| Objective: Diabetic nephropathy (DN) is a kind of chronic complicationsof diabetes due to microvascular damage.It is the leading cause of renal failure.Toll like receptor4(TLR4) is an important member of Toll protein family thatfirst discovered. It is involved in a variety of inflammatory and immuneresponses. It had shown that TLR4was closely related with diabetes anddiabetic complications. Now there was little research about the relationshipbetween TLR4and DN. So the relations between the two needs to berevealed.A20is a Cys2/Cys2cell liquid zinc finger protein. In the conditionof inflammation, it can be expressed in almost all tissue cells and it is anendogenous protein that the main role is to limit the activity of NF-κB andAP–1to protect cells from damage. Simvastatin is one of cholesterol-lowingdrugs which is prescribed widely and has affirmative therapeutic effect inclinical work. Recent researches suggested that simvastatin could attenuate theinflammatory response independent of lipid-lowering effect which wasplaying a protective role in diabetic individuals. In this experiment type2diabetic model was copied by giving Sprague-Dawlay (SD) rats high-sugar-fatdiet,then there was hyperinsulinemia and insulin resistance in the rats. In thefollowing, a low dose of streptozotocin (STZ) was given to distory thepancreatic islet cells.After14weeks, the expression of TLR4and A20in renaltissues were detected by immunohistochemical method and reversetranscriotion-polymerase chain reaction (RT-PCR) in order to discuss theassociation between the two and DN.Furthermore, simvastatin was given toexperimental rats to explore the possible mechanism of protective effects.Methods:10rats were randomly selected to be normal control group(groupA) from the40SD rats by the application of the random number tables, fed by general diet.The rest were fed with high-sugar-fat diet (mixed with15%cooked lard,2.5%cholesterin,20%sugar in general diet). Theexperimental rats have excessive weight gradually and insulin resistance.Thena small dose of STZ (30mg/kg) was given. Six weeks later, FBG and FINSwere detected,the rats whose FBG was higher than7.8mmol/L and ISIdecreased were considered as T2DM rats. The diabetic rats were randomlydivided into diabetic model group (group B) and simvastatin interventiongroup (group C). The experiment lasted for14weeks. At the end of thisexperiment, body weight, FBG, TC, TG, LDL-C, HDL-C, Cr, BUN,24h UAlbwere detected. Kidney weight was measured for calculating kidneyhypertrophy index. The expression of TLR4and A20in renal tissue wasdetected by immunohistochemical mothed. The results were analyed withcomputer image-analysis system and IHS of TLR4and A20were calculated.Meanwile, the expression of TLR4was detected by RT-PCR. The results wereanalyed with Bandscan5.0. The data were dealt with SPSS13.0.Results:1Various indexes after6weeks:12.02±1.83mmol/L of test rats FBG washigher than6.16±0.38mmol/L of controls FBG (P<0.01),18.37(18.08,19.43)mIU/L of test rats FINS compared to17.75(17.08,18.53) mIU/L ofcontrol rats was unstatistics distinction（P＞0.05）,0.0046(0.0038,0.0052) oftest rats ISI was lower than0.0094(0.0088,0.0095) of control group (P<0.01).Thus far,the T2DM rat model was accomplished.2Various indexes after14weeksGroup A：FBG5.72±0.43mmol/L, TG0.78±0.45mmol/L, TC2.40±0.21mmol/L, LDL-C1.23±0.16mmol/L, HDL-C1.29±0.25mmol/L, UAlb2.67±0.21ug/24h, Cr59.02±3.02μmol/L, BUN5.22±0.31mmol/L, Ccr0.79±0.36ml/min, RW/BW1.60±0.10.Group B：FBG12.37±3.0mmol/L, TG1.81±0.24mmol/L, TC5.50±0.82mmol/L, LDL-C2.12±0.1mmol/L, HDL-C0.53±0.15mmol/L, UAlb26.31±0.62ug/24h, Cr96.20±10.01μmol/L, BUN9.54±0.72mmol/L, Ccr0.19±0.28ml/min, RW/BW1.90±0.22. Group C：FBG11.5±2.0mmol/L, TG1.06±0.31mmol/L, TC2.93±0.42mmol/L, LDL-C1.63±0.04mmol/L, HDL-C1.03±0.11mmol/L, UAlb16.61±1.02ug/24h, Cr71.21±5.01μmol/L, BUN6.11±0.72mmol/L, Ccr0.79±0.35ml/min, RW/BW1.60±0.22.FBG、TG、TC、LDL-C、UAlb、BUN、Cr、RW/BW of Group B rats werehigher than that of Group A rats (P<0.05). HDL-C and Ccr were lower thanthat of Group A rats (P<0.01). FBG、TG、TC、LDL-C、UAlb、BUN、Cr、RW/BW of Group C rats were lower than that of Group B rats (P<0.05).HDL-C and Ccr were higher than that of Group B rats (P<0.01).3HE staining: The glomerular morphology and glomerular cells of group Arats were in order, basement of glomerular capillary were clear andregular.While in T2DM rats, expansed glomerular, excessive glomerular cellscould be observed.The changes in group C rats were relieved than that ofgroup B rats.4The protein expression of TLR4and A20in renal tissue: The IHS7.8±1.3ofTLR4in group B rats was higher compared to2.0±1.0of group A rats(P<0.01). The IHS4.2±1.3of TLR4in group C rats was lower compared tothat of group B (P<0.01). The IHS4.8±.0.8of A20in group B rats was highercompared to2.0±1.0of group A rats (P<0.05). The IHS2.8±0.4of A20ingroup C rats was lower compared to that of group B (P<0.05).5RT-PCR: The Optical density (OD) of mRNA was detected by Bandscan5.0analysis system. The OD of TLR4mRNA/β-action mRNA in group B was0.82±0.05.It was higher compared to0.378±0.025of group A rats (P<0.01).The OD of TLR4mRNA/β-action mRNA in group C was0.616±0.043.It waslower than that in group B.6The correlation analysis: TLR4was positively correlated with24hour UAlb,Cr, A20.The correlation coefficient was0.679,0.586, and0.576respectively.(P<0.05)Conclusions:1T2DM rats had higher Urine albumin, BUN, Scr, KW/BW. HE stainingshowed that T2DM rats had expansed glomerular and excessive glomerular cells.They had suggested that Type2diabetic rats had been in existence ofdiabetic nephropathy.2The expression of TLR4and A20was increased in renal tissue and it waslower after simvastatin intervention.TLR4may participate in the developmentof diabetic nephropathy. As the negative regulation protein of NF–κB,increased A20expression had a protective effect in inflammatory response.There would be a theoretical basis for the treatment of DN.3Simvastatin may have kidney protective effect on DN, partly via thedownregulation of TLR4expression. |
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