Renoprotection Of Berberine On Diabetic Nephropathy Rats And The Effect On The AGEs-RAGE Signal Pathway In Rat's Kidney

Aim: In the vivo experiments on the SD rats,to observe the effects of berberine on renal histopathological and biochemical indexes of renal function in diabetic nephropathy rats,to investigate the protective effect of BBR on renal function and pathological changes in DN rats.To detect the expression of signal proteins secreted by some tissues and cells in the kidney of rats and the regulation of the corresponding pathway and the changes which affected by the BBR administration.Explore the renal protective effect of BBR is generated by what mechanisms.In vitro cell test,stimulation of mesangial cells and podocytes by simulating DN conditions,and then,to detect abnormal changes of AGEs-RAGE signal pathway in mesangial cells and regulatory changes of the signaling pathway VEGF-VEGFR2 interacting with podocytes and mesangial cells.At the same time,we observed the effects of BBR,the corresponding stimulus and blocking agent administration.To further investigate the protective effect of BBR on DN and its potential molecular mechanisms and important molecular targets,to provide a theoretical basis for the rational development and utilization of BBR in the prevention and treatment of DN.Moreover,offering new research directions and ideas for explore the drugs of DN treatment.Methods: Preparation of DN rat model,induced by long term high glucose and high fat diet combined with low dose of streptozotocin(STZ,35mg/kg)intraperitoneal injection.Measurement DN rats fasting blood glucose(FBG)level after injection 72 h,SD rats with FBG greater than or equal to 11.1mmol/L were included in the DN model group.The experimental animals were grouped as follows: normal group,DN model group,BBR low dose group(50mg/kg),BBR medium dose group(100mg/kg),BBR high dose group(200mg/kg),metformin group(200mg/kg)and captopril group(15mg/kg),12 rats in each group.The normal group rats were fed with normal diet,model group and drug group to continue to give high sugar and high fat diet,and free drinking water all group.All drug groups according to the body weight of rats once daily intragastric administration,normal group and model group were given the same solvent Sodium carboxymethylcellulose solution according to the weight parallel group gavage.FBG values were measured every two weeks,weight was measured once a week,the day before the rats were killed to collect 24 h urine to test the content of total urinary protein and urine creatinine.Blood samples were collected from the femoral artery after anesthesia.Blood urea nitrogen,serum creatinine and AGEs were detected by serum separation.The ipsilateral kidney was weighed and the renal pathological examination was performed,including HE staining and PAS staining.Immunohistochemical method was used to observe the distribution and expression of AGEs,RAGE,TGF-?1 and PKC-? in the renal cortex;western blot method was used to detect the expression of signal pathway proteins in renal cortex;enzyme linked immunosorbent assay(ELISA)was used to detect the expression level of AGEs in serum of each group.Glomerular mesangial cells and podocytes were studied in vitro.First,the rat mesangial cells and podocytes were isolated for primary culture and subculture,establishment of DN model cell line.The effect of BBR on proliferation of mesangial cells and podocytes in DN state by CCK-8 Kit.Detection of AGEs secretion in mesangial cells by ELISA.For proteins from the AGEs-RAGE pathway,first,the western blot method was used to observe the expression and changes of the signal proteins under the normal and the model condition and after various concentration of BBR administration,AGEs stimulation and Ab-RAGE blocking.The distribution,expression types and changes of RAGE protein were observed by laser scanning confocal microscope.To the VEGF-VEGFR2 signal pathway proteins.First,the protein chip was used to detect the secretion of podocytes in DN state,and the key factors were determined.Western bolt method to verify the change of VEGF,the distribution and expression of protein was determining by laser scanning confocal microscope.CCK-8 method for determining the optimal concentration of VEGF stimulate in mesangial cells.The expression of phosphorylated VEGFR2 were observed by western bolt method under the condition of DN,high glucose,BBR treatment,VEGF protein and podocyte supernatant stimulation.Distribution and expression of p-VEGFR2 by laser scanning confocal microscope.Results: 1.Effects of BBR on body weight and fasting blood glucose in DN rats Compared with the normal group,the fasting blood glucose level of DN rats was significantly increased after the model was successful.There was no significant hypoglycemic effect in each BBR treatment group in the previous four weeks.In 6 to8 weeks,the FBG of the middle dose and high dose of BBR were significantly lower than that of DN model group.The metformin group significantly reduced blood glucose after 4 weeks.For weight,only high dose group had improvement in eighth weeks.2.Effects of BBR on renal function and biochemical indexes in DN rats Compared with the normal group,the kidney weight/body weight ratio,urinary protein/creatinine ratio,blood urea nitrogen and serum creatinine were significantly higher in the DN group.The middle dose BBR group and the high dose BBR group,and the positive drug metformin group and the captopril group can significantly improve renal function and reduce biochemical indicators.3.Effects of BBR on renal histopathology in DN rats The histopathological examination showed that,compared with the normal group,model group rat's kidney emerges glomerular hypertrophy,hyalinization,basement membrane thickened,extracellular matrix accumulation,more serious glomerular sclerosis,fibrosis and associated with a large number of inflammatory cell infiltration.When DN rats were treated with the middle dose and high dose of BBR,the above symptoms were significantly improved,positive drug metformin group and captopril group also has obvious effect to relieve symptoms.4.Effects of BBR on the distribution and expression of AGEs,RAGE,p-PKC-?and TGF-?1 protein in renal tissues of DN rats The results of immunohistochemistry showed in DN rat kidney tissue,AGEs diffuse distribution throughout the glomerulus and its surrounding;RAGE was mainly distributed on the cell membrane of the mesangial cells,and few scattered in the cytoplasm;The cytoplasm,nucleus and cell membrane all of them p-PKC-? has distributed in,but mainly overexpressed in cytoplasm;TGF-?1 is mainly located in extracellular fluid and extracellular matrix.Immunohistochemistry showed proteins expression that compared with the normal group,the expression of above proteins was significantly increased in the kidney tissue of DN model rats.In the BBR administration group,low dose of BBR can effectively reduce the expression of abnormal protein,the middle dose and high dose of BBR can more significantly improve and reduce the excessive production.Based on the above results,the western bolt method was used to verify the results.It was found that the all dose BBR could reduce the over expression of the proteins.Among them,the low dose BBR was decreased the expression of TGF-?1,but not statistically significant.The effect of middle dose BBR was the most obvious on p-PKC-?.Influence of BBR on all proteins,middle and high dose of BBR effect was stronger than that of the low dose of BBR.5.Effects of BBR on the growth and proliferation of glomerular mesangial cells and podocytes CCK-8 kits assay showed that the over proliferation of mesangial cells in DN state.Since 15 ?mol/L,BBR has a significant inhibitory effect on the proliferation of mesangial cells.The IC50 of mesangial cells by BBR is about 73 ?mol/L.According to the results,the concentration of the subsequent mesangial cells tests was 30 ?mol/60 ?mol/L and 90 ?mol/L.But for podocytes,the number of podocytes decreased significantly under DN,when the concentration of BBR was 60 ?mol/L,the absorbance of podocytes was the highest,at this concentration and before,BBR had significant protective effect on podocytes.And then,with the increase of BBR concentration,the absorbance decreased gradually,it is speculated that the cytotoxicity of BBR may increase with the increase of concentration,which may lead to the damage of podocytes.Therefore,the follow-up of the BBR concentration of podocytes was determined to be 30 ?mol/L,60 ?mol/L and 90 ?mol/L.6.Effects of VEGF on the growth and proliferation of glomerular mesangial cells The results of CCK-8 assay showed the excessive proliferation of mesangial cells induced by high glucose and VEGF.When the concentration of VEGF reached 2ng/ml,the proliferation of mesangial cells reached the highest value.And then,with the increase of VEGF concentration,the proliferation of mesangial cells was inhibited.7.Effect of BBR on the level of AGEs in the rats serum and supernatant of mesangial cells The results of ELISA assay showed that the AGEs content in DN model rats serum and the supernatant of mesangial cells induced by high glucose was significantly higher than that in normal group.Each dose and concentration of BBR had significant inhibitory effect.Among them,the effect of BBR in middle dose and high dose group was stronger than that in low dose group.8.Effect of BBR on AGEs-RAGE pathway in mesangial cells The results of western bolt showed that the expression of AGEs,RAGE,p-PKC-? and TGF-?1 in mesangial cells under the DN state were significantly higher than those in normal group.Different concentrations of BBR had significant inhibitory effect.Among them,the middle dose BBR group had the strongest inhibitory effect.When AGEs stimulated mesangial cells,the expression of RAGE,p-PKC-? and TGF-?1increased significantly,but BBR had no inhibitory effect at this time.And blocking agent Ab-RAGE can significantly reduce the expression of p-PKC-? and TGF-?1.The results of laser scanning confocal microscope showed that RAGE was mainly expressed as the membranous protein in mesangial cell under DN state.At the same time,the expression of RAGE on the cell membrane was significantly higher than that in the normal group.And BBR can effectively inhibit the over expression of membrane RAGE.9.Effects of BBR on VEGF-VEGFR2 pathway between podocytes and mesangial cells The results from the protein chip was used to detect the supernatant of podocytes showed,the expression level of VEGF was significantly increased in DN.The results of western bolt assay showed that the VEGF protein secreted by podocytes in model group was significantly higher than that in normal group,BBR administration group can effectively reduce the content of VEGF.The expression of p-VEGFR2 in mesangial cells induced by high-glucose was increased obviously compared with normal group.And after VEGF stimulation,the p-VEGFR2 of mesangial cells was increased more significantly,different concentrations of BBR have different degrees of improvement.The expression of p-VEGFR2 was significantly increased in the mesangial cells stimulated by the supernatant of podocytes,both BBR and VEGF inhibitors can effectively inhibit the over expression of p-VEGFR2.The results showed that the distribution of VEGF was diffuse in podocytes was detected by confocal laser scanning method,it is a secretory factor.P-VEGFR2 is mainly distributed on the cell membrane.BBR can significantly reduce the phosphorylation of VEGFR2.Conclusions: 1.BBR can effectively improve the renal function of DN rats,reduce blood glucose,protect the kidney and improve the renal pathology in DN rats.2.The downstream factors of AGEs-RAGE pathway in rat kidney including PKC-?and TGF-?1 under the DN condition,and the whole AGEs-RAGE-PKC-?-TGF-?1pathway plays an important role in DN.BBR can significantly affect this pathway,the important target factor is AGEs.3.In the DN state,the podocyte affects the mesangial cells by secreting VEGF and activates the VEGFR2 on the cell membrane,thus affecting the development of DN.BBR can effectively decrease the secretion of VEGF and inhibit the over phosphorylation of VEGFR2,and then affect the whole VEGF-VEGFR2 pathway to protect the kidneys.