To Study The Effect And Mechanism Of Dietary Fiber Inulin On Diabetic Kidney Injury Based On Metabonomics

Background:Diabetic nephropathy(DN)accounts for almost one third of advanced nephropathy,and its increasing incidence is attracting widespread attention in recent years.Hyperglycemia was once thought to be a major driver of diabetic complications,but subsequent studies have shown that advanced generation end products(AGEs)are more critical triggers of chronic inflammatory responses.Short-chain fatty acids(SCFAs)are monocarboxylic acids with 1-6 carbon chain length which are abundant in the gut,including acetate(Na Ac),propionate(Na Pc)and butyrate(Na B).SCFAs are beneficial regulators of the “ gut-renal axis ”.Based on methodologies technology,this thesis studied the mechanism by which soluble dietary fiber inulin alleviates systemic inflammatory response and renal injury in diabetic patients through the "enteric-renal axis",and further explored the mechanism of three important SCFAs alleviating AGEs induced inflammatory response,providing a new idea for the prevention and treatment of DN.Methods:1.Serum samples from 30 diabetic patients,30 diabetic patients with nephropathy and 30 normal subjects were collected,serum levels of AGEs,IL-1β and TNF-α by ELISA,urine microalbumin,blood creatinine and blood urea nitrogen were detected by automatic biochemical analyzer.and to analyze the correlation between AGEs and renal injury indicators,and the correlation between AGEs levels and inflammation.2.Diabetic mouse model was established,and the control group(NC group),diabetic group(T2DM group)and inulin group(INU group)were set up.The NC group received normal diet until the end of the experiment while the diabetic group and inulin group were given high-sugar and high-fat diet(HFD)for 4 weeks.Starting from the 5th week,Mice in both groups were intrabitoneally injected with 50 mg/kg STZ for 5 consecutive days to induce diabetes.The fasting blood glucose(FBG)of mice was monitored with glucose meter,the model was successfully established when FBG≥11.1 mmol/L was determined for 3 consecutive days.With continued HFD feeding for 6 weeks,the INU group received inulin by gavage at 500 mg/kg daily.After the experiment,mice were chloral hydrate anesthetized and sacrificed,and serum and renal cortex tissues were collected.The FBG levels were monitored weekly with a blood glucose meter.The levels of BUN,SCr,TC,TG,and LDL-C were determined by automated biochemistry analyzer;The m RNA levels of α-SMA,NLRP3,IL-1β and IL-10 in kidney were detected by q RT-PCR.The renal changes were observed by HE,PAS and Masson staining.The distribution and levels of α-SMA and RAGE in kidney were identified by immunohistochemistry(IHC);LC-MS/MS was used to detected serum SCFAs;UPLC-QTof-MS/MS was used to detected the amount of metabolites in serum.3.THP-1 cells were pretreated with 100 μmol/L sodium acetate,sodium propionate,and sodium butyrate and stimulated at 400 μmol/L AGEs for 24 h.The m RNA expression of IL-1β,TNF-α,IL-10,NLRP 3 and Caspase-1 was measured by q RT-PCR;the expression of NLRP3 inflammatory complex,the activation of NF-κ B signaling pathway and cellular autophagy;ROS were detected by flow cytometry;and cell metabolome were detected by UPLC-QTof-M S/MS technology.4.HK-2 cells were pretreated with low,medium and high concentrations of sodium butyrate and stimulated for 6 h by AGEs.The m RNA expression of ZO-1,Occludin,E-cadherin and α-SMA was detected by q RT-PCR.The protein expression of PI3 K,Akt,Beclin-1,LC3 B and α-SMA were detected by Western blotting.Results:1.Compared with diabetic patients alone,serum AGEs and inflammation were significantly increased in patients with diabetic nephropathy(P < 0.05),and serum AGEs were significantly associated with renal injury indicators and serum inflammation levels.2.The levels of TC,TG,LDL-C,SCr,BUN,FBG and AGEs in T2 DM group were obviously increased(P < 0.05).The levels of acetic acid,propionate,and butyric acid were significantly reduced(P < 0.05).In mice,the volume of glomerulus increased,mesangial matrix increased significantly,renal tubular epithelial cells showed vacuolation,some cells shed to form tubular.In kidney tissues,the expressions of renal inflammatory cytokines(NLRP3,IL-1β)andα-SMA in T2 DM were also increased(P < 0.05),the m RNA expression level of antiinflammatory factor IL-10 was decreased(P < 0.05).After the inulin intervention,Serum lipid and SCr levels in INU group were significantly improved(P < 0.05).The levels of serum AGEs were decreased and inulin increased the expression of acetic acid,propionic acid and butyric acid(P < 0.05).In addition,the epithelial cell shedding of renal tubules was decreased,and the tubular shape of renal tubules was decreased.At the same time,the m RNA expression of NLRP3,IL-1β and α-SMA were obviously inhibited(P < 0.05),and IL-10 content increased(P < 0.05).IHC showed that inulin could also improve the levels of α-SMA and RAGE in the kidney of mice.The results of UPLC-QTof-MS/MS showed that inulin could improve the arachidonic acid metabolism,glycerophospholipid metabolism,vitamin B6 synthesis and retinol metabolism in diabetic mice.3.The results of THP-1 cell experiment showed that ROS expression in THP-1 cells was significantly increased after AGEs treatment(P < 0.05).The m RNA levels of IL-1β,TNF-α,NLRP3 and Caspase-1 were enhanced,and AGEs treatment also inhibited the levels of IL-10(P< 0.05).Meanwhile,AGEs induced increased the protein level of NLRP3 and Caspase-1 and increased NF-κB pp65 and p-iκBα in THP-1 cells(P < 0.05).At the same time,AGEs induced increased levels of LC3 B and Beclin-1(P < 0.05).After the intervention with SCFAs,the m RNA expressions of IL-1β and TNF-α in AGEs+Na Pc and AGEs+Na B groups were significantly decreased(P < 0.05),the m RNA and protein levels of NLRP3 and Caspase-1 were obviously inhibited by three SCFAs(P < 0.05).At the same time,the m RNA expression of IL-10 was significantly increased(P < 0.05).All three SCFAs could inhibit ROS expression(P < 0.05).In addition,three SCFAs inhibited the levels of NF-κB pp65 and p-iκBα(P < 0.05),and the levels of LC3 B and Beclin-1 in AGEs+Na Pc and AGEs+Na B group were significantly decreased(P <0.05).The metabolomics results showed that the three SCFAs differently improved linoleic acid metabolism,glycerophospholipid metabolism,and D-glutamine and D-glutamate metabolism in THP-1 cells.4.The results of HK-2 cell experiment showed that after intervention with AGEs,the levels of Occludin,ZO-1 and E-cadherin showed a decreasing trend,while α-SMA increased(P < 0.05).In addition,AGEs significantly increased the expression of p-PI3 K and p-Akt and inhibited the expression of LC3 B and Beclin-1(P < 0.05),the autophagy was inhibited.After incubation with high concentration of Na B,Na B promoted the rise of Occludin,ZO-1 and E-cadherin levels,and also inhibited m RNA expression of α-SMA(P < 0.05).In addition,Na B also inhibited the activation of PI3K/Akt signaling pathway and promoted autophagy of HK-2 cells.Conclusions:Our results showed that serum AGEs levels were significantly associated with kidney damage and inflammation levels in DN patients.Animal experiments showed that inulin can increase serum SCFAs concentration in diabetic mice,reduce renal inflammation level and serum AGEs level,and alleviate renal injury,which may play a role through arachidonic acid metabolism,glycerophospholipid metabolism,vitamin B6 metabolism and retinol metabolism,Further in vivo studies showed that three major SCFAs could inhibit AGEs-induced macrophage inflammatory response,and sodium butyrate could induce autophagy to protect HK-2 cells through PI3K/Akt signaling pathway and improve renal fibrosis.