The Effects Of TGF-β1/PI3K/Akt Pathway On Diabetic Nephropathy

Diabetic nephropathy is one of the most common and severe diabetic complications,and it is characterized by glomerular basement membrane thickening and fibrosis of the kidney.Hyperglycemia is the key initiating factor in the development of DN,and it generates a cascade of events in the kidney,which eventually results in chronic renal failure.Oxidative stress and the overproduction of ROS in DN are the direct consequences of hyperglycemia,promoting the pathological process of DN in several different ways.Transforming growth factor-β1（TGF-β1）is thought to be the core factor in many fibrotic disorders,including DN.Recent experimental evidence suggests that TGF-β1 participates in the activation of the phosphoinositide-3-kinase（PI3K）/Akt pathway.PI3K plays a crucial role in cell growth and cell survival.PI3K phosphorylates and activates Akt.Activated Akt can phosphorylate and inactivate FoxO3a protein,leading to reduction of manganese superoxide dismutase（MnSOD）.MnSOD scavenges superoxide radicals produced within the organelle and can protect mitochondria from the harmful effects of ROS.Altered function or expression of MnSOD can have remarkable consequences on mitochondrial function and the cells due to oxidative damage,leading to the development of different diseases.Although it has been well established that excessive generation of ROS is a critical upstream factor in DN,the interrelationships between glucose-induced ROS production,the TGF-β1/PI3K/Akt/FoxO3a pathway and downstream MnSOD have not been fully demonstrated in vitro or in vivo.Tubulointersitial fibrosis is thought to be the final common pathway in most cases of end-stage renal disease.Renal tubular epithelial-mesenchymal transition（EMT）is an important pathogenesis of tubulointersitial fibrosis.A variety of studies have implicated TGF-β1 as an inducer or essential mediator of EMT in fibrosis.TGF-β1 engages type II and type I receptors at the plasma membrane,triggering phosphorylation of Smad2/3,which then form dimers with Smad4 that accumulate in the nucleus,driving transcriptional events that mediate the TGF-β1 response.TGF-β1also activates non-Smad-dependent signaling events that may contribute to fibrosis.Recently,among of non-Smad signaling,activation of the PI3K/Akt pathway is emerging as a central feature of EMT.However,the mechanism of TGF-β1-mediated PI3K/Akt activation and the promotion of EMT are not completely clear.The mammalian target of rapamycin（mTOR）is an important downstream protein of PI3K/Akt pathway,which plays a pivotal role in mediating cell size and mass,proliferation,and survival.Whether mTOR could promote EMT under high glucose conditions is also unknown.If the activated TGF-β1/PI3K/Akt signaling pathway promotes the process of EMT via the important element mTOR,then inhabitation of mTOR expression and activity to suppress EMT can be recognized as a novel train of thought to develop medicines against diabetic nephropahy.Ginkgo biloba extract（EGb）,extracted from Ginkgo biloba leaves,is a defined,complex mixture containing Ginkgo flavone glycoside and terpene lactones.Evidence accumulated shows that EGb has a number of benefits,including ameliorating hemodynamics,suppressing the platelet-activating factor,scavenging ROS,and relaxing vascular smooth muscles.Furthermore,EGb was found to inhibit TGF-β1-induced EMT.However,the interrelationships between the effect of EGb on EMT,the TGF-β1/PI3K/Akt pathway and downstream mTOR have not been fully demonstrated.To explore the effect of TGF-β1/PI3K/Akt pathway in diabetic nephropathy,we investigated the mechanism of ROS overproduction via TGF-β1/PI3K/Akt/FoxO3a pathway in vitro and in vivo,and the role of TGF-β1/PI3K/Akt in tubular EMT.Furthermore,we discussed the effects of EGb on TGF-β1/PI3K/Akt pathway.These results will provide new therapeutic targets for the novel DN drugs,and theoretical and experimental base for the clinical therapy.PartⅠ The Effects of TGF-β1/PI3K/Akt Pathway on Oxidative Stress in Rat Mesangial Cell Cultured in High GlucoseObjective To investigate the effects of TGF-β1/PI3K/Akt pathway on ROS and the potential mechanism on rat mesangial cell（MC）cultured in high glucose.Methods The line HBZY-1 of rat MC was cultivated in DMEM media with normal glucose(final concentration of 5.56 mmol·L^-1 glucose).The cultured cells were divided as follows:normal standard group(NS group,5.56 mmol·L^-1 glucose),manniol group(MA group,5.56 mmol·L^-1 glucose+24.44 mmol·L^-1 mannitol),high glucose group(HG group,30 mmol·L^-1 glucose),solvent control group(DMSO group,30 mmol·L^-1 glucose+0.1%DMSO),hydrogen peroxide group(H₂O₂ group,10^-5 mmol·L^-1 H₂O₂+5.56 mmol·L^-1 glucose),transforming Growth Factor-beta1inhibitor group(SB 431542 group,30 mmol·L^-1 glucose+10μmol·L^-1 SB 431542),PI3K inhibitor group(LY294002 group,30 mmol·L^-1 glucose+20μmol·L^-1LY-294,002),antioxidant group(NAC group,30 mmol·L^-1 glucose+50μmol·L^-1N-acetyl-L-cysteine).24 h after cultivation,the following data were detected:the level of reactive oxygen species（ROS）of MCs by flow cytometry;the amount of TGF-β1 secreted by the cells through ELISA;the expression of Akt,p-Akt（Ser473）,FoxO3a,p-FoxO3a,MnSOD by western blotting;the relative mRNA amounts of TGF-β1,MnSOD by RT-PCR;and the distribution of FoxO3a in MC by immunofluorescence microscopy.Results Compared with the NS group,intracellular ROS production,the TGF-β1 mRNA and protein levels,the p-Akt/T-Akt and p-FoxO3a/T-FoxO3a proteins ratio were increased in the high glucose group,while the MnSOD mRNA and protein were significantly decreased.In the NS group,FoxO3a was predominantly detected in the nucleus.In contrast,high glucose strongly promoted FoxO3a localization to the cytoplasm.Compared with the HG group,the p-Akt/T-Akt and p-FoxO3a/T-FoxO3a protein ratio were significantly decreased,the MnSOD mRNA and protein were increased in the SB431542 group,LY294002 group,and the NAC group.Furthermore,after treatment with the TGF-β1 inhibitor or the PI3K inhibitor,FoxO3a was retained in the nucleus,and intracellular ROS production were significantly decreased.Conclusions High glucose concentrations induced excessive ROS production,which stimulated the PI3K/Akt/Fox O3a signaling pathway via the up-regulation of TGF-β1,resulting in the phosphorylation and the inactivation of FoxO3a and the reduction in the expression of MnSOD,finally leads to further excessive ROS production.Part Ⅱ The Effects of TGF-β1/PI3K/Akt Pathway on Oxidative Stress in db/db miceObjective To investigate the effects of TGF-β1/PI3K/Akt pathway on reactive oxygen species in db/db mice.Methods The db/db mice were randomly divided into 3 groups（8 mice/group）,which received intraperitoneal injections of vehicle（8%DMSO）,SB431542(6.3μg·g^-1 dissolved in 8%DMSO),or LY294002(3.0μg·g^-1 dissolved in 8%DMSO),respectively.The db/m mice also received intraperitoneal injection of 8%DMSO（NS group,n=8）.The four groups of mice were treated for 7 days,and blood glucose was measured by glucose oxidase method;creatinine（Cr）and blood urea nitrogen（BUN）were measured by chemical colorimetry;microalbuminuria was determined by ELISA;the protein of relative ratio of p-Akt,p-FoxO3a and MnSOD was detected western blot;the distribution of p-FoxO3a in the kidney cortical tissues was determined by immunofluorescence microscopy,MDA levels in the kidney and serum were determined by high performance liquid chromatograph（HPLC）.Results Compared with the NS group,the blood glucose level,Cr,BUN,microalbuminuria and kidney weight in the DN group were significantly increased,the phosphorylation of Akt and FoxO3a proteins,the MDA level were significantly increased,while the expression of MnSOD protein in the DN group was significantly decreased.Furthermore,p-FoxO3a immunofluorescence staining was markedly increased in the kidney cortical tissues of DN group.Compared with the DN group,the inhibitor of TGF-β1（SB431542）reduced BUN and Cr in the DN mice,and the inhibitor of PI3K（LY294002）reduced BUN.The phosphorylation of Akt and FoxO3a protein,the level of MDA in the SB431542 group and LY294002 group were significantly decreased,and the MnSOD protein was significantly increased.Moreover,p-FoxO3a immunofluorescence staining was markedly decreased.Conclusions Hyperglycemia induced oxidative stress,which stimulated the TGF-β1/PI3K/Akt/FoxO3a signaling pathway,resulting in the phosphorylation and the inactivation of FoxO3a,and the reduction in the expression of MnSOD.The reduction of MnSOD expression increased the end products of lipid peroxidation（MDA）,which aggravated the oxidative stress injury.Part Ⅲ The Effects of TGF-β1/PI3K/Akt Pathway in Tubular epithelial-mesenchymal transition under high glucose conditionsObjective To investigate the effect of high glucose-induced ROS on the activation of TGF-β1/PI3K/Akt pathway in tubular epithelial-mesenchymal transition（EMT）and its potential mechanism.Methods A normal rat kidney tubular epithelial cell line（NRK-52E）was cultured in vitro for the present research.The cultured cells were divided as follows:normal glucose group(NG group,5.56 mmol·L^-1 glucose),high glucose group(HG group,60 mmol·L^-1 glucose),hydrogen peroxide group(H₂O₂ group,10^-5 mmol·L^-1H₂O₂+5.56 mmol·L^-1 glucose),transforming growth factor-β1 group(TGF-β1 group,10 ng·ml^-1 TGF-β1+5.56 mmol·L^-1 glucose).transforming growth factor-β1inhibitor group(SB431542 group,60 mmol·L^-1 glucose+10μmol·L^-1 SB431542),antioxidant group(NAC group,60 mmol·L^-1 glucose+50μmol·L^-1N-acetyl-L-cysteine),PI3K inhibitor group(LY294002 group,60 mmol·L^-1 glucose+20μmol·L^-1 LY294002),mTOR inhibitor group(Rapamycin group,60 mmol·L^-1glucose+20 nmol·L^-1 Rapamycin).48 h after cultivation,the following data were detected:the morphological changes of NRK-52E cells observed under a inverted microscope;the level of reactive oxygen species（ROS）of NRK-52E cells by flow cytometry;the amount of TGF-β1 secreted by the cells through ELISA;the expression of Akt（protein kinase B,Akt/PKB）,p-Akt（Ser473）,mTOR,p-mTOR（Ser2448）,E-cadherin andα-SMA by western blotting;and the relative mRNA amounts of TGF-β1,E-cadherin andα-SMA by real time RT-PCR.Results Compared with the NG group,the cells of the HG group changed from being fibroblast-like shaped to being spindle-shaped,intracellular ROS production,the TGF-β1 mRNA and secretion levels,the p-Akt/T-Akt and p-mTOR/T-mTOR proteins ratio,the mRNA and protein levels ofα-SMA were increased in the high glucose group,while the mRNA and protein levels of E-cadherin were significantly decreased.Compared with the HG group,the cells in the NAC,SB-431542,LY-294002 and rapamycin groups presented a cobblestone-like appearance,the p-Akt/T-Akt and p-mTOR/T-mTOR protein ratio,the mRNA and protein levels ofα-SMA were significantly decreased,the mRNA and protein levels of E-cadherin were increased in the SB431542 group,LY294002 group,and the NAC group.Conclusions Under high glucose conditions,large amounts of ROS was generated in NRK-52E cells followed by the up-regulated expression of TGF-β1,which activated the PI3K/Akt signaling pathway and ultimately promoted the phosphorylation of mTOR.The activated mTOR could promote tubular EMT and aggravate the development of interstitial fibrosis.Part Ⅳ Preventative Effects of Ginkgo Biloba extract on Tubular epithelial-myofibroblast Transition through TGF-β1/PI3K/Akt/mTOR PathwayObjective To observe the preventative effects of Ginkgo biloba extract（EGb）on TGF-β1/PI3K/Akt pathway and mTOR,and clarify the mechanism of EGb on the prevention and treatment of renal tubular epithelial-mesenchymal transition.Methods The cultured NRK-52E cells were divided into normal glucose group(NG group,5.56 mmol·L^-1 glucose),high glucose group(HG group,60 mmol·L^-1glucose),low dose of EGb group(GL group,10μg·ml^-1 EGb),moderate dose of EGb group(GM group,20μg·ml^-1 EGb),high dose of EGb group(GH group,40μg·ml^-1EGb),and mTOR inhibitor group(Rapamycin group,20 nmol·L^-1 Rapamycin).After the cells were given different factors for 48h,Flow cytometry was used to detect the level of reactive oxygen species（ROS）of NRK-52E.Enzyme linked immunosorbent assay（ELISA）was applied to determine the protein concentration of TGF-β1 in culture medium.Western blot was used to examine the protein of relative ratio of Akt（protein kinase B,Akt/PKB）,p-Akt,mTOR,p-mTOR,E-cadherin andα-SMA.Real time RT-PCR was used to detect TGF-β1 mRNA,E-cadherin mRNA andα-SMA mRNA of NRK-52E.Results Compared with the HG group,ROS production,the TGF-β1 mRNA and secretion levels,p-Akt/T-Akt and p-mTOR/T-mTOR protein ratio,and the expression ofα-SMA mRNA and protein of GM and GH group were significantly decreased,the levels of E-cadherin mRNA and protein were markedly increased,while ROS production,the TGF-β1 m RNA and secretion levels,p-Akt/T-Akt protein ratio of Rapamycin group were no change.Conclusions In NRK-52E cells cultured with high glucose,EGb could reduce ROS production,inhibition of the activation of TGF-β1/PI3K/Akt signaling pathway,reduction mTOR phosphorylation,increase the expression of E-cadherin,reductionα-SMA expression,which prevetive high glucose-induced renal tubular epithelial-mesenchymal transition.