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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 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 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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AIM To investigate that Danhong Injection may decrease inflammation and oxidative stress response in rat peritoneal mesothelial cells (RPMCs) cultured with high glucose.METHODS The third passage of RPMCs were used for the experiment.After being incubated with DMEM for 24 hours,RPMCs were divided into normal control group,high glucose group (RPMCs were incubate with 100 mmol/L glucose for 12 h,24 h,48 h,72 h),Danhong Injection group (complete medium with 80 mL/L Danhong Injection for 48 h) and high glucose + Danhong Injection group (100 mmol/L high glucose with 40 mL/L,80 mL/L,160 mL/L Danhong Injection for 48 h respectively).The cell proliferation was detected by MTT method.The levels of interleukin-18 (IL-18),interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in culture fluid were determined by enzyme linked immunosorbent assay.The contents of malondialdehyde (MDA),superoxide dismutase (SOD),glutathione (GSH),in the culture fluid and intracellular reactive oxygen species (ROS) were measured by kits.The expressions of Bcl-2,Bax,fibronectin (FN),endothelin-1 (EDN1) and haem oxygenase-1 (HMOX1) mRNA were detected by quantitative real-time PCR.The protein expressions of EDN1 and HMOX1 were measured by Western blot.RESULTS High glucose can significantly inhibit RPMCs' viability.High glucose can also up-regulate the expressions of IL-18,IL-6,TNF-α,ROS,MDA,Bax mRNA,FN mRNA,EDN1 mRNA and down-regulate the levels of SOD,GSH,Bcl-2 mRNA,HMOX1 mRNA in RPMCs.Treatment with Danhong Injection can effectively reverse aforementioned effect induced by high glucose.CONCLUSION Danhong Injection can reverse the inhibition of cell viability,the inflammation and oxidative stress response induced by high glucose in RPMCs,thus protect peritoneal membrane.
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Objective To study the protective effect of Shenkang injection on peritoneal mesothelial cells (PMCs) of continuous ambulatory peritoneal dialysis (CAPD) mice, and explore the possible mechanism. Methods Forty ICR mice were randomly divided into blank control (A) group, peritoneal dialysis (B) group, low dose of Shenkang (C) group ( 2.5%dialysate+5 mL/kg Shenkang injection), medium (D) group (2.5% dialysate+10 mL/kg Shenkang injection) and high (E) group (2.5% dialysate+ 20 mL/kg Shenkang injection). Mice were observed for 4 weeks. Fasting blood glucose, total cholesterol, triglyceride and C-reactive protein (CRP) were detected by biochemical assay. Tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta 1 (TGF-β1), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) levels of serum and dialysate were detected by ELISA. Pathological changes of peritoneal tissue were observed by HE staining. Expression and mRNA transcription levels of these four cytokines in the peritoneal tissue were detected by immunohistochemical staining and real-time PCR respectively. Results There were no significant differences in body weight, fasting blood glucose, total cholesterol and triglyceride between 5 groups of mice (P>0.05). Compared with group B, there was no significant difference in CRP level between group C and group E, but which was significantly decreased in group D (P<0.05). The serum and dialysate levels of TNF-α, TGF-β1, VEGF and CTGF were decreased in group C and group D. The serum and dialysate levels of TNF-αand TGF-β1 were significantly increased in group E (P<0.05), but there was no significant difference between VEGF and CTGF in group E. Compared with group E, except for CTGF in dialysate of group C, the serum and dialysate levels of TNF-α, TGF-β1, VEGF and CTGF were significant decreased in group C and group D (P<0.05). Damaged PMCs were found in group B, which were improved in various degrees in group C, group D and group E. Compared with group B, the protein expression and mRNA relative transcription levels of TNF-α, TGF-β1, VEGF and CTGF tended to decrease gradually in group C, group D and group E (P<0.05). Conclusion A certain concentration of Shenkang injection can protect PMCs by inhibiting the expression of TNF-α, TGF-β1, VEGF and CTGF in CAPD mice, so as to control the occurrence and development of peritoneal fibrosis.
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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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<p><b>OBJECTIVE</b>To study the effect of ligustrazine nanoparticles nano spray (LNNS) on transforming growth factor β (TGF-β)/Smad signal protein of rat peritoneal mesothelial cells (RPMC) induced by tumor necrosis factor α (TNF-α), and the anti-adhesion mechanism of LNNS in the abdominal cavity.</p><p><b>METHODS</b>The primary culture and subculture of rat peritoneal mesothelial cells (RPMC) was processed by trypsin digestion method in vitro. The third generation was identifified for experiment and divided into 5 groups: a blank group: RPMC without treatment; a control group: RPMC stimulated with TNF-α; RPMC treated by a low-dosage LNNS group (2.5 mg/L); RPMC treated by a medium-dosage LNNS group (5 mg/L); and RPMC treated by a high-dosage LNNS group (10 mg/L). Reverse transcription-polymerase chain reaction was applied to test the expression of fifibronectin, collagen I (COL-I), TGF-β mRNA, and Western blot method to test the Smad protein 7 expression of RPMC.</p><p><b>RESULTS</b>Compared with the blank group, a signifificant elevation in fifibronectin (FN), COL-I and TGF-β mRNA expression of RPMC were observed in the control group (P<0.05). Compared with the control group, LNNS suppressed the expressions of FN, COL-I and TGF-β mRNA in a concentrationdependent manner (P<0.05). The expression of Smad7 protein of RPMC was down-regulated by TNF-α stimulation, and up-regulated with the increase of LNNS dose (P<0.05).</p><p><b>CONCLUSIONS</b>TNF-α may induce changes in RPMC's viability, leading to peritoneal injury. LNNS could reverse the induction of fifibrosis related cytokine FN, COL-I and TGF-β, up-regulating the expression of Smad7 by TNF-α in RPMC, thus attenuate peritoneal injury by repairing mesothelial cells.</p>
Subject(s)
Animals , Male , Collagen Type I , Genetics , Metabolism , Epithelium , Metabolism , Fibronectins , Metabolism , Nanoparticles , Chemistry , Particle Size , Peritoneal Cavity , Cell Biology , Pyrazines , Pharmacology , RNA, Messenger , Genetics , Metabolism , Rats, Sprague-Dawley , Signal Transduction , Smad Proteins , Metabolism , Transforming Growth Factor beta , Genetics , Metabolism , Tumor Necrosis Factor-alpha , PharmacologyABSTRACT
Objective This study was designed to investigate the effect of matrine on high glucose‐induced tumor necrosis factor‐α(TNF‐α)and transforming growth factor‐β1 (TGF‐β1 ) overexpression in rat peritoneal mesothelial cells(PMCs) ,and to ex‐plore the possible mechanism in the peritoneal fibrosis .Methods ology The rat PMCs were incubated in vitro ,and then assigned in‐to 5 groups on the basis of the added drug concentrations :2 .5% glucose group (n=15 ,A);Glucose + 2 .5% matrine group (n=15 ,B);4 .25% glucose group (n=15 ,C);Glucose + 4 .25% matrine group (n=15 ,D);Blank control group (n=15 ,control group , added the DMEM/F12 medium) .The levels of TNF‐αand TGF‐β1 in the supernatants were measured by ELISA method at the dif‐ferent time (24h ,48h ,72h) ,and observed the changes of PMCs under the microscope .Results Compared with the groups of A ,C and D ,the PMCs in the control group and B group were small ,round ,spindle ,irregular or intensive .The expression of TNF‐αand TGF‐β1 in the A group was lightly higher than that of the control group ,but the level in the C group was significantly higher than that of the control group (P 0 .05 ) ,but the lever in the D group was significantly lower than that of the C group ( P< 0 .05 ) . Conclusion Matrine might be helpful to resist the overexpression of TNF‐αand TGF‐β1 induced by high glucose ,and then protect the PMCs .
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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 role of PI3K/Akt signaling in the regulation of epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) in peritoneal dialysis in vitro and in vivo.Methods The level of Phosphorylated serine/threonine kinase Akt and the expression of EMT associated gene and protein,including ZO-1,Vimentin and FN,were measured in mice EMT model.In vitro study,phosphorylation level and nuclear translocation of Akt,ZO-1 and Vimentin expression induced by TGF-β1 in human peritoneal mesothelial cells (HPMCs) were also observed.Moreover,HPMCs were pre-treated by one of PI3K/Akt inhibitor,LY294002,or transfected with dominant-negative Akt plasmid to inhibit PI3K/Akt signaling,then analyzed its effect on Zo-1 and Vimentin expression induced by TGF-β1.Results Compared with the control,thickened parietal peritoneum and remarkable decrease in mRNA and protein of the epithelial marker ZO-1,and notable increased in the expression of mesenchymal markers Vimentin and FN were observed in PD mice (all P ＜ 0.01).Moreover,the phosphorylation of Akt also significantly increased under above condition (P ＜ 0.01).In vitro study,with the stimulation of TGF-β1,the expression of Zo-l was down-regulated,while the expression of Vimentin increased (all P ＜ 0.01).In addition,TGF-β1 remarkably increased pAkt in HPMCs (all P ＜ 0.01) in dose-dependent.However,LY294002 and DN-Akt dramatically inhibited the vimentin expression in HPMCs induced by TGF-β1 after inhibition of pAkt.On the other hand,the expression of ZO-1 also was restored (P ＜ 0.01).Conclusion PI3K/Akt signaling is involved in EMT of peritoneal mesothelial cells in peritoneal dialysis,and may be a new target for the prevention and treatment of peritoneal fibrosis during PD.
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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 explore the effect of suramin on the epithelial-mesenchymal transition (EMT) and the excretion of transforming growth factor-β1 (TGF-β1) in peritoneal mesothelial cells (PMCs) induced by high concentrations of glucose solution (GS).Methods Cultured PMCs were divided into three groups:(1) normal control group; (2) GS-treated group:cells were treated with 1.5％,2.5％,4.25％ GS for 12 h,24 h,48 h,respectively; (3) Suramin-treated group:PMCs cultured with 4.25％ GS were exposed to different doses of suramin (25,50,100 μmol/L) for 48 h.Expression levels of α-smooth muscle actin (α-SMA) and E-cadherin were detected by Western blotting and the concentration of TGF-β1 in the culture supernatant was determined by ELISA.Results Compared with normal control group,GS-treated PMCs exhibited a time-dependent increase in the expression of α-SMA,and decrease in the expression of E-cadherin.GS also stimulated PMCs to secrete TGF-β1.In the presence of suramin,GS-induced α-SMA expression and TGF-β1 production were reduced,E-cadherin expression was increased.Conclusions Suramin can inhibit high glucose-induced EMT of PMCs by down-regulating the expression of TGF-β1.Suramin may be a novel therapeutic agent for the treatment of peritoneal fibrosis.
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Objective To observe the effect of MG132 on the expression of extracellular regulated kinase 1/2 (ERK1/2) and connective tissue growth factor (CTGF) in rat peritoneal mesothelial cells (RPMCs) induced by high glucose.Methods RPMCs were isolated,cultured and passaged by trypsin,then identified.The second 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 24 hours,high glucose (2.5％) for 0,12,24,48 hours,incubated with MG132 (0.5,1,2 μmol/L) for half an hour and then with high glucose (2.5％) for 24 hours.ERK1/2 protein was detected by Western blotting,and CTGF protein in supernatant was detected by ELISA.Results Compared with the control group,the expression of p-ERK1/2 was significantly increased in the groups stimulated by high glucose (P ＜0.01),reached the peak at 24th hour (P ＜ 0.01),and then the expression decreased at 48th hour,but still was higher than that in the normal control group (P ＜ 0.01).CTGF protein expression of RPMCs induced by high glucose increased,in time-and dose-dependent manner (P ＜ 0.05).MG132 could significantly decrease the expression of ERK1/2 and CTGF induced by high glucose (P＜0.05).Conclusions MG132 can decrease the expression of p-ERK1/2 and CTGF in RPMCs induced by high glucose.The ubiquitin proteasome pathway participates in the development of peritoneal fibrosis,and blocking the way may contribute to the prevention 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: This study was performed to compare the expression of CD44 in endometrial stromal cells (ESCs) of women with and without endometriosis and to evaluate the role of CD44 in the adherence of ESCs to peritoneal mesothelial cells (PMCs). METHODS: A PMC adherence assay was performed to evaluate the adherence of ESCs to PMCs in women with and without endometriosis. The expression of CD44 mRNA was measured by reverse transcription-polymerase chain reaction. CD44 protein was evaluated by Western blot analysis. RESULTS: There were no significant differences in the expression of CD44 mRNA and protein in ESCs or in the binding of ESCs to PMCs between patients with endometriosis and controls. Although the expression of CD44 protein was decreased in both women with endometriosis and controls after anti-CD44 antibody treatment, there was no effect on binding of ESCs to PMCs. Treatment of ESCs with peritoneal fluid from endometriosis patients resulted in a significant increase in binding of ESCs to PMCs compared to untreated ESCs in the endometriosis group. CONCLUSION: This study demonstrates that the expression of CD44 protein in ESCs from women with endometriosis might not be directly associated with adherence to PMCs.
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Female , Humans , Ascitic Fluid , Blotting, Western , Endometriosis , RNA, Messenger , Stromal CellsABSTRACT
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.