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Objective To investigate the role of oxidative stress in the epithelial-to-mesenchymal transdifferentiation (EMT) of peritoneal mesothelial cells in rat model of peritoneal fibrosis and the effect of probucol on peritoneal fibrosis. Methods The rat model of peritoneal fibrosis was induced by 4.25% high glucose peritoneal dialysis fluid (PDF). The rats were randomly divided into 4 groups:the control group, the saline group, the peritoneal fibrosis group, and the probucol group. A 4 hour peritoneal equilibration test (PET) was performed 4 weeks later. The peritoneal function and net ultrafiltration (UF) volume were determined. The level of malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in peritoneal tissue were examined. The histology of peritoneal membrane was evaluated by light microscopy. E-cadherin and α-smooth muscle actin (α-SMA) protein expression was evaluated by immunohistochemical method and Western blot.Results The mesothelial cells were detached from peritoneal membrane in peritoneal firbosis rats. Comparing with the control rats, the thickness of visceral peritoneum, the level of MDA, and the-SMA protein expression were increased while the net ultrafiltration volume, the level of GSH-Px and E-cadherin protein expression were decreased in peritoneal firbosis rats. All these changes were reversed in the rats treated with probucol.Conclusion Oxidative stress plays an important role in transdifferentiation of peritoneal mesothelial cell in the peritoneal fibrosis rats. Probucol can improve structure and function of peritoneum, and partially reverse the EMT by reducing the oxidative stress.
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Objective To investigate the role of mitochondrial respiratory chain in the hyperpermeability of human peritoneal mesothelial cells (HPMCs) induced by high glucose peritoneal glucose PDS was also added. Transmesothelial electrical resistance (TER) measurement was examined for detection of permeability damage in HPMCs. Immunostaining and Western blotting analysis were used to detect claudin-1 expression. Mitochondrial superoxide (MitoSOX) Red staining and respiratory chain complexes activities were determined for detection of mitochondrial reactive oxygen species (ROS) production and mitochondrial complexes activities. Results TER was decreased in a time- and concentration-dependent manner after culture with high glucose PDS for was also down-regulated significantly by high glucose PDS (P＜0.01). Complex Ⅲ activity was inhibited (10.8% of control, P＜0.01) accompanied with increased mitochondrial ROS generation.These changes were partially prevented by glutathione. Conclusion Mitochondrial respiratory complex Ⅲ pathway has crucial importance in maintaining TER of HPMCs, which may reveal a valuable target for novel therapies to fight hyperpermeability of peritoneum during the prolonged PD treatment.
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Objective To observe the effect of ulinastatin on the expression of interleukin 15 (IL-15), connective tissue growth factor (CTGF) and malondialdehyde (MDA) in rat peritoneal mesothelial cells (RPMCs) induced by high glucose. Methods RPMCs were isolated, cultured and passaged by trypsin, then identified. The third generation of cultured RPMCs were used in the experiment. RPMCs were divided into normal control group, high glucose (1.5%, 2.5%, 4.25%) for 6 hours and 12 hours, high glucose (2.5%) for 3, 6, 12, 24 hours or ulinastatin (160, 320, 640U/ml) for 12 hours. IL-15 mRNA was detected by real-time PCR. IL-15 and CTGF protein in supernatants was detected by ELISA. MDA protein was detected by TBAS. Results Compared with the control group, the expression of IL-15, CTGF and MDA was significantly increased in the groups stimulated by high glucose (P＜0.05) in dose- and time-dependent manner. Ulinastatin could significantly decrease the expression of IL-15, CTGF and MDA induced by high glucose in dosedependent manner both in protein and gene levels (P＜0.05). Conclusions High glucose can up-regulate the expression of IL-15, CTGF and MDA in RPMCs. Ulinastatin can reverse these changes.
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Objective To explore the effect of transforming growth factor β1 (TGF-β1) on epithelial-mesenchymal transition in rat peritoneal mesothelial cells(RPMCs) and its mechanism.Methods Primary peritoneal mesothelial cells of SP rats were cultured in vitro. After synchronization for 24 h, RPMCs were randomly divided into 2 groups: Group A (control), Group B (TGF-β1, 10 μg/L). RPMCs were stimulated by 10 μg/L TGF-β1 for different time. The mRNA and protein expression levels of E-cadherin, α-smooth muscle actin (α-SMA) and collagenⅠwere measured by RT-PCR and Western blot, respectively. The protein expression level of total RhoA was measured by Western blot. Active RhoA was extracted by Plasma Membrane Protein Extraction Kit, and assessed by Western blot. Results TGF-β1 down-regulated mRNA and protein expression of E-cadherin in RPMCs, and upregulated mRNA and protein expression of α-SMA and CollagenⅠ. TGF-β1 stimulation elicited a robust increase in RhoA activity in a time-dependent manner. RhoA activity peaked at 1 h.Conclusion RPMCs can be transdifferentiated into myofibroblast under the effect of TGF-(β1,)and the mechanism may be related to the activation of RhoA associated signal pathway.
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Objective To investigate the expression of CD40 and intercellular adhesion molecule-1 (ICAM-1) treated with lipopolysaccharide (LPS) in rat peritoneal mesothelial cells(RPMC) and the role of NF-κB signal transduction pathway. Methods RPMCs were harvested from Sprague-Dawley rat peritoneal cavity and maintained under defined in vitro conditions. The cells were exposed respectively to different concentrations of LPS for 12 h or treated with LPS (5 μg/ml) for different time points. To observe the effect of LPS on the expression of CD40 and ICAM-1, the RPMCs were treated with LPS (5 μg/ml) for different time points. To observe the effect of LPS on the expression of NF-κB and p-NF-κB protein, the RPMCs were treated by LPS or pretreated with BAY11-7085 (5 μmol/L or 1 μmol/L ) for 3 h, then treated with LPS for another 3 h, respectively. Expression of CD40 and ICAM-1 mRNA was examined by RT-PCR. Expression of NF-κB and p-NF-κB protein was detected by Western blot. Results Compared with medium control group, stimulation of RPMCs with 1 μg/ml and 5 μg/ml of LPS resulted in a significant increase in the expression of CD40 and ICAM-1 mRNA(P<0.05). 10 μg/ml of LPS had strongest effect on CD40 and ICAM-1 expression compared with that of 1 μg/ml and 5 μg/ml of LPS. Treatment with 5 μg/ml of LPS resulted in time-dependent increase in the gene level of CD40 and ICAM-1, with the peak at 3 h. However, after that time point, the gene level of them was gradually attenuated. Following treatment with LPS (5 μg/ml), the level of p-NF-κB began to increase at 15 min, gradually reached the peak at 1 h, and then decreased. But the level of p-NF-κB at 2 h was still significantly higher than that of medium control. 5 μmol/L of BAY11-7085 decreased significantly the up-regulation of CD40 and ICAM-1 induced by LPS. Conclusion LPS enhanced the expression of CD40 and ICAM-1 on RPMCs in a concentration-dependent and a time-dependent manner. LPS induced expression of CD40 and ICAM-1 depend on the NF-κB signal transduction pathway.
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Objective To investigate the effects of connective tissue growth factor (CTGF) siRNA delivered by pRetro-Super (PRS) retrovirus vector on extracellular matrix and VEGF expression in human peritoneal mesothelial cells (HPMC). Methods Four pairs of oligonucleotides including 64 bp DNA were designed and synthesized in vitro according to siRNA target sequence and PRS retrovirus desire.PRS-CTGF-siRNA1-4 recombinant retrovirus vectors were constructed.The recombinant retrovirus vectors containing CTGF-siRNA were transferred into PT67 packaging cell lines with lipefectamine 2000,then infected HPMC.mRNA expression was determined by semi-quantitative RT-PCR and protein expression was determined by Western blot.Results Both mRNA and protein expressions of CTGF,FN,Col I,laminin (LN) and VEGF were significantly increased in HPMC with 5 μg/L TGF-β1 stimulation (P＜0.01,respectively).CTGF,FN,Col I,LN mRNA and protein and VEGF mRNA expression stimulated by TGF-β1 were significantly decreased in HPMC infected with PRS-CTGF-siRNA1～4 retrovirus vectors (P＜0.01,respectively).The inhibitory rates on CTGF were 69.3%,22.2%,27.4% and 38.8%,respectively (P＜0.01).At the same time,there was also a significant reduction of VEGF protein expression in HPMC infected with PRS-CTGF-siRNA1 vector (P＜0.01).There was no significant difference in HPMC infected with PRS void vector. Conclusion CTGF siRNA delivered by PRS retrovirus vector can effectively inhibit the enhancement of extracellular matrix and VEGF expression stimulated by TGF-β1 in HPMC.
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BACKGROUND: Protein kinase C (PKC)s consist of three groups of isoenzyme; conventional, novel and atypical PKCs. Diacylglycerol (DAG) activates both conventional and novel PKCs, but not atypical PKCs. High glucose-induced fibronection production was shown to be mediated by activation of DAG-sensitive PKCs. In this study, we investigated whether PKC mediates IL-1beta-induced fibronectin mRNA expression, and the subtypes of PKC involved in the process. METHODS: Fibronectin mRNA level and phosphorylated PKC zeta/iota in total cell lysate were measured by Northern blot and Western blot, respectively. RESULTS: Pretreatment of HPMCs with calphostin C, a pan-PKC inhibitor, at doses of 500, 750 and 1, 000 nM caused dose-dependent inhibition of IL- 1beta (1 ng/mL)-induced fibronectin mRNA level. GF109203X, another pan-PKC inhibitor, at doses of 1, 5 and 10 microM also downregulated IL-1beta (1 ng/ mL)-induced fibronectin mRNA level in a dose-dependent manner. Phorbol 12-myristate 13-acetate (PMA), an activator of conventional and novel PKCs, stimulated fibronectin mRNA level at doses of 1, 10 and 100 nM. After prolonged treatment of the cells for 72 hr with PMA, another dose of PMA did not increase fibronectin mRNA level, while IL-1beta (1 ng/mL) still stimulated it. Pretreatment of the cells with 5, 10, 15 and 20 microM of myristoylated PKC zeta/iota pseudosubstrate inhibited IL-1beta (1 ng/mL)-induced fibronectin mRNA level in a dose-dependent manner, while 20 microM of myristoylated PKC [19-27] pseudosubstrate, given as a control, had no effect. Stimulation of fibronectin mRNA level by IL-1beta (1 ng/mL) was completely prevented by 20 microM of my ristoylated PKC zeta/iotapseudosubstrate. IL-1beta (1 ng/ mL) increased phosphorylated PKC zeta/iota, an active form of the enzyme. CONCLUSION: IL-1beta-induced fibronectin production in HPMCs occurs by way of activation of atypical PKCs (PKC zeta/iota).
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Humans , Blotting, Northern , Blotting, Western , Fibronectins , Interleukin-1beta , Protein Kinase C , Protein Kinases , RNA, MessengerABSTRACT
Objective: To investigate the effect of Astragalus mongholicus injection(AM) on the secretion and mRNA expression of transforming growth factor((TGF-?1)) and basic fibroblast growth factor(bFGF) in cultured human peritoneal mesothelial cells((HPMC).) Methods: HPMC got from patients underwent surgical operation were cultured in vitro.At first,cells from the third passage were incubated with RPMI1640 culture medium containing 0.1% FBS for 24 hours.Then,they were divided into control group,PDS group,AM group 1,AM group 2 and AM group 3 for testing.Each group was supplemented with equal volum of RPMI1640 culture medium containing 20% FBS.After 24 hour incubating,mitochondrial dehydrogenases activity(MTT assay),the levels of TGF-?1 and bFGF in supernatant of the cell culture and the mRNA expression of TGF-?1 and bFGF were detected.Results: Significant decrease of mitochondrial dehydrogenase activities were observed in PDS group as compared with those in control group and AM groups(P0.05).The levels of TGF-?1 and bFGF in supernatant of the cell culture were significantly lower in control group than those in PDS group.Marked lower levels of TGF-?1 and bFGF were found in AM group 1,AM group 2 and AM group 3 as compared with those in PDS group(P0.05).The mRNA expressions of TGF-?1 and bFGF in PDS group increased significantly as compared with those in control group.And significant mRNA expressions of TGF-?1 and bFGF were found in PDS group when compared with those in AM groups(P
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BACKGROUND: In early phase of peritonitis, mononuclear cells as well as polymorphonuclear leukocytes migrate rapidly into peritoneal cavity. For the migration of mononuclear cells, the expression of VCAM-1 on peritoneal mesothelial cells is important. In this study, we investigated the effect of TGF-beta1 on tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta(IL-1beta) induced VCAM-1 expression in the cultured HPMCs. METHODS: HPMCs were cultured in the presence of TNF-alpha, IL-1beta and/or TGF-beta1. VCAM-1 mRNA level was measured by Northern blot. VCAM-1 in total cell lysate and VCAM-1 expressed on cell surface were measured by Western blot and cellular ELISA, respectively. RESULTS: Incubation of the cultured HPMCs with TNF-alpha (10 ng/mL) or IL-1beta (1 ng/mL) caused an increased level of VCAM-1 mRNA, VCAM-1 protein in total cell lysate, and VCAM-1 expressed on cell surface. This stimulatory effects of TNF-alpha or IL- 1beta were inhibited by TGF-beta1 (0.1, 1, 10 ng/mL), dose-dependently. The level of VCAM-1 mRNA, VCAM-1 protein in total cell lysate, and VCAM-1 expressed on cell surface in the unstimulated cells were also inhibited by TGF-beta1 (10 ng/mL). The rate of VCAM-1 mRNA degradation after an application of actinomycin D was not affected by TGF-beta1. CONCLUSION: TGF-beta1 inhibited inflammatory cytokine induced VCAM-1 production and expression in the cultured HPMCs. Treatment of the cells with TGF-beta1 seems to suppress TNF-alpha or IL-1beta induced VCAM-1 mRNA transcription rather than decrease stabilization of VCAM-1 mRNA.
Subject(s)
Humans , Blotting, Northern , Blotting, Western , Dactinomycin , Enzyme-Linked Immunosorbent Assay , Neutrophils , Peritoneal Cavity , Peritonitis , RNA Stability , RNA, Messenger , Transforming Growth Factor beta1 , Tumor Necrosis Factor-alpha , Vascular Cell Adhesion Molecule-1ABSTRACT
BACKGROUND: High glucose in peritoneal dialysis solution has been implicated in the pathogenesis of peritoneal fibrosis. Macrophages in peritoneal cavity seem to participate in the process of peritoneal fibrosis through the production of various cytokines and growth factors. Monocyte chemoattractant protein-1(MCP-1) plays a key role in the recruitment of monocytes toward the peritoneal cavity. Vascular cell adhesion molecule-1(VCAM-1) is assumed to be important in the transmigration of monocytes. MCP-1 and VCAM-1 can be induced by various cytokines and growth factors in human peritoneal mesothelial cells(HPMC). However, effect of high glucose on the expression of MCP-1 and VCAM-1 in HPMC has not been known well. METHODS: Cultured HPMC were conditioned with glucose(5-100mM) or mannitol for varying periods up to 7 days. Cell proliferation and mRNA expression of MCP-1 and VCAM-1 were assessed by MTT assay and Northern blot analysis respectively. MCP-1 protein was measured using ELISA. Chemotactic activity of high glucose-conditioned culture supernant were evaluated by chemotactic assay. Effect of protein tyrosine kinase(PTK) inhibitor on the high glucose-induced MCP-1 mRNA expression was examined. RESULTS: Glucose inhibited the cell proliferation in a time and dose dependent manner. Northern blot analysis showed that high glucose increased the MCP-1 mRNA expression in a time(2-7days) and dose(15-100mM) dependent manner, but not VCAM-1 mRNA expression. MCP-1 protein in cell culture supernant was also increased. Equivalent osmotic concentration of mannitol had no significant effect. High glucose-conditioned supernant had an increased chemotactic activity for monocyte, which was neutralized by specific anti-MCP-1 antibody. PTK inhibitors such as genistein and herbimycin A suppressed the high glucose-induced MCP-1 mRNA expression in a dose dependent manner. CONCLUSION: High glucose induced MCP-1 expression in HPMC partly via pathways involving PTK.
Subject(s)
Humans , Blotting, Northern , Cell Adhesion , Cell Culture Techniques , Cell Proliferation , Cytokines , Enzyme-Linked Immunosorbent Assay , Genistein , Glucose , Intercellular Signaling Peptides and Proteins , Macrophages , Mannitol , Monocytes , Peritoneal Cavity , Peritoneal Dialysis , Peritoneal Fibrosis , Protein-Tyrosine Kinases , RNA, Messenger , Tyrosine , Vascular Cell Adhesion Molecule-1ABSTRACT
Leukocyte adhesion to mesothelium is an important step during peritonitis, which may be mediated by adhesion molecules including vascular cell adhesion molecule-1(VCAM-1). Nitric oxide(NO) is known to be an endogenous inhibitor of leukocyte adhesion. We investigated the effect of NO on VCAM-1 expression in cultured human peritoneal mesothelial cells and the possible role of cGMP and NF-kappa B. Cells were exposed to tumor necrosis factor-alpha(TNF-alpha) for the indicated periods in the presence or absence of NO donors, 3-morpholino-sydnonimine (SIN-1) or nitroprusside(NP). The expression of VCAM-1 mRNA and cell surface VCAM-1 molecule was measured by Northern blot analysis and flow cytometry. To detect NF-kappa B binding activity, electrophoretic mobility shift assay(EMSA) was performed. To determine the role of guanylate cyclase or cGMP, inhibitor of guanylate cyclase, 1H-[1, 2, 4] oxadiazolo [4, 3-a] quinoxalin-1-one(ODQ) or analogue of cGMP, 8-bromo-cGMP was used. Both SIN-1 and NP inhibited the TNF-alpha-induced VCAM-1 mRNA expression in a dose dependent manner. SIN-1 also inhibited the expression of cell surface VCAM-1 molecule. Furthermore, SIN-1 and NP inhibited the expression of VCAM-1 mRNA induced by interleukin-1(IL-1beta) or lipopolysaccharide(LPS) as well. By EMSA, SIN-1 inhibited the TNF-alpha-induced NF-kappa B activity. The 8-bromo-cGMP had no significant effect on TNF-alpha-induced VCAM-1 mRNA expression and ODQ also had no significant influence on the inhibitory effect of SIN-1. In conclusion, NO may play an important role in mediating the inflammatory process during peritonitis by down-regulating the mesothelial VCAM-1 expression via suppression of NF-kappa B activity through cGMP- independent pathway.
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Humans , Blotting, Northern , Cell Adhesion , Cyclic GMP , Epithelium , Flow Cytometry , Guanylate Cyclase , Leukocytes , Necrosis , Negotiating , NF-kappa B , Nitric Oxide , Peritonitis , RNA, Messenger , Tissue Donors , Vascular Cell Adhesion Molecule-1ABSTRACT
Interleukin-1beta(IL-1beta), which is released into the peritoneal cavity during peritonitis in continuous ambulatory peritoneal dialysis(CAPD) patients, stimulates the production of extracellular matrix proteins, including fibronectin and type, I collagen, in the cultured peritoneal mesothelial cells(HPMCs). This may participate in the development of peritoneal fibrosis developing after episodes of peritonitis. Heparin stimulates the fibrinolysis and it is a routine practice to put 0.5-1U/mL of heparin into the dialysis fluid to prevent the formation of fibrin clot. Besides the anticoagulant acivity, heparin has diverse biologic effects on the cells. In this study, we investigated the effect of heparin on the production of fibronectin, one of the extracellular matrix proteins, in the cultured human peritoneal mesothelial cells. Incubation of the cultured HPMCs with IL-1beta(1 ng/mL) caused an increased expression of fibronectin mRNA by the cells. This stimulatory effect of IL-18 on the expression of fibronectin mRNA was inhibited by heparin, dose-dependently as well as time-dependently. The expression of fibronectin mRNA in the unstimulated cells was also inhibited by heparin. The amount of secreted fibronectin in the culture supernatant was decreased by heparin(1U/mL). This inhibitory effect was present regardless of the addition of IL-lbeta. However, IL-lbeta-induced increase in the expression of TGF bata1 mRNA in the cultured HPMCs was not inhibited by heparin. In conclusion, heparin inhibited the production of fibronectin by the cultured HPMCs at the concentration used clinically during peritonitis in CAPD patients. This suggests that heparin may have a preventive effect on the development of peritonitis associated peritoneal fibrosis as well as decrease the formation of fibrin clot.
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Humans , Collagen , Dialysis , Extracellular Matrix Proteins , Fibrin , Fibrinolysis , Fibronectins , Heparin , Interleukin-18 , Interleukin-1beta , Peritoneal Cavity , Peritoneal Dialysis, Continuous Ambulatory , Peritoneal Fibrosis , Peritonitis , RNA, MessengerABSTRACT
The longevity of the peritoneum has been an object of interests and speculation since the inception of CAPD. Peritoneal mesothelial cells(MC) are the most important intraperitoneal cell quantitatively and have the capability to secret different types of substances including lubricant and various cytokines. It may therefore be essential to have information on the MC mass during peritoneal dialysis. However, there were no specific tools to evaluate the exact status of peritoneal membrane. CA125 is a 22kDa glycoprotein which is a clinically useful tumor marker of non-mucinous epithelial ovarian carcinoma. Recently, other cells including pleural and peritoneal MC have been proved to express CA125. This study was undertaken to determine whether CA125 can be used as a bulk marker of MC mass in clinically stable 23 CAPD patients. We also analyzed whether the observed intraperitoneal production of CA125 can be attributed to MC using the cultured human peritoneal MC from omentum. The CA125 production by MC in vitro was estimated with cultured MC of various number(10,000-5,000,000/well) for different period of time. The median concentration of CA125 was significantly higher in 24-hour spent dialysate than in serum(5.5 vs. 17.3U/ml, P<0.05). There was signifcant negative correlation between the dialysate CA125 level and the incidence of peritonitis. In-vitro experiment using cultured MC cell showed an exponential increase of CA125 level during confluence(6th day of culture), and persistently increased till 15 days of culture, reaching a plateau. A linear relation between the number of MC and the amount of CA125 in supernatant was also observed. In conclusion, CA125 is locally produced in the peritoneal cavity and can be an useful marker of MC mass in stable CAPD patients. Since the clinical significance of the inverse correlation between the CA125 level and peritonitis incidence is uncertain(low CA125 as a second result of repeated peritonitis or the importance of MC mass to regulate the antibacterial defense mechanism?), a prospective follow-up study is necessary to confirm the relation between dialysate CA125 and the incidence of peritonitis.