Astragalus extract inhibits destruction of gastric cancer cells to mesothelial cells by anti-apoptosis

AIM： To determine the inhibitory effect of Astragalus memebranaceushas on gastric cancer cell supernatant- induced apoptosis of human peritoneal mesothelial cells. METHODS： Human peritoneal mesothelial cell （HPMC） line HMrSV5 was co-incubated with gastric cancer cell supernatant （MKN45） and/or Astragalus memebranaceushas. Morphological changes in gastric cancer cells were observed under phase-contrast microscope. Quantitative cell damage was determined by MTT assay. Apoptosis was determined under transmission electron microscope and quantified by detectinr, acridine orange/ethidium bromide-stained （AO/EB） condensed nuclei under fluorescent microscope or by flow cytometry. Expressions of Bcl-2 and Bax were evaluated with immunostaining.

Destruction of Gastric Cancer Cells to Mesothelial Cells by Apoptosis in the Early Peritoneal Metastasis

This study examined the mechanism by which the gastric cancer cells lead to early peritoneal metastasis. HMrSV5 cells, a human peritoneal mesothelial cell line, were co-incubated with the supernatants of gastric cancer cells. Morphological changes of HMrSV5 cells were observed. The cell damage was quantitatively determined by MTT assay. The apoptosis of HMrSV5 cells was observed under transmission electron microscope. Acridine orange/ethidium bromide-stained condensed nuclei was detected by fluorescent microscopy and flow cytometry. The expressions of Bcl-2 and Bax was immunochemically evaluated. The results showed that conspicuous morphological changes of apoptosis were observed in HMrSV5 cells 24 h after treatment with the supernatants of gastric cancer cells. The supematants could induce apoptosis of HMrSV5 cells in a time-dependent manner. The supernatants could up-regulate the expression of Bax and suppress that of Bcl-2 in HMrSV5 cells. These findings demonstrated that gastric cancer cells can induce the apoptosis of HPMCs through supernatants in the early peritoneal metastasis, The abnormal expressions of Bcl-2 and Bax may contribute to the apoptosis. Anti-apoptosis drugs promise to be adjuvant chemotherapeutic agents in the treatment of peritoneal metastasis of gastric cancer.

Gene expression of glucose transporters and its regulation by glucose in mesothelial cells

Objective To observe the influence of glucose on the expression of glucose transporters (GluTs) in peritoneal tissues.Methods Mesothelial cells (MsCs) from Sprague-Dawley (SD) rats were cultured in medium with glucose 214.4 mmol/L or 75.5 mmol/L. The normal medium with glucose 17.5 mmol/L was used as control. Total RNA was extracted from each sample after 24 hours incubation. Reverse transcript polymerase chain reaction (RT-PCR) was performed with primers corresponding to sodium-glucose transporter (SGIT1) and GluT1 -GluT4. mRNA expression of the above GluTs from each sample was measured with quantitative PCR.Results GluT1 and GluT2 mRNA can be detected in MsCs from SD rats, while no positive bands can be found specificaly for GluT3, GluT4 and SGIT1. Quantitating the amount of PCR products indicated that the levels of GluT1 mRNA in MsCs cultured 24 h in both 214.4 mmol/L glucose and 75.5 mmol/L glucose medium decreased dramatically compared with that in normal medium ( P≤0.01 ). While under the same conditions, the levels of GluT2 mRNA in MsCs cultured 24 h in 214.4 mmol/L and 75.5 mmol/L glucose medium both increased significantly ( P ＜ 0.01 ).Conclusions GluT1 is strongly expressed in MsCs under normal glucose levels and decreased dramatically under high glucose conditions, while GluT2 expressed at a low level in normal medium and increased greatly after incubation in high glucose conditions. This may play a great role in glucose absorportion during peritoneal dialysis and have some connection with ultrafiltration failure due to the alteration of glucose absorption after long-term dialysis.

Expression of aquaporin-1 in rat pleural mesothelial cells and its specific inhibition by RNA interference in vitro

Background The discovery of water channel aquaporins （AQPs） has greatly expanded the understanding of the regulation of the water permeability of biological membranes. Aquaporin-1 （AQP1） may be involved in fluid transport in numerous pathological conditions. The objective of the present study was to examine whether AQP1 is present in cultured rat pleural mesothelial cells （PMCs） and to investigate the specific inhibitory effect of RNA interference （RNAi） on AQP1 expression in PMCs, which may provide a new method for the further studies on the relation between expression of AQP1 in PMCs and pleural fluid removal in vivo. Methods PMCs were isolated and cultured from rat pleura. The expression of AQP1 in PMCs was confirmed by immunocytochemical staining and reverse transcriptase-polymerase chain reaction （RT-PCR）. Two eukaryotic expression plasmid vectors of short hairpin RNA （shRNA） specific for the AQP1 gene of rat sapien were designed and constructed. The recombinant plasmid vectors were transfected into cultured rat PMCs by cation liposomes. Flow cytometry was used to screen the most effective shRNA at 48 hours after transfection. The expressions of AQP1 mRNA and protein were detected by RT-PCR and Western blotting method at 48 hours after transfection. Results RT-PCR and immunostaining revealed that AQP1 mRNA and protein were present in cultured rat PMCs. Two effective eukaryotic expression plasmid vectors of shRNA specific for the AQP1 gene were constructed successfully. The levels of the expression of AQP1 were inhibited by 83.45%, 90.93%, respectively, at mRNA level and 41.24%, 67.60%, respectively at protein level by two recombinant plasmids at 48 hours after transfection. The expression of AQP1 in PMCs transfected with plasmid was significantly lower than that of the cells transfected with the control plasmid HK and that of the untransfected cells （P〈0.01）. There was no significant difference in AQP1 expression between the control group and the group transfected with AQP1 nonspecific shRNAs （P〈0.05）. Conclusions The expression of AQP1 was present in rat PMCs. The application of shRNA-AQP1 could markedly inhibit the expression of AQP1 in cultured rat PMCs. The use of RNAi is a promising tool for future research into the mechanisms of pleural fluid in vivo.

Inhibiting effect of short hairpin RNA on expression of transforming growth factor-β1 in human peritoneal mesothelial cells induced by peritoneal dialysis solution

The peritoneum response to peritoneal dialysis can lead to fibrosis. The transforming growth factor-β1 （TGF-β1） plays an important role in regulating tissue repair and remodeling after injury. Excessive synthesis and deposition of matrix proteins by peritoneal mesothelial cells can lead to structural and functional changes in the peritoneal membrane, jeopardizing the long-term efficacy of peritoneal dialysis （PD）. Prolonged exposure to high glucose concentrations in PD fluid has been implicated as a major stimulus to matrix accumulation, through the induction of transforming growth factor-β1 （TGF-β1）.

Effect of Ligustrazine Nanoparticles Nano Spray on Transforming Growth Factor-β/Smad Signal Pathway of Rat Peritoneal Mesothelial Cells Induced by Tumor Necrosis Factor-α

Objective： 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. Methods： The primary culture and subculture of rat peritoneal mesothelial cells（RPMC） was processed by trypsin digestion method in vitro. The third generation was identified 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 fibronectin, collagen Ⅰ（COL-Ⅰ）, TGF-β m RNA, and Western blot method to test the Smad protein 7 expression of RPMC. Results： Compared with the blank group, a significant elevation in fibronectin（FN）, COL-Ⅰ and TGF-β m RNA 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 m RNA 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）. Conclusions： TNF-α may induce changes in RPMC＇s viability, leading to peritoneal injury. LNNS could reverse the induction of fibrosis 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.

Evaluation of the effectiveness of alginate-based hydrogels in preventing peritoneal adhesions

Infertility and intestinal blockage are just two examples of the postoperative consequences that can arise from peritoneal damage,which can also result in severe peritoneal fibrosis and peritoneal adhesions.Peritoneal adhesions are still not effectively treated,and both pharmaceutical therapy and biomaterial barriers have only had modest preventative effects.In this work,we looked into the effectiveness of in-place injectable sodium alginate hydrogel for peritoneal adhesion prevention.The findings demonstrated that sodium alginate hydrogel promoted human peritoneal mesothelial cell proliferation and migration,prevented peritoneal fibrosis by suppressing the production of transforming growth factor-β1,and,most importantly,promoted mesothelium self-repair.These findings imply that this brand-new sodium alginate hydrogel is a good candidate material for peritoneal adhesion prevention.

Morphological changes of the peritoneum in peritoneal dialysis patients

Background Long-term peritoneal dialysis (PD) requires that the peritoneal membrane remain effective for dialysis. Research directed toward human peritoneal morphology and structure is limited. The present study was performed to investigate morphological changes of the human peritoneal membrane during PD and to elucidate the possible mechanisms of its functional deterioration. Methods A total of 32 peritoneal biopsies were performed in normal subjects (n=10),uremic nondialysis patients (n=12) at the time of catheter insertion,and PD patients (n=10) at the time of catheter removal or reinsertion or at the time of renal transplantation. Peritoneal morphology was examined by light microscopy,scanning electron microscopy,and transmission electron microscopy. Results The peritoneal membrane in normal subjects consisted of a monolayer of mesothelial cells on a basement membrane and a layer of connective tissue containing cells,blood vessels,and lymphatic vessels. Mesothelial cells were polygonal,often elongated,and had numerous microvilli on their luminal surface. There were lots of oval or roundish pinocytotic vesicles in the cytoplasm of the mesothelial cells. The peritoneal morphology of uremic nondialysis patients was similar to that of normal subjects. However,significant abnormalities of the peritoneal membrane were observed in PD patients,and the changes were found to be progressive. Microvilli were the first site of damage which involved microvilli shortening,a gradual reduction in their number,and,eventually,the total disappearance of microvilli. Mesothelial cells then detached from the basement membrane, disappearing completely in some cases. In the end,the peritoneal membrane consisted only of submesothelial connective tissue without any cells.Conclusions PD can modify peritoneal morphology and structure. The morphological change is progressive and may be one of the important causes of peritoneal failure. Peritoneal biopsies can provide lots of valuable information about the effects of PD. Studying the relationship between peritoneal structure and its function proved very useful for understanding the physiopathology of the peritoneum during PD.