Effects Of Berberine On Promoter Of QDPR Gene In Rats With Type 2 Diabetic Nephropathy And Its Mechanism

BackgroundDiabetes mellitus is a heterogeneous disease characterized by microvascular pathology leading to long-term complications clinically manifested principally in the kidney. The epidemic study published on JAMA revealed over 11.6% adult DM patients in China. Type 2 diabetes mellitus is characterized by insulin resistance, and associated with hyperlipidemia. Clinically, nephropathy induced by diabetes is appeared to microalbuminuria followed by overt proteinuria. Histologically, mesangial expansion is followed by glomerular sclerosis. However, nearly no effective treatment on DN since overt urine protein occurs. This study was based on an ideal model for type 2 DN, which has not such severe renal damage by uninephrectomized plus STZ-induced DN model, but would closely reflect the natural history and metabolic characteristics of human type 2 diabetic nephropathy and respond to the pharmacological treatment.Berberine (BBR) is an isoquinoline alkaloid isolated from Coptis or Cortex Phellodendri Chinensis which has been commonly used as an oral drug to treat gastroenteritis and secretory diarrhea for more than thousand years in China. Recent studies show that BBR has benefit to diabetes, hypertension and cardiovascular disease. There are many studies showed BBR reduce blood glucose, improve insulin resistance and decrease lipid. Although the therapeutic efficacy of BBR on DM was confirmed, the effect and mechanism on diabetic nephropathy is still not fully understood.Professor Ping Li has reported that there was the modification of dihydropteridine reductase (QDPR) gene in renal cortex of spontaneous OLETF diabetic rats, which suggests a role of QDPR in diabetic nephropathy. In the further study, Doctor Yanting Gu found that over expression of QDPR inhibited TGF-β/Smad signaling pathway and reduced NOX1/NOX4 in type 1 diabetic rats. In addition, QDPR activated autophagy in HEK 293T cell. Therefore, QDPR might be a potent biomarker and therapy target in diabetic kidney disease. In the present study, we plan to construct promoters of rat QDPR gene and identify the core working zone by dual-luciferase reporter assay system. In further, we decided to detect whether BBR working on QDPR promoter.ObjectiveTo observe the renoprotective effect of BBR on high-fat diet puls low-dose-STZ induced type 2 diabetes rat model and its mechanism; to construct and identify rat QDPR gene core promotes and detect its activity treated with BBR in HEK 293T cell.Method1. Renoprotective Effect of BBR on Type 2 DN in Rats and its mechanism1.1 Renoprotective effect of BBR on type 2 DN in rats:Male Wistar rats (8-wk old, 200-250g) were purchased from Beijing HFK Bio-Technology Co. Ltd. (Beijing, China, Certificate No. SCXK 2002-0010) and randomly divided into normal control and diabetic groups. The type 2 diabetic rat model was established a high-fat diet (consisting of 50% carbohydrate,12% protein, and 38% fat) and intraperitoneal injection with a low-dose of streptozotocin (STZ,25mg/kg). the treatment was gavaged by BBR initiated on the third day after STZ injection and continued for 20 wk. Body weight was recorded at 4-wk intervals and levels of blood glucose were measured every 4 wk by tail-vein blood sampling. Rats were housed individually in metabolic cages for 24-h urinary collection at 4-wk intervals. At the end of study, blood was collected from the abdominal aorta by syringe puncture. Serum was separated for detection of total cholesterol (TC), triglycerides (TG), low density lipid cholesterol (LDL-C), high density lipid cholesterol (HDL-C), urea nitrogen (BUN), creatinine (Cre), total protein, and albumin by an automatic biochemistry analyzer. Kidney tissues were collected for histology staining and morphological study.1.2 BBR attenuated renal inflammation by inhibiting NF-κB signaling pathway on rats:based on the type 2 DN rat model, we detected inflammation feature, such as macrophages, IL-1β、TNF-α and MCP-1 by IHC and realtime PCR. In the further, we studied on NF-κB signaling pathway. We detected p-p65, p65 by IHC, western blot, and detected p-IκBα and IκBα by western blot.1.3 BBR attenuated renal fibrosis by inhibiting TGF-β/Smad signaling pathway on rats:based on the type 2 DN rat model, we detected inflammation feature, such as collagen Ⅰ、collagen Ⅳ and fibronectin by IHC, western blot and realtime PCR. In the further, we studied on TGF-β/Smad signaling pathway. We detected TGFβ. p-Smad2/3, Smad2/3 by IHC, western blot and detected Smad7, Smurf2 by realtime PCR and western blot, and detected p-TβRI, TβRI, TβRII by western blot1.4 BBR attenuated renal lipid deposition by balancing cholesterol intake and effusion on rats:based on the type DN rat model, we detected lipid indices, such as TG, TC, HDL-C, LDL-C by automatic biochemistry analyzer. Renal lipid deposition was detected by oil red O staining. In the further, we studied on cholesterol intake and effusion. We detected expression of LOX-I、RXRα、RXRβand ABCA-1 by western blot and realtime PCR.2. Study on rat QDPR gene promoters influenced by BBR2.1 Construct of rat QDPR gene promoters:We obtained rat QDPR gene genomic sequence from GenBank, and then analysisd 5’upstream sequences from ATG to-2092. Regional series truncated fragments of rat QDPR promoter gene were recombined with luciferase reporter vector pGL3-Basic. The recombinant plasmids were confirmed by enzyme cut and sequencing.2.2 The effect of rat QDPR gene promoters by BBR treatment stimulated by high glucose:The recombinant plasmids of QDPR promoters were transiently transfected into HEK293T cells respectively. BBR (5μM, 10μM) was added into the high glucose cultured condition (30mM). The transcriptional activity was tested by dual-luciferase reporter assay system to locate the core regulatory element.Results1. Renoprotective Effect of BBR on Type 2 DN in Rats and its mechanism1.1 Renoprotective effect of BBR on type 2 DN in rats:DN associated hyperglycemia was significantly reduced by a 4-week BBR treatment, whereas which was maintained in the untreated group throughout the experiment. The lower levels of total blood protein and albumin, but higher levels of serum triglyceride, cholesterol, LDL-cholesterol, and BUN, which was significantly improved by the BBR treatment. Furthermore, BBR treatment significantly attenuated 24h-microalbuminuria in diabetic rats, associated with a marked inhibition of extracellular matrix deposition in both glomeruli and tubulointerstitium, thickening of glomerular basement membrane, and tubular atrophy1.2 BBR attenuated renal inflammation by inhibiting NF-κB signaling pathway on rats:According to the results of immunohistochemistry, the number of ED-1 positive cells was markedly higher in DN group; however, almost no expression of ED-1 was found in the normal group, and BBR-treated reduces macrophage infiltration (ED-1-positive cells). According to the results of immunohistochemistry, Western blot, and quantitative real-time PCR, renal inflammation was largely decreased in the diabetic kidney, including a marked downregulation of proinflammatory cytokines (TNFα, IL-1β) and macrophage chemotactic molecule-1 (MCP-1) in the BBR-treated rats compared to the control group. We further elucidated the working mechanism by which BBR protects the rats with type 2 DN from renal inflammation. The results of immunohistochemistry and Western blotting analysis demonstrated that NF-κB signaling was hyperactivated in the diabetic kidney as supported by the subcellular translocation of phosphorylated NF-κB/p65, which was largely inhibited by BBR. In addition, the same tendency of phosphorylated IκBα.1.3 BBR attenuated renal fibrosis by inhibiting TGF-β/Smad signaling pathway on rats:According to the results of immunohistochemistry, western blot and realtime PCR, renal fibrosis such as upregulation and accumulation of collagen I, collagen IV, and fibronectin developed in the diabetic kidney was significantly attenuated by BBR treatment. We further elucidated the working mechanism by which BBR protects the rats with type 2 DN from renal fibrosis. BBR treatment significantly suppressed the activation of TGF-β/Smad3 signaling, including downregulation of TGF-β1, TβRII, phosphorylated T(3RI, phosphorylated Smad3 and Smurf2, and upregulation of Smad7 meanwhile.1.4 BBR attenuated renal lipid deposition by balancing cholesterol intake and effusion on rats:The type 2 DN rats showed higher levels of serum triglyceride, cholesterol and LDL-cholesterol, which was significantly improved by the BBR treatment. Development of hyperlipidemia in diabetic rats was associated with moderate deposition of lipids in the kidney, particularly in tubular epithelial cells as demonstrated by Oil Red O staining, which was also attenuated by BBR treatment. According to the results of western blot and realtime PCR, the expression of LOX-I was increased, while the expression of RXRα、RXRβ and ABCA-1 was decreased in DN rats. BBR treatment rebalanced the cholesterol intake and effusion.2. Study on rat QDPR gene promoters influenced by BBR2.1 Construct of rat QDPR gene promoters:5’upstream sequences from ATG to-2092, regional series truncated fragments of rat QDPR promoter gene were recombined with luciferase reporter vector. The recombinant plasmids were confirmed by enzyme cut and sequencing.2.2 The effect of rat QDPR gene promoters by BBR treatment stimulated by high glucose:The expression of QDPR was decreased in the kidney of type 2 DN rats, the same finding in HBZY cell stimulated by high glucose. High glucose repressed activity of QDPR promoter full length fragment, which was increased by BBR treatment; however, high glucose stimulated significantly activity of QDPR promoter deletion fragment, which was repressed by BBR treatment.Conclusion1. Renoprotective Effect of BBR on Type 2 DN in Rats and its mechanismBBR has a renoprotective effect on Type 2 diabetic nephropathy. Blockade of NF-κB-driven renal inflammation and TGF-β/Smad3-mediated renal fibrosis may be the possible mechanisms by which BBR protects the kidney from injury in type 2 diabetes. In addition, the imbalancel of cholesterol intake and effusion might exacerbate the renal inflammation in diabetic kidney disease.2. Study on rat QDPR gene promoters influenced by BBRThe expression of QDPR on kidney is repression both in vivo and in vitro under diabetes. Based on the promoter study, we identify the rat QDPR core promoter location. In addition, we find BBR regulates activity of QDPR promoter.