| Pentamethylquercetin (PMQ) is a methylation product of quercetin. It has significant pharmacokinetic and pharmacodynamic advantages. It is confirmed that PMQ has a variety of effects, including anti-inflammation, antioxidation, anti-myocardial ischemia reperfusion injury, anti-arrhythmia activity, anti-metabolic syndrome effect, etc. It has been reported that flavonoid compounds have notable effects on diabetes and its major complication, diabetic nephropathy (DN). Additionally, mesangial cells (MC) play a key role in DN. In this study, rat model of type 2 diabetic nephropathy was successfully established to investigate the renal protective effect of PMQ and the possible mechanism. The cell model of high glucose-induced proliferation of MC was prepared to further research the anti-DN mechanism of PMQ.Part 1 Establishment of rat model of type 2 diabetic nephropathyObjective: To establish rat model of type 2 diabetic nephropathy. Methods: At the age of 2 days, male neonate rats were treated with STZ and control group with the vehicle (i.p.). Body weight and the consumption of water and food were recorded every day from the age of 6 weeks. At the age of 12 and 16 weeks, fed and fasting blood glucose, triglyceride (TG), fasting serum insulin level were tested respectively. At the age of 16 weeks, oral glucose tolerance test (OGTT) was performed and serum creatinine was tested. At the age of 16 weeks, each group of rats were put into metabolic cages for the measurement of urine volume, urine specific gravity, urinary albumin and urinary creatinine (Ucr), and then the endogenous creatinine clearance rate (Ccr) and urinary albumin excretion rate (UAER) were calculated. After decapitation, both kidneys of the rats were isolated and kidney index was calculated. Pathological changes were also observed. Results: 1) Treating neonate rat with STZ could successfully establish type 2 diabetic rat model. During the whole growing period, the model group gained less body weight but consumed more water and food compared with the control group. At the age of 16 weeks, increased level of fed blood glucose, TG and enlarged area under the OGTT curve were observed, while serum insulin level decreased. 2) At the age of 16 weeks, significant characteristics of diabetic nephropathy have been shown. The parameters of renal function were significant changed in model group compared with control group. Increased level of UAER (from 63.50 mg/24h to 148.20 mg/24h), Ccr (from 3.25 to 3.96) and kidney index (from 0.98 ml/min to 1.49 ml/min) and pathological results showed that glomerular hypertrophy, fibrosis and basement membrane thickening were obvious in the model group.Part 2 Investigation of the effects of PMQ on rats with type 2 diabetic nephropathy and the related mechanismsObjective: To study the effect of PMQ on rats with type 2 diabetic nephropathy. Methods: Method of modeling was the same as part 1. At the age of 6 weeks, STZ- injected rats were divided into four groups randomly as follows (n=9): model group, treatment groups with low, medium or high dose of PMQ (PMQ 5, 10 or 20 mg/kg, respectively). Rats were treated with PMQ or vehicle once a day. Parameters and methods were the same as part 1.SOD and MDA levels of kidney tissues were determined. RT-PCR assays were employed to monitor the mRNA expression of PPARs (PPAR-α, PPAR-β, PPAR-γ), inflammatory cytokines (IL-6), GluT1 and TGF-β1 in the kidney tissues of each group. Results: 1) Fed blood glucose, TG decreased and serum insulin level increased in PMQ treatment groups compared with model group. 2) UAER, Ccr and kidney index decreased in PMQ treatment groups compared with model group. PMQ partially attenuated glomerular hypertrophy, fibrosis and basement membrane thickening. 3) The related mechanisms: PMQ can normalize SOD and MDA levels in kidney tissues. RT-PCR analysis suggested the mRNA expression of PPAR-α, TGF-β1, GluT1, IL-6 were up-regulated, and the PPAR-γmRNA expression were down-regulated in model group. The changes could be attenuated by PMQ treatment.Part 3 Investigation of the effects of PMQ on the changes of mesangial cells induced by high glucose and the related mechanismsObjective: To study the effect of PMQ on mesangial cells (MC) under high-glucose conditions. Methods: MC were cultured with L-DMEM+10%FBS and incubated with 30.5 mM glucose and PMQ for 48 h. PMQ concentration was 0.1, 0.3, 1, 3 or 10μM, respectively. Cell activity was tested using MTT method. Total collagen was measured with Sirus Red. SOD and MDA levels of intra-cellular were determined. RT-PCR was employed to monitor the mRNA expression of PPAR-α, PPAR-β, PPAR-γ, TGF-β1, GluT1 and IL-6. Results: 1) High glucose significantlly promoted MC proliferation. PMQ inhibited it in a dose-dependent manner. 2) Total collagen increased significantly in high-glucose group compared with control group. This level was reduced in PMQ treated groups. 3) SOD level decreased and MDA level increased in high-glucose group. PMQ can normalize them. 4) RT-PCR analysis suggested that high-glucose group up-regulated mRNA expression of TGF-β1, GluT1 and IL-6 and down-regulated that of PPAR-α, PPAR-β, PPAR-γ. PMQ treatment can attenuate these changes.Conclusions: 1) Treating neonate rat with STZ could successfully establish type 2 diabetic rat model. At the age of 16 weeks, significant characteristics of diabetic nephropathy have been shown. 2) PMQ ameliorated type 2 diabetes and DN in rats. This includes: the decrease of fed blood glucose, TG, UAER, Ccr and kidney index as well as the increase of serum insulin. 3) On cellular level, PMQ significantly inhibited high-glucose induced proliferation of MC, reduced total collagen, raised SOD and lowered MDA level. 3) The result of both models suggested that the therapeutic mechanism of PMQ in DN includes: inhibition of fibrosis, antioxidation, anti-inflammation and regulation of the mRNA expression of PPARs family, etc. |
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