Effect Of Pioglitazone On Expression Of The TLR4-TRAF6 Pathway In Renal Tissues Of Diabetic Rats And Mechanism

Objective: 1. To examine the expressions of TLR4(Toll-like receptor 4)and TRAF6(TNF receptor-associated factor 6,TRAF6)in renal tissues of diabetic rats; 2. To explore the effect of pioglitazone on expressions of TLR4-TRAF6 in renal tissues of diabetic rats.Methods: 58 5-week-old specific pathogen free male SD(Sprague Dawley) rats(with a body weight of about 180-200g) were selected and randomly divided into the normal control group(group A, n=10) and the model group(n=48). Rats in the normal control group were given conventional diet and free drinking water; rats in the model group were given high-fat and high-carbohydrate diet and free drinking water. Fasting blood glucose(FBG), fasting insulin(FINS), Triglyceride(TG), total cholesterol(TC) and low density lipoprotein(LDL-C) were detected 4 weeks later. Aftersuccessfully establishing the insulin resistance rat models, an intraperitoneal injection of STZ(35mg/kg) was given after they had been fasted for 12 hours; 72 hours after injection, blood from caudal vein of rat was obtained to measure random blood sugar(BS), and those with a BS ≥ 16.7 mmol/L for three times in succession were confirmed as diabetic rats(two rats failed in modeling). Rats in the normal control group were injected with citric acid buffer solution of the same volume. Rats who failed in modeling were removed from the experiment group. Rats in group A were given conventional diet; after successful modeling of diabetic SD rats, they were given high-fat diet and randomly divided into 5 groups: the non-intervention group(group B, n=10), the group treated with pioglitazone(2.5mg/kg.d)(group C, n=8), the group treated with pioglitazone(10 mg/kg.d)(group D, n=8), the group treated with pioglitazone(2.5 mg/kg.d) + specific TRAF6 ingibitor―MC132(0.1mg/kg.d)(group E, n=9) and the group treated with pioglitazone(10 mg/kg.d) + specific TRAF6 ingibitor―MC132(0.1mg/kg.d)(group F, n=9); rats in group A were given intragastric administration of normal saline of the same volume; rats in all the groups were intervened for 8 weeks. Rats in group E and group F were given intraperitoneal injection of specific TRAF6 ingibitor―MC132(0.1mg/kg.d) every day since the 10 th week of the experiment; at the end of the 12 th week, body weight and 24-hour urine microalbumin of rats in each group were measured;and after their blood was obtained to measure the levels of fasting blood glucose(FBG), fasting insulin(FINS), serum creatinine(SCr), blood urea nitrogen(BUN), C reactive protein, TG, TC and LDL-C, rats were dissected and their right and left kidneys were taken out; the real-time PCR method was used to measure the m RNA expression levels of TRAF6 in renal tissues of diabetic rat models; HE staining and immunohistochemistry staining were used for staining; morphology of renal tissues and protein expressions of TLR4, TRAF6 and NF-κB were observed under light microscope.Results:(1) Body weights and biochemical indexes of rats at the end of week 4: Compared to group A, rats in the model group had an increased body weight, with significant statistical difference(P<0.05); insulin, blood sugar, HOMA-IR, TG, TC and LDL of rats in the model group were increased compared to those of rats in the normal group, with significant statistical difference(P<0.05).(2) Body weights and biochemical indexes of rats at the end of week 12: Compared to group A, rats in group B, group C, group D, group E and group F had a lower body weight, with significant statistical difference(P<0.05); Compared to group A, the 24-hour urine microalbumin, SCr, BUN,TG, TC and LDL-C as well as expressions of TLR4, TRAF6 and NF-κB of rats in all the other groups were obviously increased, with significant statistical difference(P<0.05); Compared to group B, all the indexes mentioned above of rats in group C, group D, group E and group F were decreased, with significant statistical difference(P<0.05); All the indexes of rats in group D were decreased compared to group C, with significant statistical difference(P<0.05); When group E was compared with group C and group F was compared with group D, excepting that expression of TLR4 was not affected, all the other indexes of rats in group E and group F were decreased, with significant statistical difference(P<0.05); When group F was compared with group E, expressions of TRAF6 and NF-κB in renal tissues of rats in group F were increased and there was no statistical difference regarding expression of TLR4(P >0.05).(3) Pearson correlation analysis showed that expression of TRAF6 in renal tissues of diabetic rats was negatively correlated with TLR4(r=-0.912, R2=0.749 P<0.05); expression of TRAF6 was positively correlated with that of NF-κB(r=0.931,R2=0.946 P<0.05); however, TLR4 was also negatively correlated with NF-κB(r=-0.975,R2=0.783 P<0.05).Conclusions: 1. Expressions of TLR4, TRAF6 and NF-κB in renal tissues of diabetic rats induced by STZ were significantly increased. 2. Pioglitazone may down-regulate the expression of TRAF6 by regulating the expression of TLR4 in renal tissues of diabetic rats, and then inhibits the expression of the downstream inflammatory factor—NF-κB, thus to relieve inflammatory response of renal tissues and protect the renal tissues of diabetic rats.