| Research objectDiabetic nephropathy (DN) is one of the most commonly found microvascular complications of diabetes, renal damage of diabetic nephropathy affects all the structure of the kidney.Once in DN patients with late, will rely on dialysis or a kidney transplant to maintain life.DN, therefore, has always been a difficult problem to be overcome in the clinical medicine, research and development mechanism, curative effect of drug is the research hot spot.Qizhu granules (QZG) was developed according to the clinical experience in the laboratory self-help compound traditional Chinese medicine:Astragalus membranaceus radix, Ructus ligustri lucidi, Huang shu sunflower, Atractylodes macrocephala, Radix paeoniae rubra, Rosa laevigata Michx., Coptis chinensis Franch, Radix dioscorea futschauensis Uline,eight compatibility and become, in the medicine, altogether plays qi and nourishing Yin, activating xue and shui.Mainly used in early diabetic nephropathy with urinary protein of spleen kidney xu, shi and zhuo stasis resistance.This research adopts the STZ damaged islet cells, high-fat feed, kidney ligation compound method to establish model of type 2 diabetic nephropathy.Observation of Qizhu granules resistance to type 2 diabetes nephropathy rats model of kidney function, blood fat, etc. the influence of the pathologic morphology, explore the protection mechanism.Using the IHC was developed for the determination of DN rat renal apoptosis related proteins expression level, using the TUNELto observe the DN rat renal apoptosis, research from two aspects of renal protection and antiapoptotic Qizhu granules DN treatment mechanism.In vitro experiments using high glucose stimulates the glomerular mesangial cells (MCs) damage model, Qizhu granules main ingredients, determined by MTT method, Specnuezhenide (SPE), Astragaloside IV (AS-IV), Hyperoside (HYP) after processing the survival rate of MCs, discuss the protective effect for MCs. the method of Enzyme-linked Immunosorbent Assay (ELISA) determination of Transforming growth factor-betal, Fibronectin, Collagen Type Ⅳ secretion level, using flow cytometry measure its influence on the MCs, apoptosis. western blot method to measure its protein level of caspase3, Bcl-2, Bax and the influence of protein level, explore the SPE, the AS-Ⅳ, HYP protective effect and its mechanism of MCs.Research content1. Qizhu granules effects on diabetic nephropathy rats.Injuried islet cells by STZ, high fat feed, kidney ligation three compound method to establish model of type 2 diabetic nephropathy, evaluation Qizhu granules renal protective effect of DN. Mainly includes:the 24 h urine Volume,24 h urine protein, urine creatinine (UCr), urea nitrogen, Blood urea nitrogen,, serum creatinine, serum creatinine, SCr), triglycerides (triglyceride, TG), cholesterol (total cholesterol, TC), Low density lipoprotein (Low density lipoprotein cholesterol, LDL-C), High density lipoprotein cholesterol, HDL-C) biochemical indexes such as detection. Wood grain-liquid eosin staining (Hematoxylin and eosin stain, HE) observed the kidney pathological morphological changes.2. Qizhu granules antiapoptotic mechanisms.Immunohistochemical staining in the diagnosis of renal caspase3, Bcl-2, Bax protein expression levels, in situ end labeling method (TdT-mediated dUTP Nick end labeling, TUNEL) determination of QZG anti diabetic nephropathy rat renal cell apoptosis.3. Qizhu granules protection mechanism study of high sugar stimulates the MCs.Determined by QZG SPE effective components, AS-IV determined by MTT method, in the role of HYP MCs largest non-toxic concentration and the experimental concentration.By CCK 8 method to detect sugar stimulation of glomerular mesangial cell proliferation.ELISA method to detect sugar stimulation of glomerular mesangial cells secrete TGF-beta 1, FN and Col-IV.Using flow cytometry technology FITC AnnexinV and PI double staining observation and stimulation of glomerular mesangial cell apoptosis rate.Western blot method to observe the sugar stimulation of glomerular mesangial cell apoptosis related proteins caspase3, Bcl-2, Bax expression.Research results1. Qizhu granules effect on DN rats (1) The effects on renal function:Qizhu granules can significantly the 24 h urine protein, urine creatinine vs model group,20 g · kg-1 Qizhu granules and valsartan on the improvement of the DN is best; The reduce in serum creatinine and urea nitrogen had a tendency to cut, but no significant difference. (2) The effects on blood lipids:Qizhu granules in reducing serum levels of TG, TC, LDL-C.20 g · kg-1 Qizhu granules and valsartan on the improvement of the DN is superior to other groups. (3) The effects on DN kidney pathological changes:Qizhu granules can effectively restrain the DN rat glomerular shrivel, glomerular cells decreases.2. Qizhu granules sustain rat diabetic nephropathy mechanism research (1) The effects on kidney tissues relative proteins:Qizhu granules can significantly improve the model group glomerular and renal tubular lesions, Qizhu granules can significantly reduce caspase3 and Bcl-2/Bax protein expression. (2) The effects of apoptotic cells in kidney tissue:Qizhu granules can significantly restrain the kidney tissue apoptosis.3. AS-IV, HYP and SPE,which are main ingredients of Qizhu granules, protection for the MCs damage induced by high glucose (1) The SPE largest non-toxic concentration is 125 μmol · L-1;The AS-IV in the experimental range safety non-toxic;HYP largest non-toxic concentration is 125 μmol · L-1,so we chooes 100 μmol · L-1,50 μmol · L-1 two concentration of the SPE, the AS-IV, the HYP experimental concentrations. Glucose concentrations of building choice tendency for 25 mmol·L-1, duration of 48 h. (2) The effects on glomerular mesangial cell proliferation:The influence of the SPE, the AS-IV, the HYP duration of 12 h,24 h,48 h,after incubation can significantly reduce the abnormal proliferation of MCs induced by high glucose, have clear concentration-response relationship. (3) The influence of glomerular mesangial cell secretion of TGF-beta 1 and ECM: the AS-IV, HYP incubation after 48 h of high glucose stimulate abnormal secretion of glomerular mesangial cells TGF-beta 1, FN and Col-Ⅳ has a significant inhibitory effect.SPE incubation after 48 h of high glucose stimulate abnormal secretion of glomerular mesangial cells TGF-beta 1, FN has a significantly inhibitory effect. (4) Effect on glomerular mesangial cell apoptosis:the Influence of the SPE, the AS-IV, the HYP can reduce high glucose stimulation of glomerular mesangial cell apoptosis rate in the model, significantly inhibiting apoptosis.The SPE, the AS-IV, HYP can raise caspase3 and Bcl-2/Bax protein expression level.Conclusions1. Qizhu granules have the function of the prevention and treatment of diabetic nephropathy rats, the role of link may be related to improve renal function, reducing blood lipid, inhibiting renal cell apoptosis.2. AS-IV, HYP and SPE,which are main ingredients of Qizhu granules, action on the MCs has protective effect induced by high glucose, its mechanism may be related to reduce TGF-beta 1, FN and Col-Ⅳ secretion, inhibit apoptosis and apoptosis related protein. |
