Improvement Of Kidney Podocytes Injury And Hepatic Lipid Accumulation By Polyphenols And The Underlying Molecular Mechanisms In Metabolic Syndrome

Fructose corn syrup is mainly used as an industrial sweetener in all kind of foods with the rapid development of the society,thus its intake has increased in the world.A large number of researches have demonstrated that excess fructose consumption is closely related with the development of metabolic syndrome(MS)in humans and rodents.High fructose intake causes insulin resistance,oxidative stress and inflammatory response,involving the pathological development of renal podocyte injury and non-alcoholic fatty liver disease(NAFLD).Therefore,it is of great important significance in theory and practice to investigate the complex relationship of insulin resistance,oxidative stress and inflammatory response,and the new pathological mechanisms,as well as to find new drug trgets in high fructose-induced metabolic diseases.Polyphenolic compounds in traditional Chinese medicine have attracted much attention for they have widely biological properties.More studies have showed that curcumin,polydatin,pterostilbene and quercetin,with anti-oxidative and anti-inflammatory activities,can improve metabolic diseases such as diabetes,cardiovascular disease,chronic kidney disease and NAFLD.Our previous studies have demonstrated that these polyphenols ameliorated metabolic syndrome in animals.Based on MS characteristic pathology(such as insulin resistance,oxidative stress and inflammatory response),we mainly investigated the potential link between high fructose level and glomerular podocyte injury,and the underlying pathological mechanisms in animal and cell models,by using methods and technologies of pharmacology,biochemistry,cell biology and molecular biology.We also explored the new pathological way of high fructose and/or glucose-induced liver inflammation and lipid accumulation.Respectively,the protective effects of curcumin,polydatin,pterostilbene and quercetin on glomerular podocyte injury and/or NAFLD were investigated to find new targets and their possible molecular mechanisms in the prevention and treatment of metabolic diseases.The present study observed pathological changes of glomerular podocytes in fructose-induced MS in rats.Excessive fructose feeding induced the effacement of foot processes,and increased serum levels of uric acid and microalbuminuria,accompanying with the reduction of glomerular Podocin protein expression levels in rats.These results further confirmed that high fructose intake was associated with renal glomerular podocyte injury and dysfunction.Curcumin,polydatin and pterostilbene could ameliorate fructose-induced podocyte injury,high serum uric acid levels and microalbuminuria,as well as glomerular Podocin protein dysexpression in rats,providing protection against fructose-induced glomerular podocyte injury.To investigate the relationship of fructose-induced podocyte injury and miR-206 dysexpression with insulin resistance,and the mechanisms by which the polyphenols protected against glomerular podocyte injury,this study further confirmed hyperinsulinemia and insulin resistance in fructose-induced MS rats.Excessive fructose feeding was found to suppress glomerular miR-206 expression and up-regulate tyrosine phosphatase 1B(PTP1B)protein expression,as well as to impair insulin signaling pathway-insulin receptor substrate-1(IRS-1)/Akt/extracellular signal-regulated kinase 1/2(ERK1/2)in rats.These disturbance effects wereconfirmed in podocyte(HSMPs)model.5 mM Fructose stimulation down-regulated miR-206 expression levels in podocytes for 4 h and up-regulated lysate PTP1B protein levels in podocytes for 8 h.Next,transfection of podocytes with miR-206 mimic for 48 h diminished PTP1B expression,and abrogated the responses of podocytes to 5 mM fructose-induced over-expression of lysate PTP1B,indicating that podocytes miR-206 may mediate PTP1B expression in high fructose-exposed podocytes.Moreover,fructose and insulin stimulation significantly down-regulated lysate IRS 1,Akt and ERK1/2 phosphorylation in podocytes at different time points,indicating that high fructose may impair podocyte insulin sensitivity in vitro.Therefore,excessive fructose intake inhibited podocyte miR-206 expression,increased PTP1B expression and then altered IRS-1/Akt/ERKl/2 signal pathway,causing podocyte insulin sensitivity reduction and insulin resistance to induce podocyte injury and proteinuria in rats.Among of the polyphenolic components,curcumin was found to ameliorate fructose-induced podocyte injury and proteinuria by up-regulation of miR-206 expression levels,restoration of PTP1B expression.to improve IRS-1/Akt/ERKl/2 signal pathway and insulin sensitivity in vivo and in vitrro.To investigate the association of podocyte injury with oxidative stress and inflammatory response,and miR-377 dysexpression in fructose-induced MS in rats,the present study confirmed that excessive fructose ingestion could induce renal oxidative stress in rats,with elevation of malondialdehyde(MDA)and reactive oxygen species(ROS)levels,reduction of superoxidase dismutase(SOD)activity,and down-regulation of glomerular SOD1 and SOD2,as well as up-regulation of glomerular p38 MAPK phosphorylation and thioredoxin-interacting protein(TXNIP)levels.Simultaneously,renal inflammation and the elevation of inflammatory factors,such as NOD-like receptor pyrin domain containing 3(NLRP3),caspase-1 and inflammatory cytokines interleukin-1β(IL-1β)were detected in this animal model.Furthermore,high fructose intake was found to up-regulate renal miR-206 levels in rats.These observations were further confirmed in podocyte(HSMPs)models.5 mM Fructose stimulation for 4 h remarkably up-regulated miR-206 levels and down-regulated lysate SOD1 and SOD2 levels,as well as increased intracellular ROS and uric acid levels in podocytes.It significantly increased lysate p38 MAPK phosphorylation(48 h)and TXNIP protein levels(72 h)in podocytes.Co-incubation with fructose(5 mM)and p38 MAPK inhibitor SB203580 for 48 h blocked fructose-induced cell lysate TXNIP activation in podocytes.These results indicate that p38 MAPK activation may mediate over-expression of TXNIP in podocytes under high fructose condition.High fructose level also activated podocyte NLRP3 inflammasome(72 h)and increased culture supernatant and lysate IL-1β levels(96 h).Podocytes were transfected with siRNA against TXNIP for 48h,and then co-incubated with fructose.This TXNIP silencing abrogated the responses of podocytes to high fructose-induced over-expression of lysate caspase-1 and secretion of IL-1β,indicating that podocyte TXNIP over-expression may mediate activate the NLRP3 inflammasome in high fructose-exposed cells.Thus,up-regulation of podocyte miR-377 was involved in reduction of SOD1 and SOD2 protein levels and activity,which increased intracellular ROS levels and activated the ROS/p38/TXNIP/NLRP3 pathway to induce podocyte inflammation,leading to podocyte dysfunction and proteinuria in fructose-fed rats.Among of the polyphenolic components,polydatin and pterostilbene could significantly decrease kidney MDA,ROS and uric acid levels,and increase SOD activity in vivo and in vitro.Moreover,they suppressed glomerular/podocyte miR-377 levels to increase SOD1 and SOD2 expression involved in the reduction of oxidative stress and uric acid level,and then inhibited ROS/p38/TXNIP/NLRP3 pathway activation to improve fructose-induced podocyte inflammation and injury.To further investigate the mechanisms of NAFLD,and the intervention actions of the polyphenolic components,the present study further confirmed lipid metabolism disorders such as high serum and hepatic levels of total cholesterol(TC)and triglycerides(TG),high serum levels of LDL-cholesterol(LDLC)and low serum levels of HDL-cholesterol(HDLC),hyperinsulinemia,hyperleptinemia,insulin and leptin resistance,hepatic inflammation and steatosis,the hallmarks of the spectrum of NAFLD in fructose-induced MS rate.Moreover,excessive fructose intake was found to inhibit hepatic miR-203 expression,possibly resulting in up-regulation of suppressor of cytokine signaling 3(SOCS3)in rats.Simultaneously,over-expression of SOCS3 could impair leptin signaling signal transducer and activator of transcription 3(STAT3)/j anus-activated kinase-signal transducer 2(JAK2)pathway,and induce IRS1-Akt-ERK1/2 insulin signaling pathway disorder in the liver of fructose-fed rats,which subsequently altered lipid metabolism-related protein expression such as suppression of peroxisome proliferator-activated receptor α(PPARa)and up-regulation of sterol regulatory element binding protein lc(SREBP-1c).These abnormalities caused liver lipid metabolism disorder and lipid accumulation associated NAFLD in fructose-fed rats.Pterostilbene was found to increase hepatic miR-203 expression,decrease SOCS3 expression,and subsequently enhance insulin and leptin sensitivity to regulate PPARa and SREBP-lc in the liver of fructose-fed rats,showing its protection against fructose-induced lipid metabolism disorder and hepatic steatosis.On the other hand,the present study further investigated the improvement of quercetin on hepatic inflammation and lipid accumulation in liver cell models by incubating BRL-3A and HepG2 cells with high glucose(30 mM).High glucose stimulation significantly up-regulated TXNIP protein levels and activated the NLRP3 inflammasome in BRL-3A cells for 48 h and HepG2 cells for 24 h,which were significantly restored by the treatment of quercetin.TXNIP silencing abrogated the responses of BRL-3A and HepG2 cells to high glucose-induced NLRP3 inflammasome activation,indicating that down-regulation of hepatic TXNIP may be involved in the inhibition of hepatic NLRP3 inflammasome by quercetin under high glucose condition.Furthermore,siRNAi-TXNIP blocked high glucose-induced lysate PPARa down-regulation in BRL-3A and HepG2 cells.Co-treatment of quercetin was unable to alter lysate PPARα expression in TXNIP-siRNA transfecting hepatocyte cells with high glucose stimulation,indicating that improvement of quercetin on diabetic hepatic steatosis may be mediated by regulating hepatic TXNIP-PPARa pathway.Quercetin was found to effectively suppress hepatic SREBP-lc,SREBP-2,fatty acid synthase(FAS)and liver X receptor a(LXRa)expression levels in high glucose-exposed cells.It also inhibited over-expression of FAS and LXRa in caspase-1 inhibitor Ac-YVAD-CMK and high glucose co-incubated hepatocyte cells.These results suggest that regulation of TXNIP-NLRP3-SREBPs pathway by quercetin may partly mediate the improvement of diabetic hepatic steatosis.In conclusion,high fructose level inhibited podocyte miR-206 expression to up-regulate PTP1B expression,which disrupted podocyte insulin signaling IRS-1/Akt/ERKl/2 and reduced insulin sensitivity to induce insulin resistance.Moreover,it up-regulated podocyte miR-377 expression and inhibited SOD1 and SOD2 protein level and activity,activated the ROS/p38/TXNIP/NLRP3 pathway,causing podocyte oxidative stress and inflammtion response.These disturbance effects induced podocyte injury and proteinuria.Curcumin ameliorated fructose-induced podocyte injury and proteinuria by up-regulating podocytes miR-206 expression to decrease PTP1B expression,and improving IRS1-Akt-ERKl/2 pathway to enhance insulin sensitivity and reduce insulin resistance.Polydatin and pterostilbene down-regulated miR-377 expression,up-regulated SOD1 and SOD2 expression,suppressed ROS/p38/TXNIP/NLRP3 pathway activation,resulting in the improvement of podocytes inflammation and injury driven by fructose.These results suggest that curcumin,polydatin and pterostilbene have podocyte protection under high fructose condition.In addition,pterostilbene protected against fructose-induced hepatic steatosis possibly by elevation of miR-203 levels,inhibition of SOCS3 expression and subsequent improvement of insulin and leptin sensitivity to reduce lipid accumulation in the liver of rats.Quercetin inhibited liver cell TXNIP over-expression to suppress the NLRP3 inflammasome,and subsequently regulated lipid metabolism-related gene expression under high glucose condition,showing its improvement of diabetic hepatic steatosis and NAFLD.These results provide experimental and theoretical evidences for these polyphenolic components in the prevention and treatment of podocytes injury and liver lipid accumulation in metabolism diseases.