.