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Objective: To investigate the mechanism of emodin ( EM) in the expression of related protein for the fibrosis of the transforming growth factor-β1(TGF-β1)-stimulated human renal tubular epithelial (HK-2) cells. Methods: HK-2 cells were randomly divided into the normal control group, TGF-β1-stimulated model control group and emodin ( TGF-β1 +EM) group. The contents of Collage Ⅰ and fibronectin in the culture supernatant were determined by ELISA. After HK-2 cells were stimulated with TGF-β1 for 24 h, the cells were collected for immunofluorescence, Western blot and RT-PCR analysis. RT-PCR was used to detect PI3K, p-Akt and mTOR. The protein expressions of PI3K, p-Akt and mTOR were detected by Western blot. Immunofluorescence was used to detect PI3K. Results: Compared with those in the model control group, the contents of CollageⅠand fibronectin in the supernatant of emod-in group significantly decreased (P<0. 05), the expression of PI3K protein was inhibited, the expression of downstream p-Akt protein decreased, and the downstream mTOR decreased (P<0. 05), the expression levels of PI3K, p-Akt and mTOR mRNA decreased, the differences were statistically significant (P<0. 05), and the expression of PI3K decreased. Conclusion: Emodin can alleviate fibrosis of HK-2 cells stimulated by TGF-β1 through the classical Akt/mTOR pathway of autophagy.
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Objective To observe the changes of neutrophil gelatinase associated lipocalin,NGAL after endoplasmic reticulum stress by thapsigargin in renal tubular epithelial cells.Methods The renal tubular epithelial cells were treated with thapsigargin to establish the model of endoplasmic reticulum stress (ER group).CHOP and GRP78 expression were detected by Western-blot to confirm endoplasmic reticulum stress in renal tubular epithelial cells by thapsigargin.NGAL expression were detected by Western-blot in ER group and control group(normal renal tubular epithelial cells).Results After treated with 2.5 μmol/L and 5 μmol/L thapsigargin for 4 hours and 8 hours respectively,endoplasmic reticulum stress in the renal tubular epithelial cells were significantly increased comparing with control group (0.585 ± 0.045 to 0.523 ± 0.030;0.785 ± 0.049 to 0.728 ± 0.064),which was approved by the increased expression of CHOP and GRP78 (P ＜ 0.05).The expression of NGAL was significantly increased in ER group (0.567 ± 0.024 to 0.826 ± 0.057,P ＜ 0.05) and it was corresponded with the changes of CHOP and GRP78,which increased significantly with the severity of endoplasmic reticulum stress.Conclusion Endoplasmic reticulum stress can be induced by thapsigargin in renal tubular epithelial cells.The expression of CHOP,GRP78 and NGAL were significantly increased correspondingly with the severity of endoplasmic reticulum stress.It is speculated that the expression of NGAL may be related with endoplasmic reticulum stress in tubular epithelial cells.
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Objective To observe he effects of new Tangshenkang on α-SMA and E-cadherin of human renal tubular epithelial cell HK-2 in high concentrations of glucose; To explore the mechanism of new Tangshenkang on the prevention and treatment of diabetic renal fibrosis. Methods The HK-2 cells were cultured and divided into control group, high glucose group, animal serum control group, new Tangshenkang low-, medium-, and high-dosage group. After medicine intervention, cell proliferation was tested by MTT assay, and contents of α-SMA and E-cadherin were observed by ELISA assay. Results Compared with control group, α-SMA of HK-2 cultured with high glucose was much notable, but the content of E-cadherin significantly decreased, with statistical significance (P<0.05). The content of α-SMA of HK-2 cultured with new Tangshenkang decreased, and the content of E-cadherin increased; cell proliferation was markedly inhibited in culture medium supernatant of HK-2 cells cultured with high glucose plus new Tangshenkang compared with only high glucose, with statistical significance. Conclusion New Tangshenkang can inhibit cell proliferation and epithelial-myofibroblast transdifferentiation of HK-2 cell induced by high glucose, and prevent the development of diabetic renal fibrosis to a certain extent.
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Objective;To explore the mechanism ofSimiaosan drug-containing serum on prevention and treatment of renal fibrosis.Methods Human renal tubular epithelial cells (HK-2) were divided into blank group, model group, control group, and drug-containing serum high dosage group, medium dosage group and low dosage group. The expressions of CTGF and BMP-7 were determined by real-time PCR assay and immunocytochemistry.Results After the HK-2 cell was induced by uric acid (UA), the expression of CTGF significantly increased (P<0.05), the expression of BMP-7 significantly decreased (P<0.05). After intervened bySimiaosan, the expression of CTGF significantly decreased (P<0.05), the expression of BMP-7 significantly increased (P<0.05) and present dosage dependent.Conclusion Reducing the expression of CTGF and up-regulation the expression of BMP-7 and controlling epithelial-myofibroblast transdifferentiation may be the mechanism ofSimiaosan on prevention and treatment of renal fibrosis.
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Objective To explore the mechanism of Tangshenkang in the prevention and treatment of diabetic nephropathy. Methods HK-2 cells were cultured in vitro and divided into control group, high glucose group (30 mmol/L D-glucose), control group (30 mmol/L D-glucose+10% animal serum), and Tangshenkang drug-containing serum therapy groups (30 mmol/L D-glucose+5%low concentration Tangshenkang, 30 mmol/L D-glucose+10%middle concentration Tangshenkang, 30 mmol/L D-glucose+20% high concentration Tangshenkang). After 24 h and 48 h treatment, MMP-9 and TIMP-1 in cell cultural supernatant were observed by ELISA. Results MMP-9 of HK-2 cultured with high glucose was much decreased and TIMP-1 increased significantly than the control group, with statistical significance (P<0.05). TIMP-1 significantly decreased and MMP-9 increased in HK-2 cultured with high glucose plus Tangshenkang compared with those only induced by high glucose, with statistical significance (P<0.05). Conclusion Tangshenkang could regulate the secretion of fibrosis cell factor of HK-2 cell induced by high glucose, which may be one of the mechanisms in its treatment of diabetic nephropathy.
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Objective Through studying the effect of Tangnaikang (TNK) on the expression of Smad 2, 3, 7 mRNA of human renal tubular epithelial cells HK-2 induced by transforming growth factor-β1 (TGF-β1), to explore the mechanism of TNK on prevention and treatment of renal fibrosis. Methods The HK-2 cells were cultured by DMEM/F12 (1∶1) with 10% fetal bovine serum and divided into control group, TGF-β1 group (TGF-β1 10 ng/mL), blank serum control group (TGF-β1 10 ng/mL + 10% animal serum), TNK drug-containing serum therapy groups (TGF-β1 10 ng/mL + 5% TNK or + 10% TNK or + 20% TNK). After 24 h, the expression of Smad 2, 3, 7 mRNA were tested by fluorescence quantitatiye PCR assay. Results After the HK-2 cells were induced by TGF-β1, the expression of Smad 2, 3 mRNA were increased and the expression of Smad 7 mRNA was decreased compared with the control group (P<0.05). The expression of Smad 2, 3 mRNA were decreased and the expression of Smad 7 mRNA was increased in TNK drug-containing serum therapy groups compared with TGF-β1 group (P<0.05), but blank serum control group had no such effect. Conclution TNK could prevent the development of renal fibrosis to some extent through regulating the expression of Smads signaling pathway.
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Objective To explore the effect of Tangnaikang (TNK) on extracellular matrix expression of human tubular epithelial cell HK-2 induced by TGF-β1 and explore its mechanism on the renal fibrosis. Methods The HK-2 cells were cultured by DMEM/F12 (1∶1) with 10%fetal bovine serum and divided into control group, TGF-β1 group (TGF-β110 ng/mL), rat serum control group (TGF-β110 ng/mL +10% rat serum), TNK-containing rat serum therapy groups (TGF-β110 ng/mL+5% Tangnaikang, or +10%Tangnaikang, or +20% TNK). After 24 h of administration, the expression of ColⅠ, Col Ⅲ and FN mRNA were tested by fluorescence quantitative PCR assay. Results The expression of ColⅠ, Col Ⅲ and FN mRNA of HK-2 cultured with TGF-β1 were much higher than the control, and significantly decreased in HK-2 cultured with TGF-β1 plus Tangnaikang compared with only TGF-β1 (P<0.05), but rat serum control had no effect. Conclusion TNK could inhibit the expression of ColⅠ, Col Ⅲ and FN mRNA of HK-2 cell induced by TGF-β1, and prevent the development of renal fibrosis to some extent.