| BackgroundWith the development of global economy and the uplift of people’s living standard,we are facing more serious public health threat though we enjoy the convenience brought by the rich material life.In such a modernized and aging era, diabetes mellitus(DM) has risen in an astonishing speed.It has become the third serious non-infective chronic disease,the rates of its prevalence,morbidity and mortality are only behind cancer and CVD(Cardiac Vascular Disease).If the growth trend of DM could not be controlled,the global number of DM will have one to two times increase until2050. A variety of chronic complications due to the progression of DM including eye,kidney,heart and brain,will significantly increased at the same time.Then they will leading to unpredictable social and economic burden.Diabetic nephropathy(DN)is a specific and also the most common chronic microvascular complication of DM.at the same time,it is the main reason for end-stage renal disease(ESRD) abroad. According to statistics,about30%patients of type1diabetes and20%to50%patients of type2diabetes mellitus will occur DN,the5-year survival rate of progression to ESRD patients will be less than20%.The early pathological changes of DN is mesangial expansion caused by the deposition of extracellular matrix and the proliferation of mesangial cells.Extracellular matrix increased and excretion decreased induced glomerular basement membrane thickening is also visible at an early age. They both lead to the appearance of proteinuria and glomerular filtration rate decreased,eventually leading to glomerulosclerosis and renal interstitial fibrosis.There are mainly three types of pathological change in glomerulus:nodular glomerular sclerosis,diffuse glomerular sclerosis and exudative lesion.Nodular glomerular sclerosis is also known as intercapillary glomerulosclerosis and Kimnel-Steil-Wilson nodule(K-W nodule).Thus,mesangial cells appeared to change in the early pathogenesis of diabetic nephropathy and cause further histopathological abnormalities and dysfunction in disease progression or even failure,acts as a very important role in the occurrence and development of DN.DN is a multifactor disorder caused by the interaction between environmental and genetic factors,the precise mechanism is not yet fully elucidated, current studies found the classic mechanism mainly includes two main aspects, namely, metabolic disorders and hemodynamic abnormalities.More specifically, the risk factors as follows:1.Biochemical metabolic abnormalities caused by hyperglycemia,including non-enzymatic glycosylation,polyol pathway,hexose amine biosynthesis pathway,glucose toxicity,etc.;2.Glomerular injury caused by high blood pressure;3.Proteinuria impact,protein overload induced damage of tubulointerstitium and promote the progress of the disease;4.Molecular medium including transforming growth factor beta1(TGF-β1),connective tissue growth factor(CTGF),vascular endothelial growth factor(VEGF),as well as transcription factors and intracellular signaling pathways involved;5.Moreover,DN was confirmed by genetic susceptibility.In recent years, people pay more and more attention to "metabolism inflammation" beside traditional pathogenesis of chronic complications of DM,and committed to explore inflammatory cytokines and signaling pathways of DN.Inflammation may be a risk factor of DN was stated as early as in the1990s,but until2006,the concept of "metabolic inflammation" was first defined by Hotmamisilil in Nature and elaborated the participation of metabolic metabolism in the occurrence and development of diabetes and its chronic complications.Hotamisiligil pointed out that metabolic inflammation was a chronic and low-grade inflammation caused by long-tern nutrients and metabolic surplus,although its clinical symptoms were different from the traditional characteristic of acute inflammation, such as redness, swelling, heat and pain,it has a similar set of molecules and signaling pathways to those involved in classical inflammation.Since then,numerous studies started to focus on the mechanisms of inflammation involved in DM and its complications.Juan F reported that immune-mediated inflammatory is a indispensable factor in the development of DN,diverse cells involve in Inflammatory processes,including leukocytes,monocytes,and macrophages,as well as other molecules including monocyte chemoattractant protein-1(MCP-1),intercellular adhesion molecule-1(ICAM-1),Transforming growth factor-β(TGF-β),tumor necrosis factor (TNF-α),interleukin(ILs) and nuclear factor-κB (NF-κB).MCP-1, TGF-β1and IL-6have a close relationship with the development of the disease process of DN.In the course of disease,they have independent role respectively,and also can contact with each other to accelerate the progress of DN.MCP-1expressed in the vast majority of cells,including mesangial cells and monocytes,and has a strong chemotactic activity for monocytes and macrophages.Previous studies have shown that in animal models of diabetic nephropathy,as well as in the urine and blood of patients with diabetic nephropathy,the expression level of MCP-1is significantly increased.High levels of MCP-1could promote the deposition of extracellular matrix and then leading to fibrosis.TGF-β1is another important inflammatory factor and has a crucial role on the process of diabetic glomerulosclerosis.TGF-β1has an important feature of promoting hardening which induces hypertrophy and apoptosis.In vitro,TGF-β1can increase the produce of collagen, fibronectin and laminin synthesis in the glomerular mesangial cells and tubular epithelial cells,and also can inhibit the synthesis of collagenase and stimulate metal protein inhibitors,decrease extracellular matrix degradation,and then increased matrix accumulation.In addition to induced fibrosis,TGF-β1has function of the regulation of the immune and inflammatory responses. Studies have shown that the severity of glomerular damage in DM is associate to the expression level of IL-6mRNA in mesangial cells and podocytes,suggesting that IL-6may promote the secretion of extracellular matrix in these cells.Many recent studies found that the degree of glomerular basement membrane thickening patients in type2diabetes mellitus have a close relationship with IL-6levels. As we all known,thickening of the glomerular basement membrane is considered to be a strong indicator to judge the development of diabetic nephropathy,it follows that the IL-6could predict the development of DN.Nuclear factor-κB(NF-κB) protein family is a pleiotropic Transcription factors,it is also the regulation hub of mesangial cell express multiple genes related to immune inflammation and have closely relationship with mesangial cell proliferation and secrete inflammation cytokines.Generally,NF-KB being in the form of Homodimer or heterodimer.In the resting state,NF-κB combine with IκB-α with a non-covalent bonds to form a complex. After stimulate by a variety of factors,NF-κB will activate through IκBs degradation,complex depolymerization and transferred to the nucleus,followed a series of reactions to mediated downstream.NF-KB can promote the mRNA expression of various cell adhesion molecule and cytokine and plays a key role in the early gene response.Recent research has shown that NF-κB mediate the production of MCP-1in mesangial cell induced by stretching and high glucose, at the same time,NF-κB involved in intracellular signaling pathways regulating TGF-β1.These preliminary evidences shows that NF-κB is very likely to participate in the pathogenesis of diabetic glomerular injury through the regulation of the inflammatory response.Thus,drugs which could achieve the anti-inflammatory effects through suppressing the activation of NF-κB pathway has been widely developed.At present,The major interventions for the prevention of the progression of diabetic nephropathy are glycemic control, dietary protein restriction, and blood pressure control, especially by the administration of angiotensin-converting enzyme inhibitors and/or angiotensin-II receptor blockers.In addition,the improvement of intra-renal hyper-coagulation is also an important target for the management of diabetic nephropathy.Based on this purpose,antiplatelet agents or anticoagulant drugs such as prostaglandin E1(PGE1) has been widely used.Li et alreported that a quantitative decrease in urinary albumin concentration was observed in diabetic nephropathy patients at different periods after short-term Lipo-PGE1administration and followed up for1year,Even urinary albumin concentration of patients in Ⅳ period and V period also decreased with different degree.In addition,some studies found that PGE1reduced blood plasma CRP and ICAM-1levels of diabetic nephropathy patients as well as the decrease of urinary protein excretion rate,suggesting its may be efficacy in inprove inflammation state beside ameliorate hemodynamics and microcirculation.However,whether the effect of anti-inflammation associate to microcirculation,or PGE1could act in local kidey to reverse inflammation,these studies did not investigate In vitro experiment,Dell et al. Reported that a low PGE1concentration decreased cyclosporine A induced production of PAI-1in rat mesangial cells.The he found PGE1could reduce the expression of TGF-β1mRNA induced by cyclosporine A in another vitro experiment.M.Kishida et al. reported that PGE1restrained the expression of PAI-1mRNA in human mesangial cell induced by TNF-a,indicated that its efficacy in DN.Here, our studied aim to investigate the protective effect of PGE1in the damage of HBZY-1cell induced by TNF-a and high glucose. The potential mechanisms were also discussing.this study selected HBZY-1cell line as the research object.To observed the influence of TNF-a and high glucose in HBZY-1cell proliferation, and protein contents and genetic expression of inflammatory factors including MCP-1, TGF-β1, and IL-6in the cell cultural supernatants. Furthermore, we investigate whether suppression were occurred in cell proliferation and protein and genetic expression of MCP-1、TGF-β1and IL-6after different concentration additions of PGE1. And the role of reverse NF-kB activation in suppression were also been observed.Two specific contents are as follows:Partl.PGE1ameliorates injury of rat mesangial cells induced by high glucose and TNF-aObjective:To investigate the protective effect of PGE1for injury of rat mesangial cells induced by high glucose and TNF-a.Methods:1.Experimental object:HBZY-1cell line was selected as the experiment object,cultured in DMEM(low glucose) containing10%fetal bovine serum(FBS),100μg/mL penicillin and100μg/mL streptomycin at37℃,the cells were passaged every3days.2.Experimental groups:The cells were divided into7groups according to different intervention: NG group:Normal glucose (5.6mmol/L)control group; HT group:High glucose(30mmol/L)+TNF-α(10ng/mL) intervention group; HTP1group.High glucose(30mmol/L)+TNF-α(10ng/mL)+PGE1(0.01ng/mL); HTP2group:High glucose(30mmol/L)+TNF-α(10ng/mL)+PGE1(0.05ng/mL); HTP3group:High glucose(30mmol/L)+TNF-α(10ng/mL)+PGE1(0. lng/mL); HTP4group:High glucose(30mmol/L)+TNF-α(10ng/mL)+PGE1(0.5ng/mL); HTP5group:High glucose(30mmol/L)+TNF-α(10ng/mL)+PGE1(lng/mL);HBZY-1cells of all these groups were cultured in DMEM(normal glucose) containing0.5%FBS for24hours before intervention to keep synchronized growth of cells.3.Experimental methods:Tetramethyl azo salt infiltration method(MTT method):To measure optical density(OD) of different groups. Enzyme-linked immuno sorbent assay(ELISA method):To investigate MCP-1,TGF-β1and IL-6level of HBZY-1cells in the supernatant of cell culture.Real-time fluorescent quantitative PCR(qRT-PCR):To assayed the expression of MCP-1,TGF-β1and IL-6in HBZY-1cells.4.Statistical analysis Statistical analysis were performed using the SPSS13.0software Package.All data are expressed as mean±SD(x±s),Statistical significance of differences among groups was evaluated by one-way ANOVA.Any comparison between the two groups using the LSD(Least-significant difference test) method when meet the homogeneity of variance,and using Dunnett’s T3method when equal variances not assumed.P<0.05was considered as significant.Results:1.The effect of PGE1on the HBZY-1cell proliferation induced by high glucose and TNF-a.In comparison with normal glucose group,high glucose and TNF-a obvious promoted HBZY-1cell proliferation both at24hour and48hour(0.704±0.096vs.0.840±0.040;0.789±0.104vs.1.122±0.136),differences were statistical significance(P=0.004; P=0.001);In comparison with high glucose group,the inhibition effect of low concentration PGE1intervention goups(0.01,0.05,0.1ng/mL) on cell proliferation was not obvious at24hour,differences were not statistical significance(P>0.05);But high concentration of PGE1(0.5and lng/mL) could reduce cell OD value obvious(0.708±0.095vs0.840±0.040;0.687±0.082vs0.840±0.040),differences were statistical significance(P=0.005,; P=0.002).As the culture time prolonged to48hour,the cell OD value of all groups decreased obvious except HTP1(0.862±0.239vs1.122±0.136;0.800±0.211vs1.122±0.136;0.720±0.146vs1.122±0.136;0.678±0.137vs1.122±0.136),differences were statistical significance(P=0.010; P=0.002; P=0.000; P=0.000).2.The effect of PGE1on the secretion of MCP-1,TGF-β1and IL-6protein induced by high glucose and TNF-a in HBZY-1cells culture supernatants:2.1The MCP-1protein concentration in each group:In comparison with NG group,the MCP-1protein concentration in HT group increased obviously at24and48hour(51.639±6.699vs.79.932±17.543),differences were statistical significance(P=0.030; P=0.000);In comparison with HT group,there was no significant differences between groups intervention with PGE1and HT groups at24hours(P>0.05);As the culture time prolonged to48hour,HTP4and HTP5groups has significant differences compare with HT groups(82.159±5.389vs.151.353±12.229,P=0.014; P=0.009),but HTP2and HTP3groups still has no significant differences compare with HT groups(P>0.05).2.2、The TGF-P1protein concentration in each group: In comparison with NG group,the TGF-β1protein concentration in HT group did not increased obviously at24hour,statistical differences were not significance(P>0.05), as the culture time prolong to48hour, the MCP-1protein concentration in HT group increased obviously(644.939±97.461vs947.349±144.847),differences were statistical significance(P=0.004);In comparison with HT group,there was no significant differences between groups intervention with PGE1and HT groups at24hours(P>0.05);As the culture time prolonged to48hour,HTP4and HTP5groups has significant differences compare with HT groups(709.691±130.684vs947.349±144.847;615.691±75.904vs947.349±144.847,P=0.017;P=0.002),but HTP2to HTP3groups still has no significant differences compare with HT groups(P>0.05).2.3The IL-6protein concentration in each group:The concentration of IL-6protein in HBZY-1cells culture supernatants was lower than the ELISA kit,conside there was no IL-6protein secrete in the cell culture supernatants.3.The effect of PGE1on the expression of MCP-1,TGF-β1and IL-6mRNA induced by high glucose and TNF-a in HBZY-1cells:3.1The expression of MCP-1mRNA in each group:In comparison of NG group,the expression of MCP-1mRNA in HT group increased(0.240±0.056vs1.000±0.207) significantly(P=0.000);Compare with HT group,the inhibition effect of PGE1in HTP2and HTP3groups were unconspicuous(P>0.05),but HTP4and HTP5groups could inhibit the expression of MCP-1(0.736±0.073vs1.000±0.207,0.656±0.107vs1.000±0.207) significant(P=0.049; P=0.015).3.2The expression of TGF-β1mRNA in each group:In comparison of NG group,the expression of TGF-β1mRNA in HT group increased(0.429±0.150vs1.000±0.202) significantly(P=0.002);Compare with HT group,the inhibition effect of PGE1in HTP2and HTP3groups were unconspicuous(P>0.05),but HTP4and HTP5groups could inhibit the expression of TGF-β1(0.702±0.095vs1.000±0.202;0.671±0.113vs1.000±0.202) significantly(P=0.039;P=0.025).3.3、The expression of IL-6mRNA in each group:In comparison of NG group,the expression of IL-6mRNA in HT group increased(0.436±0.044vs1.000±0.221) significantly(P=0.001);Compare with HT group,the inhibition effect of PGE1in HTP2to HTP4groups were unconspicuous(P>0.05),but HTP5group could inhibit the expression of IL-6(0.701±0.027vs1.000±0.221) significant(P=0.023).Conclusions:1.In the condition of high glucose and TNF-a,rat mesangial cell proliferate obvious at48h,PGE1could inhibit cell proliferation.2.The concentration of MCP-1,TGF-β1and IL-6protein induced by high glucose and TNF-a in HBZY-1cell culture supernatants increased obviously,high concentration PGE1could reduce cell secrete MCP-1,TGF-β1and IL-6.3. The expression of MCP-1/TGF-β1and IL-6mRNA induced by high glucose and TNF-a in HBZY-1cells increased obviously, high concentration PGE1could reduce cell express MCP-1,TGF-β1and IL-6mRNA. Part2The potential mechanism of PGE1for the injury of rat mesangial cells induced by high glucose and TNF-aObjectiveTo explore the potential mechanism of PGE1for the injury of rat mesangial cells induced by high glucose with TNF-a.Methods:1.Experimental object:HBZY-1cell line was selected as the experiment object,cultured in DMEM(low glucose) containing10%fetal bovine serum(FBS),100μg/mL penicillin and100μg/mL streptomycin at37℃,the cells were passaged every3days.2.Experimental groups:The cells were divided into3groups according to different intervention: NG group:The normal glucose control group;HT group:High glucose(30mmol/L)+TNF-α(10ng/mL) intervention group; HTP5group:High glucose(30mmol/L)+TNF-α(10ng/mL)+PGE1(lng/mL);HBZY-1cells of all these groups were cultured in DMEM(low glucose) containing0.5%FBS for24hours before intervention to keep synchronized growth of cells.3.Experimental methods: Immunofluorescence technique:Detected the translocation of NF-κB p65.Results:In comparison with NG group,the fluorescence intensity of NF-κB p65in HT groups was increased.In comparison with HT groups,the fluorescence intensity of NF-κB p65in HTP5group was decreased.Conclusion:PGE1inhibited NF-κB p65translocation in high glucose with TNF-a induced HBZY-1cells,which indicates that inhibition of NF-κB pathway activation may involved in PGE1protection on mesangial cell. |
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