25 research outputs found

    Upregulation of TLR4 via PKC activation contributes to impaired wound healing in high-glucose-treated kidney proximal tubular cells

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    <div><p>Acute kidney injury (AKI) leads to a worse prognosis in diabetic patients compared with prognoses in non-diabetic patients, but whether and how diabetes affects kidney repair after AKI remains unknown. Here, we used scratch-wound healing and transwell migration models to examine whether and how wound healing is affected by high glucose levels in cultured kidney proximal tubular cells (RPTC). The results show that scratch-wound healing and transwell migration were significantly slower in high-glucose-treated kidney tubular cells (30 mM glucose) than in low-glucose-treated cells (5.5 mM). Toll-like receptor 4 (TLR4), MyD88, phospho-protein kinase C (PKC), phospho-p38 MAPK and monocyte chemoattractant protein-1 (MCP-1) mRNA levels were upregulated after high glucose treatments. Staurosporine, a selective PKC inhibitor, inhibited TLR4, MyD88 and p-p38 upregulation in the high-glucose-treated cells, indicating the involvement of PKC in high-glucose-induced TLR4 upregulation. The pharmacological inhibition of TLR4 or shRNA-mediated TLR4 knockdown improved wound healing and transwell migration in high-glucose-treated RPTC. In contrast, the overexpression of TLR4 in low-glucose-treated RPTC suppressed wound healing, mimicking the effects of high glucose levels. These results suggest that the upregulation of TLR4 expression via PKC activation contributes to defective wound healing in high-glucose-treated kidney tubular cells.</p></div

    High glucose inhibits transwell migration in cultured kidney tubular cells.

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    <p>RPTC, NRK-52E and HEK 293 cells were cultured for 2 days in low glucose (5.5 mM) or high glucose (30 mM) medium, and then used for a transwell migration experiment. (A) Representative PI staining of migratory cells was recorded with a fluorescence microscope. A total of 3x10<sup>5</sup> RPTC were seeded in a transwell insert, which was put in a 24-well plate containing 600 μL low glucose or high glucose medium for 6 h. The cells that migrated to the undersurface were stained with PI and counted. (B) Migratory cells attached to the undersurface were counted after PI staining in RPTC. (C) Migratory cells attached to the undersurface were counted after PI staining in HEK 293 cells. (D) Migratory cells attached to the undersurface were counted after PI staining in NRK-52E. Data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group.</p

    Effect of PKC on high glucose induced TLR4 expression.

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    <p>RPTC were treated for 24 hours with low glucose (5.5 mM), high glucose (30 mM), or the PKC inhibitor staurosporine (10 nM, added 1 hour before high glucose treatment), and the cell lysates were collected, TLR4 mRNA, phospho-PKC (pan), p-p38, p38 levels were examined via real-time PCR and Western blotting. (A) PKC phosphorylation and p-p38 upregulation after high glucose treatment. The PKC inhibitor staurosporine decreased phospho-PKC and p-p38 levels but not total PKC levels. (B) Staurosporine reduced TLR4 mRNA levels under the high glucose condition. (C) RPTC were pretreated with staurosporine (10 nM), then treated with high glucose (30mM) for 6, 12, 24 h, and cell lysates were collected at different time points. phospho-PKC (pan), p-p38 levels were examined via Western blotting. (D) Effect of staurosporine on scratch wound healing in low glucose and high glucose. Data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group; **, p<0.05 versus the high glucose group without staurosporine treatment.</p

    TLR4 inhibitor promotes scratch-wound healing and migration in high-glucose-treated RPTC.

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    <p>RPTC were cultured for 2 days in low glucose (5.5 mM) or high glucose (30 mM) medium, and then used for following experiments. (A) Titration of TAK-242 concentration. RPTC cells were cultured for 12 h in low glucose or high glucose medium with or without TAK-242. (B) MyD88 upregulation was also inhibited by TAK-242 under high glucose conditions by Western blotting. (C) TLR4 mRNA upregulation was also inhibited by TAK-242 under high glucose conditions by real-time PCR analysis. (D) Enhanced wound healing in the high glucose condition with the TLR4 inhibitor. RPTC cells were scratch-wounded and incubated in low glucose or high glucose medium with or without 100 nM TAK-242 for 18 h and then the healing distance was measured. (E) Enhanced cell migration under the high glucose condition with the TLR4 inhibitor. A total of 3x10<sup>5</sup> RPTC were seeded in a transwell insert, which was put in a 24-well plate containing 600 μL of medium with or without 100 nM TAK-242 in low glucose or high glucose medium for 6 h. The cells that migrated to the undersurface of the insert were stained with PI and counted. In C, D and E, data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group; **, p<0.05 versus the high glucose group without TAK-242 treatment.</p

    TLR4 upregulation during high glucose treatment in RPTC.

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    <p>TLR4 mRNA and protein levels were examined via real-time PCR and Western blotting after low gucose or high glucose treatment. (A and B) TLR4 mRNA upregulation in the high glucose medium. (C) TLR4 protein levels increased after the high glucose treatment. (D) Experiments were analyzed via densitometry and normalized to the control (0 h). (E) MCP-1 mRNA upregulation in high glucose medium for 24 h. (F) Immunofluorescence analysis of TLR4 expression. PRTC cells were cultured for 2 days in low glucose (5.5 mM) or high glucose (30 mM) medium.The cells were fixed for immunofluorescent staining of TLR4 (red). Data are expressed as the mean ± S.D. (n≥3). *, p<0.05 versus the low glucose group; **, p<0.05 versus the control (0 h).</p

    TLR4 shRNA abrogates impaired wound healing in high-glucose-treated RPTC.

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    <p>RPTC were infected with lentiviruses containing a scrambled control sequence (Scr) or the TLR4 shRNA sequence (shRNA) and then cultured for 2 days in low glucose or high glucose medium for the following experiments. (A) TLR4 knockdown caused by the TLR4 shRNA lentivirus. After infection and a high glucose treatment, whole-cell lysates were collected for an immunoblot analysis of TLR4 and MyD88. (B) Scratch-wound healing. After lentiviral infection, RPTC were scratch-wounded and incubated in low glucose or high glucose medium for 18 h to measure the distance over which healing occurred. (C) Transwell cell migration. After lentiviral infection, a total of 3x10<sup>5</sup> RPTC were seeded in a transwell insert, which was put in a 24-well plate containing 600 μL of low glucose or high glucose medium for 6 h. The cells that migrated to the undersurface were stained with PI and counted. In B and C, data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group; **, p<0.05 versus high glucose group infected with the scrambled sequence.</p

    Increased Expression of Cathepsin L: A Novel Independent Prognostic Marker of Worse Outcome in Hepatocellular Carcinoma Patients

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    <div><p>Objectives</p><p>To investigate the expression and role of Cathepsin L (CTSL) in Hepatocellular carcinoma (HCC) tissue and cell line (MHCC-97H), and to evaluate the clinical and prognostic significance of CTSL protein in patients with HCC.</p><p>Methods</p><p>The expression of CTSL was examined in HCC tissue and MHCC-97H cells by Western-blotting, Real-time PCR and immunohistochemical staining. Cell growth curve assay and colony formation assay were used to verify the effect of CTSL on the proliferation and tumor progression ability of MHCC-97H cells. Tumor formation assay in nude mice was used to analyze the effect of CTSL on the tumorigenicity of MHCC-97H cells.</p><p>Results</p><p>The status of CTSL protein in carcinoma tissues is much higher than that in paracarcinoma tissues. The overall survival of the patients with high CTSL expression was significantly shorter than the low CTSL expression group. high CTSL expression was significantly correlated with advanced clinical staging, histological grade and tumor recurrence. In vitro experiments demonstrated that over-expression of CTSL in MHCC-97H cells promoted cell proliferation and tumor progression ability. Down-regulation of CTSL showed the opposite effects. Over-expression of CTSL increase the tumorigenicity of MHCC-97H cells by in vivo experiments. Moreover, multivariate analysis suggested that CTSL expression might be an independent prognostic indicator for the survival of HCC patients after curative surgery.</p><p>Conclusions</p><p>CTSL might involve in the development and progression of HCC as a oncogene, and thereby may be a valuable prognostic marker for HCC patients.</p></div

    Relationship between CTSL expression and clinicopathologic features of HCC patients.

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    <p>Δ: The largest dimension of the tumor specimen.</p><p>Relationship between CTSL expression and clinicopathologic features of HCC patients.</p

    Univariate survival analysis of 82 patients with HCC.

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    <p>Δ: The largest dimension of the tumor specimen.</p><p>Univariate survival analysis of 82 patients with HCC.</p

    Analysis of CTSL protein in tissues by immunohistochemistry.

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    <p>A and B, CTSL expression is negative in normal liver cells. C and D, CTSL expression is weak in well-differentiated HCC cells. E and F, CTSL expression is moderate in moderately differentiated HCC cells. G and H, CTSL expression is strong in poorly differentiated HCC cells. (A, C, E, G ×200; B, D, F, H ×400).</p
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