Effects Of Folic Acid On Intestinal And Renal Function Injury In Hyperuricemia Mice

ObjectiveTo investigate the improvement effect and mechanism of Folic acid(FA)on intestinal renal function injury in Hyperuricemia(HUA)mice.Method1.Animal grouping and intervention methods: Sixty 7-week-old healthy male C57 BL/6 J mice were randomly divided into the following six groups: a control group(CON),a model group(MOD),a low-dose FA group(L-FA),a high-dose FA group(H-FA),folic acid control group(FAC)and positive drug control group(PC),10 mice per each group;The mice in each group were free to eat and drink during the experiment.Mice in CON group and FAC group were given 0.5% sodium carboxymethyl cellulose solution by gavage daily.Mice in MOD group,L-FA group,H-FA group and PC group were given suspension of purine-rich substance(containing 1 g/kg yeast extract powder and 100 mg/kg adenine)and uricase inhibitor(containing 250 mg/kg potassium oxonate)prepared by 0.5% sodium carboxymethyl cellulose solution.After 2 h,the mice in each group were continued to receive normal saline(CON,MOD),2.5 mg/kg folic acid(L-FA),5 mg/kg folic acid(H-FA,FAC)and 25 mg/kg allopurinol(PC),respectively.After the experiment lasted for 10 weeks,mice were anesthetized with pentobarbital sodium,and blood,kidney tissue,intestinal tissue and contents were collected for further analysis.2.The levels of serum uric acid,serum creatinine(Scr),blood urea nitrogen(BUN)was detected by automatic biochemical analyzer;Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6)in colon tissue and lipopolysaccharide(LPS)in kidney tissue.3.Hematoxylin-Eosin(HE)staining was used to observe the histopathological changes of small intestine and kidney.4.Immunofluorescence was used to detect the expression of α-SMA,E-cadherin and the distributions of NF-κB p65 in kidney cells.4.The protein expression levels of Toll-like receptor 4(TLR4),Myeloid differentiation protein 88(My D88),phosphorylated inhibitory Kappa B(p-IκB),uric acid transporter ABCG2,GLUT9 and URAT1 in kidney and ZO-1,Claudin-1 and Occludin in intestinal tissue were detected by Western blot(WB).5.16 S rDNA gene sequencing was used to analyze the differences of intestinal flora in the intestinal contents of mice.The contents and variations of Short chain fatty acids(SCFAs)in mouse intestinal contents were analyzed by gas chromatography-mass spectrometry(GC-MS).Results1.Body weight and serum biochemical indexes: The body weight of MOD group was significantly lower than that of CON group from week 2(P<0.05),while high-dose folic acid intervention could improve the weight loss of HUA mice.Results of serum biochemical indices showed that serum uric acid,Scr and BUN levels in MOD group were significantly higher than those in CON group(P<0.05),and serum uric acid,Scr and BUN levels in MOD group were significantly lower after folic acid intervention than those in MOD group(P<0.05).2.Related factors of renal injury indicators: Folic acid intervention significantly improved glomerular morphology and epithelial necrotizing lesions.Compared with CON group,the expression level of α-SMA protein in the kidney of MOD group was significantly increased(P<0.05),and the expression level of E-Cad protein was significantly decreased(P<0.05),and high dose of folic acid supplementation can contribute to normalize the expression of these proteins.In addition,compared with CON group,ABCG2 protein expression level in MOD group was significantly decreased(P<0.05),and GLUT9 and URAT1 protein expression levels were significantly increased(P<0.05).High dose of folic acid supplementation could significantly reverse the expression levels of ABCG2 and GLUT9(P<0.05).There was no significant difference in the expression of URAT1 protein(P>0.05).3.Intestinal mucosal barrier function index and 16 S rDNA sequencing results: Folic acid intervention significantly improved the morphology and arrangement of intestinal mucosal barrier cells in HUA mice,and the ratio of intestinal villus height and villus height to crypt depth in H-FA group was significantly higher than that in MOD group(P<0.05).Compared with CON group,the levels of IL-1β,IL-6 and TNF-α in intestinal tissue of mice in MOD group were significantly increased(P<0.05).Folic acid and allopurinol intervention could significantly reduce the levels of proinflammatory factors in HUA mice(P<0.05).WB results showed that the protein expression levels of ZO-1,Claudin-1 and Occludin in intestinal tissues of L-FA and H-FA groups were significantly higher than those of MOD group(P<0.05).The results of 16 S r DNA sequencing showed that the diversity and structural composition of intestinal microbiota of mice in MOD group were significantly changed compared with those in CON and folic acid intervention groups.The levels of total SCFAs,acetic acid and propionic acid in colon of HUA mice were significantly lower than those in CON group and high-dose folic acid treatment group(P<0.05).4.TLR4/NF-κB signaling pathway: The levels of LPS,TLR4,My D88 and p-IκB in the kidney tissue of MOD group were significantly higher than those of CON group(P<0.05),immunofluorescence results showed that the NF-κB p65 protein level in nucleus of MOD group was increased.However,high-dose folic acid intervention could significantly improve the expression levels of LPS,TLR4,My D88 and p-IκB protein in the kidney of HUA mice,and restore the quantitative balance of NF-κB p65 between cytoplasm and nucleus.ConclusionBy regulating intestinal flora and mucosal barrier function,folic acid intervention can alleviate the cascade of renal inflammation caused by circulating endotoxin and improve intestinalrenal dysfunction in hyperuricemia mice.