| Diabetes mellitus (DM) is a common endocrine metabolic disease, which clinical manifestations are including high blood sugar, high blood fat and progressive renal damage and so on. Diabetic nephropathy (DN) is a serious endocrine disorder caused by diabetes mellitus, is one of the largest harmfulness microvascular complications and main cause of end-stage renal failure (ESRD).Berberine (BBR) is mainly used in the treatment of intestinal infection such as diarrhea. Except for using as plant antimicrobial, BBR could reduce increased blood glucose level, regulate blood lipid level, relaxed blood vessels endothelium-dependent, reduce inflammation, improve insulin resistance etc.Podocytes, which mainly through podocyte foot process attach to glomerular basement membrane (GBM), are combined with endothelial cells and glomerular basement membrane constitute the glomerular filtration barrier, maintaining the integrity of the glomerular filtration barrier structure and function. Related literature and our previous research study showed that BBR could exert renoprotective effects on DN rats, the occurrence and development of DN are closely related to podocyte damage, which characteristic protein expression are including nephrin, podocin and adhesion molecule alpha 3 beta 1 integrin, so it is appriciate to explore the connection between the abnormal expression of podocyte surface characteristic protein and podocyte injury as well as the effects of BBR, which is beneficial for clinical prevention and treatment of DN, but at present the internal relationship between them are vapoury.Our research object is focused on DN rats model, we want to observe the effects of BBR on the rats overall index changes and the expression of podocyte characteristic proteins in kidney cortex tissue. At the same time, we want to discusses the role of BBR on the improvement of podocyte damage and explore its possible mechanism.Objective:In the vivo experiments, we observe the DN rat kidney overall index changes and the expression of of podocyte surface characteristic protein nephrin, podocin and adhesion molecule alpha 3 beta 1 integrin to explore the protective effect of BBR on DN rat kidney and its potencial mechanism. In vitro experiments, we used high glucose and TGF beta 1 induced podocyte damage, to detect the expression of podocyte characteristics protein, p-Akt and ILK protein expression, to explore the internal relationship between renal protective effects of BBR in DN rats and PI3K/Akt pathway, so as to provide theoretical basis for reasonable applyment of BBR in prevention and treatment for DN.Methods:We focused on SD rats, feeding with high glucose, high lipid diet combination with low dosage of streptozotocin (STZ,35 mg/kg). After 72 h induction, we divided rats of fasting blood glucose (FBG) values greater than 11.1 mol/L into experimental group, the experimental group are as follows:The successfully established experimental diabetic rat models were randomly divided as into 6 groups (10 rats in each group):DN rats group; low dosage of BBR (50 mg/kg) group; medium dosage of BBR (100 mg/kg) group; high dose of BBR (200 mg/kg) group; metformin (1 mg/kg) group and captopril (15 mg/kg) group as positive group, which continued to fed with high glucose and high fat feed. In addition, we also set up a normal control group (NC group). We have determined the overall indexes of DN rats, including blood glucose levels, renal function indexes, renal pathology examination including HE staining and PAS staining and glomerular ultrastructure observation. We adopted immunohistochemical method to detect distribution and protein expression of nephrin, podocin and alpha 3 beta 1 integrin of rat renal cortical, We also adopted western blot method to detect the expression level of nephrin, podocin, alpha 3 beta 1 integrin protein, p-Akt, Akt, ILK and TGF beta 1 of rat renal cortex.In vitro experiments we use podocyte as the research object, the related experiments groups are as following:Podocyte were grouped to:Normal control group (5.5 mmol/L glucose, NG group), high glucose+TGF beta 1 stimulation group (30 mmol/L glucose +5 ng/ml TGF beta 1, HG+TGF beta 1 group), BBR treatment group (15μM,30μM and 60μM), LY294002 intervention group (HG+TGF beta 1+LY294002 (40μM) group). Podocyte were identified by morphologic observation, immunofluorescence experiments and immunohistochemical experiments. Cell adhesion experiments were carried out to detect podocyte adhesion function. We used ELISA method to detect the effect of BBR on TGF-beta 1 expression in glomerular mesangial cells supernate. Protein-chip technique method was applied to detect the inflammatory cytokines expression levels in glomerular mesangial cells. Laser confocal method (LSCM) was used to detect the expression and distribution of TGF beta RI and TGF beta RⅡ in podocyte. Flow cytometry detection was used to detect podocyte apoptosis. Western blot method was employed to detect the expression of podocyte related protein.Results:1. The effects of BBR on the overall indexes of DN ratsCompared with NC group, the levels of blood glucose, serum blood urea nitrogen (BUN) and serum creatinine (SCr) of DN model group rats are increased significantly. BBR (100 mg/kg and 200 mg/kg) treatment group could significantly reduce blood glucose levels and BUN and SCr levels of DN rats. HE and PAS staining results showed that BBR (100 mg/kg and 200 mg/kg) treatment group could improve the abnormal changes of DN rat kidney to some degree, which could lower the hypertrophy of glomerular, decrease hyperplasia of GBM significantly, reduce extracellular matrix deposition. Electron microscope results showed that after BBR treatment, GBM structure was distinct, irregular thickened of GBM was reduced significantly as well as podocyte foot process fusion, but foot process segment fusion structure and disordered arrangement were also existed.2. The effects of BBR on distribution and expression of nephrin, podocin, alpha 3 beta 1 integrin, p-Akt, Akt, ILK and TGF beta RI protein in kidney tissue of DN ratsNephrin, podocin and alpha 3 beta 1 integrin protein were distributed evenly in the glomerular podocyte of normal kidney tissue, compared with NC group, kidney the expression of nephrin and podocin and alpha 3 beta 1 integrin protein are decreased significantly DN model group rats, p-Akt, ILK and TGF beta RI protein expression level increased significantly. BBR (100 and 200 mg/kg) treatment group could significantly increased the protein expression of nephrin and podocin and alpha 3 beta 1 integrin. High dosage of BBR (200 mg/kg) group could significantly reduce the p-Akt expression level, BBR medium and high dosage (200 mg/kg) group could significantly reduce the expression level ILK and TGF betaRI protein.3. Podocyte identificationBright field microscopy results showed that the microscopic observation of cell growth early after batches for no obvious bulge cells, closely arranged a cobblestone appearance,3 to 5 days later, the visible part of the cell cytoplasm extended, cells had obvious feet chug, connected to each other between foot process, after growth gradually merged, cells growth became retardation. Moreover, GLEPP1, podocin, nephrin and alpha 3 beta 1 integrin were expressed positive staining of cultured cells, the staining of desmin protein were negative, which could infer the cells for podocyte.4. The effects of BBR on podocyte adhesion ability and podocyte apoptosisHG plus TGF beta 1 stimulation could significantly reduce podocyte adhesion function, BBR treatment group (15μM,30μM, μM 60 and 90 μM) could improve the adhesion function of podocyte at different level; Compared with normal group, the HG plus TGF beta 1 stimulation group could significantly increase the rate of podocyte apoptosis, BBR (30μM and 60μM) treatment group could significantly reduce the podocyte apoptosis rate.5. The expression level of inflammatory factor in glomerular mesangial cells supernatantUnder the culture condition in vitro, DN glomerular mesangial cells could secrete excessive TGF beta 1, IL-6, IL-8, TNF alpha and MCP-1 significantly, while the expression of IL-alpha, IL lbeta, IL-14, IL-10, IL-13 and IFN-gamma have no apparent influence.6. The effects of BBR on the expression of nephrin, podocin, alpha 3 beta 1 integrin protein of podocyteCompared with normal group, the HG plus TGF beta 1 stimulation group could significantly reduce the expression of nephrin, podocin and adhesion molecules alpha 3 beta 1, BBR high concentration treatment group (60μM) and LY294002 treatment group could obviously increase the nephrin, podocin and alpha 3 beta 1 level, BBR medium concentration treatment group (30μM) could raise alpha 3 beta 1 integrin expression level.7. The effects of BBR on the expression of p-Akt, Akt, ILK and TGF beta RI protein of podocyteCompared with normal group, the HG plus TGF beta 1 stimulation group could significantly improve the expression level of p-Akt of podocyte, and BBR (60μM) and LY294002 treatment group could obviously reduce the expression level of p-Akt, BBR (30μM) treatment group could also obviously reduce the expression level p-Akt of podocyte. Compared with normal group, the HG plus TGF beta 1 stimulation group could significantly improve ILK of podocyte and the expression of TGF betal RI level, and BBR treatment group (30μM and 60μM) could significantly reduce ILK of podocyte and the expression of TGF betal RI level. But LY294002 treatment group could not influence the expression of ILK and TGF beta RI of podocyte significantly.Conclusions:1. BBR (100 and 200 mg/kg) could improve the overall indexes of DN model rats, upregulate the expression of podocyte protein nephrin, podocin and alpha 3 beta 1 protein of renal cortical tissue in DN rats, which infer that BBR exert renoprotective effects in DN rats partly through protecting podocyte;2. Under DN condition, glomerular mesangial cells could secret superfluous inflammatory cytokines such as TGF beta 1, IL-6, IL-8, TNF alpha and MCP-1, which may be related to accelerating podocyte injury.3. BBR could improve podocyte injury induced by high glucose and TGF beta 1, uprugulate the expression of podocyte surface characteristic proteins such as nephrin, podocin and alpha 3 beta 1 integrin and descend excessive expression of p-Akt protein, which suggest that the possible mechanism of BBR relieve podocyte damage might be associated with PI3K/Akt pathway, so that BBR could exert renoprotective effects in DN rats. |
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