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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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Aim To study the role of marein mediated AMPK signaling pathway in delaying oxidative stress, inflammation and fibrotic protein expression in diabetic nephropathy ( DN ). Methods In vitro diabetic nephropathy model was established by HG + PA induced rat mesangial cells ( HBZY-1 ) , and the cultured HBZY-1 cells were divided into normal control group (NG), HG + PA( GlucoselOO mmol • L'1 + Palmitic acid 250 [imo\ • L'1, HG +PA) model group, HG + PA + marein with different doses of 25 p,mol • L"1, 50 p,mol • L"1, 100 jimol • L"1, and 200 junol • L"1 groups. MTS was used to detect the effect of marein on HBZY-1 cell proliferation, and the optimal concentration was selected. Western blot was used to test the protein expression of NOX4, TGF-fU, MCP-1, a-SMA, FN, Collagen VI. Adenosine monophosphate activated( AMPK) protein kinase family of AMPK7I, p-AMPK a expression were measured. Results Marein inhibited high glucose palmitate-induced proliferation of HBZY-1 cells, down-regulated NOX4, TGF-(31, MCP-1, cx-SMA, FN and Collagen VI expression in model cells. Meanwhile, marein up-regulated both AMPK 7I and p-AMPKa expression. Conclusions Marein may inhibit the HBZY-1 cell proliferation, oxidative stress, inflammation and fibrosis factors expression in HG + PA induced HBZY-1 cell by activating of both AMPK 7I and AMPK signaling pathway, thus delaying renal injury in diabetic nephropathy.
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[ ABSTRACT] AIM:To explore the effects and mechanism of eleutheroside ( ETS) B or E on the proliferation of HBZY-1 cells treated with high glucose.METHODS:The HBZY-1 cells were cultured under high glucose condition.The 4th generation of HBZY-1 cells was used for determining the optimal cell density, which was consistent with the growth reg-ulation curve of the cells.The cells were divided into 6 groups:low glucose ( LG) group, high glucose ( HG) group, high glucose plus ETS-B/E ( low dose, medium dose and high dose) groups, and high glucose plus losartan ( LTG) group.Af-ter all cells were treated with the corresponding drugs at 24 h, 48 h and 72 h, the inhibitory rate of the proliferation was measured, and the expression of TGF-β1 and PPARγwas detected by immunocytochemistry and Western blotting.RE-SULTS:The best cell density was 2 000 cells/well, which was complied with the basic rules of the cell growth, and high glucose significantly promoted the HBZY-1 cell proliferation.At each time point, the inhibitory effects of ETS-B/E were significantly different between HG group and LTG group on the proliferation of the HBZY-1 cells ( P<0.05) .The expres-sion of TGF-β1 was significantly inhibited, and the expression of PPARγwas significantly promoted by ETS-B/E ( P<0.05).ETS-E showed stronger effect than ETS-B (P<0.05) in a concentration-and time-dependent manner.CONCLU-SION:ETS-B/E significantly inhibits the proliferation of HBZY-1 cells under high glucose condition by decreasing TGF-β1 expression and promoting PPARγexpression.
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Aim To investigate the influence of gly-cyrrhizic acid( GA) on cell proliferation and cell cycle and secretion of extracellular matrix ( ECM) , the level of fibronection ( FN) , type Ⅳ collagen ( C-Ⅳ) in the rats′ glomerular mesangial cells ( HBZY-1 ) cultured with advanced glycation endproducts ( AGEs ) . Meth-ods HBZY-1 were incubated in culture medium con-taining AGEs in the presence of GA and treated for 48 h. At the same time, the normal and control groups were established. Methylthiazoletetrazolium ( MTT) as-say was used to measure cell proliferation;cell cycle a-nalysis was performed using a flow cytometry; FN and C-Ⅳwere analyzed by enzyme-linked immunosorbent assay ( ELISA ) . Results Cell counts increased markedly in AGEs group. Cell cycle analysis showed that cell percentage of S phase increased, G1 reduced and PI increased from 35. 01% ± 4. 21% to 44. 93%± 0. 25% ( P <0. 05 ) . FN and C-Ⅳ secretion in-creased ( P <0. 05 ) . Whereas GA was added, cell counts decreased, and the percentage of S phase also decreased and G1 increased, PI decreased from 44. 93% ± 0. 25% to 42. 16% ± 1. 04% ( P<0. 05 ) . In addition, the secretion of FN and C-Ⅳ decreased ( P<0. 05 ) . Conclusion GA could prevent AGEs-induced HBZY-1 . GA may protect HBZY-1 from in-hibiting the abnormal cell proliferation, changing the cell cycle. GA can reduce the synthesis of FN and C-Ⅳ.
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Objective To investigate the effects of different immunosuppressive agents on mesangial cell proliferation through a mesangial cell injury model in vitro. Methods Mesangial cell line (HBZY-1) in period of proliferation was cultured in vitro with cytochalasin B for 2 h, then HBZY-1 cells were divided into 5 groups: blank (control) group, cyclosporine A (CsA) group, Tacrolimus (Tac) group, mycophelonate mofetil (MMF) group and rapamycin (RAPA) group. Subsequently,the number of HBZY-1 cells at different time points was measured by using the professional image analysis software after treatment for 6, 12 and 24 h, respectively. Results Damaged HBZY-1 cells recovered in all groups. At 6 h, the number of HBZY-1 cells in Tac group was significantly more than that in control group (P＜0.05), but the difference had no significance between the other treatment groups and control group (P＞0. 05). At 12 h, there was no significant difference in of the number of HBZY-1 cells among the all groups (P＞0. 05). At 24 h, there was no significant difference in the cell number between MMF and control groups (P＞0. 05). CsA, Tac and RAPA resulted in HBZY-1 cell proliferation, and the cell number in CsA and Tac groups was significantly more than that in the other groups (P＜0. 05). As compared with the control group, the cell number in RAPA group was significantly increased (P＜0. 05). Conclusion CsA, Tac, MMF and RAPA contribute to recovery of damaged HBZY-1 cells, but CsA and Tac result in over-proliferation of HBZY-1 cells. RAPA and MMF can prevent HBZY-1 cells against over-proliferation, and MMF scarcely results in HBZY-1 cell proliferation.
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Objective To investigate the relationship among p38 mitogen-activated protein kinase (p38MAPK), NF-KB and monocyte chemoattractant protein-1 (MCP-1), and to study the role of p38MAPK, NF-κB and MCP-1 in diabetic nephropathy. Methods Protein expressions of p38MAPK and NF-κB, and mRNA expression of MCP-1 were initially investigated in rat mesangial cell line HBZY-1, which were incubated separately with 25mmol/L glucose, 100nmol/L insulin, 100 μmol/L H2 O2 and 100mg/L advanced glycosylation end products (AGEs). The relationship among p38MAPK, NF-κB and MCP-1 expression Was observed by usingSB203580, a specific inhibitor of p38MAPK. Results The expressions of p38MAPK, NF-κB and MCP-1 were increased in HBZY-1 cells incubated separately with 25mmol/L glucose, 100nmol/L insulin, 100μmol/L H2 O2and 100 mg/L AGEs. Expressions of NF-κB and MCP-1 were significantly reduced when p38MAPK was inhibited by SB203580. Conclusion p38MAPK, NF-κB and MCP-1 are involved in development of diabetic nephropathy, and p38MAPK stimulation is essential for the expressions of NF-κB and MCP-1.