Exploring The Role And Mechanism Of Armillariella Tabescens Polysaccharides Interference In 5-FU-Induced Intestinal Mucosal Injury Based On β-Arrestin1

Objective:This study aims to investigate the role and mechanism ofβ-arrestin1（ARRB1）in the reversal of 5-FU-induced chemotherapeutic intestinal mucosal injury（CIMI）by Armillariella tabescens polysaccharides（ATPS）.Methods:Twelve ARRB1 gene knockout mice(ARRB1^-/-)and twelve wild-type C57BL/6J mice were randomly divided into control group（Control）,model group（Model）,and Armillariella tabescens polysaccharides（ATPS）group（200 mg/kg）.The model of CIMI was established by intraperitoneal injection of 5-fluorouracil（5-FU）at 50 mg/kg for 7consecutive days.The mental status,food intake,diarrhea,and activity of the mice were observed during modeling and treatment,and changes in body weight were compared among the groups.Macroscopic observation of the small intestine tissue was performed,and HE staining was used to evaluate the pathological damage.Special kits were used to detect hematological parameters,including Superoxide Dismutase（SOD）,FITC-dextran,endotoxin,and Diamine oxidase（DAO）.Immunohistochemistry was performed to detect the expression of tight junction protein markers,including zonula occludens proteins（ZO-1）,Occludin,Claudin-1,and proliferation-related protein Ki-67.Real-time PCR was used to detect the expression of Lgr5 m RNA,and small intestine crypt isolation and organoid culture were used to detect the formation and growth of intestinal organoids.Results:The general condition of the wild-type and ARRB1^-/-control mice performed well,while the model group showed a significant decrease in body weight.The weight of the wild-type mice recovered after ATPS treatment,but the body weight of the ARRB1^-/-mice continued to decline after ATPS treatment.Macroscopic observation of the small intestine showed a smooth surface and pink color in the control group,while the model group showed obvious inflammatory reactions,with dark red congestion,edema,and ulceration,compared to the control group.The WT-ATPS group showed reduced congestion and edema compared to the model group,while the ARRB1^-/--ATPS group still showed severe congestion and edema.HE staining of the jejunum tissue showed that the crypts and glands in the control group were intact and well arranged,with a small amount of scattered inflammatory cells present.In the model group,the structure was severely damaged,with shortened villi,deepened crypts,disordered gland arrangement,and obvious inflammatory cell infiltration,indicating a successful construction of the chemotherapy-induced intestinal mucositis model.Compared with the model group,the structure of the WT-ATPS group was clear,and the pathological damage was partially restored.However,the ARRB1^-/--ATPS group did not show similar histological recovery compared to the WT-ATPS group.The results of the hematological experiments showed that compared with the control group,5-FU decreased the activity of SOD,increased the levels of serum FITC-dextran,endotoxin,and DAO,and decreased intestinal permeability.ATPS increased the activity of SOD and intestinal permeability but failed to increase the activity of SOD in ARRBI^-/-mice nor decrease the intestinal permeability in ARRBI^-/-mice.The immunohistochemistry results showed that compared with the control group,the expression levels of tight junction（TJ）proteins（Claudin-1,Occludin,and ZO-1）and proliferation-related protein Ki-67 in the model group were decreased to a certain extent.Compared with the model group,the expression levels of TJ proteins and Ki-67 protein were significantly increased in the WT-ATPS group.However,compared with the WT-ATPS group,the expression levels of these proteins were lower in the ARRB1^-/--ATPS group,and the expression levels of TJ proteins Claudin-1 and ZO-1 were significantly reduced.The results of the organoid cultures show that compared with the control group,the formation rate of intestinal spheroids and organoids in the model group decreased,indicating that the evolution and growth of organoids in the intestinal tissue damage caused by chemotherapy were impaired.The formation rate of spherical structures and organoids in the WT-ATPS group was significantly increased,indicating recovery.The formation rate of spherical structures and organoids in the ARRB1^-/--ATPS group was lower than that in the WT-ATPS group,and the number of buds was less.Conclusion:1.ATPS plays a protective role in the mucosa by reducing intestinal permeability,protecting intestinal barrier integrity,and maintaining stem cell properties in 5-FU-induced intestinal mucositis.2.The important role of ATPS in the prevention and treatment of chemotherapy-induced intestinal mucositis may depend on ARRB1.