Shp-2／NF-κB Pathway Mediates the Inhibition of Lipoxin A4 onIL-1β-induced Synthesis of IL-6 in Glomerular Mesangial Cells

Objective: To examine whether lipoxin A4 (LXA4) has an antagonistic effect on IL-1β-induced synthesis of IL-6 in glomerular mesangial cells, and to explore the molecular mechanisms of signal pathway in LXA4 actions. Methods: The glomerular mesangial cells of rat were cultured and treated with IL-1β with or without preincubation with LXA4 at different concentrations. The amount of IL-6 in the supernatant of cells was analyzed by enzymelinked immunosorbent assay(ELISA). The expressions of mRNA of IL-6 were determined by RT-PCR. The expressions of Src homology 2( SH2 ) containing protein-tyrosine phosphatase 2(Shp-2) were assessed by immunoprecipitation and immunoblotting. Activities of DNA-binding of nuclear factor-kappa B(NF-κB) were measured by electrophoretic mobility shift assay(EMSA). Results:IL-1β- snulated secretion of protein and expression of mRNA of IL-6 in mesangial cells were inhibited by LXA4 in a dose-dependent manner. LXA4 antagonizes the phosphorylation of Shp-2 and activities of NF-κB induced by IL-1β Conclusion: LXA4 antagonists IL-1β-induced synthesis of IL-6 in glomerular mesangial cellsthrough the mechanism of Shp-2/NF-κB pathway-dependent signal transduction.     

Effect of N-tosyl-L-phenylalanylchloromethyl Ketone on Tumor Necrosis Factor-alpha -induced NF-κB Activation and Apoptosis in U937 Cell Line

Summary: To investigate the effect of N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) on tumor necrosis factor-alpha-induced NF-κB activation and apoptosis in U937 cell line, changes and subcellular localization of NF-κB/p65 and IκB-α were observed by fluorescencemicroscopy and expression and degradation of IκB-α by flow cytometry. The apoptosis of U937 cells was measured by flow cytometry and electrophoresis of DNA. Immunolfluorescence assay showed that NF-κB/p65,IκB-α only localized in cytoplasm. After TNF-α stimulation, p65 was localized only in nuclei, and IκB-α was only localized in cytoplasm and decreased. The changes of TNF-α stimulation were specifically inhibited by TPCK. Flow cytometry also revealed the downregulation of IκB-α protein during TNF-α-induced apoptosis and the down-regulation was specifically inhibited by TPCK. Flow cytometry also showed the apoptosis of U937 cells after TNF-α induction. DNA ladder can be detected in cells treated by TNF-α. It is concluded that degradation of IκB-α protein and NF-κB/p65 translocation occur during TNF-α-induced apoptosis of U937 cells, suggesting the activation of NF-κB.TPCK-sensitive protease plays an important role in the degradation of IκB-α protein induced by TNF-α in U937 cells. TPCK sensitive protease also plays an important role in the apoptosis of U937 cells induced by TNF-α.     

Inhibition of NF-κB by mutant IκBα enhances TNF-α-induced apoptosis in HL-60 cells by controlling bcl-xL expression

Backgound The aim of this study was to explore whether the inhibition of nuclear factor-κB (NF-κB)activation by mutant IκBα (S32,36→A) can enhance TNF-α-induced apoptosis of leukemia cells and to investigate the possible mechanism. Methods The mutant IκBα gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IκBα were obtained by the limiting dilution method. TNF-α-induced NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-α. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM). Results Mutant IκBα protein was confirmed to exist by Western blot. The results of EMSA showed that NF-κB activation by TNF-α in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IκBα repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-α, but changed very little in transfected HL-60 cells. The inhibition of NF-κB activation by mutant IκBα enhanced TNF-α-induced apoptosis. Thecytotoxic effects of TNF-α were amplified in a time- and dose-dependent manner. Conclusions NF-κB activation plays an important role in the resistance to TNF-α-induced apoptosis. The inhibition of NF-κB by mutant IκBα could provide a new approach that may enhance the antileukemia effects of TNF-α or even of other cytotoxic agents.     

Effects of vitamin E on the proliferation and collagen synthesis of rat hepatic stellate cells treated with IL-2 or TNF-α

Objective To study the effects of vitamin E on the proliferation and collagen synthesis of rat hepatic stellate cells treated with interleukin-2 (IL-2 ) or tumor necrosis factor-α (TNF-α). Methods Hepatic stellate cells were isolated from male Sprague-Dawley rats by using modified Friedman’s method. Using the isolated cells cultured and treated with IL-2 or TNF-α, we studied the effects of vitamin E on their proliferation and collagen synthesis through an (3)H-thymidine and (3)H-proline incorporation assay, as well as through observation of these cells under a contrary phase microscope. Results Adding IL-2 increased the both proliferation and collagen synthesis of hepatic stellate cells. Their proliferation was also increased by the addition of TNF-α, although it decreased collagen synthesis. Vitamin E had marked inhibitory effects on the ability of cells treated with IL-2 or TNF-α to reproduce or synthesize collagen. Conclusion Vitamin E can inhibit the proliferation and collagen synthesis of hepatic stellate cells. It is possible that vitamin E affects liver fibrosis through these activities.     

Dahuang Zhechong Pill (大黄虫丸) containing serum inhibited platelet-derived growth factor-stimulated vascular smooth muscle cells proliferation by inducing G1 arrest partly via suppressing protein kinase C α-Extracellular regulated kinase 1/2 signaling pathway

Objective:To investigate effects of Dahuang Zhechong Pill（大黄（?）虫,DHZCP） on the cell cycle and the related signal pathways in vascular smooth muscle cells（VSMCs） stimulated by platelet-derived growth factor（PDGF） with the method of serum pharmacology.Methods:DNA synthesis in VSMCs was examined by detecting 5’-bromo-2’-deoxyuridine incorporation with the immunocytochemical method.The cycle of VSMCs was evaluated with flow cytometry.Expressions of cyclin D1,p27,protein kinase Cα（PKCα）,and phosphorylated extracellular signal regulated kinase 1/2（ERK1/2） were quantified by Western blot method. Results:DHZCP containing serum significantly inhibited DNA synthesis of PDGF-stimulated VSMCs,arrested the cells in G₁ phase,modulated the protein expressions of cyclin D₁ and p27,and suppressed the activation of PKCαand ERK1/2.Conclusion:DHZCP containing serum inhibits VSMCs proliferation via modulating the expressions of cell cycle proteins to arrest the cell in G₁ phase,which is attributed to,at least in part,suppressing PKCα-ERK1/2 signaling in VSMCs.     

Telmisartan but not Valsartan Inhibits TGF-β-mediated Accumulation of Extracelluar Matrix via Activation of PPARγ

Glomerulosclerosis, defined as phenotype transition of mesangial cell and deposition of extracelluar matrix, remains a chronic disease with excessive morbidity and mortality. The molecular mechanism underlying the suppression of mesangial cell activation is not fully understood. Since activation of peroxisome proliferators-activated receptor γ (PPARγ) has been proposed to decrease the effects of transforming growth factor-β (TGF-β) on glomerulosclerosis, we examined here whether and how telmisartan, an angiotensin Ⅱ type 1 receptor blocker with PPARγ-modulating activity, inhibited TGF-β-induced glomerulosclerosis in rat glomerular mesangial cells. Protein levels of PPARγ were detected by Western blot. Activation of PPARγ response element (PPRE) was analyzed by luciferase assays. Deposition of extracelluar matrix was tested by confocol laser scanning. The results showed that telmisartan, but not valsartan, another angiotensin Ⅱ type 1 receptor blocker, up-regulated PPARγ protein levels in a dose-dependent manner (P<0.05). Activation of PPRE, represented by luciferase activity, was also increased with higher concentration of telmisartan in a dose-dependent manner (P<0.05). Furthermore, telmisartan inhibited TGF-β-induced α-smooth muscle actin expression and collagen IV secretion in mesangial cells. GW9662, an inhibitor of PPAR-γ, blocked the inhibitory effects of telmisartan on TGF-β-induced glomerulosclerosis in mesangial cells. Our study indicates a benefit of telmisartan as a PPARγ agonist against TGF-β-induced mesangial cells activation in renal glomerulus. It may provide possibility that telmisartan works as a potential agent against diabetic nephropathy and hypertensive renal disease.     

Role of p38MAPK in mediating TNF-α-induced apoptosis of rat glioma cell line C6

Objective: To study the role of p38MAPK in mediating TNF-α-induced apoptosis of rat glioma cell line C6. Methods: Effect of TNF-α on the proliferation of C6 cells was determined by MTT assay. The TNF-α induced apoptosis was detected by transmission electron microscopy and flow cytometry. The expression of p38MAPK was detected by SABC method and Western-blot. The effect of SB202190, a specific inhibitor of p38MAPK, on TNF-α-induced apoptosis was observed by flow cytometry and SABC method. Results: Inhibitory rate of TNF-α(2×105 U/L) on C6 cells was 43. 75% . In the TNF-α treated group, apoptotic cells were observed by transmission electron microscopy and the apoptotic rate was 37. 5% by flow cytometry. p38MAPK positive signals were detected by SABC method and Western-blot. In the SB202190 treated group, the apoptotic rate was 7. 0% and no p38MAPK signals were found. Conclusion: Apoptosis of C6 cells and expression of p38MAPK can be induced by TNF-α. The activation of p38MAPK promotes the apoptosi     

Effect of Sodium Tanshinone ⅡA Sulfonate on Phosphorylation of Extracellular Signal-regulated Kinasel/2 in Angiotensin Ⅱ-induced Hypertrophy of Myocardial Cells

Objective： To observe the effects of sodium tanshinone ⅡA sulfonate （STS） on angiotensin Ⅱ （Ang Ⅱ）-induced hypertrophy of myocardial cells through the expression of phosphorylated extracellular signal-regulated kinase （p-ERK1/2）. Methods： In the primary culture of neonatal rat myocardial cells, the total protein content in myocardial cells was determined by coomassie brilliant blue and the protein synthesis rate was measured by [3H]-Leucine incorporation as indexes for hypertrophy of myocardial cells. The expression of p-ERK1/2 was determined using Western blot and immunofluorescence labeling. Results： （1） The total protein and protein synthesis rate increased significantly in contrast to the control group after the myocardial cells were stimulated by Ang Ⅱ （1 μ mol/L） for 24 h; STS markedly inhibited the increment of the total protein level induced by Ang Ⅱ and the syntheses of protein. （2） After pretreatment of myocardial cells with Ang Ⅱ （1 μmol/L） for 5 min, the p-ERK1/2 protein expression was increased, with the most obvious effect shown at about 10 min; pretreatment of myocardial cells with STS at different doses （2, 10, 50μmol/L） for 30 min resulted in obvious inhibition of the expression of p-ERK1/2 stimulated by Ang Ⅱ in a dose-dependent manner. （3） After the myocardial cells were stimulated by AngⅡ （1 μ mol/L）, the immunofluorescence of ERK1/2 rapidly appeared in the nucleus. The activation and translocation process of ERK1/2 induced by Ang Ⅱ was blocked distinctly by STS. （Conclusion： STS inhibited the myocardial cell hypertrophy induced by Ang Ⅱ, and the mechanism may be associated with the inhibition of p-ERK1/2 expression.     

Effect of serum renotropin on rat glomerular mesangial cell growth in vitro.

In vitro study of rat glomerular mesangial cell growth was carried out to investigate the pattern of serum renotropic stimulation in 5/6 nephrectomized (5/6NT) rats. Incorporation rates of 3H-TdR, 3H-UR and 3H-Leu into mesangial cells were used as parameters to measure serum renotropic stimulation. Renotropin activity increased immediately after 5/6NT, reaching peak at 48 h, and was succeeded by a rapid decline at 96 h postoperatively. A second rise occurred on the 7th day, followed by gradual decrement. It seemed evident that the serum obtained 48 h after 5/6NT surgery was capable of stimulating the uptake of 3H-TdR, 3H-UR and 3H-Leu by the mesangial cells (MCs). It may be concluded that renotropin promotes DNA, RNA and protein synthesis of MCs. The prolonged and constant effect of serum renotropin on MCs might be one of the important mechanisms in progressive sclerosis of the remaining kidney after subtotal nephrectomy.

     

Effect of Sodium Tanshinone Ⅱ A Sulfonate on Phosphorylation of Extracellular Signal-regulated Kinase1/2 in Angiotensin Ⅱ-induced Hypertrophy of Myocardial Cells

Objective:To observe the effects of sodium tanshinone Ⅱ A sulfonate(STS)on angiotensin Ⅱ(Ang Ⅱ)-induced hypertrophy of myocardial cells through the expression of phosphorylated extracellular signal-regulated kinase(P-ERK1/2).Methods:In the primary culture of neonatal rat myocardial cells.the total protein content in myocardial cells was determined by coomassie brilliant blue and the protein synthesis rate was measured by[3H]-Leucine incorporation as indexes for hypertrophy of myocardial cells.The expression of p-ERK1/2 was determined using Western blot and immunofluorescence Iabeling.Results:(1)The totaI protein and protein synthesis rate increased significantly in contrast to the control group after the myocardial cells were stimulated by Ang Ⅱ (1 μmol/L)for 24 h;STS markedly inhibited the increment of the total protein level induced by Ang Ⅱ and the syntheses of protein.(2)After pretreatment of myocardial cells with Ang Ⅱ(1 μ mol/L)for 5 min,the p-ERK1/2 protein expression was increased,with the most obvious effect shown at about 10 min;pretreatment of myocardial cells with STS at different doses(2,10,50 μ mol/L)for 30 min resulted in obvious inhibition of the expression of p-ERK1/2 stimulated by Ang Ⅱ in a dose-dependent manner.(3)After the myocardial cells were stimulated by Ang Ⅱ(1 μ mol/L),the immunofluorescence of ERK1/2 rapidly appeared in the nucleus.The activation and translocation process of ERK1/2 induced by Ang Ⅱ was blocked distinctly by STS.Conclusion:STS inhibited the myocardial cell hypertrophy induced by Ang Ⅱ,and the mechanism may be associated with the inhibition of p-ERK1/2 expression.     

Antitumor Effect of Interferon-α on U937 Human Acute Leukemia Cells in vitro and Its Molecular Mechanism

In order to investigate the antitumor effect and molecular mechanism of interferon-α(IFN-α) on human acute myeloid leukemia cell line U937 cells in vitro,the proliferation of U937 cells was determined by MTT assay,the apoptosis rate was analyzed by flow cytometry(FCM),and the mRNA expression of cell cycle regulatory protein cyclin E was detected by RT-PCR.The results showed that IFN-α could inhibit the proliferation of U937 cells significantly in a dose-and time-dependent way(P<0.01),and induce the apoptosis of U937 cells also in a dose-and time-dependent manner at the concentration of 1000-4000 U/L(P<0.01).The apoptosis rate of U937 cells was even over 50% when cultured with IFN-α for 36-48 h at the concentration of 2000-4000 U/L.Moreover,the expression of cyclin E mRNA was markedly inhibited by the addition of IFN-α,and the inhibition was time-dependent(P<0.01).It was concluded that the anti-leukemia mechanism of IFN-α might be correlated with its antiproliferative and apoptotic inducing effects,and the down-regulation of the cyclin E expression might be one of its molecular mechanisms.     

Xiayuxue Decoction(下瘀血汤)Attenuates Hepatic Stellate Cell Activation and Sinusoidal Endothelium Defenestration in CCI_4-Induced Fibrotic Liver of Mice

Objective:To investigate the effects of ancient Chinese medical formula Xiayuxue Decoction(下瘀血汤,XYXD) on activation of hepatic stellate cells(HSCs) and defenestration of sinusoidal endothelial cells(SECs) in CCI_4-induced fibrotic liver of mice.Methods:High performance liquid chromatography was used to identify the main components of XYXD and control the quality of extraction.C57BL/6 mice were induced liver fibrosis by CCl_4 exposure and administered with XYXD for 6 weeks simultaneously.Liver tissue was investigated by hematoxylin-eosin and Sirius-red staining.Sinusoidal fenestrations were observed by scanning electronic microscopy and fluorescent immunohistochemistry of PECAM-1(CD31).Whole liver lysates were detected of α-smooth muscle actin(α-SMA) and type-1 collagen by Western blot.Primary rat HSCs-T6 cells were analyzed by detecting a-SMA,F-actin,DNA fragmentation through confocal microscopy,Western blot,terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL) assay and cellomics arrayscan,respectively.Results:Amygdalin and emodin in XYXD were identified.XYXD(993 mg/kg) inhibited Sinus red positive area up to 70.1%(P0.01),as well as protein levels of α-SMA and type-1 collagen by42.0%and 18.5%(P0.05) respectively.In vitro,XYXD(12.5 μg/mL,50 μg/mL) suppressed the activation of HSCs and reversed the myofibroblastic HSCs into quiescent,demonstrated as inhibition of fluorescent F-actin by 32.3%and 46.6%(P0.05).Besides,XYXD induced the apoptosis of HSC-T6 cells by 20.0%(P0.05)and 49.5%(P0.01),evidenced by enhanced TUNEL positivity.Moreover,ultrastructural observation suggested XYXD inhibited defenestration of SECs,which was confirmed by 31.1%reduction of protein level of CD31(P0.05).Conclusions:XYXD inhibited both HSCs activation and SECs defenestration which accompany chronic liver injuries.These data may help to understand the underlying mechanisms of XYXD for prevetion of chronic liver diseases.     

Role of Extracelluar Regulated Protein Kinases in FTY720-induced Apoptosis of Leukemia Cell Lines HL-60 and U937

The effects of a novel immunosuppressive agent FTY720 on proliferation inhibition and apoptosis of acute leukemia cell lines HL-60 and U937, and the role of extracelluar regulated protein kinase (ERK) in the course of proliferation inhibition and apoptosis induced by FTY720 were studied. The proliferation inhibition rate of HL-60 and U937 cells by various concentrations of FTY720 was detected by MTT assay. Cell apoptosis was detected by DNA fragmem analysis and flow cytometry. The phosphorylated ERK1/2 protein expression was observed by Western blotting. The change of intracellular distribution of ERK1/2 protein was identified by SP immunohistochemical staining. The results showed that FTY720 could inhibit the growth of HL-60 and U937cells effectively in a dose-dependent manner. After incubation with FTY720 for 24 h, apoptosis wasobserved in HL-60 and U937 cells. The intracellular expression of phosphorylated ERK1/2 protein was also down-regulated and the distribution of ERK1/2 protein in cell‘ nuclear was reduced during FTY720-induced apoptosis. So, that FTY720 inhibited ERK1/2 phosphorylation might mediate the role of FTY720-induced apoptosis and proliferation inhibition of leukemia cells.