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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 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.