ABSTRACT
OBJECTIVE To investigate the mechanism of nicotinamide mononucleotide (NMN) on inflammation and fibrosis between endogenous nicotinamide phosphoribosyltransferase (NAMPT) and bone morphogenetic protein 7 (BMP-7) in diabetic glomerular cells.METHODS ① In vivo,spontaneous diabetic C57/BL6 mice and wild C57/BL6 mice were divided into two groups.When blood glucose was above (34.2±1.9) mmol· L-1,renal histology of diabetic mice became obvious.The protein expressions of Nampt and nuclear transcription factors-kappa B p65 (NF-κB p65),silent mating type information regulation 2 homolog 1 (SIRT1) and BMP7 were analyzed by lengths of immunofluorescence.② In vitro,rats' glomerular cells HBZY-1 were incubated with glucose 200 mmol· L-1 for different lengths of time (0,24,48 and 72 h) and at different concentrations of NMN (0,50,100 and 200 iμmol· L-1).The protein levels of Nampt and BMP7 were detected by Western blotting and the protein expressions of NF-κB p65 and α-SMA were measured by immunofluorescence assay.The protein levels of Nampt,BMP7 and NF-κB p65 were detected by Western blotting after HBZY-1 cells were treated with NMN 100 μmol· L-1 and FK866 10 μmol· L-1 for 24 h.RESULTS ① In vivo,the glomeruli of diabetic C57/BL6 mice showed obvious atrophy.Fluorescence intensity of Nampt was increased (P＜0.05),but that of BMP7 and SIRT1 in renal glomeruli cells was decreased compared with the wild type (P＜0.01).② In vitro,HBZY-1 cells were cultured in glucose 200 mmol· L-1 for 48 and 72 h.The protein expression of NAMPT was increased,but that of BMP7 was decreased (P＜0.05,P＜0.01).Expressions of NF-κB p65 and α-SMA were increased (P＜0.01) by immunofluorescence.The expression of BMP7 was increased after treatment with glucose 200 mmol· L-1,followed by NMN 50,100 and 200 μmol · L-1 for 24 h (P＜0.01).The expressions of NAMPT and NF-κB p65 were decreased (P＜0.01).The expressions of Nampt and NF-κB p65 in glucose 5.6 mmol· L1 +FK866 and glucose 5.6 mmol· L-1+ NMN groups were increased (P＜0.01),but the expression of BMP7 did not change.CONCLUSION Upregulation of endogenous Nampt obviously intervenes in BMP7 expression in the process of glomerular inflammatory fibrosis in severe diabetes.NMN can affect the protein expression of BMP7 via a special Nampt signaling pathway.
ABSTRACT
Objective:To investigate the regulation effect of endogenous nicotinamide phosphoribosyl transferase (Nampt) on the Vimentin expression of glomerular cells in high concentration glucose,and to clarify the mechanism of formation of diabetic kidney inflammation fibrosis.Methods:The C57/BL6 diabetic mice were selected and the kidney tissues were collected,and the wild C57/L86 mice were used as control group;the pathological section and tissue fluorescence staining were performed.The expression and location of endogenous Nampt and Vimentin in the glomerular cells were detected by immuno-focused technology.The HBZY-1 cells were randomly divided into 4 groups:low concentration of glucose (LG,0.56 mmol · L-1) control group,high concentration glucose (HG,200 mmol · L-1) group,HG +-FK866 group and HG+nicotinamide mononucleotide (NMN) group.In HG group,the cells were treated with FK866 (10 μmol · L-1) and NMN (1 mmol · L-1) for 24 h after cultured with HG for 5 d.The expression levels of Nampt,Vimentin,nuclear factor-kappa B p65 (NF-κBp65) and sirtuin type 1 (Sirt1) were detected by immunofluorescence and Western blotting methods.The expression levels of Nampt and Vimentin were detected by RT-PCR and Western blotting methods.Results:The shape and size of glomerulus had obvious atrophy of the mice in severe diabetic group compared with normal C57/BL6 mice group.The expression level of Vimentin in glomerular cells was increased with the increasing of endogenous Nampt (P＜0.01).When the HBZY-1 cells were cultured in HG condition,the exprssion levels of Nampt,Vimentin and NF-kB p65 were obviously increased while the Sirt1 expression levels was significantly decreased compared with control group (P＜ 0.01).The expression levels of Nampt,Vimentin and NF-κB p65 in glomerular cells in HG+FK866 HG+ NMN groups were singnificartyly decreased compared with control group (P＜0.01).Conclusion:The endogenous Nampt over-expression in glomerular cells can enhance the expression of Vimentin under high concentration of glucose stress through NF-κBp65 and Sirt1 signal pathway.
ABSTRACT
Objective:To investigate the regulation effect of endogenous nicotinamide phosphoribosyl transferase (Nampt) on the Vimentin expression of glomerular cells in high concentration glucose,and to clarify the mechanism of formation of diabetic kidney inflammation fibrosis.Methods:The C57/BL6 diabetic mice were selected and the kidney tissues were collected,and the wild C57/L86 mice were used as control group;the pathological section and tissue fluorescence staining were performed.The expression and location of endogenous Nampt and Vimentin in the glomerular cells were detected by immuno-focused technology.The HBZY-1 cells were randomly divided into 4 groups:low concentration of glucose (LG,0.56 mmol · L-1) control group,high concentration glucose (HG,200 mmol · L-1) group,HG +-FK866 group and HG+nicotinamide mononucleotide (NMN) group.In HG group,the cells were treated with FK866 (10 μmol · L-1) and NMN (1 mmol · L-1) for 24 h after cultured with HG for 5 d.The expression levels of Nampt,Vimentin,nuclear factor-kappa B p65 (NF-κBp65) and sirtuin type 1 (Sirt1) were detected by immunofluorescence and Western blotting methods.The expression levels of Nampt and Vimentin were detected by RT-PCR and Western blotting methods.Results:The shape and size of glomerulus had obvious atrophy of the mice in severe diabetic group compared with normal C57/BL6 mice group.The expression level of Vimentin in glomerular cells was increased with the increasing of endogenous Nampt (P＜0.01).When the HBZY-1 cells were cultured in HG condition,the exprssion levels of Nampt,Vimentin and NF-kB p65 were obviously increased while the Sirt1 expression levels was significantly decreased compared with control group (P＜ 0.01).The expression levels of Nampt,Vimentin and NF-κB p65 in glomerular cells in HG+FK866 HG+ NMN groups were singnificartyly decreased compared with control group (P＜0.01).Conclusion:The endogenous Nampt over-expression in glomerular cells can enhance the expression of Vimentin under high concentration of glucose stress through NF-κBp65 and Sirt1 signal pathway.
ABSTRACT
Objective To study the expressions of nicotinamide phosphoribosyl transferase (Nampt)in main energy metabolism organs (liver, pancreas, skeletal muscle, and kidney ) of the rats with type 2 diabetes mellitus (T2DM),and to explore the correlation between the expression and distribution of Nampt and the occurrence of diabetes.Methods The SD rat diabetes model was established by injecting with streptozotcin (STZ).The SD rats were randomly divided into diabetes group and control group. Immunohistochemical Envision staining assay was used to detect the distribution and protein expressions of Nampt in liver,pancreas,muscle,and kidney tissues of the rats,at the same time the blood glucose and serum insulin levels were also be detected. Results The blood glucose level of the rats in diabetes group was significantly higher than that in control group (P<0.01),and the fasting insulin level was lower than that in control group (P<0.01).The Nampt expression in the liver tissue of the rats in diabets group was significantly increased,which distributed near the hepatic sinus in diabetes rats,and the Nampt expression was also increased in skeletal muscle in which the whole cell was thick dying;the Nampt expression mainly distributed in the renal tubular epithelial cells. Compared with control group, the positive expression rates of Nampt in liver,skeletal muscle,and kidney tissues of the rats in diabets group were significantly increased (P<0.05 or P<0.01).There was nearly no Nampt expression in pancreas tissue of the rats in diabetes group and the Nampt expression level was significantly lower than that in control group (P<0.01). Conclusion The Nampt expressions are much different in main energy metabolic organs of the rats with diabetes.It is suggested that Nampt may be used as a specific indicative marker in the process of diabetes.
ABSTRACT
Objective To study the effects of benzyl propionate nandrolone (BPN ) on the nicotinamide phosphoribosyl transferase (Nampt), insulin receptor substeate-2 (IRS-2 )and pancreatic duodenal homeobox-1 (PDX-1)expressions, cell cycle changes as well as insulin secretion in pancreatic islet cell NIT-1 lines, and to explore the influence of BPN in the Nampte xpression in NIT-1 cells and insulin signaling molecules in high glucose oxidation stress.Methods The NIT-1 cells were cultured with different concentrations (5.6,11.1,16.7,and 27.6 mmol·L-1)of glucose,then they were treated with 10 mg·L-1 BPN for 48 h with no BPN treatment as corresponding control groups.The expression levels of Nampt,IRS-2,and PDX-1 were tested by Western blotting assay.The changes of cell cycle were determined by FCM and the cell insulin secretion levels were measured with radioimmunoassay.Results Compared with corresponding control groups,the expression levels of Nampt,IRS-2, and PDX-1 proteins in the NIT-1 cells in various BPM groups were increased (P<0.05 or P<0.01).The G0/G1 phase arrest was relieved (P<0.01)when the cells was cultured in low glucose (5.6 mmol·L-1 )condition,and the G2/M block was remitted significantly in high glucose (27.6 mmol·L-1 )condition (P<0.01),furthermore, the cell insulin secretion was promoted compared with control groups except 1 1.1 mmol· L-1 glucose group (P<0.01).Conclusion BPN can promote the expression levels of Nampt,ISR-2 and PDX-1 proteins in NIT-1 cells. There is close relationship between the Nampt expression in NIT-1 cells and insulin signaling pathway and BPN prevents the cells from insulin resistance.
ABSTRACT
The roles of NF-kappaB (NF-kappaB) expression, Bax activity and cytochrome C (Cyt C) release, apoptosis of islet cells induced by high concentration glucose were explored in vitro. Pancreatic islet cells, which were isolated from Kunming mice, were cultured with different concentrations of glucose in DMEM, and divided into the following groups: G1, G2, G3, G4, G5, and G6 groups, corresponding to the glucose concentrations of 5.6, 7.8, 11.1, 16.7, 22.5, and 27.6 mmol/L, respectively. After culture for 120 h, insulin secretion was evaluated by radioimmunoassay, and the NF-kappaB expression was detected by immunocytochemistry. Bax activity and Cyt C release were measured by immunofluorescence, and apoptosis was examined by Hoechst33342 assay. The results showed that in G1, G2 and G3 groups, insulin secretion was enhanced with the increase of glucose concentration, and the NF-kappaB expression was also increased (P<0.05), but Bax activity, Cyt C release and apoptosis rate showed no significant difference among them. However, in G4, G5, and G6 groups, apoptosis rate of islet cells, NF-kappaB expression, Bax activity, and Cyt C release were all significantly increased, and insulin secretion was impaired as compared with G1, G2, and G3 groups (P<0.05). It was concluded that the exposure of islet cells to high glucose could induce islet cells apoptosis as well as impaired insulin secretion. The NF-kappaB signaling pathway and mitochondria pathway in islet cells might play some roles in the progressive loss of islet cells in diabetes. The inhibition of the NF-kappaB expression could be an effective strategy for protecting pancreatic islet cells.
ABSTRACT
Objective To study the signal pathway of apoptosis of renal proximal tubular cells (RPTC) caused by cisplatin for prevention of toxic effects of cisplatin on kidney. Methods pcDNA3.1/Hygromycin vector and pEGFP-C3 vector including Bcl-2(Bcl-acta,Bcl-cb5 and Bcl-nt) were co-transfected in RPTC.After treated with cisplatin, Bax was activated,cytochrom C release and apoptosis were analyzed with confocal microscope and immunofluoresence technique. Apoptotic cells stained with Hoechst33258 were also counted and statistically analyzed. Results The percent of cytochrom C release (35.74%) in Bcl-cb5 transfected group was higher than those in Bcl-nt group (18.7%) and Bcl-acta group(24.6%)(P
ABSTRACT
Objective To explore the mechnism of toxicity induced by cisplatin on renal proximal tubular cells(RPTC) line and anti-apoptosis mechanism of BCL-2 with transfection of different mutant BCL-2 in vitro.MethodsRPTC was translocated with different mutants BCL-2(s).Cell apoptosis induced by cisplatin on RPTC was analyzed with confocal and flurencent microscope.The cell apoptosis was measured with Hoechst33258 after treatment with cisplatin.Results Different mutant BCL-2(BCL-acta,BCL-cb5)were translocated on mitochondrial and Endoplasmic Reticulum(ER) respectively.BCL-acta protected RPTC from apoptosis induced by cisplatin more easily than BCL-cb5 group in a time-dependant manner(P
ABSTRACT
Objective To study the function of Bak in mitochondrial signaling pathway and interaction between Bax and Bak during apoptosis.Methods Bax/Bak double knock out(Bax-/- and Bak-/-)MEF cells from mouse embryo fibroblasts(MEF) and Hela cells were divided into four groups according the cell different genotypes(wild type,Bax-/-,Bak-/- and double knock out) and treated with different chemical reagents after co-transfection with Bax,Bak,Mito-Red and empty pEGFP vector.Apoptosis,mitochondrial morphology and cytochrome C release were detected with confocal microscope,immunofluoresence and Western blotting techniques.Results There were correlations between the percentage of Hela cell apoptosis and mitochondrial fission(%) as well as cytochrome C(%)(P