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Objective:To investigate the effect of Banxia Xiexintang on the epithelial-mesenchymal transition （EMT） of human peritoneal mesothelial cell line （HMrSV5） induced by gastric cancer-derived exosomes （Exo）. Method:Banxia Xiexintang-containing serum was prepared and the human gastric cancer NCI-N87-derived exosomes （NCI-N87-Exo） were extracted， followed by their identification by transmission electron microscopy and Western blotting and labeling with 1，1-dioctadecyl-3，3，3，3- tetramethylindocarbocyanine perchlorate （Dil）. The cells were divided into the blank group， model group， and low-， medium-， and high-dose （13.5，27，54 g·kg<sup>-1</sup>） Banxia Xiexintang groups. HMrSV5 cells in the blank group were cultured alone， the ones in the model group with 100 mg·L<sup>-1</sup> NCI-N87-Exo， and those in the low-， medium-， and high-dose Banxia Xiexintang groups with 100 mg·L<sup>-1</sup> NCI-N87-Exo plus low-， medium-， and high-dose 10% Banxia Xiexintang-containing serum， respectively. Confocal laser microscope was used to observe the uptake of NCI-N87-Exo by HMrSV5 cells at 24 h， 48 h and 72 h. Seventy-two hours later， the morphological changes in HMrSV5 cells were observed. The protein expression levels of E-cadherin， cytokeratin 19 （CK19）， <italic>α</italic>-smooth muscle actin （<italic>α</italic>-SMA）， elastin， and transforming growth factor-<italic>β</italic><sub>1</sub> （TGF-<italic>β</italic><sub>1</sub>）， Smad2/3， and p-Smad2/3 were assayed by Western blot. Result:It was observed under the transmission electron microscope that NCI-N87-Exo showed an oval or dish-shaped vesicle structure with a particle size ranging from 40 to 80 nm. Exo marker proteins CD9 and CD63 were highly expressed while calreticulin was not expressed， implying that the NCI-N87-Exo was confirmed. After 24 h， 48 h， 72 h of co-culture， it was observed under the fluorescence microscope that NCI-N87-Exo were taken up by HMrSV5 cells， which was positively correlated with time. Compared with the blank group， Banxia Xiexintang significantly inhibited the uptake of NCI-N87-Exo by HMrSV5 cells， with better effect noticed in the middle- and high-dose Banxia Xiexintang groups（<italic>P</italic><0.05，<italic>P</italic><0.01）. After intervention with Banxia Xiexintang-containing serum， the HMrSV5 cells were arranged densely， and the intercellular space was significantly reduced， with the most obvious changes present in the high-dose Banxia Xiexintang group. Western blot revealed that the protein expression levels of E-cadherin and CK19 in HMrSV5 cells after being intervened with the medium- and high-dose Banxia Xiexintang-containing serum were increased significantly as compared with those in the blank group， whereas the levels of <italic>α</italic>-SMA and Elastin were decreased significantly （<italic>P</italic><0.01）. Banxia Xiexintang-containing serum at the low， medium， and high doses remarkably down-regulated TGF-<italic>β</italic><sub>1</sub> and p-Smad2/3 protein expression（<italic>P</italic><0.05，<italic>P</italic><0.01）. However， there was no significant change in Smad2/3. Conclusion:NCI-N87-Exo can be taken up by HMrSV5 cells to induce EMT. Banxia Xiexintang can inhibit the uptake of NCI-N87-Exo by HMrSV5 cells and the resulting EMT induced by NCI-N87-Exo， which is related to the regulation of TGF-<italic>β</italic><sub>1</sub>/Smads signaling pathway.
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[Abstract] Objective To explore the relationship between Kruppel-like factor 4 (KLF-4) and E-cadherin in human peritoneal mesothelial cells (HPMCs), and the expression and function of KLF-4 in the animal model of peritoneal fibrosis induced by high glucose peritoneal dialysate. Methods Co-transfection in HPMCs with the plasmid of KLF-4 and the bind site or mutant in the promoter region of E-cadherin, and then the luciferase activity was measured of the each bind site and its matched mutants to estimate whether KLF-4 can combine with the bind site in the promoter region of E-cadherin; Chromatin immunocoprecipitation (CHIP) was exploited to verify if KLF-4 can combine with the bind site in the promoter region of E-cadherin; Real-time PCR and Western blotting were performed to detect the expression of E-cadherin at the bind site and matched mutants of b, d, f and g. Thirty SD rats were randomly divided into saline group, peritoneal dialysate group and experimental group (10 each). Rats in saline group were given intraperitoneal injection with 0.9% NaCl, in peritoneal dialysate group were given with 4.25% high glucose peritoneal dialysate, and in experimental group were given via tail vein with 4.25% high glucose peritoneal dialysate and the mixture of KLF-4 plasmid suspension containing ultrasound microbubble. To observe the peritoneal tissue thickness of the 3 groups of rats by Hematoxylin and Eosin staining. Masson trichrome staining was performed to detect the deposition of collagen fibers in peritoneal tissue of the 3 groups of rats. Immunohistochemistry was used to detect the expression level of KLF-4, E-cadherin, α-SMA and fibronectin (FN) in peritoneal tissue of the 3 groups of rats. Results Promoter luciferase reporter gene and CHIP results showed that KLF-4 can combine with the bind site in the promoter region of E-cadherin in HPMCs. Real-time PCR and Western blotting showed that KLF-4 can positively regulate the expression of E-cadherin. HE staining showed that the peritoneal tissue was obviously thickened in rats of peritoneal dialysate group [(105.91±12.0) μm] than in rats of saline group [(20.89±5.39) μm] and of experimental group [(23.05±6.07) μm] with statistical significance (P0.05). Masson staining showed that the deposition of collagen fiber significantly increased in peritoneal dialysate group (0.89±0.09) than in saline group (0.19±0.03) and experimental group (0.15±0.06) with statistical significance (P0.05). Immunohistochemistry results showed that the expressions of KLF-4 and E-cadherin were obviously lower in peritoneal dialysate group (0.27±0.09, 0.31±0.03) than in saline group (0.79±0.19, 0.83±0.13) and experimental group (0.85±0.11, 0.76±0.11) with statistically significant difference (P0.05). In contrast, the expressions of α-SMA and FN were evidently higher in peritoneal dialysate group (0.83±0.09, 0.63±0.09) than in saline group (0.22±0.08, 0.30±0.07) and experimental group (0.19±0.05, 0.11±0.03) with statistically significant difference (P0.05). Conclusion KLF-4 may positive regulate the expression of E-cadherin by combining with the bind site in the promoter region of E-cadherin, and inhibit the peritoneal fibrosis induced via high glucose peritoneal dialysate.
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Objective To explore the role of long noncoding RNA-MGC (lnc-MGC) on the trans-differentiation of human peritoneal mesothelial cells (HPMCs) induced by high glucose.Methods The immortalized HPMCs were used to establish control group and high glucose group (60mmol/L) respectively.Cells in control group were cultured with ordinary cell medium,and in high glucose group were stimulated with high glucose medium for 72h.Changes of lnc-MGC expression in the both groups were measured by RT-PCR,and the changes of mRNA and protein expression of E-Cadherin,α smooth muscle actin (α-SMA),connective tissue growth factor (CTGF),type Ⅰ collagen (COL-1) and type Ⅲ collagen (COL-3) in epithelial cells of both groups were measured by RT-PCR and Western blotting.The HPMCs were transfected with lentivirus,and then the changes of the above indexes were observed after up-and down-regulation of lnc-MGC.Results By high glucose stimulation of HPMCs for 72h,RTPCR results showed that the expressions oflnc-MGC,α-SMA,CTGF,COL-1 and COL-3 mRNA increased obviously (P＜0.05),and the expression of E-Cadherin mRNA decreased markedly (P＜0.05) in high glucose group than in control group;Western-blotting results indicated that the expression of protein was consistent with that of mRNA,and the differences were statistically significant (P＜0.05).After lentivirus transfection and down-regulation oflnc-MGC,RT-PCR results showed that the expressions of lnc-MGC,α-SMA,CTGF,COL-1 and COL-3 mRNA decreased obviously (P＜0.05),and the expression of E-Cadherin mRNA increased markedly (P＜0.05) in high glucose group than in control group;Western-blotting results showed that the expression of protein was consistent with that of mRNA,and the differences were statistically significant (P＜0.05).After lentivirus transfection and upregulation of lnc-MGC,RT-PCR results showed that the expressions of lnc-MGC,α-SMA,CTGF,COL-1 and COL-3 mRNA increased significantly (P＜0.05),and the expression of E-Cadherin mRNA decreased obviously (P＜0.05) in high glucose group than in control group;Western blotting results showed that the expression of protein was consistent with that of mRNA,and the differences were statistically significant (P＜0.05).The downstream target was predicted as miRNA126-3p,and compared with the control group,the expression ofmiRNA126-3p increased (P＜0.05) after high glucose stimulation,and after transfection with down regulated lnc-MGC lentivirus,the expression of miRNA126-3p decreased obviously (P＜0.05),and transfection with up regulated lnc-MGC lentivirus,the expression ofmiRNA126-3p increased obviously (P＜0.05).Conclusions lnc-MGC participates in the process of HPMCs transdifferentiation through regulating miRNA126-3p.Regulation oflnc-MGC expression level may control the phenotype transition of HPMCs,and delay the development of peritoneal fibrosis.
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Objective:To explore the effect of high glucose-based peritoneal dialysis fluids on NLRP3-IL-1β in human peritoneal mesothelial cells.Methods:HMrSV5 cells (SV40 immortalized human peritoneal mesothelial cell line) were grown in type Ⅰ collagen-coated dishes in DMEM/F12 containing 10％ fetal calf serum (FCS).All experiments on HMrSV5 cells were performed between passages 5 and 10.The cells were divided into 7 groups:control,1.5％ dextrose,2.5％ dextrose,4.25％ dextrose,rotenone,thenoyltrifluoroacetone (TTFA),and antimycin A.Immunoblotting was used to evaluate the expression of IL-1 β.Small interfering RNA (siRNA) targeting NLRP3 was used to downregulate the expression of NLRP3 and Western blot was used to evaluate the expression of IL-1 β in human peritoneal mesothelial cells exposed to 4.25％ dextrose.In the meanwhile,resveratrol (RSV) was used to induce autophagy,3-methyladenine (3-MA) and siRNA against Beclin 1 or ATG5 were used to block autophagy,flow cytometric was used to analyze the respiring (mitotracker deep red),total (mitotracker green) and reactive oxygen species (ROS)-generating mitochondria (mitoSOX);Western blot was used to evaluate the expression of IL-1β.Results:The IL-1β relative expressions were 0,0.175 ±0.082,0.418 ± 0.163,2.357 ±0.288,2.642 ±0.358,3.271 ±0.462,and 0.123 ±0.091,indicating that the cells exposed to high glucose-based peritoneal dialysis fluids and cells treated with mitochondria respiratory chain key enzyme complex Ⅰ,and complex Ⅲ inhibitors increased the IL-1β expression.And we found that NLRP3 knock-down significantly blocked the upregulation of IL-1 β.In addition,the fluorescence intensity of total mitochondria and ROS-generating mitochondria in the following groups:control,negative control,RSV,3-MA,ATG5 siRNA,Beclin1 siRNA were 1.76 ± 0.42,1.83 ± 0.55,1.85 ± 0.62,7.36 ± 0.92,5.35 ± 0.77,5.06 ± 0.62 and 821.68 ± 95.12,868.15 ± 102.82,723.39 ± 92.56,1 660.08 ± 113.65,1 433.01 ± 107.24,1 562.36 ± 112.88 respectively.The increased concentrations of mitochondrial ROS and IL-1β upregulation were confirmed in the inhibition but not the induction of autophagy.We also found that downregulation of ATG5 and Beclin1 sensitized cells for the release of IL-1β induced by MSU (monosodium urate) or nigericin which was the NLRP3 inflammasome activator.RSV treatment attentuated this effect.Conclusion:Long-term application of high glucose-based peritoneal dialysis fluids can trigger the consistent activation of NLRP3-IL-1ββ in peritoneal mesothelial cells.Timely initiation of autophagy may block the NLRP3-IL-1ββ activation and provide a basis for the further development of a potential therapeutic strategy for delay of chronic inflammation and peritoneal fibrosis associated with peritoneal dialysis.
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Objective To explore the effect of long noncoding RNA-ATB (LncRNA-ATB) on phenotypic transition and proliferation of human peritoneal mesothelial cells (HPMCs) induced by high glucose.Methods HPMCs used in experiment were divided into three groups:control group,mannitol group and hypertonic glucose group.HPMCs in control group received no treatment,and in hypertonic glucose group and mannitol group were treated with 50mmol/L D-glucose and isotonic mannitol for 72 hours,respectively.Real-time PCR was employed to detect the mRNA expression of LncRNA-ATB,E-cadherin,α-smooth muscle actin (α-SMA),connective tissue growth factor (CTGF),Cyclin D1,cyclin dependent kinase inhibitor 4 (CDK4),protein 27 (p27)and proliferating cell nuclear antigen (PCNA).Western blotting was performed to detect the proteins expression of E-cadherin,α-SMA,CTGF,Cyclin D1,CDK4,p27 and PCNA,and flow cytometry was used to test the cell cycle.Lentivirus artifice was used to up-or down-regulate the expression of LncRNA-ATB in untreated HPMCs.Real-time PCR was employed to detect the mRNA expression of E-cadherin,α-SMA and CTGF,Western blotting was performed to detect the proteins expression of E-cadherin,α-SMA and CTGF,and flow cytometry was used to test the cell cycle.Results It is revealed by Real-time PCR,Western blotting and flow cytometry that the expressions increased of LncRNA-ATB,α-SMA,CTGF,Cyclin D1,CDK4 and PCNA induced by hypertonic glucose,and decreased of E-cadherin and p27 (P＜0.05).Up-regulation of LncRNA-ATB promoted HPMCs phenotypic transition and proliferation,while down-regulation alleviated HPMCs phenotypic transition and proliferation.Conclusion Hypertonic glucose may accelerate HPMCs phenotypic transition and proliferation by up-regulating the expression of LncRNA-ATB.
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Objective To explore the effects of p53 up-regulated modulator of apoptosis (PUMA)-α protein on the apoptosis and fibrosis of human peritoneal mesothelial cells (HPMCs) induced by high glucose.Methods HPMCs were induced by 50mmol/L D type glucose or mannitol for 72 hours respectively,flow cytometry was employed to detect the rate of apoptotic cells,and theexpression levels of apoptosis-and fibrosis-related proteins were detected by Western blotting.The untreated HPMCs were transfected with Lenti-PUMA-α,and the treated cells were transfected with shRNA-PUMA-α,the number of apoptotic cells and the expression levels of apoptosis-and fibrosis-related proteins were detected with the methods mentioned above.Results Flow cytometry showed that the rate of apoptotic HPMCs increased after being induced by high glucose for 72 hours,and Western blotting revealed that the expression levels of pro-apoptotic and pro-fibrotic related proteins increased,but the arrestins of apoptosis and fibrosis-related proteins decreased.Up-regulation of PUMA-α promoted apoptosis and fibrosis,while down-regulation of PUMA-α alleviated apoptosis and fibrosis of HPMCs.Conclusion High glucose may accelerate apoptosis and fibrosis of HPMCs by up-regulating the expression of PUMA-α.
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Objective To explore the role of Kruppel-like factor 4 (KLF-4) in phenotypic transition of human peritoneal mesothelial cells (HPMCs) induced via high glucose. Methods HPMCs were induced by 50mmol/L glucose for 72 hours, the expressions of epithelium-cadherin (E-cadherin), KLF-4, α-smooth muscle actin (-SMA), connective tissue growth factor (CTGF) and miRNA-143 were detected by Real-time PCR and Western blotting, respectively. The treated cells were transfected with LVKLF-4 and inhibitor, the untreated cells were transfected with shRNA-KLF-4 and mimic. The mRNA and protein expressions of KLF-4, E-cadherin, α-SMA, CTGF and miRNA-143 were detected by Real-time PCR and Western blotting, respectively. Results Real-time PCR showed that the expression of E-cadherin decreased and of α-SMA, CTGF and miRNA-143 increased, but of KLF-4 not changed in high glucose treated cells. Western blotting showed that the expression of KLF-4 and E-cadherin decreased. Upregulating KLF-4 increased the expression of E-cadherin, but decreased the expression of α-SMA and CTGF. Down-regulating KLF-4 decreased the expression of E-cadherin, but augment the expression of α-SMA and CTGF. Conclusion High glucose may induce the down-regulation of KLF-4 protein, and SRF- miRNA-143-KLF-4 signal pathway axis may be involved in the process of HPMC phenotypic transition.
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Objective To investigate the effect of glucose-based peritoneal dialysis fluids and icodextrin-based peritoneal dial-ysis fluids on the expression of TLR2 and TLR4 on huamn peritoneal mesothelial cells .Methods Human peritoneal mesothelial cell line 5 - 10 generations(HMrSV5) was cultured in DMEM /F12 medium supplemented with 10% (v/v) fetal calf serum (FCS) .Cell viability and cell proliferation were assessed using M TT method .The experiment were divided into 5 different groups :group A (control group) ,1 .5% dextrose group ,2 .5% dextrose group ,4 .25% dextrose group and 7 .5% Lcodextrin group .Icodextrin group (aikau dextrin) ,TLR2 and TLR4 expression were detected by Western blot .Results Treatment with different concentrations of glucose-based peritoneal dialysis fluids for 24 h did not affect the expression of TLR2 and TLR4 protein .In addition ,after stimula-tion for 48 h ,1 .5% dextrose ,2 .5% dextrose ,4 .25% dextrose decreased TLR2 expression by (5 .5 ± 2 .8)% ,(31 .4 ± 7 .5)% , (54 .9 ± 1 .9)% respectively ,TLR4 expression by (32 .9 ± 17 .6)% ,(47 .7 ± 13 .5)% ,(66 .4 ± 13 .5)% respectively .Stimulation for 72 h ,decreased TLR2 expression by (29 .4 ± 14 .7)% ,(38 .9 ± 9 .9)% ,(63 .5 ± 16 .5)% respectively ,TLR4 expression by(59 .5 ± 16 .8)% ,(63 .1 ± 9 .5)% ,(79 .2 ± 14 .0)% respectively .There was no significant change in TLR2 and TLR4 protein expression on 7 .5% icodextrin group .Conclusion Glucose-based peritoneal dialysis fluids ,but not icodextrin-based peritoneal dialysis fluids downregulates expression of TLR2 and TLR4 by HM rSV5 .
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Objective To investigate the effects of AstragalosideⅣ (AST) on the expression of high glucose peritoneal dialysis solution (PDS)-induced transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF) in cultured human peritoneal mesothelial cells. To discuss the regulating effect of AST on induced fibrosis cytokines.Methods The HMrSV5 cell line was cultivated to the fifth generation and divided into normal group (10%FBS cultivation solution), model group (4.25% PDS and 10% FBS cultivation solution) and the low, medium and high doses AST groups (4.25% PDS with a respective 10, 20, 40 μg/mL AST). MTT colorimetric assay was employed to detect cell activity and ELISA was applied to detect expression of TGF-β1, CTGF and VEGF in cultured supernatants.Results Except for 5 μg/mL group, HPMCs activity of high glucose plus different concentration AST groups were enhanced in different degrees, especially with 40, 45, and 50 μg/mL (P<0.05). The expression of TGF-β1, CTGF, and VEGF in model group increased. Compared with the control group, expression of the three AST groups significantly decreased and showed dose-effect relationship (P<0.05). Conclusion AST could reduce the expression of TGF-β1, CTGF and VEGF in high glucose-induced HPMCs and was useful in slowing down the progress of peritoneal fibrosis.
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Objective To investingate the effect of low-density lipoprotein (LDL) on epithelial -mesenchymal transition and extracellular matrix (ECM) accumulation in human peritoneal mesothelial cells (HPMCs).Methods (1)HPMCs were randomly divided into control group,LDL group (100 mg/L) and LDL (100 mg/L) + lactoferrin (100 mg/L,LDL receptor blocking agent) group.After co-cultured for 24 h,the expression of LDL receptor in HPMCs was examined by immunofluorescence staining,and the LDL uptake by HPMCs was observed with oil red O staining.(2)HPMCs were cultured with different concentrations of LDL (0,25,50,100 mg/L).After co-cultured for 24 h,the change of cell morphology was observed by inverted phase contrast microscope,and the expression of α-smooth muscle actin (α-SMA) was examined by immunofluorescence.(3) HPMCs were randomly divided into control group (5.6 mmol/L glucose),mannitol group (M,2.18％ mannitol),low glucose group (LG,30 mmol/L),high glucose group (HG,120 mmol/L) and HG + LDL group (120 mmol/L glucose + 100 mg/L LDL).Cocultured for 48 h,the mRNA expression of α-SMA,E-cadherin and type 1 plasminogen activator inhibitor (PAI-1) was detected by real-time quantitative PCR,the protein expression of α-SMA was detected by Western blotting,the content of type I collagen (Col I) and PAI-1 in supernatant was detected by ELISA.Results (1) After co-cultured with LDL for 24 h,the expressin of LDL receptor was found on the cell membrane of HPMCs.Oil red staining showed that LDL could be uptaken into the cells and abolished by LDL receptor blocker.(2) HPMCs tended to be loosely intercellular connected to each ofher,and prsesnted significant formation of fibroblast-like spindle morphology.The cytoplasm immunofluorescence intensity of α-SMA gradually increased with the increase of LDL concentration.Compared to the control group,the expressions of α-SMA mRNA and protein were significantly increased,and the expression of E-cadherin mRNA was decreased in HG + LDL group(all P ＜ 0.05).But the expressions of the parameters above-mentioned were not significant different between HG group and HG + LDL group or between HG group and control group.(3) Compared with HG group or control group,the concentrations of Col Ⅰ [(19.27±0.17) μg/L vs (14.09±0.30) μg/L or (14.81±0.91) μg/L,all P ＜ 0.05] and PAI-1 [(498.24±76.91) ng/L vs (342.19±30.43) ng/L or (220.39±33.82) ng/L,all P ＜ 0.05] in supernatant of HPMCs were significantly up-regulated in HG + LDL group,meanwhile the expression of PAI-1 mRNA was significantly higer than that in control group (P =0.022).Conclusions HPMCs uptake LDL into cells via LDL receptors.LDL can induce HPMCs transdifferentiation in the condition of high glucose,increase the secretion of Col Ⅰ,inhibit the degradation of ECM through up-regulating the expression of PAI-1,and lead to ECM accumulation.
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Objective To explore the effect and mechanism of fluvastatin on the expression of fibronectin(FN) in human peritoneal mesothelial cells (HPMCs) induced by high-glucose peritoneal dialysate (HGPDS).Methods Cultured HPMCs were randomly divided into control,HGPDS,HGPDS plus GSK650394 10-5 mol/L (the competitive inhibitor of SGK1),different concentrations of fluvastatin,fluvastatin 10-6 mol/L and GSK650394 10-5 mol/L alone.The morphology change of HPMC was observed by light microscopy.The cellular viability was detected by MTT colorimetry.The mRNA and protein expressions of serum and glucocorticoid-inducible kinase 1 (SGK1) and FN were detected by RT-PCR,Western blotting or ELISA.Results After incubation with HGPDS,the cell morphology changed from typical cobblestone-like appearance to fibroblast-like appearance,and the cell viability was inhibited significantly (P＜0.05).Fluvastatin 10-6mol/L and GSK650394 could improved the cell morphology and the cell viability injured by HGPDS (P＜0.05).Compared with the normal control group,the mRNA and protein expressions of SGK1 and FN increased significantly in HPMC treated with HGPDS(P＜0.05).GSK650394 significantly decreased the high expression of SGK1 and FN (P＜0.05),also the fluvastatin had same effects as GSK650394 in dose-dependent manner (P＜0.05).Conclusions High-glucose peritoneal dialysate can increase FN expression in human peritoneal mesothelial cells,which can be attenuated by fluvastatin.The protective role of fluvastatin in HPMC may be partially achieved through the signal pathway of SGK1.
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Objective To investigate the effects of Ligustrazine on TNF-α-induced TGF-β and CTGF expression in human peritoneal mesothelial cells (HPMCs). Methods HPMCs were isolated from human omenta by trypsin di-gestion. Then, the subcultured HPMCs were divided into control group, TNF-α-induced (1 μg/L) group and TNF-α-induced plus low-, medium-and high-dose Ligustrazine (10, 20 and 40 mg/L Ligustrazine respectively) groups. The viability of HPMCs was measured by MTT assay. RT-PCR was used to detect the expressions of TGF-β1 and CTGF mRNAs in HPMCs. TGF-β1 and CTGF in supernatants were measured by ELISA. Cell protein concentration was measured by trace bicinchoninic acid (BCA) method to validate the ELISA assay results. Results Ligustrazine significantly decreases TNF-α-induced TGF-β1 and CTGF expression in a dose-dependent manner at both protein and gene levels ( P < 0. 05 ). In addition, medium-and high-dose Ligustrazine injection significantly ameliorates the viability of HPMCs inhibited by TNF-α ( P < 0. 05 ). Conclusion Ligustrazine inhibits expressions of TGF-β and CTGF of HPMCs in an inflammatory conditions.
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Objective To explore the effects of high glucose on oxidative stress of human peritoneal mesothelial cells(HPMCs).Methods HPMCs were cultured in vitro and identified by immunohistochemistry, and cells of second generation were selected. After HPMCs were treated by glucose with different concentrations for some time, MTT method was employed to detect the cell viability. 2’,7’-dichlorofluorescein diacetate (DCFH-DA) was used as a reactive oxygen species (ROS) capture. The cell viability of HPMCs and ROS level were analysed after being intervened by glucose with different concentrations and for different time. Results Viability of HPMCs was significantly inhibited in a dose-and time-dependent manner by high glucose(P
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Objective To investigate the effects of Ligustrazine on TNF-?-induced TGF-?1 and CTGF expression in human peritoneal mesothelial cells (HPMCs). Methods HPMCs were isolated from human omenta by trypsin digestion. Then,the subcultured HPMCs were divided into control group,TNF-?-induced (1 ?g/L) group and TNF-?-induced plus low-,medium-and high-dose Ligustrazine (10,20 and 40 mg/L Ligustrazine respectively) groups. The viability of HPMCs was measured by MTT assay. RT-PCR was used to detect the expressions of TGF-?1 and CTGF mRNAs in HPMCs. TGF-?1 and CTGF in supernatants were measured by ELISA. Cell protein concentration was measured by trace bicinchoninic acid (BCA) method to validate the ELISA assay results.ResultsLigustrazine significantly decreases TNF-?-induced TGF-?1 and CTGF expression in a dose-dependent manner at both protein and gene levels (P
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Objective To study the effects of lactate peritoneal dialysis solution(L-PDS) with different concentrations on apoptosis of human peritoneal mesothelial cells(HPMC),the expressions of bcl-2,bax and activity of caspase-3.Methods HPMC were separated using enzyme digestion and cultivated stably in vitro.After HPMC were co-cultivated with different concentrations(1.50%,2.50%,4.25%) L-PDS,flow cytometry was used to test the apoptosis of HPMC,RT-PCR was used to observe the expressions of bcl-2 and bax,fluorometric method was used to detect the activity of caspase-3.Results Compared with control group, L-PDS could induce the apoptosis of HPMC,especially in high concentration.With the increasing of L-PDS concentration,the expression of bcl-2 mRNA decreased,the expression of bax mRNA increased,the activity of caspase3 raised.There were significant differences of the indexes mentioned above between 4.25%,2.50% L-PDS groups and control group(P0.05).Conclusion L-PDS could induce HPMC apoptosis,which may be executed by alternating of the expressions of bcl-2,bax and activating of caspase3.
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Objective To make sure the effect of dialysate composition on HPMCs.Methods Cell strains were subculturing.There are six groups in this experiment: Group 1(control);group 2(4.25% Glucose);group 3(1.75mmol/L Ca~(2+));group 4(1.25 mmol/L Ca~(2+));group 5(4.25% Glucose+1.75mmol/L Ca~(2+));group 6(4.25% Glucose+1.25mmol/L Ca~(2+)).The capacity of proliferation of HPMCs was assessed by MTT assay. Results Proliferation of HPMCs was inhibited in 4.25% glucose group in time dependence.Ca~(2+) induce proliferation of HPMCs,however,no effect on proliferation of HPMCs exists in different Ca~(2+) group.Conclusion High glucose can inhibit cell proliferation;Ca~(2+)(1.75mmol/L,1.25mmol/L) can promote the proliferation and 1.25mmol/L is better.
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High glucose activates protein kinase C, induces reactive oxygen species generation, and upregulates expression of transforming growth factor-beta1(TGF-beta1) and fibronectin by human peritoneal mesothelial cells(HPMC). High glucose also induces premature senescence in mesothelial cells. Mesothelial cells shrink after exposure to hypertonic medium and intracellular uptake of amino acids increase to ensure subsequent volume increase. Based on these observations, new and more biocompatible peritoneal dialysis solutions that are glucose free and/or iso-osmolar have been developed. We investigated the effects of different osmolality and different osmotic agents including glucose, mannitol, and icodextrin on viability and proliferation of HPMC. HPMC were obtained from the omental tissues of consenting patients undergoing Cesarean section or elective abdominal surgery. All experiments were performed using cells in the 2nd or 3rd passage. Near-confluent HPMC grown in culture dishes were incubated with serum-free medium for 48 hours to arrest and synchronize cell growth. Lactate dehydrogenase(LDH) release was measured for cell viability and [3H]-thymidine incorporation for proliferation of cultured HPMC, after exposing HPMC to different concentrations of glucose, mannitol, and icodextrin for up to 96 hours. High glucose and mannitol at concentrations up to 100 mM(375 mOsm) did not increase LDH release up to 96 hours compared to control M199. When HPMC were exposed to 2, 4, 7.5, and 9% of icodextrin for 24-96 hours, LDH release did not increase. Glucose at 30, 50, and 100 mM significantly inhibited [3H]-thymidine incorporation by HPMC at 24 and 48 hours. Mannitol at 30, 50, and 100 mM for 24 hours and at only 100 mM for 48 hours also significantly inhibited cell proliferation. Icodextrin 9% (305 mOsm) inhibited cell proliferation compared with control M-199 at 24 hours. In conclusion, high osmolality per se dose not appear to increase HPMC death. However, high osmolality appears to inhibit HPMC proliferation at early stage. In addition, high glucose appears to inhibit HPMC proliferation independent of osmolality since high glucose continues to inhibit cell proliferation at 48 and 72 hours when mannitol at the same concentration did not. Icodextrin 9% of which osmolality is 305 mOsm inhibits HPMC proliferation at early stage but does not appear to increase HPMC death.
Subject(s)
HumansABSTRACT
Preservation of peritoneal membrane function is important in the success of long-term peritoneal dialysis (PD). During PD, human peritoneal mesothelial cells (HPMC) are continuously exposed to unphysiological peritoneal dialysis solution(PDS) charaterized by high glucose and lactate concentrations, low pH, and hyperosmolality. Since few studies have examined the effects of lactate and pH on HPMC biology, the present study investigated the effects of lactate and pH on the viability and proliferation of cultured HPMC and on the production of TGF-beta1, a fibrogenic cytokine, and fibronectin by cultured HPMC. HPMC were obtained from the omental tissue of pregnant women who were undergoing Cesarean section. Cells at confluence were utilized to determine the viability(LDH release), proliferation([3H]-thymidine incorporation), and the production of fibronectin and TGF-beta1(ELISA) after synchronizing the cell growth by incubating with serum free media for 24 hours. After exposure to the media containing lactate and pH, LDH release increased in dose- and time-dependent manner. Both 1.5% and 4.25% commercial PD solutions were cytotoxic and induced more than 80% LDH release within 24 hours. LDH release decreased with increasing dilution of commercial peritoneal dialysate, but there was no significant difference in LDH release between 1.5% and 4.25% PDS. LDH release increased in response to pH 5.5. Thymidine incorporation assay revealed that lactate and low pH significantly inhibited proliferation of HPMC. ELISA revealed that exposure of HPMC to lactate and low pH decreased fibronectin protein synthesis, when compared to cell exposed to bicarbonate containing M199 media. Our results clearly show that lactate and low pH lead to dose- and time-dependent cell death and reduce proliferation of cultured HPMC. Lactate and low pH per se appear to decrease fibronectin production by HPMC but may set a stage for other factors to promote progressive fibrosis during the healing stage in long-term PD.
Subject(s)
Female , Humans , Pregnancy , Biology , Cell Death , Cesarean Section , Culture Media, Serum-Free , Enzyme-Linked Immunosorbent Assay , Fibronectins , Fibrosis , Glucose , Hydrogen-Ion Concentration , Lactic Acid , Membranes , Peritoneal Dialysis , Pregnant Women , Thymidine , Transforming Growth Factor beta1ABSTRACT
Human peritoneal mesothelial cells may have a great potential to secrete chemokines, growth factors, adhesion molecules, and various cytokines stimulated with proinflammatory cytokines during peritoneal infection. In the course of peritonitis, rapid neutrophil cell influx and subsequent monocytic cell influx can be observed. It has been demonstrated that human peritoneal mesothelial cells secrete a C-X-C chemokine, IL-8, which contributes to the recruitment of neutrophil influx during peritoneal infection. However, the production and role of C-C chemokines have not been fully defined in human peritoneal mesothelial cells. This study was performed to evaluate the production of MCP-1 and RANTES and their influence on the chemotaxis of monocytes when human peritoneal mesothelial cells were stimulated with IL-1beta. Mesothelial cells obtained by enzymatic digestion of pieces of human omentum and stimulated with a various doses and times of IL-1beta. The expression of MCP-1 and RANTES mRNA was measured by Northern blot assay and the expression of their proteins was analyzed by ELISA. To evaluate their function, monocytes chemotaxis assay was performed using a 48-well chemotactic chamber. Cultured human peritoneal mesothelial cells appeared to be polygonal at confluence using phase contrast microscope. Indirect immunofluorescent staining demonstrated that the mesothelial cells reacted positively with anti-cytokeratin antibody and anti-vimentin antibody. The expression of MCP-1 and RANTES mRNA increased in response to IL-1beta in time and dose dependent manner. The protein levels of MCP-1 and RANTES with stimulation of 1.0ng/mL of IL-1beta for 24 hours were higher than those without(30.0+/-2.22 vs 3.55+/-0.74ng/105cells and 1.53+/-0.41 vs 0.11+/-0.02ng/105cells respectively, p<0.05, n=6). Chemotaxis assay showed that the supernatants from human peritoneal mesothelial cells with stimulation of IL-1beta for 24 hours had significantly higher chemotaxis of monocytes than those without(71+/-3.4% vs 50+/-2.9%, p<0.05, n=6). Coincubation of supernatants with stimulation and antibodies to MCP-1 or RANTES(20 micro L/mL, 10 micro L/mL, respectively) resulted in a significant inhibition of chemotaxis of monocytes by 33% and 12%(47+/-3.1% and 62+/-3.0% respectively, p<0.05, n=6). Human peritoneal mesothelial cells are capable of the expression of MCP-1 and RANTES mRNA and the production of their proteins in response to IL-1beta. Functionally, mesothelial cells derived Mand RANTES may contribute to the recruitment of monocytes and amplify the inflammatory process. Thus, human peritoneal mesothelial cells play an important role during peritoneal infection.