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【Objective】 To explore the role and mechanism of TRPC in promoting extracellular matrix (ECM) deposition in rat glomerular mesangial cells (HBZY-1). Methods Immunofluorescence staining was performed to observe the distribution and expression of TRPC1 and TRPC6 in HBZY-1 cells. After AngⅡ stimulation, qRT-PCR and Western blotting were used to detect the mRNA and protein expressions of Gαq/PLCβ4/TRPC signaling pathway main proteins and ECM deposition indicators (α-SMA, collagenⅢ and fibronectin). By silencing the expressions of TRPC1 and TRPC6 by RNA interference, the expressions of ECM deposition indicators were detected. Changes in [Ca2+]i influx were determined through Fluo-4AM Ca2+ imaging. 【Results】 Both TRPC1 and TRPC6 were expressed in HBZY-1, and were mainly located in cell membrane and cytoplasm. After AngⅡ stimulation, Gαq/PLCβ4/TRPC signaling pathway was activated, and the mRNA and protein expressions of Gαq, PLCβ4, TRPC1 and TRPC6 were all increased (P<0.05). [Ca2+]i influx also increased (P<0.01), and the mRNA and protein expressions of ECM deposition indicators (α-SMA, ColⅢ and Fn) were upregulated (P<0.05). Silencing the expressions of TRPC1 and TRPC6 by RNA interference led to decreased [Ca2+]i influx (P<0.05), and downregulated mRNA and protein expressions of ECM deposition indicators in HBZY-1 cells (P<0.05). The results suggested that inhibition of TRPC expressions could inhibit AngⅡ induced ECM deposition in HBZY-1 cells, which might be associated with decreased [Ca2+]i influx. 【Conclusion】 TRPC may be a novel therapeutic target of renal fibrosis.
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【Objective】 To explore the role and mechanism of TRPC6 in apoptosis of glomerular mesangial cells (HBZY-1) induced by endoplasmic reticulum stress (ERS). 【Methods】 The experiment groups were classified as follows: normal control (NC), thapsigargin (TG), TG+SKF96365, and TG+TRPC6 siRNA groups. Transcription and protein expressions of TRPC6 and ERS related proteins (GRP78 and Caspase12) were detected by qRT-PCR and Western blotting. Additionally, cell apoptosis was measured by flow cytometry and Hoechst33258. Finally, Fluo-4 AM Ca2+ imaging technique was used to determine changes of intracellular calcium ( [Ca2+] i) by laser scanning confocal microscope. 【Results】 Morphological changes of apoptotic cells were characterized by nuclear enrichment or nuclear fragmentation, and the apoptosis rate was increased after TG stimulation. The expressions of TRPC6 and ERS related proteins (GRP78 and Caspase12) were elevated in TG group compared with NC group (P<0.05). Pre-incubation of HBZY-1 cells with SKF96365 and TRPC6 siRNA decreased cell apoptosis (P<0.05). The entry of [Ca2+] i also increased after TG stimulation (P<0.05). The expressions of TRPC6, GRP78 and Caspase12 were downregulated compared with TG group after treatment with SKF96365 and TRPC6 siRNA accompanied by decreased [Ca2+] i (P<0.05). 【Conclusion】 Taken together, this study suggests that inhibition of TRPC6 can alleviate TG-induced HBZY-1 cell apoptosis.
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Renal fibrosis， the final pathological outcome of end-stage chronic kidney diseases， is associated with inflammation， oxidative stress， epithelial-mesenchymal transdifferentiation （EMT）， and extracellular matrix deposition. It belongs to the categories of edema， ischuria， anuria and vomiting， and consumptive disease in traditional Chinese medicine （TCM）， with the key pathogenesis of Qi deficiency and blood stasis and the primary treatment principle of replenishing Qi and activating blood. Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma mainly contains astragalosides， polysaccharides， calycosin， salvianolic acid， and tanshinone， with the effect of tonifying Qi and activating blood. Studies have shown that this herb pair and its active components can delay the progress of renal fibrosis by regulating multiple signaling pathways. With consideration to the pathogenesis of Qi deficiency and blood stasis， this article reviews the research progress in the mitigation of renal fibrosis by Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma from the aspects of protecting glomerular filtration barrier， inhibiting EMT and mesangial cell proliferation， improving renal hemodynamics， and protecting renal function. Furthermore， the mechanisms were summarized. Specifically， Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma and its effective components can improve mitochondrial function and fatty acid metabolism， alleviate endoplasmic reticulum stress and autophagy disorders， and inhibit immune inflammation and oxidative stress by regulating nuclear factor E2-related factor 2 （Nrf2）/PTEN-induced kinase 1 （Pink1）， Nrf2/antioxidant response element （ARE）， tumor necrosis factor-α （TNF-α）/nuclear transcription factor-κB （NF-κB）， miR-21/Smad7/transforming growth factor beta （TGF-β）， Wnt/β-catenin， long non-coding RNA-taurine up-regulated gene 1 （lncRNA-TUG1）/tumor necrosis factor receptor-associated factor 5 （TRAF5）， Ras-related C3 botulinum toxin substrate 1 （Rac1）/cell division cycle protein 42 （CDC42）， Ras homolog （Rho）/Rho-associated coiled-coil containing protein kinase （ROCK）， phosphatidylinositol-3-kinase （PI3K）/protein kinase B （Akt）， Janus kinase （JAK）/signal transducer and activator of transcription （STAT）， peroxisome proliferator-activated receptor α （PPARα）/peroxisome proliferator-activated receptor γ coactivator l alpha （PGC-1α）， and p38 mitogen-activated protein kinase （p38 MAPK）. This review aims to provide references for the relevant research， give play to the role of Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma， and provide guidance for the clinical treatment of renal fibrosis.
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Hyperglycemic kidney injury (HKI) is a common complication of diabetic patients. We examined the relationship between HKI and the abnormal expression of 5-hydroxytryptamine (5-HT) system induced by hyperglycemia in type 2 diabetes mellitus (T2DM). In animal experiments, a T2DM model was established in mice by feeding a high-fat diet with intraperitoneal injection of streptozotocin. The mice were treated with the 5-HT2A receptor (5-HT2AR) antagonist sarpogrelate hydrochloride (SH) and 5-HT synthesis inhibitor carbidopa (CDP) (respectively or in combination). In cell culture experiments, human glomerular mesangial cells (HMC) were stimulated with D-glucose (D-Glu), and 5-HT2AR, 5-HT synthesis, and 5-HT degradation were inhibited by SH, CDP, or monoamine oxidase A (MAO-A) inhibitor clorgyline. Periodic acid-Schiff (PAS) staining and Masson staining, immunohistochemistry and Western blot, fluorescent probe, and enzyme linked immunosorbent assay (ELISA) and enzyme reagent were respectively used to detect histopathology, protein expression, intracellular reactive oxygen species (ROS), and biochemical indexes. The animal experiments were in accordance with the regulations of the Animal Ethics Committee of China Pharmaceutical University. The results showed that 5-HT2AR, 5-HT synthases, and MAO-A were expressed in glomerular basement membrane and kidney tubular epithelial cells of mouse kidney and HMC. The expression of these proteins was significantly up-regulated in T2DM mice or when HMC cells were exposed to high concentration of D-Glu. HKI, characterized by abnormal renal function, glomerular swelling, and glomerular basement membrane thickening and fibrosis, is closely associated with an increase in kidney 5-HT2AR, 5-HT synthesis, and 5-HT degradation. Among them, 5-HT2AR can mediate the expression of 5-HT synthases and MAO-A; MAO-A can catalyze the degradation of 5-HT to increase the production of mitochondrial ROS, leading to the phosphorylation of nuclear factor kappa B (NF-κB) with the production of inflammatory cytokines, and the up-regulation of matrix metalloproteinase-2 (MMP-2) and α-smooth muscle actin (α-SMA) with the production of collagens. SH and CDP can effectively treat HKI, and the combination of SH and CDP has a clear synergistic effect.
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To explore the affect and mechanisms of rapamycin on mesangial cell proliferation and cell cycle, rat mesangial cells (HBZY-1) were cultured and divided into the six groups: normal; normal with platelet derived growth factor (PDGF) 20 ng·mL-1; PDGF + rapamycin 1, 10, 100, 1 000 nmol·L-1. The cell proliferation was measured by MTT in 24 and 48 h; flow cytometry was used to detect the cell cycle phase. Western blot was performed to determine cyclin D1,cyclin E, cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), p27, p70S6K/p-p70S6K protein expression. The p27 mRNA was detect by Real-time PCR. The results showed that rapamycin significantly suppressed PDGF induced glomerular mesangial cells (MCs) proliferation in a dose and time-dependent manner, but with the dose increased (1 to 1 000 nmol·L-1), the time dependence gradually weakened. Rapamycin inhibited mesangial cell proliferation and arrested the cell cycle in the G0/G1 phase. PDGF at 20 ng·mL-1 significantly increased the expression of cyclin D1, cyclin E and CDK2, CDK4 (P < 0.05), but rapamycin did not affect the expression of cyclin D1, cyclin E and CDK2, CDK4. Rapamycin can significantly inhibited p70S6K phosphorylation, up-regulated the expression of p27 protein and mRNA. Collectively, rapamycin has the effect of inhibiting the glomerular mesangial cells proliferation of mesangial cells by regulating the transcription of p27 mRNA, increasing its protein expression through the mTORC1/p70S6K pathway, resulting in decreased activity of cyclin-CDK, and blocking cell cycle in G0/G1 phase.
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Objective To investigate the effect of different concentration of uric acid on proliferation and phentyic change of HBZY-1 cells in vitro. Methods The morphological change of HBZY-1 cells exposed to uric acid was observed under the inverted microscope and transmission electronmicroscope. MTT bioassay was used to assess the effect of uric acid on the proliferation of HBZY-1 cell. ELISA method was used to assess the level of angiotensin Ⅱ in HBZY-1 cell incubated with different concentration of uric acid for different treatment duration.The protein and mRNA expression of α-smooth muscle actin (α-SMA) , transforming growth factor-β1 (TGF-β1) and fibronectin in HBZY-1 cells was measured by Western blot and RT-PCR assay, respectively. Results Uric acid (0.4 mmol/L, 24 h) could induce phentypic change of messangial cell from triangle to fibroblast-like morphology.Uric acid could significantly stimulate the proliferation of HBZY-1 cells for 24 h in a concentration-dependent manner (P < 0.05). Uric acid could stimulate the secration of angiotensin Ⅱ in a dose-and time-dependent manner.RT-PCR and Western blot results showed that the markers of myofibroblast transdifferentiation, including a-smooth muscle actin (α-SMA) , transforming growth factor-β1 (TGF-β1) and fibronectin, were significantly upregulated in HBZY-1 cell treated with over 0.1 mmol/L uric acid for 48 h compared with the control group (P < 0.05, respectively). Conclusion Uric acid can induce the proliferation and phentypic change of HBZY-1 in vitro.
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Objective • To extract serum IgA1 from patients with IgA nephropathy (IgAN) (end stage renal disease vs long-term stable renal function) to explore the effect on proliferation rate of human mesangial cells (HMCs) and the level of transforming growth factor-β1 (TGF-β1). Methods • Nine patients with primary IgAN were divided into rapidly progressive group (n=6) and long-term stable group (n=3). Jacalin affinity chromatography and sephacryl S-200HR molecular sieves were used to distract serum IgA1. HMCs were cultured and co-cultivated with different IgA1 concentration (10, 50, 250 and 1 000 μg/mL)at point of 12 h and 24 h respectively. The proliferation rate was measured by cell counting kit-8 (CCK8). The expression of TGF-β1 mRNA was measured via quantitative real-time PCR (qRT-PCR). Enzyme-linked immunosorbent assay (ELISA) was used to detect TGF-β1 protein. Results • Serum IgA1 from IgAN patients with different renal functions (end stage renal disease vs long-term stable renal function) activated proliferation of HMC significantly, presenting with time-dependence and concentration-dependence. The highest value showed at 250 μg/mL and 1 000 μg/mL respectively. Serum IgA1 in two groups of patients statistically increased the expression of TGF-β1 mRNA and protein. In group with end stage renal disease, the summit stood at 10 μg/mL and started to decrease by degrees afterwards; while in group with long-term stable renal function, the level of TGF-β1 increased in a parallel manner with the serum IgA1. Conclusion • Serum IgA1 from IgAN patients with different renal functions (end stage renal disease vs long-term stable renal function) can both promote the proliferation of HMC. There is no dramatical difference observed with in10-50 μg/mL, but the IgA1 from group with end stage renal disease reveals a stronger effect on TGF-β1, in accordance with the pathological type of the patients (IgA sclerosis), suggesting the prognostic value of serum IgA.
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Objective·To extract serum IgA1 from patients with IgA nephropathy (IgAN) (end stage renal disease vs long-term stable renal function) to explore the effect on proliferation rate of human mesangial cells (HMCs) and the level of transforming growth factor-β1 (TGF-β1). Methods?·?Nine patients with primary IgAN were divided into rapidly progressive group (n=6) and long-term stable group (n=3). Jacalin affinity chromatography and sephacryl S-200HR molecular sieves were used to distract serum IgA1. HMCs were cultured and co-cultivated with different IgA1 concentration (10, 50, 250 and 1?000?μg/mL)at point of 12?h and 24?h respectively. The proliferation rate was measured by cell counting kit-8 (CCK8). The expression of TGF-β1 mRNA was measured via quantitative real-time PCR (qRT-PCR). Enzyme-linked immunosorbent assay (ELISA) was used to detect TGF-β1 protein. Results?·?Serum IgA1 from IgAN patients with different renal functions (end stage renal disease vs long-term stable renal function) activated proliferation of HMC significantly, presenting with time-dependence and concentration-dependence. The highest value showed at 250?μg/mL and 1?000?μg/mL respectively. Serum IgA1 in two groups of patients statistically increased the expression of TGF-β1 mRNA and protein. In group with end stage renal disease, the summit stood at 10?μg/mL and started to decrease by degrees afterwards; while in group with long-term stable renal function, the level of TGF-β1 increased in a parallel manner with the serum IgA1. Conclusion?·?Serum IgA1 from IgAN patients with different renal functions (end stage renal disease vs long-term stable renal function) can both promote the proliferation of HMC. There is no dramatical difference observed with in10-50?μg/mL, but the IgA1 from group with end stage renal disease reveals a stronger effect on TGF-β1, in accordance with the pathological type of the patients (IgA sclerosis), suggesting the prognostic value of serum IgA.
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Objective To investigate the effect and mechanism of all-trans retinoic acid (ATRA) on the cyclosporin (CsA)-induced proliferation and apoptosis of glomerular mesangial cells in rat models. Methods The glomerular mesangial cells induced by different doses of CsA were treated with different doses of ATRA. MTT assay was carried out to detect cell proliferation. Hoechst 33258 fluorescent staining was adopted to observe the morphology of the apoptotic cells. Flow cytometry was conducted to detect the cellular apoptosis rate. Immunofluorescent staining was employed to quantitatively measure the expression level of mitochondria-derived pro-apoptotic Smac protein. Results Compared with the control group, administration of CsA at a dose of 0.5 μg/mL and above could suppress cellular proliferation, and use of CsA at a dose of 1.0 μg/mL and above could induce cellular apoptosis. The expression level of Smac protein was significantly up-regulated by CsA administration with a dose and time dependence (all P<0.05).Compared with the CsA group, combined administration of CsA and ATRA exerted a more significant inhibitory effect on cellular proliferation. Supplement of ATRA could significantly inhibit glomerular mesangial cellular apoptosis induced by CsA and down-regulate the expression of Smac protein with a dose dependence (both P<0.05). Conclusions CsA can inhibit cellular proliferation, induce cellular apoptosis and up-regulate the expression of Smac protein of glomerular mesangial cells. ATRA is capable of suppressing glomerular mesangial cellular apoptosis induced by CsA, which is probably mediated by the Smac signaling pathway.
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To investigate the effect of estrogen receptor (ER) agonist 17β-estradiol and G protein-coupled estrogen receptor 1 (GPER) agonist fulvestrant on masangial cell fibrogenesis under protein kinase C (PKC),we quantified type Ⅳ collagen (COL4A1),fibronectin (FN1),connective tissue growth factor (CTGF) and transforming growth factor-β1 (TGFβ1) gene transcription and semi-quantified phosphorylation of Akt signal upon Phorbol 12-myristate 13-acetate stimulation (which increased COL4A1,FN1,CTGF and TGFβ1 gene transcription to 2.5-0.5,1.4 ±0.2,26 ± 11 and 1.9 ±0.3 times compared with baseline,P ＜0.05) when incubated with the two drugs.It was found that 17β-estradiol and fulvestrant down-regulated COL4A1,FN1,CTGF and TGFβ1 genes transcription (P ＜0.05) and Akt signaling under PKC activation via ER and GPER.ER and GPER agonists are beneficial in protecting the mesangial cells from fibrogenic stimuli by inhibiting PKC signaling and excessive extracellular matrix production.
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Objective:To investigate the expression of immunoglobulin A (IgA) in human mesangial cells (HMCs).Methods:The HMCs were cultured.The subcellular location of IgA was detected by immunofluorescence staining;the transcripts of Ig α,Ig κ and Ig λ constant region were detected by reverse transcription-polymerase chain reaction (RT-PCR) and further analyzed by DNA sequencing.The expressions of Igαt and Ig λ were detected at transcription level by Western blot after the cytoplasmic protein extraction.The culture supernatant was collected to explore whether IgA could be secreted out of the cell and the protein was further analyzed by mass spectrometry after being purified by affinity chromatography with jacalin-sepharose.The results of DNA sequencing and mass spectrometry were aligned with the mRNA and amino acid sequences in the National Center of Biotechnology Information (NCBI) data-base.Results:By immunofluorescence staining,we detected the presence of IgA heavy chain Ig α,light chain,both Ig κ and Ig λ in expressions of transcripts of Ig α1,Ig α2,Ig κ and Ig λ in the HMCs and the alignment of the sequences of the RT-PCR products with those of the Ig Cα1,Ig Cα2,Ig κ and Ig λ mRNA in the NCBI database exhibited that the similarities were 99％,97％,98％ and 97％,respectively.Western blot showed Ig α and Ig λ expressions in the cell lysate and secretion of Ig α1 and Ig α2 heavy chains in cell culture supernatant.To further explore the protein that secreted into the supernatant,after supernatant affinity chromatography with jacalin-sepharose,the proteins were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and the band approximating to 65 000 was cut and sent to mass spectrometry.The results were aligned with the amino acid sequences of Ig α1 and Ig α2 constant region in NCBI database,showing that amino acids between No.52 and No.104,amino acids between No.154 and No.221,amino acids between No.276 and No.327 from Ig Cα1 and amino acids between No.52 and No.113,amino acids between No.151 and No.204,amino acids between No.251 and No.314 from Ig Cα2 were the same with those derived from B cells.Conclusion:Our findings suggested that HMCs could synthesize and secret IgA.
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Objective To investigate the influence of glycyrrhizic acid (GA) on oxidative stress injury in glomerular mesangial cells (HBZY-1) induced high glucose.Methods HBZY-1 was cultured and divided into the normal control group,high glucose group and high glucose +-GA group.The cell proliferation activity was measured by MTT assay.UV spectrophotometry was used to detect the SOD and MDA levels.And the ROS changes were detected by laser confocal microscopy.Immunohistochemistry and Western blot were used to detect the expression of Mn-SOD protein in various groups.Mn-SOD mRNA was detected by Q-PCR.Results (1) The morphological changes in each group:HBZY-1 cells were diamond-shaped,cells were normal and the structure was clear in the NG group.In the HG+-GA group,the number of cells was increased slightly,individual cell body was slightly hypertrophy.The cell structure in the HG group was not clear,the cells appeared flat,the number was increased significantly,the cell body was slightly hypertrophy.(2)The proliferative effect of HBZY-1 in each group:compared with the NG group,the OD value in the HG group was significantly increased,and the OD value in the HG+GA group was decreased compared with the HG group(P＜0.05).(3)The relative content of ROS in the HG group was higher than that in the NG group,and which in the HG+GA group was decreased (P＜0.05).(4)The expression of Mn-SOD in each group:the relative expression of Mn-SOD in the HG group was significantly lower than that in the NG group (P＜0.05),and the expression of Mn-SOD in the HG+-GA group was increased compared with the HG group(P＜0.05).Conclusion Glycyrrhizic acid has a certain inhibiting effect on the abnormal proliferation of HBZY-1 induced by high glucose.Glycyrrhizic acid can protect the cell hypertrophy and cell damage caused by glomerular mesangial cells via oxidative stress,and glycyrrhizic acid can regulate the Mn-SOD expression induced by high glucose.
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Objective To investigate the influence of glycyrrhizic acid (GA) on oxidative stress injury in glomerular mesangial cells (HBZY-1) induced high glucose.Methods HBZY-1 was cultured and divided into the normal control group,high glucose group and high glucose +-GA group.The cell proliferation activity was measured by MTT assay.UV spectrophotometry was used to detect the SOD and MDA levels.And the ROS changes were detected by laser confocal microscopy.Immunohistochemistry and Western blot were used to detect the expression of Mn-SOD protein in various groups.Mn-SOD mRNA was detected by Q-PCR.Results (1) The morphological changes in each group:HBZY-1 cells were diamond-shaped,cells were normal and the structure was clear in the NG group.In the HG+-GA group,the number of cells was increased slightly,individual cell body was slightly hypertrophy.The cell structure in the HG group was not clear,the cells appeared flat,the number was increased significantly,the cell body was slightly hypertrophy.(2)The proliferative effect of HBZY-1 in each group:compared with the NG group,the OD value in the HG group was significantly increased,and the OD value in the HG+GA group was decreased compared with the HG group(P＜0.05).(3)The relative content of ROS in the HG group was higher than that in the NG group,and which in the HG+GA group was decreased (P＜0.05).(4)The expression of Mn-SOD in each group:the relative expression of Mn-SOD in the HG group was significantly lower than that in the NG group (P＜0.05),and the expression of Mn-SOD in the HG+-GA group was increased compared with the HG group(P＜0.05).Conclusion Glycyrrhizic acid has a certain inhibiting effect on the abnormal proliferation of HBZY-1 induced by high glucose.Glycyrrhizic acid can protect the cell hypertrophy and cell damage caused by glomerular mesangial cells via oxidative stress,and glycyrrhizic acid can regulate the Mn-SOD expression induced by high glucose.
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Objective To observe the influence of serum containing esculentoside A(EsA) on the proliferation of glomerular mesangial cell (GMC) and the activation of ERK1/2-AP-1 pathway of glomerular mesangial cell induced by IL-1β.Methods SD rats were gavaged by different doses of EsA(5,10,20,40 mg/kg) for getting medicated sera.The control group was set (gavage by 0.5sodium carboxymethylcellulose);the EsA medicated serum was used to treat rGMC.The control serum group was set.The influence of EsA medicated serum in each group on rGMC proliferation was detected by MTT;the rGMC was divided into the blank control group,IL-1β single action group,IL-1β+ EsA double action group,IL-1β+ U016 double action group and IL-1β+ U0126 + EsA combined action group,which were synchronized and then cultured for 48 h.Western blot was used to detectthe expression of p-ERK1/2 and AP-1 an the imaging analysis was performed.Results The EsA medicated serum(5-10 mg/kg gavage) inhibited the cellular proliferation(P＜0.05 or P＜0.01);the IL-1β group promoted the expression of p-ERK1/2 and AP-1 in rGMC(P＜ 0.05),after acting on rGMC in the IL-1β+EsA double action group,IL-1β+U0126 double action group and IL-1β+U0126+EsA combined action group,the expression of p-ERK1/2 and AP-1 was decreased(P＜0.05).Conclusion Serum containing EsA (5～10 mg/kg gavage) significantly inhibits the rGMC proliferation;EsA inhibits IL-1β induced rGMC proliferation,its action pathway on ERK1/2-AP-1 is one of mechanisms for inhibiting rGMC proliferation.
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Objective:To evaluate the effects of rapamycin on the proliferation,apoptosis and cell cycle of glomerular mesangial cells induced by high glucose,and to explore its significance in the prevention and treatment of diabetic nephropathy. Methods:The rat GMC HBZY-1 was divided into four groups:control group,high glucose group,the first group of high glucose plus rapamycin,the second group of high glucose plus rapamycin. CCK-8 assay was used to detect the proliferation of cells, flow cytometry was introduced to evaluate the apoptosis and cell cycle of HBZY-1,Real-time PCR was used to detect the mRNA of AngiotensinⅡ(ANGⅡ),transfor ming growth factor beta1 ( TGF-β1 ) and vascular endothelial growth factor ( VEGF ) . Results: The proliferation level of HBZY-1 induced by high glucose was significantly increased,and the level of apoptosis decreased,and the expression level of ANGⅡ,TGF-β1 and VEGF was increased. Rapamycin significantly inhibited,and there was a dose dependent,and down regulated the expression of ANGⅡ,TGF-β1,and VEGF. For the cell cycle,the S phase cells in the high glucose group were significantly higher than those in the normal group (P<0. 05),and the S phase cell proportion was decreased after rapamycin intervention (P<0. 05). Conclusion:Rapamycin can inhibit the proliferation of HBZY-1 in high glucose,promote its apoptosis and lead to G1/S arrest,and down regulate the expression of ANGⅡ,TGF-β1 and VEGF.
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BACKGROUND: In China, mesangial proliferative glomerulonephritis (MsPGN) is one of the most common kidney diseases. In this study, we treated a rat model of chronic anti-Thy-1 MsPGN with Shenhua Tablet and evaluated whether the tablet was able to protect the kidney function. Thirty-six Wistar rats were randomly divided into six groups: (1) Sham surgery (Sham); (2) anti-Thy-1 nephritis model (Thy-1); (3) anti-Thy-1 nephritis model + irbesartan-treated (Irb); (4) anti-Thy-1 nephritis model + low-dose of Shenhua Tablet (SHL); (5) anti-Thy-1 nephritis model + medium-dose of Shenhua Tablet (SHM); (6) anti-Thy-1 nephritis model + high-dose of Shenhua Tablet (SHH). RESULTS: Thirteen weeks after drug treatment, urinary proteins were quantified and renal pathological changes were thoroughly examined at the time point of 24 h. Meanwhile, the expression levels of p-Erk1/2, cyclin D1 and p21 at the renal cortex were also tested. The levels of urinary proteins and total cholesterol in the blood were significantly reduced in rats treated with any drug tested in this study. The level of triglyceride was significantly reduced in all three Shenhua Tablet-treated groups. Renal pathomorphological scores were significantly improved in groups of Irb, SHM and SHH. Mesangial cell proliferation was significantly inhibited in any drug-treated group. p-Erk1/2 and cyclin D1 were downregulated whereas p21 was upregulated in the renal cortex. CONCLUSIONS: Our study indicated that Shenhua Tablet is able to inhibit the abnormal proliferation of mesangial cells and to prevent kidney damage, which is likely associated with downregulation of p-Erk1/2 and reduced activity of its downstream target-cyclin D1.
Subject(s)
Animals , Male , Drugs, Chinese Herbal/pharmacology , Glomerulonephritis, Membranoproliferative/drug therapy , Cell Proliferation/drug effects , Mesangial Cells/drug effects , Isoantibodies , Time Factors , Serum Albumin/analysis , Drugs, Chinese Herbal/therapeutic use , Glomerulonephritis, Membranoproliferative/pathology , Chronic Disease , Reproducibility of Results , Rats, Wistar , Mitogen-Activated Protein Kinase 1/analysis , Cyclin D1/analysis , Computers, Handheld , p21-Activated Kinases/analysisABSTRACT
MicroRNA could participate in a variety of physiological and pathological processes via iden-tifying the target genes by their seed sequences and regulating gene expression at post-transcriptional level. Glo-merular mesangial cell is one of the most chief cells of the glomerulus and plays an important role in the renal fi-brosis. The mesangial cellular proliferation and dysfunction may appear once attacked by pathogenic factors. Then,the extracellular matrix increases in the stimulation of inflammatory cytokines,eventually,resulting in glo-merular sclerosis and renal fibrosis. Reports have shown that series of microRNA could take part in the process of injury of mesangial cells and lead to renal fibrosis. MicroRNA could be a potential therapeutic target for early chronic kidney diseases.
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Objective Long noncoding RNA (lncRNA) has been identified to regulate DNA methylation, histone acetylation, and gene post-transcriptional regulation in kinds of diseases, including tumorigenesis, obesity and so on.Therefore, lncRNAs might be the potential targets of mesangial cells proliferation.Methods Mesangial cells were exposed to suitable concentration of TGF-β through cell proliferation assay;then the lncRNAs expression levels were detected by microarray in experimental group and control group separately;finally the differentially expressed lncRNAs were identified by RT-PCR;meanwhile, and the expression levels of target genes were also detected by RT-PCR.Results Cell viability assay confirmed that 10 ng/ml TGF-β could promote mesangial cell proliferation significantly.Totally, over 30 000 lncRNAs were detected in TGF-β treated MCs and control group cells separately.Compared to the control group, 5550 lncRNAs differentially expressed in TGF-β treated MCs, including 119 up-regulated and 147 down-regulated over 2 fold.RT-PCR results appeared that uc.60, MRAK079149, MRAK029456, XR_005507, XR_007641, uc.14, and uc.412 were significantly up-regulated in TGF-β treated MCs, and BC088254, DQ402472, BC098733, BC158832,BC098746 were stably down-regulated.Compared to the control group, the mRNA expression levels of AATF and NEK were increased in the TGF-β treated mesangial cells (P ＜ 0.05).AATF and NEK were downstream target genes of uc.412 and MRAK079149 respectively.Conclusion The differential expression of long noncoding RNAs presents in the experimental mesangial cells proliferation induced by TGF-β.
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Objective To investigate the effects of Hui-hui Gan-song Yin(HGY) on the accumulation of extracellular matrix of rat glomerular mesangial cells(MCs) induced by high glucose.Methods The 40 SD rats were randomly divided into normal control group(distilled water), glurenorm group(10 mg/kg), HGY high-dose group(10 g/kg) and HGY low-dose group(5 g/kg), 10 rats in each group.The rats in each group were treated with corresponding drugs, twice a day.After 3 days, the serum containing each drug were prepared to culture rat MCs in vitro.The MCs were divided into the normal control group( 10% serum of rats in normal control group ) , high glucose group ( 30 mmol/L glucose +10% serum of rats in normal control group), glurenorm group(30 mmol/L glucose+10% serum of rats in glurenorm group), HGY high-dose group(30 mmol/L glucose+10% serum of rats in HGY high-dose group) and HGY low-dose group(30 mmol/L glucose+10% serum of rats in HGY low-dose group).The fibronectin(FN), ColⅠand ColⅣ levels were detected by Western blot.Results Compared with normal control group, the expression of FN, ColⅠand ColⅣ in high glucose group increased(P<0.01).The HCY suppressed the protein expression of FN, ColⅠand ColⅣ significantly(P<0.05).Conclusion The serum containing HGY could suppressed protein expression of FN , ColⅠand ColⅣ and inhibit the accumulation of extracellular matrix of MCs induced by high glucose, which could protect glomerulus and delay the development of diabetic nephropathy.
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Objective To investigate the effects of β-adrenoceptor (β-AR) activation on the apoptosis in human mesangial cells and it's mechanism.Methods Cultured HMC were used in experiments and were divied into four groups:the control group; β-AR activation (β-AR agonist NE/Pra) group; β-AR inhibitor (Prop) group; antioxidants group.The experiments technology including PCR,confocal scanning microscope,immunofluorescence and Tunel.Results The results of RTPCR and confocal scanning microscope showed that β1-AR and β2-AR were expressed in human HMC.β-AR activation induced reactive oxygen species (ROS) increase in human MCs,the relative levels of ROS were elevated as early as 0.5 h after β-AR activation,and gradually increased and peaked at 4 h on a concentration and time dependent manner.Tunel results demonstrated that β-AR activation induced apoptosis with ROS on a concentration and time dependent manner,β-AR blocking agent-propranolol significantly inhibited β-AR activation induced apoptosis.Antioxidants including vitamin C and NAC could inhibited β-AR activation induced apoptosis (all P ＜ 0.01).Conclusions β-AR is functionally expressed in human mesangial cell,furthermore β-AR activation-induced ROS increase mediate apoptosis.Antioxidants can inhibit β-AR activation induced apoptosis.