Inhibitory Effect of Resveratrol on LPS-induced Glomerular Mesangial Cells Proliferation and TGF-β1 Expression via Sphingosine Kinase 1 Pathway / 中国结合医学杂志

OBJECTIVE@#To elucidate the renoprotective effect of resveratrol (RSV) on sphingosine kinase 1 (SphK1) signaling pathway and expression of its downstream molecules including activator protein 1 (AP-1) and transformation growth factor-β1 (TGF-β1) in lipopolysaccharide (LPS)-induced glomerular mesangial cells (GMCs).@*METHODS@#The rat GMCs line (HBZY-1) were cultured and randomly divided into 5 groups, including control, LPS (100 ng/mL), and 5, 10, 20 µmol/L RSV-treated groups. In addition, SphK1 inhibitor (SK-II) was used as positive control. GMCs were pretreated with RSV for 2 h and treated with LPS for another 24 h. GMCs proliferation was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The proteins expression of SphK1, p-c-Jun and TGF-β1 in GMCs were detected by Western blot, and DNA-binding activity of AP-1 was performed by electrophoretic mobility shift assay (EMSA). The binding activity between RSV and SphK1 protein was detected by AutoDock Vina and visualized by Discovery Studio 2016.@*RESULTS@#LPS could obviously stimulate GMCs proliferation, elevate SphK1, p-c-Jun and TGF-β1 expression levels and increase the DNA-binding activity of AP-1 (P<0.05 or P<0.01), whereas these effects were significantly blocked by RSV pretreatment. It was also suggested that the effect of RSV was similar to SK-II (P>0.05). Moreover, RSV exhibited good binding affinity towards SphK1, with docking scores of -8.1 kcal/moL and formed hydrogen bonds with ASP-178 and LEU-268 in SphK1.@*CONCLUSION@#RSV inhibited LPS-induced GMCs proliferation and TGF-β1 expression, which may be independent of its hypoglycemic effect on preventing the development of mesangial cell fibrosis and closely related to the direct inhibition of SphK1 pathway.

Effect of salvianolic acid B on high glucose-induced phenotypic transition and extracellular matrix secretion in human glomerular mesangial cells / 中国病理生理杂志

AIM:To investigate the effect of salvianolic acid B (Sal B) on high glucose-induced phenotypic transition and extracellular matrix (ECM) secretion in human glomerular mesangial cells (HGMCs) and the underlying mechanisms.METHODS:HGMCs were randomly divided into control group, high glucose group and high glucose plus high dose, medium dose and low dose of Sal B groups.The HGMCs except those in control group were exposed to high glucose (33.3 mmol/L) for 72 h, while those in Sal B groups were co-incubated with indicated concentrations of Sal B.The protein levels ofα-smooth muscle actin (α-SMA) , transforming growth () and phosphorylated Smad2 and p38 mitogen-activated protein kinase (MAPK) were determined by Western blot.The secretion levels of collagen type I (Col I) , collagen type III (Col III) , fibronectin (FN) and laminin (LN) were measured by ELISA.RESULTS:Exposure to high glucose markedly increased the protein expression ofI, Col III, FN and LN in the HG-MCs (P<0.01).The phosphorylation levels of Smad2 and p38 MAPK were also significantly increased (P<0.01).Coincubation with Sal B evidently decreased the protein expression ofI, Col III, FN and LN in the HGMCs induced by high glucose (P<0.05 or P<0.01).The phosphorylated levels of Smad2 and p38 MAPK were also reduced noticeably (P<0.05 or P<0.01).CONCLUSION:Sal B significantly suppresses high glucose-induced phenotypic transition and ECM secretion in the HGMCs, which might be attributed, at least partly, to inhibition ofSmad signaling pathway and p38 MAPK activation.

Tea polyphenols delays human glomerular mesangial cells senescence induced by high glucose via regulating STAT3/miR-126/telomere signaling pathway activation / 中国中药杂志

The aim of this paper was to explore the effects and possible mechanisms in vitro of tea polyphenols (TP) delaying human glomerular mesangial cells (HGMCs) senescence induced by high glucose (HG). HGMCs were cultured in vitro and divided into the normal group (N, 5.5 mmol·L⁻¹ glucose), the mannitol group(MNT, 5.5 mmol·L⁻¹ glucose plus 24.5 mmol·L⁻¹ mannitol), the high dose of D-glucose group (HG, 30 mmol·L⁻¹ glucose), the low dose of TP group (L-TP, 30 mmol·L⁻¹ glucose plus 5 mg·L⁻¹ TP) and the high dose of TP group (H-TP, 30 mmol·L⁻¹ glucose plus 20 mg·L⁻¹ TP), which were cultured in 5% CO₂ at 37 °C, respectively. Firstly, the effects of TP on the cell morphology of HGMCs were observed after 72 h-intervention. Secondly, the cell cycle, the positive rate of senescence-associated-β-galactosidase (SA-β-gal) staining and the telomere length were detected, respectively. Finally, the protein expressions of p53, p21 and Rb in the p53-p21-Rb signaling pathway were investigated, respectively. And the expressions of p-STAT3 and miR-126 were examined severally. The results indicated that HG not only arrested the cell cycle in G₁ phase but also increased the positive rate of SA-β-gal staining, and shortened the telomere length. HG led to the protein over-expressions of p53, p21 and Rb and HGMCs senescence by activating the p53-p21-Rb signaling pathway. In addition, L-TP delayed HGMCs senescence by improving the cell cycle G₁ arrest, reducing SA-β-gal staining positive rate and lengthening the telomere length. L-TP reduced the protein over-expressions of p53, P21 and Rb induced by HG and inhibited the telomere-p53-p21-Rb signaling pathway. Moreover, the expression of p-STAT3 was increased and the expression of miR-126 was decreased in HGMCs induced by HG. L-TP reduced the expression of p-STAT3 and increased the expression of miR-126 in HGMCs. In conclusion, HG could induce HGMCs senescence by activating the telomere-p53-p21-Rb signaling pathway in vitro. L-TP could delay HGMCs senescence through regulating STAT3/miR-126 expressions and inhibiting the telomere-p53-p21-Rb signaling pathway activation. These findings could provide the effective interventions in clinic for preventing and treating renal cell senescence in diabetic kidney disease.

Effects of astragaloside-IV on the expression of inflammatory factor and proliferation of glomerular mesangial cells induced by angiotensin Ⅱ / 中国应用生理学杂志

OBJECTIVE@#To study the effects of astragaloside-IV (As-IV) on the expression of inflammatory factor and proliferation of glomerular mesangial cells (GMCs) induced by angiotensin Ⅱ(AngⅡ).@*METHODS@#The model of diabetic nephropathy(DN) was mimic by angiotensin Ⅱ (10mol/L)inducing GMCs injury. Then the GMCs were treated with As-IV at different concentrations(25,50,100 μmol/L)for 48 hours. The proliferation of GMCs was detected by MTT. The level of reactive oxidative species (ROS) was measured by flow cytometry. The expression of monocyte chemoattractant protein-1(MCP-1) protein in supernatant was detected by enzyme- linked immunosorbent assay (ELISA). The expression of transforming growth factor-β1(TGF-β1) in GMCs was measured by Western blot.@*RESULTS@#Compared with model group, the proliferation of GMCs was inhibited in As-IV group. As-IV decreased the level of intercellular ROS, down-regulated the secretion of MCP-1 and the expression of TGF-β1 proteins.@*CONCLUSIONS@#As-IV could inhibit cell proliferation and inflammatory factors expression on GMCs induced by AngⅡ.

Expression and function of Orai1-BK Ca complex in high glucose treatment rat glomerular mesangial cells / 中国药理学通报

Aim To investigate whether Orai1 and BKCa form a physical and functional signal complex in glomerular mesangial cells ( GMCs) and reveal the role of high glucose treatment on Orai1-BKCa complex and SOCE-induced hyperpolarization. Methods The in-teraction of Orai1 and BKCa was evaluated by co-immu-noprecipitation ( co-IP ) . Western blot method was used to detect the expression levels of Orai1 and BKCa . SOCE was activated by Ca2+ depletion evoked by TG in normal glucose and high glucose. The DiBAC4(3) was employed as fluorescence indicator to measure the potential change of membrane. . Results The co-IP experiment results showed that Orai1 interacted with BKCa in GMCs. SOCE induced the hyperpolarization of GMCs. SOCE and SOCE-induced membrane hyperpol-orizaiton were enhanced by high glucose treatment for three days. In addition, the expression levels of Orai1 and BKCa were enhanced significantly in the high glu-cose-cultured cells. Conclusions Orai1 can form a signaling complex with BKCa , which participates in the regulation of hyperpolarization in GMCs and may be in-volved in the hyperpolarization in high glucose cultured GMCs.

Paeonol up-regulates CKIP-1 to resist high glucose-induced fibrosis in glomerular mesangial cells / 中国药理学通报

Aim To observe whether paeonol can in-hibit fibronectin (FN) and intercellular cell adhension molecule-1 (ICAM-1) expressions in high glucose (HG)-induced glomerular mesangial cells(GMCs) via up-regulating CKIP-1 and activating the Nrf2 signaling pathway. Methods The effects of paeonol on the ex-pressions of CKIP-1,Nrf2,FN and ICAM-1 were eval-uated in GMCs treated with HG. Small interfering RNA was used to deplete CKIP-1 protein expression, and Western bolt was used to detect the expressions of Nrf2, HO-1 and SOD1. DHE fluorescent probe tech-nique was used to determine intracellular superoxide level. Results The protein levels of CKIP-1 and Nrf2 were elevated by paeonol in HG-treated GMCs. In the meanwhile,the expressions of Nrf2 downstream antiox-idant enzymes, i.e. HO-1 and SOD1, were also up-regulated by paeonol, which was accompanied by re-ductions of superoxide and H2O2levels. Importantly, paeonol reversed the excessive accumulation of FN and ICAM-1 in HG-induced GMCs. si-CKIP-1 decreased the up-regulation of Nrf2,HO-1 and SOD1 expressions during paeonol treatment, which was accompanied by increased superoxide and H2O2levels. Furthermore, si-CKIP-1 reversed the down-regulated levels of FN and ICAM-1 induced by paeonol. Conclusion Pae-onol inhibits the expressions of FN and ICAM-1 in HG-treated GMCs possibly by up-regulating CKIP-1 and activating the Nrf2 signaling pathway.

miR-451 inhibits inflammatory responses in glomerular mesangial cells by targeting Psmb8 in diabetic nephropathy mice / 中国病理生理杂志

AIM:To investigate the effect of microRNA(miR)-451 by targeting proteasome subunit βtype 8 (Psmb8)on the inflammatory responses in mouse glomerular mesangial cells(MCs)under high-and low-glucose condi-tions.METHODS:The expression levels of miR-451,IL-18 mRNA and TNF-αmRNA were detected by qPCR.The pro-tein expression levels of IL-18,TNF-αand Psmb8 were determined by Western blot when miR-451 was over-expressed and down-expressed in the MCs.Moreover,the expression of IL-18 and TNF-αwas detected when Psmb8 was silenced by si-Psmb8 in MCs.RESULTS:The expression of miR-451 was significantly decreased in the MCs treated with high glucose compared with low glucose group(P<0.01).However,the expression of Psmb8 was increased in high glucose group as compared with low glucose group(P<0.01).Moreover, the expression levels of Psmb8, IL-18 and TNF-αwere signifi-cantly decreased when miR-451 was over-expressed in high glucose group(P<0.01).Additionally,the expression levels of IL-18 and TNF-αwere significantly reduced when Psmb8 was silenced in the MCs under high glucose condition.CON-CLUSION:miR-451 reduces the expression of inflammatory factors via targeting Psmb8 in the MCs under high glucose condition.Therefore,miR-451 may play a role in inflammation of diabetic nephropathy.

Effect of hydrogen molecule on apoptosis-related proteins and Nrf2 signa-ling pathway in glomerular mesangial cells cultured with high glucose / 中国病理生理杂志

AIM:To investigate the role of hydrogen molecule on apoptosis-related proteins in glomerular me-sangial cells cultured with high glucose and to explore its possible mechanism.METHODS:Mouse glomerular mesangial cells cultured in vitro were divided into 4 groups:normal control group(C group,5.5 mmol/L glucose),mannitol group(G group,5.5 mmol/L glucose+19.5 mmol/L mannitol),high glucose group(H group,25 mmol/L glucose),high glu-cose+hydrogen-rich water group(HH group,25 mmol/L glucose+hydrogen-rich water),and cultured for 48 h.The pro-tein levels of Bax,Bcl-2,cleaved caspase-3,nuclear factor E2-related factor-2(Nrf2),heme oxygenase-1(HO-1)and NAD(P)H:quinone oxidoreductase-1(NQO-1)were determined by Western blot ,and the mRNA expression of HO-1 and NQO-1 was determined by RT-PCR.The level of intracellular reactive oxygen species(ROS)was detected by dihydro-ethidium method,and the activity of superoxide dismutase(SOD)was measured by WST-8 assay.RESULTS:Compared with C group,the protein levels of Bax and cleaved caspase-3 were up-regulated,and Bcl-2 was down-regulated in H group(P <0.05).No significantly difference of the protein levels mentioned above between C and HH group was observed. Compared with H group,the protein levels of Bax and cleaved caspase-3 were down-regulated,and Bcl-2 was up-regulated in HH group(P <0.05).The level of intracellular ROS was higher and the activity of SOD was lower in H group than those in C group(P<0.05).However,there was no difference of the SOD activity between C group and HH group.The level of intracellular ROS decreased and the activity of SOD increased in HH group as compared with H group(P<0.05). Compared with C group,clearly reduced protein expression of Nrf2,HO-1 and NQO-1,and decreased mRNA expression of HO-1 and NQO-1 in H group were observed(P<0.05).Compared with H group,the protein levels of Nrf2,HO-1 and NQO-1 as well as the mRNA levels of HO-1 and NQO-1 were obviously increased in HH group(P<0.05 ).CONCLU-SION:Hydrogen molecule inhibits the expression of pro-apoptotic proteins and induces the expression of anti-apoptotic pro-teins in glomerular mesangial cells cultured with high glucose.The mechanism may be related to activation of Nrf 2 signaling pathway.

Effects of pirfenidone on renal fibrosis in mice with diabetic nephropathy and its mechanisms / 中华肾脏病杂志

Objective To investigate effects of pirfenidone (PFD) on diabetic nephropathy model in db/db mice and to explore its possible mechanisms.Methods (1) Wild-type mice were as the normal control group,and db/db mice were divided into model group and PFD group,with 6 mice in each group.In the PFD group mice were administered continuously by 250 mg· kg-1· d-1 PFD for 18 weeks,and mice in the other two groups were administered by 0.5％ sodium carboxymethyl cellulose.Blood glucose and 24 h urinary albumin were measured.The pathological changes of renal tissue were evaluated by PAS staining,PASM staining,Masson staining and Sirius red staining.The expression of collagen type Ⅳ in kidney tissues was detected by immunohistochemistry.(2) Mouse mesangial cells (SV40 MES-13 cells) were cultured as research objects.They were divided into control group,hyperosmolar group,high glucose (HG) group,and 50,100,200,400,800,1600 mg/L PFD+HG group.BrdU cell proliferation test was used to evaluate cell proliferation rate.Cells were divided into control group,hyperosmolar group,HG group and PFD+HG group.The mRNA expressions of α-smooth muscle actin (α-SMA),collagen type Ⅰ,collagen type Ⅳ,transforming growth factor-β1 (TGF-β1),interleukin (IL)-1β,IL-6 and monocyte chemotactic protein-1 (MCP-1) were detected by real-time PCR.Results (1) Compared with normal control group,the model mice had higher weight,blood glucose and 24 h urinary albumin,accompanied with glomerular hypertrophy,mesangial area expansion,tubulointerstitial fibrosis and deposition of collagen type Ⅳ (all P ＜ 0.05).Compared with those in model group,in PFD group 24 h urinary albumin decreased,glomerular hypertrophy,mesangial area expansion and tubulointerstitial fibrosis alleviated,and the protein expression of collagen type Ⅳ inhibited (all P＜0.05).(2) Compared with those in HG group,MES-13 cell proliferation rates of 100,200,400,800,1600 mg/L PFD+HG groups decreased (all P ＜ 0.05),and the mRNA expressions of α-SMA,collagen type Ⅰ,collagen type Ⅳ,TGF-β1,IL-1β,IL-6 and MCP-1 reduced in 400 mg/L PFD+HG group (all P ＜ 0.05).Conclusions PFD can inhibit high glucose-induced proliferation and activation of glomerular mesangial cells,decrease the expression of TGF-β1 and proinflammatory factors,as well as reduce the synthesis of collagen,which improve renal fibrosis of db/db mice.

Secretion of extracellular matrix were inhibited by Exendin-4 treatment through MAPK-NF-κB related inflammation in high glucose-induced glomerular mesangial cells / 中华内分泌代谢杂志

Objective To explore the effects and related mechanism of Exendin-4 on secretion of extracellular matrix in high glucose-induced glomerular mesangial cells(GMCs). Methods GMCs were incubated in medium with glucose or Exendin-4 for the four groups: normal glucose group(NG group): cells were treated in medium with 5.6 mmol/L glucose; NG with Exendin-4 treatment group(NGE group): cells were treated with 5.6 mmol/L glucose and Exendin-4; high glucose group(HG group): cells were cultured with 30 mmol/L glucose; HG with Exendin-4 treatment group(HGE group): cells were treated with 30 mmol/L glucose and Exendin-4 at concentration of 3, 5, 10, 15, 30 nmol/L separately, which were cultured for 12, 24, 48 hours. GMCs treated with Exendin-4 were determined by assessing proliferation using a CCK8 method. The levels of fibronectin(FN), collagen type Ⅳ(Col-Ⅳ)in the cell supernatant were measured using enzyme-linked immunosorbent assay(ELISA). The gene levels of Col-Ⅳ, FN, and expression of inflammatory mediators including monocyte chemotactic protein 1(MCP-1), tumor necrosis factor-α(TNF-α), intercellular cell adhesion molecule-1(ICAM-1), transforming growth factor-β1(TGF-β1)were evaluated using reverse transcription PCR(RT-PCR); The expression of nuclear transcription factor-κB(NF-κB), glucagon-like peptide-l receptor(GLP-1R), and phosphorylation levels of mitogen-activated protein kinases(MAPKs)were evaluated by Western blot. Results(1)After treatment with 10 nmol/L Exendin-4 for 24 hour, the proliferation rate of GMCs was significantly decreased compared with 3 nmol/L, 5 nmol/L Exendin-4 treatment(P0.05). (2)The gene expression of FN, Col-Ⅳ and the inflammatory mediators, MCP-1, TNF-α, ICAM-1, TGF-β1 in HG group were significantly increased compared with the NG group,(all P<0.05). After treatment with Exendin-4, the levels of FN, Col-Ⅳ and the gene expression of TNF-α, MCP-1, ICAM-1, TGF-β1 were decreased(all P<0.05). (3)Compared with NG group, the expression of NF-κB and the phosphorylation of extracellular regulated protein kinases(p-Erk1/2), Jun N-terminal kinase(p-JNK)and, p38MAPK(p-p38MAPK)in the group of HG group were increased significantly, accompanied by the decrease of GLP-1R protein level(all P<0.05). Importantly, Exendin-4 treatment significantly reduced protein expression of p-Erk1/2, p-JNK, and NF-κB(all P<0.05), and the level of GLP-1R protein increased(P<0.05). Furthermore, specific Erk1/2, JNK or NF-κB inhibitors markedly blocked Exendin-4-mediated decrease in the levels of FN, Col-Ⅳ. Conclusion Exendin-4 treatment inhibits the secretion of extracellular matrix potentially through Erk1/2, JNK/NF-κB signaling in higher glucose induced GMCs.

MRTF-A mediates FN and ICAM-1expressions by NF-κB pathway in AGEs-induced GMCs / 中国药理学通报

Aim To observe the expression of MRTF-A in rat glomerular mesangial cells(GMCs) induced by advanced glycation end products(AGEs) and its effect on ICAM-1 and FN;to explore whether MRTF-A is involved in the process of diabetic nephropathy by affecting NF-κB pathway.Methods Under the condition of AGEs, CCG-1423 and anti-MRTF-A small interfering RNA were used to knock down MRTF-A and MRTF-A plasmid was used to activatt MRTF-A, The expression level of MRTF-A, ICAM-1, FN and p65 in nucleus were detected by Western blot.Results The protein expressions of MRTF-A was increased in AGEs-induced GMCs.The expressions of FN and ICAM-1 and p65 in nucleus were downregulated by knocking down MRTF-A.However, the expressions of FN, ICAM-1 and p65 in nucleus were upregulated by overexpressing MRTF-A.Conclusions AGEs can upregulate the expression of MRTF-A in GMCs, and MRTF-A mediates the protein expressions of FN and ICAM-1 by affecting NF-κB signaling pathway in AGEs-induced GMCs.

Influence of fermented red ginseng extract in proliferation of rat glomerular mesangial cells and extracellular matrix degradation under high sugar stimulation / 吉林大学学报(医学版)

Objective:To explore the influence of fermented red ginseng extract (FRGE) in the proliferation of rat glomerular mesangial cells (GMCs) and the degradation of extracellular matrix(ECM)under high sugar stimulation, and to clarify the prevention and treatment effects of FRGE on diabetic nephropathy (DN) and the possible mechanism.Methods:The rat GMCs were cultured and divided into normal concentration of D-glucose (NG) group, high concentration of D-glucose (HG) group and high concentration of D-glucose plus different concentrations (3.75, 7.50, 15.00 mg·L-1) of FRGE groups. The proliferation rates of rat GMCs were detected with MTT,and the type Ⅳcollagen(Col Ⅳ) levels in supernatants of the GMCs were detected by ELISA. The protein expressions levels of matrix metalloproteinase-2(MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) were detected with Western blotting m ethod.Results:Compared with NG group, the proliferation rate of GMCs in HG group was increased(P<0.01), the Col Ⅳ level was increased(P<0.01),the MMP-2 expression level was decreased, and the TIMP-2 expression level was up-regulated(P<0.01).Compared with HG group, the proliferation rates of GMCs in various FRGE groups were decreased(P<0.01), the Col Ⅳ levels were decreased(P<0.01),the expression levels of TIMP-2 were reduced(P<0.01),and the expression levels of MMP-2 were increased(P<0.01).Conclusion:FRGE can inhibit the proliferation of rat GMCs induced by high sugar and promote the ECM degradation to delay the occurrence and development of DN.

Effect of Dihydromyricetin on High Glucose-induced Glomerular Mesangial Cell Proliferation and Fibro-nectin Accumulation / 中国药房

OBJECTIVE:To study the effect of dihydromyricetin(DMY)on high glucose(HG)-induced glomerular mesangial cell(MCs)proliferation and fibronectin(FN)accumulation,and explore its mechanism for diabetic nephropathy glomerulosclero-sis. METHODS:Cells were divided into normal group(5.5 mmol/L glucose),HG group(30 mmol/L glucose),DMY low-concen-tration,medium-concentration,high-concentration groups (30 mmol/L glucose+22.5,45,90 μmol/L DMY). After incubating 48 h,MTT was used to detect the proliferative activity [reflected by the optical density(OD)value] of cells;molecular docking meth-od was adopted to conduct simulation analysis for DMY binding state with Smad2. Cells were divided into normal group(5.5 mmol/L glucose),HG group (30 mmol/L glucose),DMY group (30 mmol/L glucose+45 μmol/L DMY) and DMY control group (5.5 mmol/L glucose+45 μmol/L DMY). After incubating 5 h,Western blot was used to detect the expression levels of phosphorylated Smad2 (p-Smad2) and extracellular matrix protein FN. RESULTS:Results of MTT detection showed,compared with normal group,OD values in HG group were significantly increased(P<0.05);compared with HG group,OD values in DMY each con-centration group were significantly reduced(P<0.05). The Gibbs free energy(ΔG)of DMY and Smad2 protein was -5.64 kJ/mol, Ki was 73.53 μmol/L,and there were hydrogen bond donor and receptor binding in No. 465,464,461,458 amino acid residues. Results of Western blot showed,compared with normal group,expression levels of p-Smad2 and extracellular matrix protein FN in HG group were significantly increased (P<0.05);compared with HG group,expression levels of p-Smad2 and extracellular ma-trix protein FN in DMY group were significantly decreased(P<0.05). CONCLUSIONS:DMY inhibits HG-induced MCs prolifera-tion and improves diabetic nephropathy glomerulosclerosis by combining with Smad2 and inhibiting Smad2 phosphorylation to re-duce the extracellular matrix protein FN expression.

Role of STAT3 in the regulation of autophagy in the glomerular mesangial cells of diabetic nephropathy / 中华肾脏病杂志

Objective To observe the changes of STAT3 signaling transduction pathway and autophagy activity in human glomerular mesangial cells cultured in high glucose, as well as the effect of STAT3 on autophagy, exploring whether SAT3 further influence extracellular matrix proteins type IV collagen secretion through the regulation of autophagy. Methods Culture human renal mesangial cells under different conditions, STAT3 pathway was inhibited with specific blocking agent S3I?201 and siRNA respectively. The experiment was divided into: (1) Control group: normal glucose concentration; (2) High glucose group: divided into 12 h, 24 h, 48 h, 72 h incubation group. (3) High glucose+S3I?201 group: pretreated cells with 30 μmol/L S3I?201 (Selleck S1155) for 1 h, then incubation with high glucose for another 24 hours. (4) High glucose+STAT3?siRNA group: siRNA transfection firstly, then incubation with high glucose for 24 hours. (5) High glucose+S3I?201+3?MA group: pretreated cells with 2 mmol/L 3?MA (Selleck S2767) and 30 μmol/L S3I?201 for 1 h, then incubation with high glucose for another 24 hours. Western blot was employed to detect the protein of STAT3, p?STAT3 and autophagy related protein LC3, p62 expressions. The changes of autophagosome quantity was observed with transmission electron microscope. The extracellular matrix protein collagen IV expression was measured with ELISA. Results Compared with the control group, glomerular mesangial cells cultured with high glucose for 24h, the expressions of STAT3 and p?STAT3 increased (P<0.01), while the expression of autophagy related proteins LC3II/LC3I decreased. The expression of p62 increased and the number of autophagosome reduced under transmission electron microscope, which all indicated the decrease of autophagy activity (P<0.05). Blocking STAT3 signaling pathway with S3I?201 and STAT3?siRNA respectively, compared with high glucose group, LC3II/LC3I was up?regulated and p62 was down?regulated, and the number of autophagosome was increased significantly, which all indicated the increase of autophagy activity (P<0.05). Extracellular matrix proteins collagen IV expression of cells cultured with high glucose was higher than the control group (P<0.05), and the application of S3I?201 blocking STAT3 pathway caused type IV collagen expression to decrease (P<0.05). The application of the autophagy inhibitor 3?MA could convert the result and lead to an increase of type IV collagen expression (P<0.01). Conclusions High glucose could active STAT3 signaling pathway of human renal mesangial cell and increase STAT3, p?STAT3 expression. High glucose could inhibit autophagy activity of human renal mesangial cells. Inhibition of STAT3 pathway activation may reduce the inhibitory effect of high glucose on autophagy of human renal mesangial cells. High glucose leads to an increase of type IV collagen secretion of human glomerular mesangial cells. The activation of STAT3 pathway may increase type IV collagen secretion through negative regulation of autophagy, which eventually leads to diabetic nephropathy.

Protective mechanism of loganin on endoplasmic reticulum stress of mesangial cells induced by advanced glycation end products / 中草药

Objective: To investigate the protective effect and the mechanism of loganin (an active component in Cornus officinalis) on the endoplasmic reticulum (ER) stress of glomerular mesangial cells (GMCs) induced by advanced glycation end products (AGEs). Methods: Human GMCs were cultured in vitro and divided into control group, model group (AGEs group), loganin group, and amino guanidine group (set as positive control, 0.1, 1.0, and 10.0 μmol/L). After being incubated with loganin (final concentration of 0.1, 1.0, and 10.0 μmol/L) for 1 h, GMCs were stimulated by AGEs (200 mg/L) for 24 h. Then, the cell proliferation was measured of using MTS method. PGE2 was investigated by Elisa. Receptors of advanced glycation end products (RAGE), and ER stress-related protein like GRP78, IRE1, XBP1, and inflammatory factor NF-κB and COX-2 in GMCs were detected by Western blotting. Results: Loganin could suppress the proliferation of GMCs induced by AGEs, improve the subcellular injury of GMCs, down-regulate the expression of ER stress-related protein GRP78, IRE1, XBP1, and RAGE, reduce the inflammation-related protein NF-κB, COX-2, and the level of PGE2. Conclusion: Loganin could improve the ER stress of GMCs induced by AGEs, lessen the inflammation and subcellular injury of GMCs, its mechanism might be related to the decreased expression of RAGE and the inhibition of the IRE1 pathway.

Regulatory effects of the bile acid membrane receptor TGR5 on FN and TGF-β1 in rat glomerular mesangial cells cultured under high glucose condition / 中国药理学通报

Aim To investigate the expression of G protein-coupled receptor TGR5 and its effects on FN and TGF-β1 expression cultured under high glucose condition in rat glomerular mesangial cells , and then to explore the role of TGR5 in diabetic nephropathy. Methods INT-777 and TGR5 plasmid were used to activate TGR5 under high glucose(HG,30 mmol·L - 1 glucose ) condition, and anti-TGR5 small interfering RNA(TGR5 siRNA) was used to knock down TGR5. The protein expression of FN and TGF-β1 in rat me-sangial cells was detected by Western blot. Results TGR5 could be detected in rat glomerular mesangial cells. Both FN and TGF-β1 protein levels could be in-creased by high glucose compared with control group(P < 0. 05),and be inhibited by activiation of TGR5(P <0. 05). On the other hand,knockdown of TGR5 could increase FN and TGF-β1 protein to abnormal levels(P< 0. 01,P < 0. 05). Conclusion TGR5 suppresses HG-induced FN and TGF-β1 expression in rat glomer-ular mesangial cells,suggesting a protective role in the process of diabetic nephropathy.

Effect and mechanism of simvastatin on proliferation and cycle of glomerular mesangial cells cultured in high-concentration glucose / 重庆医学

Objective To study the effect of simvastatin(SIM ) on the proliferation and cycle of glomerular mesangial cells (GMCs) cultured in high concentration of glucose ,and to observe the variation of inflammation factor and extracellular matrix . Methods GMCs of rats were divided into 4 groups :low glucose(LG) group(Glu 5 .6 mmol/L) ,High glucose (HG) group(Glu 30 mmol/L) ,HG + SIM (10 μg/L)group and HG + SIM group(25 μg/L) .Cells′ proliferation activity was tested by M TT ,simultane-ously compared the ratio of cells in different period (G0 /G1 ,G2 + M and M ) ,and levels of TGF-β1 ,FN and COL4 were compared a-mong different groups .Results Absorbancy (A) of different periods in HG group was higher than that in other 3 groups ,difference had statistical significance(P< 0 .05) .In LG group ,the ratio of cells in G0 /G1 were(35 .67 ± 3 .38)% while S-stage were(41 .24 ± 5 .64)% ;in HG group ,the ratio of cells in G0 /G1 were(25 .88 ± 4 .02)% while S-stage were(28 .65 ± 1 .88)% ;in HG + SIM (10 μg/L)group ,the ratio of cells in G0 /G1 were(28 .12 ± 2 .01)% while S-stage cell were(35 .55 ± 2 .09)% ;in HG + SIM (25 μg/L ) group ,the ratio of cells in G0 /G1 were(31 .85 ± 3 .52)% ,while in S-stage were(39 .82 ± 2 .23)% ,the difference had statistical sig-nificance(P< 0 .05) .Levels of TGF-β1 ,FN and COL4 in HG group were higher than those of in HG + SIM (10 μg/L)group ,HG +SIM (25 μg/L)group and LG group ,the difference had statistical significance(P< 0 .05) .Conclusion By decreasing TGF-β1 level , simvastatin can inhibit the proliferation of GMCs and regulate the cell cycle ,to suppress the hypertrophy of GMCs .

Alpha lipoic acid modulates high glucose-induced rat mesangial cell proliferation via mTOR signaling pathway / 中华内分泌代谢杂志

Objective To examine whether alpha lipoic acid (LA) regulates high glucose-induced mesangial cell proliferation via mammalian target of rapamycin (mTOR) signaling.Methods The cell proliferation and cycle were determined by methylthiazoletetrazolium(MTT) assay and flow cytometry,respectively.The mRNA expression of AMP-activated protein kinase(AMPK) was detected by realtime PCR.The phosphorylation levels of protein kinase B (Akt),mTOR,eukaryotic translation initiation factor 4E binding protein 1 (4EBP1),and 70S6 kinase (p70S6K) were measured by Western blot.Results 0.25 mmol/L LA promoted high glucose-sitmulated rat mesangial cell proliferation(P＜0.01) and entry of cell cycle into S phase(P＜0.01),along with increased phosphorylation levels of Akt,mTOR,p70S6K,and 4EBP1 (P＜0.05).These effects of 0.25 mmol/L LA disappeared when Akt activity was inhibited.On the contrary,1.0 mmol/L LA inhibited high glucose-induced cell proliferation(P＜0.01) and entry of cell cycle into S phase(P＜0.01),with the decreased phosphorylation levels of mTOR,p70S6K,and 4EBP1 (P＜ 0.05) and the enhanced activity of AMPK(P＜0.01).These effects of 1.0 mmol/L LA were prevented when AMPK activity was inhibited.Conclusions LA dose-dependently regulates mesangial cell proliferation induced by glucose via mTOR signaling pathway.

Effect and mechanism of FoxO1 overexpression in rats with diabetic nephropathy / 中华内分泌代谢杂志

Objective To study the role and molecular mechanism of forkhead transcription factor O1 (FoxO1) on proliferation of mesangial cells( MCs) in diabetic rats. Methods Empty lentiviral vector( LV-pSC-GFP) and the constitutively active FoxO1 lentiviral vector(LV-CA-FoxO1) were constructed. Diabetic rat model was established and rats were divided into diabetes group(DM group), diabetes with LV-pSC-GFP group(NC group), and diabetes with LV-CA-FoxO1 group(CA group). The normal SD rats of the same age were considered as the normal control group(NG group). The lentiviral vector was injected into the renal cortex of diabetic rats in corresponding groups. Body weight, blood glucose, 24 h urinary protein, urine albumin, serum creatinine, and blood urea nitrogen was detected at the end of 2 weeks, 4 weeks, and 8 weeks. The ratio of kidney weight/ body weight was counted and the renal cortex was reserved for light microscopy, electron microscopy and frozen section after rats were sacrificed in different groups. The mRNA level of FoxO1 and p27Kip1 were detected by real-time PCR. The protein expressions of FoxO1, p-FoxO1, and p27Kip1 were tested by Western blotting. Results The renal pathological changes were obviously ameliorated in CA group. Compared with DM group, the mRNA and protein expression of FoxO1 and p27Kip1 were significantly increased in CA group (P 0. 05). The p-FoxO1 / FoxO1 ratio was decreased ( P 0. 05). Conclusion Overexpression of FoxO1 in kidneys of diabetic rats can inhibit the proliferation of mesangial cells, and may through up-regulating the expression of p27Kip1 delay the progression of diabetic nephropathy.

Influence of tannic acid on contents of 8-OHdG and 3-NT in nephridial tissue of diabetic rats and supernatant of glomerular mesangial cells / 中国免疫学杂志

Objective:To observe the effect of tannic acid on renal morphology and function of diabetes mellitus model rats ,and to explore the mechanism of improving effect of tannic acid from oxidative stress , nitrosative stress angle.Methods: 8 rats were randomly selected from 68 6-week-old male Wistar rats as normal control group and the remaining 60 rats accepted high-sugar and high-fat diet for 4 weeks, then were injected streptozotocin ( STZ, 52 mg/kg ) intraperitoneally in order to manufacture a diabetic rat model.Further the diabetic rats were randomly divided into model group ,aminoguanidine group ,low-dose of tannic acid group and high dose of tannic acid group.The rats in aminoguanidine group were injected aminoguanidine [AG,40 mg/(kg· d)] intraperitoneally, those in low-dose of tannic acid group were injected tannic acid [TA,20 mg/(kg· d)] and those in high-dose of tannic acid group were injected tannic acid [TA,30 mg/(kg· d)].The rats in normal control group and model group were injected normal saline [NS, 30 mg/(kg· d)] and all rats were sacrificed and tissues were derived at the end of the week 10.Morphologic changes of kidney in diabetic rats were observed by HE staining and correlative biochemical indices of renal function were detected by biochemical analyzer.8-hydroxy deoxyguanosine (8-OHdG) and 3-nitrotyrosine (3-NT) content of renal tissue in rats in different groups were detected by ELISA method.Mesangial cells cultured in vitro were treated with high concentration of glucose (30 mmol/L) and AGEs (250 mg/L) and at the same time with different concentration of tannic acid (10,20,40 and 80μmol/L) on the basis of setting corre-sponding control group.The contents of 8-OHdG and 3-NT in the culture supernatant were measured by ELISA method after 48 hours.Results:Tannic acid can effectively improve the renal pathological changes and improve renal function of diabetic rats .The contents of 8-OHdG and 3-NT in kidney tissue homogenate of diabetic rats and in the supernatant of GMC cultured with high glucose or AGEs were all significantly increased and can be reduced by tannic acid.Conclusion:Tannic acid improving the structure and function damage of kidney in diabetic rats might be achieved by oxidative stress and nitrosative stress mechanism .