Xiaokeping-containing serum suppresses high-glucose-induced proliferation of mesangial cells involved in the regulation of cell cycle progression and the p38 mitogen-activated protein kinase pathway.

OBJECTIVE: To investigate the efficacy of Xiaokeping (XKP)-containing serum on the proliferation of high-glucose-induced mesangial cells (MCs) and the potential underlying mechanism. METHODS: XKP-containing serum was prepared by the intragastric administration of XKP in rats. HBZY-1 cells were cultured with normal glucose (NC group), high glucose (HG group), and high glucose with different XKP concentrations. Cell proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the cell cycle distribution was detected by flow cytometry. The expression of p38 mitogen-activated protein kinase (p38MAPK) pathway components in MCs was detected by Western blotting and quantitative real-time polymerase chain reaction. RESULTS: The MC proliferation level in the high-glucose group was significantly higher than that in the normal control group, and XKP suppressed the HG-induced proliferation of MCs dose dependently. Moreover, flow cytometry revealed that XKP blocked cell cycle progression by inducing cell cycle arrest in G1 phase and inhibiting S phase entry. XKP down-regulated the protein and mRNA expression of p38MAPK in MCs (P < 0.05 vs HG). CONCLUSION: The present study demonstrated that XKP-containing serum inhibits high-glucoseinduced proliferation of MCs by causing cell cycle arrest at G1 phase and inhibiting S phase entry. The underlying mechanism involves the down-regulation of the p38MAPK signaling pathway, providing a theoretical basis for the use of XKP to treat diabetic kidney disease.

Discovery of polypodiside as a Keap1-dependent Nrf2 activator attenuating oxidative stress and accumulation of extracellular matrix in glomerular mesangial cells under high glucose.

Diabetic nephropathy (DN) is a severe microvascular complication of diabetes mellitus. High glucose has resulted in oxidative stress and following renal fibrosis as the crucial nodes of this disease. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor regulating transcription of many antioxidant genes and suppressing synthesis of extracellular matrix. To discover Nrf2 activators targeting DN, we have evaluated polypodiside using cell-based assays. The results showed polypodiside inhibited the high glucose-induced self-limited proliferation of glomerular meangial cells. Activation of Nrf2 and enhanced transcription to antioxidant response elements were observed in the presence of polypodiside. Oxidative stress and accumulation of extracellular matrix induced by high glucose in glomerular meangial cells have been ameliorated by polypodiside. Further investigations revealed the effects of polypodiside on glomerular meangial cells were associated with activation of Nrf2. Co-immunoprecipitation of Nrf2 disclosed polypodiside disrupted the Kelch-like ECH-associated protein-1 (Keap1)-Nrf2 interaction. Molecular docking elucidated polypodiside could enter the Nrf2 binding cavity of Keap1 via interacting with the residues encompassing that cavity. These findings indicate polypodiside is a Keap1-dependent Nrf2 activator affording the catabatic effects against oxidative stress and accumulation of extracellular matrix in glomerular meangial cells under high glucose.

Danggui buxue tang inhibited mesangial cell proliferation and extracellular matrix accumulation through GAS5/NF-κB pathway.

Diabetic nephropathy (DN) is the common complications of diabetes mellitus, but the efficacy of available treatments for the prevention of DN is still unsatisfactory. In the present study, we aimed to explore the effect of Danggui buxue tang (DGT) on the proliferation of high glucose (HG)-induced mesangial cells and accumulation of extracellular matrix in mesangial cells. We found DGT up-regulated the expression of growth arrest specific transcript 5 (GAS5) and IκB kinase (IKK) dose-dependently in mouse mesangial cells (SV40 MES-13). We found DGT regulated the expression IKK and the activity of nuclear transcription factor-κB (NF-κB) via GAS5, and proved that long non-coding RNA (lncRNA) GAS5 was positively related with IKK. And we proved GAS5 regulated the expression of IKK and the activity of NF-κB. In addition, DGT inhibited the viability of MES-13 cells and extracellular matrix-related proteins (laminin (LN), fibronectin (FN) and collagen IV (Col IV)) via GAS5. Moreover, we proved GAS5 regulated the viability of SV40 MES-13 cells and extracellular matrix-related proteins through NF-κB pathway. DGT inhibited the proliferation of mesangial cells and accumulation of extracellular matrix via GAS5/NF-κB, therefore, DGT could be an effective treatment for the prevention of DN.

Effectiveness of Qingre Lishi Yishen decoction on the glomerular fibrosis of immunoglobulin A nephropathy in a rat's model.

OBJECTIVE: To investigate the effect of the clinical effective prescription of Qingre Lishi Yishen decoction (QRLS) on the activation of mesangial cells in immunoglobulin A nephropathy (IgAN) rats. METHODS: IgAN rat's model was established by combine with intragastric administration of bovine serum albumin (BSA) + intravenous injection of lipopolysaccharide (LPS) by + subcutaneous injection of carbon tetrachloride (CCL4). Then the animals were randomly divided into four groups: control group, IgAN model group, IgAN model with Valsartan (Val) treatment group and IgAN model with QRLS treatment group. To observe the indexes of 24-h urine protein, renal function, deposition of immune complexes, expression of activation factor, fibrosis marker and inflammatory cytokines in four different groups. RESULTS: The Val or QRLS treatment group: (a) it reduced the immune complexes deposition of IgA in glomerular mesangial and inhibited mesangial cell proliferation; (b) it decreased the expression of smooth muscle actin (α-SMA), fibronectin (FN) and tumor necrosis factor alpha (TNF-α). CONCLUSION: The study suggested that QRLS ameliorate renal structure and function in IgAN rat's model. Furthermore, we also observed that QRLS alleviated mesangial cells activation and matrix accumulation partly by decreasing the α-SMA, then to downregu.

Naringin ameliorates the high glucose-induced rat mesangial cell inflammatory reaction by modulating the NLRP3 Inflammasome.

BACKGROUND: The Nucleotide binding and oligomerization domain-like receptorfamily pyrin domain-containing 3 (NLRP3)-inflammasome plays an important role in various diseases, including a variety of kidney diseases. Naringin exhibits anti-inflammatory and anti-oxidation effects among others, but its specific mechanisms are not clear. We investigated the expression of the NLRP3-inflammasome under high-glucose conditions, assessed the effects of naringin on that process, and further elucidated the role of naringin in the pathogenesis of diabetic kidney disease(DKD). METHODS: To assess the therapeutic potential of naringin and the mechanisms involved, we cultured rat glomerular mesangial cells and grouped them according to different glucose concentrations, different action times, different concentrations of MCC950, and different concentrations of naringin.The cell proliferation was measured by MTT assay. The expression of Interleukin-1ß(IL-1ß) and Interleukin18 (IL-18) in the cell supernatant were detected by ELISA. The expression and activity of NLPR3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and Caspase-1 were detected by Western Blot. RESULTS: The expressions of NLRP3, ASC, caspase-1, IL-1ß, and IL-18 in rat glomerular mesangial cells were significantly higher in the high glucose (HG) group than in the control normal glucose (NG) group and exhibited time-dependence activity. The expression levels of NLRP3, caspase-1, IL-1ß, and IL-18 in different treatment groups were significantly lower compared with the HG group after 48 h of MCC950 pre-treatment (p < 0.05). Pre-treatment with naringin produced the same results. Naringin also inhibited the proliferation of cells. CONCLUSIONS: The NLRP3-inflammasome potentially plays a role in the process of activation and inflammation of glomerular mesangial cells as induced by high-glucose conditions. Naringin inhibited the proliferation of cells that were induced by high glucose. Further, it reduced the expression of inflammatory factors that are mediated by NLRP3 through the NLRP3-caspase-1-IL-1ß/IL-18 signaling pathway, which makes naringin a potentially novel treatment for DKD disease.

Effect of Huaier On the Proliferation of Mesangial Cells in Anti-Thy-1 Nephritis.

BACKGROUND/AIMS: To determine whether an aqueous extract of Trametes robiniophila Murr. (Huaier) suppresses anti-Thy-1 mesangial proliferative glomerulonephritis (MsPGN) in vivo and platelet-derived growth factor (PDGF)-BB-induced mesangial cell proliferation in vitro. METHODS: Male Wistar rats were randomly categorized into 5 groups: Sham, Thy-1, and 3 Huaier-treated groups (low, medium, and high dose). Two weeks after treatment, urinary proteins were quantified and renal pathological changes were examined. MAX interactor 1 (Mxi-1) and proliferating cell nuclear antigen (PCNA) expression levels in isolated glomeruli, rat mesangial cell viability, cell-cycle distribution, and cell-cycle pathways were assessed. RESULTS: Huaier diminished the proliferative damages and urinary protein secretion in Thy-1 rats. PCNA was downregulated, whereas Mxi-1 was upregulated in the isolated glomeruli of Huaier-treated groups compared with the Thy-1 group. Huaier inhibited PDGF-BB- stimulated proliferation of rat mesangial cells in a time- and dose-dependent manner (50% inhibitory concentration = 6.19 mg/mL) and induced G2 cell-cycle arrest. Cell-cycle pathway proteins were downregulated, whereas Mxi-1 was upregulated in Huaier-treated mesangial cells compared with PDGF-BB-stimulated cells. CONCLUSION: Huaier reduces urinary protein excretion and relieves hyperplasia in mesangial cells in anti-Thy-1 MsPGN as well as inhibits PDGF-BB-stimulated proliferation and DNA synthesis of rat mesangial cells in vitro, suggesting its novel therapeutic potential in MsPGN.

Effects of Dangguibuxue decoction on rat glomerular mesangial cells cultured under high glucose conditions.

BACKGROUND: Dysfunction of glomerular mesangial cells (GMCs) plays an important role in pathogenesis of diabetic nephropathy. Here, we investigated the effects of Dangguibuxue decoction (DBD), an herbal traditional Chinese medicinal (TCM) formula composed of Astragali Radix and Angelicae Sinensis Radix, on GMC proliferation and fibrogenesis under high-glucose (HG) conditions. METHODS: Sixty male Sprague Dawley rats were divided into 5 groups and administered intragastric 0.9% saline, low concentration DBD (DBD-L, 1.75 g/kg/d), middle concentration DBD (DBD-M, 3.5 g/kg/d), high concentration DBD (DBD-H, 7.0 g/kg/d) and gliclazide (GL, 2 mg/kg/d), respectively, for 1 week, and then their sera were obtained. Rat mesangial cells (HBZY-1 cells) were treated with these sera under HG condition (30 mmol/L). RESULTS: The proliferation of GMCs under HG conditions was significantly greater than that under normal glucose condition. Low concentration DBD (DBD-L) inhibited proliferation of GMCs after 72-h incubation (P < 0.01), while high concentration DBD (DBD-H) inhibited GMCs proliferation at 24, 48 and 72 time points (P < 0.01). There was no significant difference between the inhibitory effect of DBD-H and GL sera on GMC proliferation (P > 0.05). Furthermore, all concentrations of DBD (DBD-L, DBD-M and DBD-H) significantly decreased the protein expression of α-SMA(α-smooth muscle actin) (P < 0.01), an indicator of interstitial fibrosis of GMCs. Finally, DBD-L, DBD-M, DBD-H sera obviously inhibited the increase of HYP (hydroxyproline)secretion under HG condition (P < 0.01). CONCLUSION: Our results demonstrate an inhibitory effect of DBD extract on proliferation and fibrogenesis of GMCs under HG conditions. The potential role of DBD in the treatment of diabetic neuropathy merits further investigation.

Inhibitory effects of Shenkang injection and its main component emodin on the proliferation of high glucose­induced renal mesangial cells through cell cycle regulation and induction of apoptosis.

Increased mesangial cell proliferation is a major pathological feature of early-stage diabetic nephropathy (DN). The present study investigated the effects of the Traditional Chinese Medicine Shenkang injection (SKI) and its main component emodin (EM) on high glucose­cultured mesangial cells. The proliferation rate, cell cycle distribution, apoptosis and morphology of rat renal mesangial cells (RMCs) cultured in the presence of various concentrations of glucose (5.6 or 25 mM), SKI (25, 50 or 100 mg/l) or EM (10, 20 or 40 µM) were assessed at time­points of 12, 24 or 48 h. High­glucose treatment promoted the proliferation of RMCs, which was significantly inhibited by SKI and EM, while these drugs had no effect on RMCs under normal glucose conditions, as indicated by an MTT assay. Furthermore, flow cytometric analysis revealed that SKI and EM inhibited the cell cycle progression of RMCs and induced apoptosis. Transmission electron microscopy revealed morphological characteristics of apoptosis and western blot analysis demonstrated the upregulation of B­cell lymphoma 2­associated X protein (bax) and activation of caspases in RMCs following treatment with SKI or EM under high­glucose conditions. In conclusion, SKI and its major active component EM were shown to inhibit high­glucose­induced proliferation of RMCs via inducing cell cycle arrest at G1 phase as well as cellular apoptosis via upregulation of pro­apoptotic mediators bax and caspase activation, and may therefore be suitable for the treatment of DN.

Protective Effects of Green Tea Polyphenol Against Renal Injury Through ROS-Mediated JNK-MAPK Pathway in Lead Exposed Rats.

To investigate the potential therapeutic effects of polyphenols in treating Pb induced renal dysfunction and intoxication and to explore the detailed underlying mechanisms. Wistar rats were divided into four groups: control groups (CT), Pb exposure groups (Pb), Pb plus Polyphenols groups (Pb+PP) and Polyphenols groups (PP). Animals were kept for 60 days and sacrificed for tests of urea, serum blood urea nitrogen (BUN) and creatinine. Histological evaluations were then performed. In vitro studies were performed using primary kidney mesangial cells to reveal detailed mechanisms. Cell counting kit-8 (CCK-8) was used to evaluate cell viability. Pb induced cell apoptosis was measured by flow cytometry. Reactive oxygen species (ROS) generation and scavenging were tested by DCFH-DA. Expression level of tumor necrosis factor-α (TNF-α), interleukin-1-ß (IL-1-ß) and IL-6 were assayed by ELISA. Western blot and qPCR were used to measure the expression of ERK1/2, JNK1/2 and p38. Polyphenols have obvious protective effects on Pb induced renal dysfunction and intoxication both in vivo and in vitro. Polyphenols reduced Pb concentration and accumulation in kidney. Polyphenols also protected kidney mesangial cells from Pb induced apoptosis. Polyphenols scavenged Pb induced ROS generation and suppressed ROS-mediated ERK/JNK/p38 pathway. Downstream pro-inflammatory cytokines were inhibited in consistency. Polyphenol is protective in Pb induced renal intoxication and inflammatory responses. The underlying mechanisms lie on the antioxidant activity and ROS scavenging activity of polyphenols.

Inhibitory effects and mechanism of 25-OH-PPD on glomerular mesangial cell proliferation induced by high glucose.

OBJECTIVE: To investigate the protective effects and potential mechanism of the compound 25-OH-PPD (PPD) on the glomerular mesangial cells (GMC) under high glucose condition. METHODS: The hypertrophic GMC cells were established by DMEM containing glucose and randomly divided into five groups, including the normal control group (Control), the high glucose model group (HG, 25 mmolL(-1)), the PPD low dose group (1µmolL(-1), PPD-L), the PPD middle dose group (5µmolL(-1), PPD -M) and the PPD high dose group (10µmolL(-1), UCN-H). The GMC were incubated for 48h under different treatment factors. Total protein content was determined by Lowry method. The diameter of the single GMC and volume were measured by computer photograph analysis system. The GMC cell viability was analyzed by MTT assay. The level of malondialdehyde (MDA), the content of glutathione (GSH) and superoxide dismutase (SOD) activity were measured by ELISA. [Ca(2+)]і transient was measured by Till image system and by cell-loading Fura-2/AM. The expression of COX-1 and COX-2 were also determined using ELISA method. RESULTS: The viability of GMC and the total protein content were decreased in HG group, different dosage PPD group could increase these indexes (P<0.05). The level of MDA was increased, the content of GSH and SOD was decreased in HG group, while PPD could reduce the MDA and enhance GSH and SOD (P<0.05). Following treatment with different dosage (PPD-L, PPD-M or PPD-H), the [Ca(2+)]і transient was reduced (P<0.05 or P<0.01). Moreover, the expression of COX-1 was decreased while COX-2 expression was increased in different dosage PPD groups. CONCLUSION: The protective effects of PPD on GMC from HG-induced hypertrophy may be associated with the inhibition of [Ca(2+)]і transient and decreasing expression of COX-1 via the oxidative-stress injure pathway.

[Effect of Moutan Cortex on AGEs-induced mesangial cell proliferation and basement membrane thickening].

OBJECTIVE: To investigate the effect of Moutan Cortex on mesangial proliferation and basement membrane thickening induced by advanced glycation end products (AGEs). METHOD: The glomerular mesangial cells (MC) injury model was established by inducing by AGEs. The cell were divided into 6 groups: the blank group ( BSA, 200 mg L-1) , the model group (AGEs, 200 mg L-1), the positive control group (AG, 10 mmol L L-1), and drug administration groups, namely the Moutan Cortex-treated high-dose group (2 x 10(-4) g mL(- 1)), the Moutan Cortex-treated medium-dose group (1 x 10(-4) g mL-1 ), and the Moutan Cortex-treated low-dose group (0. 5 x 10(-4) g . mL(-1)). The MTT method was performed to observe the effect of Moutan Cortex on the proliferation of MC. The content of fibronectin (FN) and collagen secretion 1V (Col IV) in cell supernatant were detected by ELISA kits. The western blot analysis was carried out to observe the FN expression. The Real-time PCR analysis was applied to examine the Col IV mRNA expression. RESULT: AGEs significantly increased AGEs-induced MC proliferation and FN and Col 1V secretion. The western blot analysis showed that MC could down-regulate the FN expression of MC secretion. According to the results of the real-time PCR assay, MC could down-regulate AGEs-induced MC secretion Col IV mRNA expression. CONCLUSION: MC had a certain protective effect on MC cultured under AGEs conditions. MC could remarkably inhibit the composition and secretion of Col IV and FN in matrix and the basement membrane thickening, and provide an experimental basis for the treatment of diabetic nephropathy.

The attenuation of Moutan Cortex on oxidative stress for renal injury in AGEs-induced mesangial cell dysfunction and streptozotocin-induced diabetic nephropathy rats.

Oxidative stress (OS) has been regarded as one of the major pathogeneses of diabetic nephropathy (DN) through damaging kidney which is associated with renal cells dysfunction. The aim of this study was to investigate whether Moutan Cortex (MC) could protect kidney function against oxidative stress in vitro or in vivo. The compounds in MC extract were analyzed by HPLC-ESI-MS. High-glucose-fat diet and STZ (30 mg kg(-1)) were used to induce DN rats model, while 200 µg mL(-1) AGEs were for HBZY-1 mesangial cell damage. The treatment with MC could significantly increase the activity of SOD, glutathione peroxidase (GSH-PX), and catalase (CAT). However, lipid peroxidation malondialdehyde (MDA) was reduced markedly in vitro or in vivo. Furthermore, MC decreased markedly the levels of blood glucose, serum creatinine, and urine protein in DN rats. Immunohistochemical assay showed that MC downregulated significantly transforming growth factor beta 2 (TGF-ß2) protein expression in renal tissue. Our data provided evidence to support this fact that MC attenuated OS in AGEs-induced mesangial cell dysfunction and also in high-glucose-fat diet and STZ-induced DN rats.

Effects of herbal compound 861 on collagen synthesis and degradation in rat mesangial cells exposed to high glucose.

OBJECTIVE: To investigate the effects of Herbal Compound 861 (Cpd 861) on collagen synthesis and degradation in rat mesangial cells exposed to high glucose. METHODS: The third to fifth passage of rat mesangial cells were exposed to high glucose and Cpd 861 at a concentration of 0.25-4.00 g/L for 24, 48 and 72 h, respectively. Benazepril (10(-7)-10(-3) mmol/L) was selected as positive control. The methyl thiazolyl tetrazolium colorimetric assay was used to evaluate the effect of Cpd 861 on cell proliferation. After incubation with Cpd 861 at a concentration of 2.00 g/L for 48 h, the protein secretions of collagen type IV, matrix metallopeptidase 9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), transforming growth factor beta 1 (TGF-ß1), and hepatocyte growth factor (HGF) were detected by enzyme-linked immunosorbent assay method. And rat mesangial cells were harvested to determine MMP-9, TIMP-1, TGF-ß1 and HGF mRNA expression by reverse transcription polymerase chain reaction. RESULTS: Cpd 861 inhibited cell proliferation induced by high glucose in a dose- and time-dependent manner. Compared with high glucose, collagen type IV production was decreased significantly by Cpd 861 (P<0.01). Cpd 861 increased the protein secretions and mRNA expressions of MMP-9 and HGF, whereas the protein secretions and mRNA expressions of TIMP-1 and TGF-ß1 were reduced markedly (P<0.05). The ratio of MMP-9 to TIMP-1 was enhanced by Cpd 861 significantly. There was no significant difference in all above-mentioned effects between Cpd 861 (2.00 g/L) and benazepril (10(-5) mmol/L). CONCLUSION: The anti-glomerulosclerosis mechanisms of Cpd 861 were partly attributed to its effects of inhibiting mesangial cell proliferation, decreasing collagen synthesis and enhancing collagen degradation.

[Effects of parthenolide on high glucose-induced cell proliferation, NF-κB activation and MCP-1 expression in rat mesangial cells].

OBJECTIVE: To explore the effects of parthenolide (PTL) on high glucose-stimulated cell proliferation, NF-κB activation and monocyte chemoattractant protein-1 (MCP-1) expression in rat mesangial cells (MCs). METHODS: MCs were cultured in normal glucose (5.6 mmol/L), high glucose (30 mmol/L), or high glucose with PTL. MTT assay was used to detect the cell proliferation. MCP-1 content in the supernatant was measured by ELISA, and the level of IκBα was detected by Western blotting to reflect NF-κB activity. EMSA method was used to measure the activation of NF-κB. RESULTS: MC proliferation, MCP-1 expression and NF-κB activation were significantly enhanced and the expression of NF-κB-binding protein IκBα was obviously reduced in cells cultured in high glucose. Application of PTL obviously abolished the effects of high glucose. CONCLUSION: PTL can suppress high glucose-stimulated NF-κB activation and MCP-1 expression in rat MC. These data provide a theoretical basis for the clinical application of PTL in prevention and control of diabetic nephropathy.

[Protective effect and mechanism of astragaloside IV on oxidative stress injury of mesangial cells].

OBJECTIVE: To study the protective effect of astragaloside IV (AS IV) on H2O2 induced human mesangial cells (HMC), and further explore its molecular mechanism. METHOD: The cultured mesangial cells were divided into 5 groups: the normal control group, the H2O2 model group, the AS IV (12.5, 100 nmol x L(-1)) group and the Tempol (1 x 10(5) nmol x L(-1)) group. The MTT method was used to observe cell viability. Hoechst 33258 staining was used to observe the HMC apoptosis and DHE staining was used to detect the generation of reactive oxygen species (ROS). The flow cytometry was used to detect the changes in cell cycle. Western blot was used to detect the expression of Cyclin D1, CyclinA, p38, and T-p38. RESULT: H2O2 (1 x 10(5), 2 x 10(5), 3 x 10(5), and 4 x 10(5) nmol x L(-1)) could induce HMC oxidative stress injury, with significant decrease in the cell survival rate. AS IV (100 nmol x L(-1)) could significantly inhibit HMC oxidative stress injury induced by H2O2 (3 x 10(5) nmol x L(-1)), increase the survival rate of HMC cells, inhibit cell apoptosis, and decrease intracellular ROS production. AS IV could also increase the expression of Cyclin D1, recover normal cell proliferation, and decrease the expression of p38. CONCLUSION: AS IV can protect H2O2 induced oxidative stress injury in mesangial cells. Its mechanisms may be related to inhibiting the p38/MAPK signaling pathway, increasing the expression of Cyclin D1 and decreasing the intracellular ROS oxidative stress injury.

Poria cocos inhibits high glucose-induced proliferation of rat mesangial cells.

Mesangial cell proliferation is correlated with the progression of renal failure. The purpose of this study was to determine whether a water extract of Poria cocos Wolf (WPC), a well-known medicinal plant, regulates rat mesangial cell proliferation in the presence of high glucose (HG). HG significantly accelerated [(3)H]-thymidine incorporation, which was inhibited by WPC (1-50 µg/mL) in a dose-dependent manner. Cell migration and fibronectin mRNA expression data also supported the anti-proliferative effect of WPC. Western blot analysis revealed that pretreatment with WPC decreased the expression of cyclins and cyclin-dependent kinases (CDKs) and promoted the expression of p21(waf1/cip1) and p27(kip1). WPC also suppressed HG-induced p38 mitogen-activated protein kinase (p38 MAPK) and extracellular-signal-regulated kinase 1/2 (ERK 1/2) phosphorylation. Furthermore, WPC inhibited HG-induced production of dichlorofluorescein (DCF)-sensitive intracellular reactive oxygen species (ROS). In conclusion, HG promoted mesangial cell proliferation, and WPC inhibited this activity, at least in part, via induction of cell cycle arrest and activation of anti-oxidant properties. Taken together, these results suggest that P. cocos may be a potent regulator of HG-induced proliferation.

[Effects of Qufengtongluo Recipe on expressions of cell cycle regulatory proteins in rat mesangial cells].

OBJECTIVE: To observe the effects of Qufengtongluo Recipe (QFTLR) on the expressions of cell cycle regulatory proteins in rat mesangial cells (MCs) in vitro and investigate the mechanism by which QFTLR inhibits MC proliferation. METHODS: Using the methods of serum pharmacology, we studied the expressions of cell cycle regulatory proteins in rat MCs treated with QFTLR by laser scanning confocal microscope and immunohistochemistry. RESULTS: Compared with the normal control cells, the cells challenged with lipopolysaccharide (LPS) showed significantly enhanced expressions of cyclin D1, CDK2, and P21 (P<0.01) and obviously lowered protein expression of P27 (P<0.01). Treatment of the LPS-challenged cells with QFTLR and benazepril both resulted in significantly attenuated expressions of cyclin D1, CDK2, and P21 and obvious increase of P27 expression (P<0.05 or P<0.01), but QFTLR produced stronger effects than benazepril in regulating of cyclinD1, P21 and P27 protein expressions (P<0.05 or P<0.01). CONCLUSION: QFTLR inhibits rat MC proliferation in vitro possibly by down-regulating the cellular expressions of cyclin D1, CDK2, and P21 and up-regulating the expression of P27 protein.

Diabetic nephropathy-related active cyclic peptides from the roots of Brachystemma calycinum.

Three new cyclic peptides, namely duanbanhuains A-C (1-3), were isolated from the roots of Brachystemma calycinum which is a traditional medicine used to treat rheumatic diseases. Their structures were identified by means of a suite of MS and NMR experiments. These compounds were purposely evaluated for their inhibitory effects on the release of MCP-1, IL-6, collagen IV and reactive oxygen species (ROS) against high-glucose-stimulated mesangial cells. The results showed that compounds 1 and 2 exhibited potent inhibition on the production of IL-6, collagen IV and ROS at the concentration of 10 µM.

Inhibitory effects of two major isoflavonoids in Radix Astragali on high glucose-induced mesangial cells proliferation and AGEs-induced endothelial cells apoptosis.

Radix Astragali, the dried roots of Astragalus membranaceus var. mongholicus, is well known to have a protective effect on diabetic nephropathy. However, the effects of isoflavonoids in Radix Astragali on glomerular cells, which play a key role in the development of diabetic vascular complications, remain largely unknown. Thus, the purpose of this study was to investigate in vitro the effect of calycosin and calycosin-7-O-ß-D-glucoside, two major isoflavonoids in Radix Astragali, on high glucose-induced rat mesangial cells proliferation and AGEs-induced human glomerular endothelial cell apoptosis. The results indicated that both calycosin and calycosin-7-O-ß-D-glucoside (10-100 µM) could inhibit high glucose-induced mesangial cell early proliferation. Additionally, AGEs-mediated cell apoptosis was also attenuated by treatment of glomerular endothelial cells with either calycosin or calycosin-7-O-ß-D-glucoside (1-100 µM). Therefore, the results obtained in this study suggest that both calycosin and calycosin-7-O-ß-D-glucoside have a significant therapeutic potential to modulate the development and/or progression of diabetic nephropathy.

Extract of Cassiae Semen and its major compound inhibit S100b-induced TGF-beta1 and fibronectin expression in mouse glomerular mesangial cells.

Non-enzymatic glycation reactions between reducing sugar and free reactive amino groups of protein lead to the formation of advanced glycation end products, which increase under conditions of aging or diabetes. A previous study showed that extracts of Cassiae Semen (CS), the seed of Cassia tora, had inhibitory activity on advanced glycation end products formation in vitro. To examine the pharmacological effects of a butanol-soluble extract of CS under conditions of diabetic nephropathy, we evaluated the expression of transforming growth factor-beta1 (TGF-beta1) and fibronectin, key mediators of diabetic nephropathy, in mouse glomerular mesangial cells cultured in the presence of S100b (a specific ligand for receptor of advanced glycation end products). CS inhibited S100b-induced TGF-beta1 and fibronectin expression in mouse mesangial cells by suppressing activation of Smad2/3, extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), and oxidative stress. Moreover, CS suppressed nuclear factor-kappa B (NF-kappaB) activation in S100b-stimulated mouse mesangial cells. To identify the active compounds of CS, three major compounds, rubrofusarin-6-O-beta-d-gentiobioside (CS-A), toralactone-9-O-beta-d-gentiobioside (CS-B), and cassiaside (CS-C), were tested in cells. Of these compounds, CS-A significantly decreased the expression of TGF-beta1 and fibronectin and NF-kappaB DNA binding activity. These findings suggest that CS, especially CS-A, has potential as a preventive agent for advanced glycation end products-related diabetic complications.