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Aim To explore the effect of exogenous hydrogen sulfide ( H2 S ) on hypoxia/reoxygenation ( H/R) injury in glomerular mesangial cells and elucidate its relevant mechanism. Methods H/R induced mouse mesangial cell line ( SV40MES13 ) to establish cell damage model. Cell viability was detected by cell proliferation kit ( CCK8 ), the content of H
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Objective:To explore the expression of type 2 complement receptor (CR2) in mesangial cells of the renal tissue in IgA nephropathy (IgAN) and its possible mechanism involved in complement C3 deposition.Methods:The demographic data, samples of plasma and renal tissues of primary IgAN patients diagnosed by renal biopsy in the Guangdong Provincial People's Hospital from August 2021 to May 2022 were collected. According to the fluorescent intensity of mesangial complement C3 deposition, the patients were divided into complement C3 deposition ≥2+ group and complement C3 deposition <2+ group. The circulating IgA and complement C3 levels were detected by enzyme linked immunosorbent assay (ELISA). The influencing factors of kidney prognosis, plasma IgA and complement C3 levels were compared between the two groups. Immunofluorescence was used to detect the expression of IgA, complement C3 and CR2 in the renal mesangial cells of IgAN patients and normal renal tissues around renal carcinoma. Human mesangial cells were cultured in vitro and randomly divided into control group and experimental group. The experimental group was incubated with IgA protein (2 g/L) for 8 hours. The expressions of CR2 protein and mRNA were measured by Western blotting and real-time fluorescence quantitative PCR. The biological function of differential genes was analyzed by gene ontology (GO) and Kyto encyclopedia of genes and genomes (KEGG) enrichment analysis. Results:A total of 75 patients with IgAN were included in this study, including 50 patients in the complement C3 deposition ≥2+ group and 25 patients in the complement C3 deposition <2+ group. The proportions of patients with urine red blood cell count negative, 1+, 2+ and 3+-4+ in the complement C3 deposition ≥2+ group were 2.0%, 8.0%, 18.0% 72.0%, respectively, which were more serious than those in the complement C3 deposition <2+ group (4.0%, 4.0%, 52.0%, 40.0%) ( Z=-2.320, P=0.020). Meanwhile, the proportion of S1 in Oxford pathological classification in the complement C3 deposition ≥2+ group was higher than that in the complement C3 deposition <2+ group (68.0% vs. 40.0%, χ2=5.389, P=0.020), and there were no statistically significant differences in gender, age, 24-hour urinary protein, serum creatinine, other indicators of Oxford pathological classification between the two groups. ELISA results showed that plasma IgA concentration in the complement C3 deposition ≥2+ group was higher than that in the complement C3 deposition <2+ group [3.62 (2.95, 5.53) g/L vs. 2.72 (2.15, 4.24) g/L, Z=2.405, P=0.016], and the plasma complement C3 concentration was lower than that in the complement C3 deposition <2+ group [199.6 (116.0, 328.0) mg/L vs. 319.2 (158.3, 454.5) mg/L, Z=-2.383, P=0.017]. Spearman correlation analysis showed that the complement C3 deposition intensity was positively correlated with IgA deposition intensity in mesangial area ( rs=0.441, P<0.001). Immunofluorescence results showed that there was colocalization of IgA and complement C3 in the glomeruli of IgAN patients. The expression of CR2 in the kidney was consistent with complement C3 deposition, and CR2 was colocalization with complement C3. In vitro experiments, the expression of CR2 in IgA protein group was higher than that in the control group ( P<0.05). GO and KEGG enrichment analysis found that IgA protein induced active changes in various pathways of mesangial cells. Conclusion:IgA protein induces mesangial cells to express CR2 and participates in complement C3 deposition, which may be an important mechanism of complement C3 activation in IgAN.
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Objective:To investigate the effect of high glucose on intracellular cholesterol content in rat glomerular mesangial cells and the underlying mechanism.Methods:Rat glomerular mesangial cells were cultured in vitro and divided into high glucose culture group (high glucose group, medium glucose concentration of 30 mmol/L) and normal glucose culture group (normal group, medium glucose concentration of 5.5 mmol/L). Lipid content was determined by oil red O staining and spectrophotometer colorimetry at 24, 36 and 48 h of culture. Intracellular protein imprinting was used to detect the expression of low density lipoprotein cholesterol receptor (LDLR) and adenosine triphosphate binding cassette A1 (ABCA1). Results:Oil red O staining showed that the intracellular lipid drops in the high glucose group were less than those in the normal group at 36 h, and there was no significant difference between the two groups at 24 h and 48 h of culture. The total cholesterol (TC) and cholesterol ester (CE) in glomerular mesangial cells of rats in the high glucose group were significantly lower than those in the normal group ( P=0.028, 0.029), while there was no significant difference in the free cholesterol (FC) between the two groups ( P=0.306). There was no significant difference in TC, CE and FC between the two groups at 24 and 48 h of culture (all P>0.05). The expression of LDLR in mesangial cells of rats in high glucose group was significantly lower than that in normal group at 24, 36 and 48 h of culture ( P=0.043, 0.004, 0.028), and the expression of ABCA1 was significantly higher than that in normal group at 24, 36 and 48 h of culture, ( P=0.050, 0.009, 0.006). Conclusions:High glucose may reduce intracellular cholesterol content in rat glomerular mesangial cells by reducing LDLR protein expression and increasing ABCA1 protein expression.
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AIM: To investigate the injury of emodin (EMO) in reduce of glomerular mesangial cells (MCs) in lupus nephritis by targeting forkhead protein K2 (FOXK2) through miR-96-5p. METHODS: The contents of 24 h urine protein, serum urea nitrogen (BUN) and serum creatinine (Scr) in MRL / faslpr mice (lupus nephritis group) and MRL / MPJ mice (control group) were detected. MCs were separated, purified and divided into: MCs group (MCs without any treatment), L-EMO group (MCs treated with 10 μmol/L Emodin), M-EMO group (MCs treated with 25 μmol / L Emodin), H-EMO group (MCs treated with 50 μmol / L Emodin), H-EMO + miR-96-5p-NC group (MCs treated with 50 μmol / L Emodin and transfected with miR-96-5p-NC), and H-EMO + miR-96-5p-minic group (MCs treated with 50 μmol/ L Emodin and transfected with miR-96-5p-minic). Double luciferase report experiment was used to verify the targeting relationship between miR-96-5p and FOXK2. The real-time quantitative fluorescent polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-96-5p. Western blot was used to detect the expression of FOXK2 and apoptosis related proteins. The enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors in MCs. cell count kit 8 (CCK-8) was used to determine the activity of MCs. Annexin-V FITC/PI double staining was used to detect apoptosis of MCs. RESULTS: Compared with the control group, 24 h urinary protein content, serum BUN and Scr levels in the lupus nephritis group were significantly increased (P< 0.05). Compared with the MCs group, the miR-96-5p expression, interleukin1β (IL-1β), interleukin6 (IL-6), tumor necrosis factor-α (TNF-α), A450 value and B-lymphoblastoma-2 (Bcl-2) protein in the L-EMO group, M-EMO group and H-EMO group were significantly decreased (P<0.05), the FOXK2 level, cell apoptosis rate, Bcl-2 related X gene (Bax), aspartate specific cysteine proteinase-3 (cleaved Caspase-3) protein levels were significantly increased, respectively (P<0.05), the effect of Emodin was dose-dependent. Compared with the H-EMO group and H-EMO+miR-96-5p-NC group, H-EMO+miR-96-5p-minic group obviously increased the miR-96-5p expression, inflammatory factor levels, A450 value and Bcl-2 protein level (P<0.05), and obviously decreased FOXK2 level and cell apoptosis rate (P< 0.05). CONCLUSION: EMO can reduce the injury of lupus nephritis MCs by down-regulating miR-96-5p and then up-regulating FOXK2.
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Aim To investigate the inhibitory effect of ginsenoside Rg1 on sodium palmitate induced fibrosis in human glomerullar mesangial cells (HMCs) and its mechanism. Methods (1) HMCs were treated with different concentrations of PA for 24 h, the intracellular lipid accumulation was observed by oil red staining, and the intracellular ROS production was detected by H2DCFDA kit; (2) HMCs were divided into control, PA (160 μmol·L
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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.
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Animals , Rats , Lipopolysaccharides/pharmacology , Mesangial Cells , Resveratrol/pharmacology , Transcription Factor AP-1 , Transforming Growth Factor beta1 , Intercellular Signaling Peptides and Proteins , Cell Proliferation , DNA , Cells, CulturedABSTRACT
Abstract Introduction: Diabetes mellitus (DM) is a chronic disease characterized by hyperglycemia that leads to diabetic nephropathy (DN). We showed that P2X7, a purinergic receptor, was highly expressed in DM; however, when oxidative stress was controlled, renal NO recovered, and the activation of this receptor remained significantly reduced. The aim of this study was to assess the influence of NO on the P2X7 and apoptosis in mouse immortalized mesangial cells (MiMC) cultured in high glucose (HG) medium. Methods: MiMCs were cultured with DMEM and exposed to normal glucose (NG), mannitol (MA), or HG. Cell viability was assessed by an automated counter. Supernatants were collected for NO quantification, and proteins were extracted for analysis of NO synthases (iNOS and eNOS), caspase-3, and P2X7. Results: Cell viability remained above 90% in all groups. There was a significant increase in the proliferation of cells in HG compared to MA and NG. NO, iNOS, caspase-3, and P2X7 were significantly increased in HG compared to NG and MA, with no changes in eNOS. We observed that there was a strong and significant correlation between P2X7 and NO. Discussion: The main finding was that the production of NO by iNOS was positively correlated with the increase of P2X7 in MCs under HG conditions, showing that there is a common stimulus between them and that NO interacts with the P2X7 pathway, contributing to apoptosis in experimental DM. These findings could be relevant to studies of therapeutic targets for the prevention and/or treatment of hyperglycemia-induced kidney damage to delay DN progression.
Resumo Introdução: Diabetes mellitus (DM) é uma doença crônica caracterizada por hiperglicemia levando à nefropatia diabética (ND). Mostramos que P2X7, um receptor purinérgico, foi altamente expresso na DM; entretanto, quando o estresse oxidativo foi controlado, o NO renal recuperou-se, e a ativação deste receptor permaneceu significativamente reduzida. Este estudo objetivou avaliar a influência do NO no P2X7 e a apoptose em células mesangiais imortalizadas de camundongos (CMiC) cultivadas em meio de glicose elevada (GE). Métodos: CMiCs foram cultivadas em meio DMEM e expostas à glicose normal (GN), manitol (MA), ou GE. A viabilidade celular foi avaliada por contador automático. Sobrenadantes foram coletados para quantificação de NO, e foram extraídas proteínas para análise de NO sintases (iNOS e eNOS), caspase-3, e P2X7. Resultados: A viabilidade celular permaneceu acima de 90% em todos os grupos. Houve aumento significativo na proliferação de células na GE comparado com MA e GN. NO, iNOS, caspase-3 e P2X7 foram significativamente aumentados na GE comparados com GN e MA, sem alterações na eNOS. Observamos que houve correlação forte e significativa entre P2X7 e NO. Discussão: O principal achado foi que a produção de NO pela iNOS foi positivamente correlacionada com aumento de P2X7 em CMs sob condições de GE, mostrando que existe um estímulo comum entre eles e que o NO interage com a via do P2X7, contribuindo para apoptose na DM experimental. Estes achados podem ser relevantes para estudos de alvos terapêuticos para a prevenção e/ou tratamento de danos renais induzidos por hiperglicemia para retardar a progressão da ND.
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AlM: To investigate the effect of C. sinensis extracts on HBx (Hepatitis B virus X protein) induced cell proliferation and extracellular matrix (ECM) accumulation and the underlying mechanism in cultured human mesangial cells (HMCs). METHODS: Human mesangial cells were stable transfected with pCMV-HBx to establish an HBx over-expression model, and a control group was transfected with an empty vector. Cell proliferation was determined by MTT assay and DNA synthesis assay. Western blotting was used to measure the expression of PI3K/Akt signaling pathway-related proteins and extracellular matrix. RESULTS: HBx transfection induced cell proliferation, matrix accumulation. HBx-transfected mesangial cells had increased activity of the PI3K/Akt pathways, and treatment with C. sinensis suppressed this effect. CONCLUSlON: C.sinensis attenuates the HBx-induced human mesangial cell proliferation and matrix production in human mesangial cells via inhibition of the PI3K/Akt pathways.
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Aim To study the protective effect of Mon¬golian medicine rhubarb-3 decoction on renal function of CRF model rats and explore its mechanism.Meth¬ods SD rats were randomly divided into normal group and model group.The model group was established by adenine gavage.After successful modeling, they were randomly divided into model, positive, rhubarb-3 de¬coction low, medium and high dose groups, which were administered at intervals for 12 weeks.The bio¬chemical detection of CSF, BUN and Scr was per¬formed.The pathological changes of renal tissues were observed by HE and Masson staining.The expression of PCNA and a-SMA in renal tissues was detected by immunohistochemistry.The mRNA expressions of CK18, Vimentin, TGF-pi and FN in kidney tissues were detected by RT-PCR.The expressions of a-SMA, E-cadherin, PCNA, Smad2, Smad3 protein in kidney tissue were detected by Western blot.TGF-pi induced abnormal proliferation of I IMC and interstitial transfor¬ mation of HK-2, and at the same time, it was treated with serum containing rhubarb-3 decoction.The prolif¬eration of HMC was detected by CCK-8.Interstitial transformation of HK-2 was detected by RT-PCR.Re- suits Compared with the model group, BUN and Scr in CRF rats decreased, and the expression of fibrosis- related proteins and genes in renal tissue decreased af¬ter rhubarb-3 decoction treatment.The serum contai¬ning rhubarb-3 decoction significantly inhibited HMC proliferation and HK-2 interstitial transformation.Con¬clusions Rhubarb-3 Decoction can improve renal function in CRF model rats, and its mechanism may be related to inhibiting HMC proliferation and HK-2 inter¬stitial transformation and probably regulating TGF-f}/ Smad signaling pathway.
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ObjectiveTo observe the intervention of modified Sanrentang on the lipopolysaccharide-induced proliferation of rat glomerular mesangial cells， the phosphatidylinositol-3 kinase（PI3K）/protein kinase B（PKB/Akt）/nuclear factor kappa B（NF-κB） signaling pathway， and to investigate its mechanism in improving kidney inflammation in rats with immunoglobulin A nephropathy（IgAN）. MethodThe 18 rats were divided into 3 groups by serum pharmacology method： normal group， high-dose and low-dose （20.70，10.35 g·kg-1·d-1） groups with 6 rats in each group. Modified Sanrentang high- and low-dose groups were intragastric with the corresponding solution of modified Sanrentang， and normal group was intragastric with equal volume of distilled water. After 5 days of intragastric administration， blood samples were collected to prepare drug-containing serum. Rat mesangial （HBZY-1） were divided into five groups of normal group， LPS 10 mg·L-1 in the model group， benazepril（50 μmol·L-1）， modified Sanrentang high- and low-dose group. Preclude the use of methyl thiazolyl tetrazolium（MTT） method detect the proliferation activity of HBZY-1 cells， enzyme-linked immunosorbent assay（ELISA） was used to determine the content of each group type Ⅳ collagen（ColⅣ），Western blot and Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） were used to detect protein and mRNA expression levels of PI3K/Akt/NF-κB signaling pathway. ResultAs compared with the normal group， MTT assay showed that exposure to LPS significantly enhanced the proliferative activity， the ColⅣ was increased significantly of HBZY-1 cells（P<0.01）， p-Akt， p-p65 was increased significantly （P<0.01）. Compared with the model group， the proliferation and ColⅣ of rat chronic glomerulonephritis cells induced by LPS by inhibiting PI3K/Akt/NF-κB signaling pathway（P<0.01）， and the phosphorylation of Akt was significantly inhibited（P<0.01）， the expression levels of NF-κB p65 was reduced in modified Sanrentang high-dose group（P<0.01）. ConclusionModified Sanrentang could inhibit cell proliferation and the content of ColⅣ in rat mesangial cells induced by LPS， and its mechanism might be related to suppression of PI3K/Akt signaling pathway.
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ObjectiveTo study the effect and mechanism of Wuwei Xiaoduyin in treating rat renal mesangial cells （HBZY-1） induced by lipopolysaccharide （LPS） through the nuclear factor-κB （NF-κB） signaling pathway. MethodRat HBZY-1 cells were randomly assigned into the normal group， model group， benazepril （50 μmol·L-1） group， and high- and low-dose （2.75 and 0.69 g·kg-1） Wuwei Xiaoduyin groups. The normal group， model group， and benazepril group were treated with 10% normal rat serum， and the Wuwei Xiaoduyin groups with 10% medicated serum. Except the normal group， the other four groups were treated with LPS （100 ng·mL-1） for modeling in vitro. The changes of cell morphology were observed under optical microscope. The expression of NF-κB p65 was detected by immunofluorescence （IF） method. Methyl thiazolyl tetrazolium （MTT） colorimetry was employed to detect cytotoxicity and cell proliferation. The levels of interleukin-1β （IL-1β）， intercellular adhesion molecule-1 （ICAM-1）， laminin （LN）， and fibronectin （FN） in cell supernatant were determined by enzyme-linked immunosorbent assay （ELISA）. The mRNA levels of IL-1β， FN， and NF-κB p65 were measured by real-time fluorescence quantitative PCR. The protein levels of phosphorylated inhibitor of NF-κB kinase β （p-IKKβ）， phosphorylated NF-κB inhibitor （p-IκBα）， and NF-κB p65 were determined by Western blot. ResultCompared with the normal group， the modeling increased cell proliferation （P<0.01）， elevated the levels of IL-1β， ICAM-1， LN， and FN in cell supernatant （P<0.01）， and up-regulated the mRNA levels of IL-1β， FN， and NF-κB p65 （P<0.01） and the protein levels of p-IKKβ， p-IκBα， and NF-κB p65 （P<0.01）. Such changes were recovered by benazepril and Wuwei Xiaoduyin （P<0.05， P<0.01）. ConclusionWuwei Xiaoduyin can mitigate the inflammatory injury of renal mesangial cells induced by LPS by inhibiting the NF-κB signaling pathway.
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Nanosized copper particles (nano Cu) have been incorporated into products in multiple industries, although studies have demonstrated that these particles are nephrotoxic. We investigated the cytotoxicity of nanosized copper particles on rat mesangial cells and measured rates of apoptosis, the expression of caspase-3, and generation of reactive oxygen species. We also measured autophagy through the acridine orange (AO) staining and expression of Beclin-1, microtubule-associated protein 1 light chain 3, and p62 to screen the underlying mechanism of toxicity. Nanosized copper particles inhibited mesangial cell viability, up-regulated the activity of caspase-3, and increased the rates of apoptosis and the generation of reactive oxygen species in a concentration-dependent manner. Exposure to nano Cu increased the formation of acidic vesicular organelles and the expression of Beclin-1, microtubule-associated protein 1 light chain 3, and p62, and treatment with an autophagy inhibitor reduced nephrotoxicity. This indicated that the autophagy pathway is involved in the toxicity induced by nanosized copper particles to mesangial cells. This finding can contribute to the development of safety guidelines for the evaluation of nanomaterials in the future.
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Objective:To investigate the effect of pirfenidone (PFD) on the proliferation of human glomerular mesangial cells (HMC) stimulated by serum IgA1 in patients with IgA nephropathy (IgAN) and its possible mechanism.Methods:Serum IgA1 of IgAN patients was purified by Jacalin affinity chromatography combined with Sephacryl S-200 gel filtration, and then heated to aggregated form (aIgA1). CCK8 method was used to confirm the concentration and time of PFD. The cells were divided into blank control group, IgA1 (0.5 mg/ml) group and IgA1 (0.5 mg/ml)+PFD (2 mmol/L) group. The CCK8 method was used to detect proliferation of mesangial cells. The cell cycle was detected by flow cytometry, and the proliferation index of mesangial cells was calculated. The expression levels of transforming growth factor β1 (TGF-β1), Smad4, Smad7, fibronectin (FN) and collagen Ⅳ protein and mRNA were detected through Western blotting and real-time PCR.Results:Compared with blank control group, the proliferation of HMC was promoted significantly by aIgA1 ( P<0.05). After PFD treatment, the proliferation of HMC was significantly inhibited ( P<0.01). Compared with the blank control group, the number of G1 phase cells decreased, the number of S phase cells and cell proliferation index increased in IgA1 group (all P<0.05). Compared with IgA1 group, the number of cells in G1 phase increased significantly, the number of cells in S phase and G2/M phase decreased significantly, and the cell proliferation index decreased in IgA1+PFD group (all P<0.05). Western blotting and real-time PCR results showed that compared with the blank control group, the protein and mRNA expressions of collagen Ⅳ, FN and Smad4 in HMC stimulated by aIgA1 were significantly increased, while TGF-β1 protein expression was increased and Smad7 protein expression was decreased (all P<0.05). After PFD treatment, the protein and mRNA expression of collagen Ⅳ, FN and Smad4 in HMC was significantly decreased, while TGF-β1 protein expression was obviously decreased, and Smad7 protein was up-regulated (all P<0.05). There was no significant difference in the mRNA expression of TGF-β1 and Smad7 in each group before and after PFD treatment (all P>0.05). Conclusions:PFD can increase the arrest of HMC in G1 phase, inhibit the proliferation of HMC induced by aIgA1 of IgAN patients, and reduce the production of extracellular matrix. The mechanism may be related to up-regulation of Smad7 expression and down-regulation of TGF-β1/Smad4 pathway.
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Objective:To investigate the effects of Guiqi Dingnian prescription （GDP） on the expression of related molecules in Janus tyrosine kinase 2 （JAK2）/signal transducer and activator of transcription （JAK2/STAT） signaling pathway of <italic>D</italic>-galactose （<italic>D</italic>-gal）-induced senescent mesangial cells. Method:The senescent mouse mesangial cells induced by 10 g·L<sup>-1</sup> <italic>D</italic>-gal were continuously treated with 40 mg·L<sup>-1 </sup>GDP for three days. The senescence of the treated cells was determined by senescence-associated （SA）-<italic>β</italic>-gal staining. The cell cycle was detected by flow cytometry. The cell viability was analyzed using the cell counting kit-8 （CCK-8）. The mRNA expression levels of tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， interleukin-6 （IL-6）， nuclear transcription factor-<italic>κ</italic>B （NF-<italic>κ</italic>B）， and IL-1<italic>α</italic> were detected by real-time polymerase chain reaction （Real-time PCR）. The protein expression levels of STAT1， phosphorylated STAT1 （p-STAT1）， STAT3， and p-STAT3 in the JAK2/STAT signaling pathway were determined by Western blot. Result:CCK-8 results showed that the optimal concentration of GDP was 40 mg·L<sup>-1</sup>. Compared with the blank group， the positive rate of SA-<italic>β</italic>-gal in the model group was significantly higher（<italic>P</italic><0.01）， the percentage of cells in G<sub>0</sub>/G<sub>1</sub> phase was significantly increased（<italic>P</italic><0.05）， the percentage of cells in G<sub>2</sub>/M and S phase was significantly decreased（<italic>P</italic><0.01）. The mRNA expression levels of TNF-<italic>α</italic>，IL-6，NF-<italic>κ</italic>B and IL-1<italic>α </italic>were significantly increased（<italic>P</italic><0.01）. Compared with the model group， the model + GDP group exhibited significantly decreased SA-<italic>β</italic>-gal-positive cells （<italic>P</italic><0.05）， reduced cells in the G<sub>0</sub>/G<sub>1</sub> phase （<italic>P</italic><0.05）， increased cells in the G<sub>2</sub>/M and S phases （<italic>P</italic><0.01）， and down-regulated TNF-<italic>α</italic>， IL-6， NF-<italic>κ</italic>B， and IL-1<italic>α </italic>mRNA expression （<italic>P</italic><0.05） and STAT1， p-STAT1， STAT3， and p-STAT3 protein expression （<italic>P</italic><0.05）. Conclusion:GDP delays the senescence of mouse mesangial cells possibly by down-regulating the expression of related molecules in the JAK2/STAT pathway.
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Aim To investigate the therapeutic effect of SirTl agonist resveratrol on IgA nephropathy rats and its mechanisms. Methods An IgA nephropathy rat model was established. The rats were divided into four groups randomly: control group, IgA nephropathy group, control treated with Res group and IgA nephropathy treated with Res group. The urine protein was detected by Ponceau S; the biochemical indexes were detected by automatic biochemical analyzer; the pathological changes of kidney were observed by PAS and Masson staining; IgA deposition was observed by immunofluorescence; the expressions of PDGF-B and TGF-fil were detected by immunohistochemistry. Results Compared with IgA nephropathy group, the volume of 24-hour urinary protein and the expression of BUN and Scr in Res group decreased significantly, and the fluorescence of IgA in glomerulus was less in resveratrol group; mesangial cells and matrix proliferated and glomerular volume increased in IgA nephropathy group at the later stage, and both of them were significantly inhibited. Resveratrol could significantly reduce the high expression of PDGF-B and TGF-β1 in IgA nephropathy group. Conclusions Res can inhibit the deposition of IgA immune complex in mesangial region of IgA nephropathy rats and reduce glomerulosclerosis by down-regulating the expression of PDGF-B and TGF-β1, in turn it suppresses cell proliferation in mesangial region. It suggests that resveratrol plays an important role in slowing down the progression of IgA nephropathy.
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Aim To investigate the mechanism of the mediation of high mobility group protein BI (HMGB1) and Toll like receptor 4 (TLR4) in the proliferation and extracellular matrix deposition of glomerular mesangial cells in lupus nephritis. Methods Immunohistochemistry and immunocytochemistry were employed to detect the TLR4 expression levels in the LN clinical specimens and MRL/lpr mice. Western blot was used to detect the TLR4 and Myd88 expression levels in human mesangial cells stimulated by recombinant HMGB1. Cell counting kit-8, Western blot and ELISA were employed to detect the proliferation and FN expression levels in HMCs stimulated by the exchange plasma of LN patients. Results Immunohistochemistry and immunocytochemistry results showed that compared with control groups,the expression levels of TLR4 in glomeruli cells of LN patients and MRL/lpr mice were up-regulated. Western blot showed that compared with control groups, the expression levels of TLR4 and Myd88 increased in HMCs stimulated by recombinant HMGB1. While the inhibition of HMGB1 and TLR4 both improved the proliferation, FN synthesis and FN secretion of HMCs induced by the exchange plasma of LN patients (both P < 0. 05). Conclusion HMGB1 may participate in the pathogenesis of LN by activating TLR4 to mediate the proliferation and extracellular matrix deposition of mesangial cells.
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ABSTRACT Objective To analyze whether passive inhalation of cigarette smoke causes morphological, structural, and functional changes in kidneys of rats. Methods Wistar rats, aged eight weeks, weighing on average 260g, were divided into Control Group and Smoking Group. Each group was subdivided into four groups of ten animals for morphofunctional analysis, in a period of seven and 28 days. The Smoking Group was exposed to smoke of 40 cigarettes per day, at certain times and in automated equipment for cigarette burning, called smoking machine (SM-MC-01). After the exposure period, urine and blood samples were collected for the functional analyses, and the kidneys were dissected and submitted to histological procedures for morphoquantitative analyses. Results After exposure of animals of the Smoking Group, the following were observed: lower weight gain; lower water and feed intake; decreased renal weight, diameter, and volume; reduction in cortical thickness and glomerular volume density; decrease in glomerular and capsular diameter; increase in mesangial density; decreased urine volume; increased levels of glucose, serum creatinine and microalbuminuria; decreased urinary creatinine levels and creatinine clearance rate. Conclusion Passive smoking negatively influences renal morphology and glomerular filtration rate, with effects similar to those described in the literature regarding active smoking.
RESUMO Objetivo Analisar se a inalação passiva da fumaça do cigarro proporciona alterações morfológicas, estruturais e funcionais nos rins de ratos. Métodos Ratos Wistar, com oito semanas de idade, pesando, em média, 260g, foram divididos em Grupo Controle e Grupo Tabagista. Cada grupo foi subdividido em quatro grupos de dez animais para análise morfofuncional, em um período de sete e 28 dias. O Grupo Tabagista foi exposto à fumaça de 40 cigarros por dia, em horários determinados e equipamento automatizado de queima de cigarros, denominado smoking machine (SM-MC-01). Após o período de exposição, foram coletadas amostras de urina e sangue para as análises funcionais, e os rins foram dissecados e submetidos a procedimentos histológicos para análises morfoquantitativas. Resultados Após a exposição dos animais do Grupo Tabagista, observou-se menor ganho de peso; menor consumo de água e ração; menor peso, diâmetro e volume renal; redução em espessura cortical e densidade de volume glomerular; diminuição no diâmetro glomerular e capsular; aumento na densidade mesangial; volume urinário diminuído; níveis aumentados de glicose, creatinina sérica e microalbuminúria; níveis reduzidos de creatinina urinária e redução da taxa de depuração da creatinina. Conclusão O tabagismo passivo influencia negativamente na morfologia renal e na taxa de filtração glomerular, com efeitos semelhantes aos descritos na literatura em relação ao tabagismo ativo.
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Animals , Rats , Tobacco Smoke Pollution/adverse effects , Smoking/adverse effects , Rats, Wistar , Glomerular Filtration Rate , KidneyABSTRACT
Aim To investigate the effect of catalpol on the stimulation of macrophage-mediated mouse mesang-ial cells ( MMCs) injury by advanced glycation end products (AGEs). Methods MMCs and RAW264.7 macrophages were co-cultured in vitro and divided into control group, model group,catalpol group (0. 1, 1.0, 10. 0 jjimol • L"1 ) , and aminoguanidine group ( 10. 0 fimol • L"1) , which was setas the positive control. After drug administration for 1 h, RAW264. 7 was stimulated by AGEs (100 mg • L_l) for 24 h. MTT assay was employed to detect the proliferation rate of MMCs . Immunofluorescence was used to investigate the expres-sion levels of COL-IV in MMCs. Western blot was applied to assess the expression of FN,COL-IV and TGF-P in MMCs. ELISA was utilized to determine the levels of IL-6, IL-12 and TNF-ot in supernatant liquid of RAW264. 7 macrophages. Results Catalpol could inhibit RAW264.7 macrophage-mediated MMC-induced proliferation stimulated by AGEs ( P < 0. 05, P < 0.01), down-regulate the levels of FN, COL-IV and TGF-0 proteins in MMCs (P < 0. 05, P < 0. 01) , and decrease IL-6, IL-12 and TNF-a level (P < 0. 05, P < 0. 01). Conclusions Catalpol has apparent protective effect on AGEs-stimulated macrophage-mediated me-sangial cell injury. It down-regulates FN, COL-IV and TGF-p protein expression in MMCs, down-regulates inflammatory factor levels and reduces inflammation, thereby alleviating diabetic kidney damage.
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Objective To investigate effects of melatonin (MT) on high glucose-induced cell proliferation,Toll-like receptor 4 (TLR4) signaling pathway and expressions of inflammatory factor in mouse mesangial cells (SV40).Methods SV40 cells were divided into mannitol control group (30 mmol/L mannitol),normal control group (5 mmol/L glucose),control (5 mmol/L glucose)+ 1000 μmol/LMT group,high glucose group (25 mmogL glucose),high glucose +10,100,1000 μmol/L MT group and high glucose + TLR4 inhibitor (TAK242) group.(1) The cell viability was measured by CCK-8 cytotoxicity kits,and cell proliferation was measured by EdU kits.The expression of TLR4 and the nuclear translocation of nuclear factor-κB (NF-κB p65) were observed by immunofluorescence.(2) Realtime quantitative PCR was used to detect TLR4 mRNA expression.Real-time quantitative PCR and ELISA were used to determine the mRNA and protein secretion levels of the downstream inflammatory factors,such as monocyte chemoattractant-1 (MCP-1),interleukin-1β (IL-1β) and tumor necrosis factor of α (TNF-α);Western blotting was used to detect TLR4 pathway proteins,such as TLR4,myeloid differentiation factor 88 (MyD88),β interferon TIR domain adaptor (Trif),phosphorylated interferon regulatory factor 3 (p-IRF3) and phosphorylated NF-κB inhibitory protein (p-IκB).Results High glucose stimulated mesangial cell proliferation,promoted TLR4 expression and NF-κB p65 transcription activity.Both MT and TAK242 inhibited the above reactions,and the effects of MT was concentration-dependent.Compared with the normal control group,high glucose group had up-regulated expressions of TLR4,MCP-1,IL-1β and TNF-α mRNA (all P ＜ 0.05),but also significantly increased the protein expressions of MyD88,Trif,p-IRF3 and p-IκB (all P ＜ 0.05).Compared with those in the high glucose group,the expression of TLR4 was down-regulated in the high glucose+ 10,100,1000 μmol/L MT group and the high glucose+TAK242 group (all P ＜ 0.05),while the expressions of MyD88,Trif,p-IRF3,p-IκB,MCP-1,IL-1β and TNF-α decreased (all P ＜ 0.05).The effects of MT was concentration-dependent.Conclusions High glucose stimulates the proliferation of SV40,and MT can inhibit the proliferation of mesangial cells and the expressions of inflammatory factors through TLR4 signaling pathway.
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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.