Based On STAT3 Signal To Explore The Prevention And Treatment Of IgAN By Rhein Regulating Intestinal Peyer’s Patch Immunity And Its Mechanism

Objective:In this study,the effects of rhein on signal transduction and transcriptional activator3（STAT3）signaling pathway and immune function of small intestinal mucosal pyle gathering lymph nodes（Peyer’s patch）in immunoglobulin A nephropathy（IgAN）model rats were studied to explore the effect and mechanism of rhein on IgAN prevention and treatment,providing new ideas and feasibility basis for clinical prevention and treatment of IgAN.Methods:Forty SPF male SD rats were randomly divided into normal control group,IgAN model group,rhein treatment group and rhein prevention group,with 10 rats in each group.IgAN model group and rhein treatment group were gavage of bovine serum albumin（BSA）on alternate days（400 mg/kg）during the first 6 weeks,rhein prevention group was gavage with rhein（100 mg/kg）+BSA（400 mg/kg）suspension alternate days during the first 6 weeks.At week 6 and week 8,lipopolysaccharide（LPS）was injected into caudal vein once（0.05mg/time）,and carbon tetrachloride（CCl₄）castor oil solution was injected subcutaneously once a week（0.6 m L/time）for consecutive 9 weeks.The normal control group was given an equal amount of normal saline instead of the drug.At week 7,rhein treatment group and rhein prevention group were intragaically injected with rhein（100 mg/kg）daily for 4 weeks.Normal control group and IgAN model group were given the same amount of normal saline.After the last intragastric administration,the count of urinary sediment red blood cells,24h urinary protein（24 h-UTP）,serum creatinine（SCr）and serum urea nitrogen（BUN）were measured.The contents of serum immunoglobulin A（IgA）and small intestinal mucous secretory IgA（s IgA）were determined by enzyme-linked immunosorbent assay（ELISA）.Pathological changes of kidney and Peyer’s patch were observed by hematoxylin-eosin（HE）staining,and Katafuchi score and stereology were performed.IgA deposition in mesangial region was observed by immunofluorescence（IF）.The expressions of interleukin-6（IL-6）,interleukin-17（IL-17）and transforming growth factor-β（TGF-β）in Peyer’s patch of intestinal mucosa were detected by immunohistochemistry（IHC）.Real-time fluorescence quantitative polymerase chain reaction（RT-q PCR）was used to measure the m RNA expression levels of STAT3 in the Peyer’s patch of intestinal mucosa and the orphan receptorγt（RORγt）in the downstream transcription factor retinoic acid receptor.Phosphorylated STAT3（p-STAT3）and RORγt in Peyer’s patch of intestinal mucosa were measured by Western blotting（WB）.Results:1.Effects of rhein on urinary sediment red blood cell count and 24 h-UTP in IgAN model rats:Compared with normal control group,urinary sediment red blood cell count and24 h-UTP in IgAN model group were significantly increased（P<0.01）;Compared with IgAN model group,urine erythrocyte count and 24 h-UTP in rhein treatment group and rhein prevention group were significantly decreased（P<0.01）.2.Effects of rhein on SCr and BUN of IgAN model rats:Compared with normal control group,SCr and BUN of IgAN model group were significantly increased（P<0.01）;Compared with IgAN model group,SCr and BUN of rhein treatment group were decreased（P<0.05）,and SCr and BUN of rhein prevention group were significantly decreased（P<0.01）.Compared with rhein treatment group,BUN of rhein prevention group was significantly decreased（P<0.01）.3.Effects of rhein on serum IgA and small intestinal mucous s IgA of IgAN model rats:Compared with normal control group,serum IgA and small intestinal mucous s IgA concentrations of IgAN model rats were significantly increased（P<0.01）.Compared with IgAN model group,serum IgA and small intestinal mucus s IgA concentrations in rhein treatment group and rhein prevention group were significantly decreased（P<0.01）.Compared with rhein treatment group,serum IgA concentration in rhein prevention group was significantly decreased（P<0.01）.4.Effects of rhein on renal histopathological changes in IgAN model rats:Compared with normal control group,the renal corbosomes in IgAN model group were enlarged,renal capsule dilated,mesangial hyperplasia and fibrosis were obvious,and renal tubule was swollen and blocked（P<0.01）.Compared with IgAN model group,renal corelle hypertrophy,mesangial hyperplasia fibrosis and renal tubule swelling were improved in rhein treatment group and rhein prevention group（P<0.01）.Compared with rhein treatment group,renal corelle hypertrophy,mesangial hyperplasia fibrosis and renal tubule swelling in rhein prevention group were significantly improved（P<0.01）.5.Effect of rhein on IgA deposition in kidney tissue of IgAN model rats:IgA fluorescence in kidney tissue of normal control rats was not obvious.Compared with normal control group,the renal cortex of IgAN model group showed bright green granular IgA deposition（P<0.01）.Compared with IgAN model group,the fluorescence intensity of renal cortex in rhein treatment group and rhein prevention group was decreased（P<0.01）.Compared with rhein treatment group,the fluorescence attenuation of renal cortex in rhein prevention group was more distinct（P<0.01）.6.Effects of rhein on pathological morphology of small intestinal mucosa of IgAN model rats:Compared with normal control group,germinal center and body density of IgAN model rats increased significantly（P<0.01）.Compared with IgAN model group,germinal center and body density in Peyer’s patch decreased significantly in rhein treatment group and rhein prevention group（P<0.01）.7.Effects of rhein on the expression of IL-17,IL-6 and TGF-βin intestinal mucosa of IgAN model rats:Compared with normal control group,the expressions of IL-17,IL-6 and TGF-βin intestinal mucosa of IgAN model rats were significantly increased（P<0.01）;Compared with IgAN model group,the expressions of IL-17,IL-6 and TGF-βin rhein treatment group and rhein prevention group were significantly decreased（P<0.01）.8.Effects of rhein on the expression of STAT3 m RNA and RORγt m RNA in intestinal mucosa of IgAN model rats:Compared with normal control group,the expressions of STAT3m RNA and RORγt m RNA in intestinal mucosa of IgAN model rats were significantly increased（P<0.01）.Compared with IgAN model group,the expressions of STAT3 m RNA and RORγt m RNA in rhein treatment group and rhein prevention group were significantly decreased（P<0.01）.9.Effects of rhein on the expression of P-STAT3 and RORγt proteins in intestinal mucosa of IgAN model rats:Compared with normal control group,the expression of P-STAT3 and RORγt proteins in intestinal mucosa of IgAN model rats were significantly increased（P<0.01）.Compared with IgAN model group,P-STAT3 and RORγt protein expressions were significantly decreased in rhein treatment group and rhein prevention group（P<0.01）.Compared with rhein treatment group,P-STAT3 and RORγt protein expressions in rhein prevention group were significantly decreased（P<0.01）.Conclusion:Rhein can improve the immune function of small intestinal mucosa in IgAN model rats,and has certain prevention and treatment effect on IgAN model rats,and the preventive effect is better than the therapeutic effect.The mechanism of action may be related to rhein inhibiting the expression of related factors in the STAT3 signaling pathway in the Peyer’s patch of IgAN model rats,and improving the balance of immune cytokines in the Peyer’s patch of small intestinal mucosa.