ABSTRACT
Aim To investigate the mechanism of high mobility group protein B1(HMGB1)and tumor necrosis factor α-induced protein-3(TNFAIP3)involved in cell proliferation in lupus nephritis(LN)patients and human mesangial cells(HMC).Methods Immunofluorescence and immunohistochemistry technique were employed to detect HMGB1,TNFAIP3 and IκBα expression levels in glomerular cells of type Ⅳ LN patients.BrdU incorporation technology was used to detect cell proliferation level in HMC after stimulated by recombinant HMGB1.TNFAIP3 and IκBα expression levels in HMC were detected after HMGB1 stimulation by Western blot.Results The expression levels of HMGB1 and TNFAIP3 were increased in LN patients,while IκBα was decreased.HMC proliferation levels increased significantly after HMGB1 stimulation.At the same time,30 minutes after HMGB1 stimulation,the expression level of TNFAIP3 was significantly increased(P<0.05),while IκBα decreased(P<0.05)and then p65 increased significantly(P<0.05),compared with control group.Conclusion HMGB1 and TNFAIP3 are probably involved in mesangial cell proliferation by activating of NF-κB signaling pathways in LN pathogenesis.
ABSTRACT
Aim To observe the effect of gypenosides ( GP) on the expression of receptor for advanced gly-cated endproducts ( RAGE ) and transforming growth factor-β1 ( TGF-β1 ) in human mesangial cells( HMCs) induced by AGEs. Methods HMCs were cultured in DMEM of low glucose containing 15% fetal bovine ser-um in vitro, which were divided into four groups: the normal group, model group, GP group and positive control group. In addition to the normal group, the other groups were stimulated by AGEs ( 200 mg · L-1 );furthermore, GP group was intervened with dif-ferent concentrations(25,75,175 mg·L-1) of GP, while control group was given 10 mmol · L-1 of amin-oguanidine hydrochloride. The expression of RAGE and TGF-β1 protein of each group was detected by Western blot; the expression of RAGE and TGF-β1 mRNA of each group was detected by RT-PCR. Re-sults The expression of RAGE, TGF-β1 protein and mRNA in HMCs induced by AGEs in the model group was significantly higher than that of the normal group ( P<0. 01 );compared with the positive control group ( P<0. 01 ) , GP could obviously reduce the expression of RAGE, TGF-β1 protein and mRNA in a dose-de-pendent manner. Conclusion GP can reduce the ex-pression of RAGE in HMCs induced by AGEs, block AGEs-RAGE signaling pathway and decrease the ex-pression of the downstream factor TGF-β1 , therefore, it plays the role in the resistance of rennal fibrosis in DN.
ABSTRACT
Objective: To observe the effect of gypenosides (GP) on the expression of receptor for advanced glycated endproducts (RAGE) and the oxidative stress status of human mesangial cells (HMCs) induced by AGEs. Methods: HMCs were cultured in DMEM of low glucose containing 13% fetal bovine serum in vitro, which were divided into six groups: normal control group (DMEM of low glucose containing 13% fetal bovine serum), AGEs group (AGEs 200mg/L), GP low-dose group (25 mg/L and AGEs 200 mg/L), GP mid-dose group (75 mg/L and AGEs 200 mg/L), GP high dose group: (150 mg/L and AGEs 200 mg/L), and aminoguanidine positive control group (0.1 mmol/L and AGEs 200 mg/L). The expression of RAGE mRNA levels of each group was detected using semi-quantitative RT-PCR method, protein expression levels by Western blotting after cultured 72 h. Simultaneously, the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in cell supernatant, and intracellular glutathione trace (GSH) in cell were measured. Results: AGEs could significantly promote the expression of RAGE and mRNA in HMCs (P<0.01), and enhance cellular oxidative stress levels. GP could effectively inhibit the expression of RAGE and mRNA, increase the level of SOD in cell supernatant and GSH in cell, reduce the level of MDA in cell supernatant in a dose-dependent manner compared with the control group, the difference was significant (P<0.05). Conclusion: AGEs stimulation can induce the high expression of RAGE in HMCs and enhance the level of oxidative stress in vitro. GP can down the high expression of RAGE stimulated by AGEs, while improve the oxidative stress in cells, and its possible mechanism of GP may block AGEs-RAGE-oxidative stress signaling pathways mediated by RAGE, it shows the lower levels of MDA and SOD and higher levels of GSH in cell.
ABSTRACT
Objective To investigate the effects of specific protein 1 ( Sp1 ) on the TNF-αin-duced expression of inositol 1, 4, 5 trisphosphate receptor type 1 ( IP3R1 ) in human mesangial cells ( HMCs) and to further elucidate the molecular mechanism regarding the decreased glomerular filtration rate ( GFR ) during hepatorenal syndrome .Methods Quantitative real-time polymerase chain reaction and Western blot assay were used to analyze the effects of TNF-αon the expression of IP3R1 at mRNA level and the expression of IP3R1 and Sp1 at protein level in HMCs , respectively.HMCs were transfected with a re-combinant plasmid PGL3-IP3R1 promoter to determine the effects of TNF-αon the activity of IP3R1 promot-er.HMCs were treated with Mithramycin A , an inhibitor of Sp1 binding, and transfected with Sp1-siRNA plasmid respectively to evaluate the expression of IP 3R1 regulated by TNF-α.The role of TNFR1 and TNFR2 in the TNF-αinduced expression of Sp 1 and IP3R1 proteins were detected by Western blot .Results TNF-αincreased the expression of IP3R1 at mRNA level and the expression of IP3R1 and Sp1 at protein lev-el in HMCs.Moreover, the activity of IP3R1 promoter in HMCs was remarkably increased by TNF-αas well.TNF-αinduced expression of IP3R1 was inhibited by Mithramycin A in a concentration dependent manner.HMCs transfected with Sp1-siRNA plasmid showed a significantly decreased expression of IP 3R1 protein.Both anti-TNFR1 and anti-TNFR2 antibodies blocked the TNF-αinduced IP3R1 expression, while only anti-TNFR1 antibodies inhibited the TNF-αinduced Sp1 expression.Conclusion TNF-αmight in-crease the expression of IP3R1 through the TNFR1/Sp1 signaling pathways in HMCs .
ABSTRACT
Objective To discuss the effects of high glucose on human mesangial cells (HMC)Wnt-β-catenin signaling pathway,and the intervention of rhein on it.Methods High concentration of glucose (30 mmol/L) combined with different concentrations of rhein were used to intervene cultured human mesangial cells.The activity of mesangial cell proliferation after all the interventions was examined by MTT measurement.Total Wnt、β-catenin RNA was detected By RT-PCR in normal mesangial cells and cells intervened by high glucose and Rhein.Results ①Inhibition effect to human mesangial cells:compared with NG group for 24 h,48 h and 72 h (OD values were 0.169± 0.051,0.228±0.074,0.285±0.075),human mesangial cells proliferation in HG group(OD values were 0.307± 0.074,0.507 ±0.038,0.711±0.075),HG+R1 group(OD value were 0.241± 0.027,0.334±0.015,0.499±0.063),HG+R2 group (OD value were 0.244±0.081,0.386±0.033,0.531±0.011),and HG+R3 group(OD value were 0.277±0.036,0.407± 0.057,0.594±0.042) were iucreased significantly (P＜0.05、P＜0.01) ; Compared with HG group for 24 h,48 h and 72 h,the HG+R1 group,the HG+R2 group and the HG+ R3 group showed a downward trend at 24 h,but not significantly; but the trend decreased significantly at 48 h and 72 h,and the performance expressed a time,concentration-dependent (P＜0.05,P＜0.01).② In the normal state,the mesangial cells expressed s certain amount of Wnt and β-catenin,when they were stimulated by high glucose,the expression of Wnt、β-catenin in mRNA increased (P＜0.05) ; while the expression of them was significantly reduced in high glucose-induced mesangial cells in HG+R1 group,HG+R2 group and HG+R3 group(P＜0.05).Conclusion Rhein may inhibit the proliferation of high glucose-induced mesangial cells through the Wnt and β-catenin gene expression.
ABSTRACT
Objective To explore the effects of TNF-α on the expression of IP3 R1 mRNA and protein in human mesangial cells (HMCs) and elucidate the role of protein kinase C (PKC) in this signal pathway.Methods Quantitative real-time polymerase chain reaction and immunoblot assay were used to examine the effects of TNF-α on IP3R1 mRNA and protein expression.Depletion PKC,the selective inhibitor of PKCα Safingol and inhibitor of PKCδ Rottlerin,overexpression of dominant negative mutant of PKC to examine the mechanism of signal transduction of TNF-α-regulated IP3 R1 in HMCs.PKCα activation was assayed by Western blot.Results TNF-α increased IP3R1 mRNA and protein expression in HMCs,effects that were blocked by prolonged incubted chronic PMA,Safingol and also by domain negative PKCα construct.TNF-α promoted PKCα activation with maximal PKCα phosphorylation that occurred 8 h post-stimulation.Conclusion TNF-α increased the expression of IP3 R1 and this was mediated through the PKCα activation signaling pathways in HMCs.
ABSTRACT
BACKGROUND: This study aimed to explore the effects of TNF-α on the expression of IP3R1 mRNA and protein in human mesangial cells (HMCs), and to elucidate the mechanism of TNF-α relating to IP3R1 expression in the occurrence of hepatorenal syndrome (HRS). METHODS: HMCs were stimulated by tumor (TNF-α) with 100 ng/mL for different hours (2, 4, 8, and 24 hours). The expression changes of IP3R1 mRNA and protein were detected by quantitative real-time polymerase chain reaction and immunoblotting. Several inhibitors including D609, U73122, PP1, safingol, rottlerin and non-radioactive protein kinase C (PKC) were used to examine the mechanism of signal transduction of TNF-α-regulated IP3R1 in HMCs. RESULTS: The levels of IP3R1 mRNA at 2 hours after TNF-α exposure were significantly enhanced and peaked at 8 hours in HMCs (P<0.01), then descended at 24 hours (P<0.01). The levels of IP3R1 protein at 4 hours after TNF-α exposure were obviously increased and peaked at 24 hours after TNF-α exposure (P<0.01). Compared to the control group, safingol (PKCα inhibitor) and D609 (phosphatidylcholine-specific phospholipase C inhibitor) significantly blocked the TNF-α-induced expression of IP3R1 mRNA (3.30±0.81 vs. 1.95±0.13,P<0.05; 2.10±0.49,P<0.01) and IP3R1 protein (3.09±0.13 vs. 1.86+0.39,P<0.01; 1.98±0.02,P<0.01). TNF-α promoted PKCα activation with maximal PKCα phosphorylation that occurred 8 hours after stimulation measured by non-radioactive PKC assay, and the effect was markedly attenuated by pretreatment with D609 or safingol. CONCLUSION: TNF-α increased the expression of IP3R1 and this was mediated, at least in part, through the PC-PLC/PKCα signaling pathways in HMCs.
ABSTRACT
Objective To investigate the role of Rho/ROCK signaling pathway in the process of human mesangial cells (HMCs) inflammation and fibrosis induced by high glucose. Methods Synchronized HMCs were divided into following groups: ( 1 ) Normal glucose control group ( NG, 5.5 mmol/L glucose); ( 2 ) High glucose group ( HG, 30 mmol/L glucose); (3) Mannitol group( Man,5.5 mmol/L glucose+ 24.5 mmol/L mannitol); (4) NG +Y-27632 group( 10 μ mmol/L Y-27632 ); ( 5 ) HG Y-27632 group ( 10 μmmol/L Y-27632 ). The supernatant and cells were collected at 0,12,24,36,48, and 72 h. Western blot was used to detect the active RhoA and total RhoA,while RhoA, ROCK-Ⅰ, CTGF, and TNF-α mRNA expressions were determined with realtime PGR method in the cells, then ELISA method was used to check protein levels of FN, CTGF, and TNF-α in the supernatant. Results ( 1 ) RhoA activation was stimulated after treatment for with 30 mmol/L glucose, peaked at 1 h, and then decreased ( P = 0. 02). (2) RhoA, ROCK-Ⅰ, CTGF, and TNF-α mRNA expressions in HMC cultured under high glucose were higher than those in the normal group ( P ＜ 0.05 ), and there was certain time-dependence. Besides, there was no statistical significance between Man and NG groups( P＞0. 05 ). ( 3 ) After Y-27632 pretreatment and being cultured with normal glucose and high glucose for24 h or48 h, RhoA, ROCK-Ⅰ, CTGF, and TNF-α mRNA expressions were significantly decreased ( P＜0.01 ) as compared with groups without treatment. (4) High glucose increased FN, CTGF,and TNF-α protein secretion of HMC in a time-dependent manner( P＜0. 05 ). ( 5 ) After Y-27632 pretreatment and being cultured with normal and high glucose for 12,24,36,48,72 h, FN, CTGF, and TNF-α protein secretions were significantly reduced( P＜0.05 ). Conclusion Rho/ROCK signaling pathway may mediate inflammation and fibrosis induced by high glucose in HMCs, supporting a potential role for inhibitors of Rho/ROCK in the treatment of diabetic nephropathy.
ABSTRACT
To investigate the expression of vascular endothelial growth factor (VEGF)and the levels of reactive oxygen species (ROS)in human mesangial cells cultured in high glucose and being intervened by pigment epithelium-derived factor (PEDF). The results showed that with increased glucose concentration, the expressions of VEGF and ROS generation also gradually increased and PEDF significantly downregulated VEGF expression and ROS generation in a dose-dependent manner. PEDF may delay the diabetic nephropathy progress by improving vascular permeability and inhibiting oxidative stress.
ABSTRACT
Objective:To investigate the effects of Yukuiqing (YKQ) on the expression of connective tissue growth factor (CTGF) in cultured human renal mesangial cells (HRMC) incubated with AGEs. Methods:The HRMCs were incubated with AGEs (200 ?g/mL) and 1.25% YKQ which was prepared by Chinese herbal medicine serum pharmacological approach for 0,8,16,24,48 and 72h or different concentrations (0.313%,0.625% and 1.25%) of YKQ for 48h,respectively,which were incubated with DMEM and rat serum (RS) as the control. CTGF mRNA and protein were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western Blotting respectively. Results:In Yukuiqing group,the expression of CTGF mRNA and protein decreased significantly compared with control groups (P
ABSTRACT
Objective To observe the effect of oxymatrine(OM) on the expressions of p-STAT1 and PIAS1 signaling molecules at protein and mRNA levels in the proliferation of the human mesangial cells(HMC) induced by lipopolysaccharide(LPS) and explore the relationship between them.Methods HMCs were primarily cultured from a 4-month-old aborted human fetus(with informed consent and approved by the Ethics Committee of Lanzhou University),and then divided into 3 groups,that is,control group,LPS group(10 ng/ml) and OM group(LPS 10 ng/ml and OM 320 mg/L).After cultured for 12,24 and 48 h respectively,HMC proliferation were analyzed by methyl thiazolyl tetrazolium(MTT) assay and type Ⅳ collagen in the supernatants were detected by ELISA.At the same time points,the cells lysates were collected for the mRNA and protein expressions of p-STAT1 and PIAS1 by real-time quantitative RT-PCR and Western blot analysis.Results The cell proliferation of LPS group was faster and the type Ⅳ collagen protein was increased more than the control group(P
ABSTRACT
BACKGROUND: Diabetic nephropathy is a leading cause of end-stage renal disease and is charaterized by activation of some growth factors (e.g., angiotensin II, endotelin-1, IL-8, and TGF-beta) and deposition of extracellular matrix proteins. Both ACE inhibitors and AT1 receptor blockers partially prevent renal hypertrophy in diabetes. Recently, IL-6 is thought to act as an autocrine growth factor for the mesangial cells. Angiotensin II (Ang II) is one of the noninflammatory stimulators of IL-6 release from mesangial cells. IL-6 have been implicated in glomerulonephritis, including mesangioproliferative glomerulonephritis. IL-6 may be associated with renal damage, especially mesangioproliferative diabetic nephropathy. However, little is known about the pathogenetic relations between IL-6 and diabetic nephropathy. METHODS: To evaluate the effects of high glucose concentration, Ang II and its blockers on IL-6 and fibronectin production, human mesangial cells were cultured in various conditions. Normal concentration (100 mg/dL) and high concentration of D-glucose (450 mg/dL), Ang II (10(-7)M), high glucose with Ang II, captopril (10(-6)M), and losartan (10(-6)M) were added. After 48 hours, IL-6 and fibronectin concentration in the supernatant were measured by ELISA method. RESULTS: The effects of various conditions on the production of IL-6 and fibronectin in cultured human mesangial cells were as follows: 1. The concentration of IL-6 in the supernatant was significantly low in high glucose group (9.9+/-0.2 pg/mL) compared to that in control group (18.0+/-6.2 pg/mL) (p<0.05), and there was no difference in the supernatant concentration of fibronectin between the groups of high glucose and control. 2. The concentration of IL-6 in the supernatant of Ang II group (28.0+/-5.0 pg/mL) was significantly higher than that in control group (18.0+/-6.2 pg/mL) (p<0.05), and there was no difference in the supernatant concentration of fibronectin between the groups of Ang II and control. 3. In the supernatant of high glucose with Ang II group, the concentration of IL-6 (20.0+/-4.0 pg/mL) was significantly higher than that of high glucose group (9.9+/-0.2 pg/mL) (p<0.05), and the concentration of fibronectin (3,100+/-50 pg/mL) was significantly higher than that of control group (2,840+/-290 pg/mL) (p<0.05). 4. The concentration of IL-6 in the supernatant was significantly lowered after the addition of captopril (10.7+/-1.8 pg/mL) and losartan (9.3+/-2.4 pg/mL) in high glucose with Ang II group (20.0+/-4.0 pg/mL) (p<0.05). 5. The concentration of fibronectin was significantly lowered after the addition of captopril (2,640+/-30 pg/mL) and losartan (2,440+/-230 pg/mL) in high glucose with Ang II group (3,100+/-50 pg/mL) (p<0.05). 6. There was no difference in the concentration of supernatant IL-6 and fibronectin between the groups of captopril and losartan. CONCLUSION: High glucose concentration decreases and Ang II increases the production of IL-6 by cultured human mesangial cells. Captopril and losartan decrease the production of IL-6 and fibronectin which have been stimulated by high glucose concentration and Ang II. These drugs may be useful in the treatment of renal disease, especially diabetic nephropathy, in which Ang II and high blood glucose are cooperative in the progression of nephropathy.