| Pentamethylquercetin (PMQ), as one member of natural polymethoxylated flavonoids, is originally found in sea buckthorn and the rhizome of Kaempferia parviflora. We chose to synthesize it by a simple methylation reaction of the starting material quercetin with a high yield and purity, which provided satisfactory quantity and quantity required in this study. Our previous researches showed that PMQ ameliorated hyperglycemia in neonatally streptozotocin-induced diabetic rats, upregulated adiponectin level in3T3-L1cells, ameliorated metabolic disorders and prevented cardiac hypertrophy. Moreover, in our previous research on diabetic rats, we found that PMQ not only ameliorated hyperglycemia, but also effectively normalized urine volume. Therefore, in this study, we tested our hypothesis that PMQ has renal protective effect in diabetes. GK rats were chosen to establish animal model since their renal fibrosis syndromes are similar to those in diabetic nephropathy patients. Mesangial cells (MC) played an important role in diabetic nephropathy and it could mimic the diabetic condition in vivo when MCs were exposed to high glucose. Furthermore, we investigated the mechanism, which may be associated with the anti-renal fibrotic effect of PMQ.Objective:To study the effect of PMQ on renal fibrosis in spontaneous type Ⅱ diabetic rats and MCs in high glucose, as well as the related mechanism. Methods:(1)Divided the12-week aged male GK rats into five groups:GK rats group, PMQ group (5,10,20mg/kg) and metformin group (300mg/kg), while the age-matched Wistar rats used as control group (n=7-9). Body weight, consumptions of water and food of each group were monitored every week. After15-week treatment, the levels of fasting and fed glucose and insulin, fasting triglyceride, as well as cholesterol, were tested respectively. After16-week treatment, oral glucose tolerance test was performed. Before the end of the experiment, the24h urine volume, the levels of urine glucose, albumin, creatinine and blood urea nitrogen were tested. Urinary albumin excretion rate were calculated. After decapitation, both kidneys of the rats were isolated and kidney index was calculated. Pathological changes were observed by PAS-and Masson-staining. The levels of fibronectin and collagen IV on mRNA and protein levels were measured.(2) Cell fibrotic model of MCs was induced with30.5mM glucose. Treated groups were added PMQ1,3or10μM, or captopril100μM, for48h. Cell proliferation was tested using MTT method. Cell area was measured with CM-DiI. Total collagen was measured with Sirus Red. RT-PCR and The levels of fibronectin and collagen IV on mRNA and protein levels were measured.(3) The expression of TGF-β1, Smad7, Smad2/3and p-Smad2/3in the rats’kidneys of each group and MCs in high condition, on both mRNA and protein levels. Result:(1) After16-week treatment, PMQ improved the diabetic syndromes and renal fibrosis compared with GK rats, including:a. improved glucose and lipid metabolism, insulin resistance and glucose intolerance; b. reduced urine volume, urine glucose and kidney index; c. ameliorated glomerular hypertrophy and glycogen deposition; and d. reduced the expression of collagen IV and fibronectin on mRNA and protein levels.(2) Compared with high glucose group, PMQ treatment inhibited cell proliferation, reduced total collagen accumulation and alleviated cell hypertrophy. Meanwhile, on mRNA and protein levels, the expression of fibronectin and collagen IV were reduced after PMQ intervention.(3) Compared with model group, PMQ treatment reduced the expression of TGF-β1and p-Smad2/3, enhanced Smad7, both in kidneys and MCs.Conclusion:(1) GK rats had not only showed the typical type II diabetic syndromes, but also renal fibrosis;(2) PMQ significantly improved glucose and lipid metabolism, attenuated insulin resistance and glucose intolerance. Meanwhile, PMQ alleviated renal dysfunction and fibrosis in GK rats;(3) PMQ notably ameliorated glomerular hypertrophy and total collagen deposition in mesangial cells in high glucose; and (4) the therapeutic mechanism of PMQ in anti-renal fibrosis in diabetes might be associated with suppressed TGF-β/Smads signaling. |
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