Effect Of Intestinal Trimethylamine-N-oxide On Diabetic Nephropathy Via MAPK/NF-κB Signaling Pathway

Objective To investigate the effects of TMAO(trimethylamine-N-oxide)on renal function and fibrosis in diabetic nephropathy,and further study the changes of intestinal flora during the course of diabetic nephropathy.The fecal microbiota transplantation to prove the function of TMAO production in rats with diabetic nephropathy.To evaluate the proinflammatory effect of TMAO on HMC and HK-2,and further investigate the effect of TMAO on MAPK/NF-κB signaling pathway.Methods(1)A rat model of diabetic nephropathy was established by Zucker Diabetic Fatty(ZDF)rat feeding Purina #5008 feed and randomly divided into 4groups: control group(C1 group: ZDF normal rat group)and diabetic nephropathy model group(E1 group: ZDF rat diabetes model group),inhibitor group(C2 group:ZDF normal rat + DMB),diabetic nephropathy inhibition group(E2 group: ZDF rat diabetes model + DMB),3,3-dimethyl-3-butanol(DMB)is a structurally similar substance of choline that reduces the production of TMAO.The blood,feces,kidneys were taken for research when modeling is successful.(2)The blood samples were tested for renal function,and the serum TMAO content was detected by high performance liquid chromatography-tandem mass spectrometry.HE staining and Masson staining were used to observe the changes of renal tissue pathology and renal fibrosis in rats,and the fibrosis related protein P-samd3 was detected by Western blotting.(3)The feces of diabetic and diabetic nephropathy rats were detected by16 SrRNA sequencing and the function of the flora was studied by fecal microbiota transplantation.(4)HMC and HK-2 cell were treated with TMAO and NF-κB inhibitors.Cell viability was measured by CCK-8 method.inflammatory response factors IL-1,IL-6,TNF-α and MCP-1 were detected by real-time fluorescent quantitative PCR,and the expression of p65 protein,P-p65,nuclear and cytoplasmic P65,ERK and P-ERK protein were detected by Western blotting.Results(1)At 8 weeks of modeling,the blood glucose of the model group(E1,E2group)exceeded 16.7 mmol/L,and the renal function index was significantly higher than that of the control group(C1,C2 group)(P<0.05),HE staining showed significant damage to the kidney tissue structure,these indicated the diabetic nephropathy model was established successfully.(2)Renal HE staining in the ZDF diabetic nephropathy model group showed significant glomerular injury and increased renal function(P<0.05)compared with the diabetic nephropathy-suppressed group,Masson showed significant fibrosis in the kidney and the expression level of P-smad3 is increased(P<0.05),serum TMAO levels were increased in the non-inhibitory group(E1,C1 group)compared with the inhibition group(E2,C2 group)(P<0.05).(3)The sequencing results of intestinal flora showed that the diversity of intestinal flora in the process of diabetic nephropathy changed,and the area under the ROC curve was 0.83 when the intestinal flora was used as a biomarker to distinguish the diabetic nephropathy model group from the control group.The results of fecal microbiota transplantation.showed that the diabetic nephropathy model group had stronger TMAO ability than the control group(P<0.05).(4)The expression of inflammatory factors IL-1,IL-6,TNF-α and MCP-1 in HMC and HK-2 cells was significantly increased after TMAO treatment(P<0.05);Western blot results showed that the expression of P-p65 in NF-κB pathway and P-ERK in MAPK pathway was significantly increased(P<0.05),and the expression of p65 protein in nucleus was significantly increased after TMAO treatment(P<0.05).The expression level of inflammatory factor in the group of TMAO treated with NF-κB signaling pathway inhibitor was significantly lower than that treated with TMAO alone(P<0.05).Conclusions(1)In the process of diabetic nephropathy,the structure of intestinal flora will change significantly,and the ability of the flora to produce TMAO will increase,which further increase the serum TMAO,causing the degree of kidney damage and fibrosis.(2)TMAO can activate the MAPK/NF-κB signaling pathway to further produce inflammatory cytokines.