Effect Of Pioglitazone On The Expression Of TLR4of Renal Tissue In The Diabetic Rats

Objectives:1.To investigate the changes of Toll-like receptor4(TLR4) expression in renal tissue and regulating possible mechanism in rats with diabetic nephropathy.2. To explore the impact of pioglitazone on the expression of TLR4of diabetic rat renal tissue and the molecular mechanism of its anti-inflammatory.Methods:Fifty-seven male Sprague-Dawlry rats of SPF were randomly divided into normal control group (group A, n=9) and module group (n=48). Giving conventional feed,drinking free,module group was induced to hyperglycemia by injection streptozotocin (STZ,50mg/kg) into enterocolia. And group A was given citric acid buffer at the same dose. These Diabetic rats were confirmed by random blood sugar≥16.7mmol/L of three consecutive times after72hours of STZ injection (3cases failed to achieve the standard of diabetes was excluded). Then they were randomly divided into five groups:low-dose pioglitazone intervention group (5mg/kg daily[B group, n=9]),high-dose pioglitazone intervention group (10mg/kg daily [C group, n=9]), low-dose pioglitazone plus intraperitoneal injection TLR4inhibitor Eritoran5mg/kg.d intervention group (D group, n=9) and high-dose pioglitazone plus intraperitoneal injectionTLR4inhibitor Eritoran5mg/kg.d intervention group (E group, n=9), no intervention group(F group, n=9).These rats in group A were received equal volume as the other’s with the normal saline by daily gavage. At the end of the8th week,24h urine samples were collected to examine24h microalbuminuria. At the same time, body weight was measured. Then the rats were killed to collect blood and kidney. Blood sample were collected for analysis of fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), serum creatinine (SCr), C-reactive protein(CRP) and so on. Kidney was weighted to calculate the ratio of kidney weight to body weight (KW/BW). The morphological changes of kidney were observed through light microscopy after hematoxylin-eosin (HE) staining. Immunohistochemistry staining was used to examine the expression of TLR4and PPARγ protein in the kidney.. Proteinand mRNA expressions of TLR4, PPARγ were analyzed by western blot, RT-PCR,respectively.Results:(1)The KW/BW，24h urine protein, plasma concentration of CRP andcreatinine，the renal expression of TLR4increased，but decreased PPARγin Group B,C,Dand E diabetic rats, compared with A group(P<0.01). Furthermore, The KW/BW，24h urine protein, plasma concentration of CRP and Scr，the renal expression of TLR4decreased，but increased PPARγin group B, C, Dand E diabetic rats,compared with Fgroup(P<0.05). Pioglitazone down-regulated protein and mRNA expressions of TLR4and PPARγ were up-regulated of renal tissue in B, Cgroup. Compared with B, C grouphave statistical significance. The experiment also found that Pioglitazone and TLR4inhibitor clould synergistically reduced TLR4expression and significantly enhancedPPARγ expressions in D, E group diabetic rats, but Compared with D, E group have nostatistical significance.(2) Pearson correlation analysis showed that the expression of TLR4of diabetic ratrenal tissue has a positive correlation with rat’s24h urine protein, serum creatinine,blood CRP levels,the correlation coefficients were0.899,0.947,0856(P <0.05).But ithas a negative correlation with the expression of PPARγ,the correlation coefficientwas-0.919(P <0.05).Conclusions:1.Expression of TLR4increaseing in renal tissues and regulationof the PPARγ reduceing in the progression of diabetes suggest that TLR4may take partin the pathogenesis of diabetic nephropathy.2.Pioglitazone decrease pro-inflammatory mediators such as TLR4, but increaseproduction of anti-inflammatory molecules like PPARγ in rats，that can lessen thediabetic nephropathy impairment.