| Background and PurposePeritoneal dialysis(PD)is one of the methods of renal replacement therapy for patients with end-stage renal disease.Compared with HD,PD is beneficial to the protection of residual renal function,and has the advantages of little influence on hemodynamics,relatively low treatment cost and home operation.An increasing number of evidence suggests that long-term exposure to high-concentration glucosecontaining peritoneal dialysis fluid(PDF)can lead to the occurrence of chronic complications in patients with PD,such as increasing the incidence of cardiovascular disease(CVD)and the loss of residual renal function(RRF).Long-term PD may cause a significant inflammatory response to the abdominal cavity,including the infiltration of macrophages and leukocytes into the peritoneum,and the release of inflammatory cytokines is another major driver of the peritoneal structure and function deterioration,in addition to the influence of high glucose and peritoneal dialysis.Peritonitis is characterized by the infiltration of the peritoneal neutrophils,which ultimately leads to the occurrence of peritoneal-related peritonitis,which has forced the patient to withdraw from the peritoneal dialysis treatment.Studies have shown that IL-8 is a potent chemotactic cytokine secreted by neutrophils.Previous studies have shown that Inflammatory factors such as IL-6 and IL-8 were significantly increased in dialysate of patients with peritonitis,and the concentration of IL-6 in PD patients was significantly higher than that in patients with peritonitis during catheterization and peritonitis.However,the concentration of IL-8 increases only in the event of peritoneal infection.These results suggest that the determination of IL-8 concentration in dialysate can be used as a specific index for tracing peritonitis in PD patients.NF-?B is a common transcription factor that mediates the expression of multiple genes in different biological processes,including immune response,inflammation,cell growth and survival.NF-?B is also a key mediator of a variety of major inflammatory cytokines(such as TNF-? and IL-1)that stimulate signal transduction and thus participate in the inflammatory phase of inflammation.EMT is triggered by profibrotic and inflammatory cytokines,and EMT in HPMCs is thought to be an early stage of peritoneal fibrosis.Prevention of EMT in PD patients can reduce fibrosis around the peritoneum,thereby protecting the peritoneal cortex.The purpose of this study is to discuss that in high glucose environment,inflammatory cells PBMCs can promote EMT in human peritoneal mesothelial cells(HPMCs)by secreting IL-8.The PBMCs were cultured in a medium containing glucose,and the supernatant after the culture was collected,diluted 10 times,and the HPMCs were further cultured.Western blot and real-time PCR were used to detect EMT-related markers such as E-cadherin,?-SMA and Vimentin..HPMCs were stimulated with different concentrations and different times of IL-8 to observe changes in EMT-related markers;and the effect of IL-8 on NF-?B signaling pathway.The NF-?B specific inhibitor PDTC was added to the medium containing IL-8,and the EMT process of HPMCs was inhibited after the NF-?B signaling pathway was blocked.From this we can conclude that in high glucose environment,PBMCs secrete IL-8 through the NF-?B signaling pathway to promote EMT in HPMCs,which ultimately leads to peritoneal dialysis-related peritonitis.Methods 1.The relatively normal peritoneal tissues(such as cholelithiasis appendicitis and other diseases)and PD peritoneal ultrafiltration failure during extubation were collected.The peritoneal tissues were stained with macrophage marker CD68 by immunohistochemical method.Positive microscope was used to observe the infiltration of inflammatory cells in tissues and take photographs.2.RPMI-1640 medium with 5.5m M glucose concentration was used as control group,PBMCs was cultured with RPMI-1640 with high glucose concentration for 24 h,the supernatant was diluted at 1:10,and HPMCs was further cultured for 24 h with this supernatant.The method of Western blot,immunofluorescence and q RT-PCR were used to detect the protein and m RNA of E-cadherin,?-SMA and Vimentin.3.After PBMCs was cultured with 60 m M high glucose medium for 24 h,the supernatant was diluted at 1:10,which was used to culture HPMCs for 12 h,24 h,and 48 h respectively.The method of Western blot and q RT-PCR were used to detect the protein and m RNA of E-cadherin,?-SMA and Vimentin.4.PBMCs was cultured with RPMI-1640 medium with 5.5m M gluose concentration and RPMI-1640 with high glucose concentration in 60 m M for 24 h.The method of q RT-PCR was used to detect The expression of IL-8 m RNA.5.After HPMCs was stimulated with control medium,10ng/ml and 30ng/ml concentration of IL-8 for 24 hours,The method of Western blot was used to detect the protein of E-cadherin,?-SMA and Vimentin.6.After HPMCs was stimulated with 30ng/ml IL-8 for 12 h,24 h,48 h respectively,the protein expression levels of EMT markers(E-cadherin,vimentin and ?-SMA)were detected by Western blot.7.After HPMCs was stimulated with normal medium,10ng/ml and 30ng/ml concentration of IL-8 for 24 hours,the method of Western blot was used to detect phosphorylation level of NF-?B P65 protein.8.After HPMCs was stimulated with 30ng/ml IL-8 for 12 h,24 h,48 h respectively,the phosphorylation level of NF-?B p65 protein was detected by Western blot.9.Normal control group,30ng/ml IL-8 stimulation group and 30ng/ml IL-8 PDTC inhibitor group were set up respectively.After blocking the NF-?B signaling pathway,the method of Western blot was used to detect the protein expression levels of EMT markers.Results 1.The peritoneal tissues of patients with peritoneal dialysis failure were stained with CD68 and a large number of CD68 positive cells were found.2.Compared with control and 5.5m M glucose concentration,the expression level of E-cadherin protein and m RNA in 60 m M glucose was lower than that of control and 5.5m M glucose concentration.The expression levels of ?-SMA and Vimentin protein and m RNA were significantly increased(* # P < 0.05).3.With the prolongation of stimulation time,the protein and m RNA expression level of E-cadherin decreased gradually.However,the protein expression level and m RNA of ?-SMA and Vimentin increased gradually,the difference was statistically significant(*P < 0.05).4.PBMCs were stimulated by 60 m M for 24 h and the expression of m RNA of IL-8 was significantly increased compared with control(* P < 0.05).5.With the increase of stimulation concentration of IL-8,the protein expression level of E-cadherin decreased gradually.However,the protein expression level of ?-SMA and Vimentin were gradually increased,the difference was statistically significant(* P < 0.05).6.Exposured to 30ng/ml IL-8,with the prolongation of stimulation,the protein expression level of E-cadherin decreased gradually.However,the protein expression level of ?-SMA and Vimentin gradually increased,the difference was statistically significant(*P< 0.05).7.The phosphorylation level of NF-?B p65 increased gradually with the increase of IL-8 stimulation concentration(* P < 0 05).8.Exposured to 30ng/ml IL-8,with the prolongation of stimulation time,the phosphorylation level of NF-?B p65 increased gradually(* P < 0 05).9.After blocking the NF-?B signaling pathway,western blot showed that the expression of E-cadherin in the inhibitor PDTC was significantly higher than that in the IL-8 stimulation,while the protein expression levels of ?-SMA and Vimentin were significantly lower than those in the IL-8-stimulated(* P < 0.05).Conclusions 1.High glucose-stimulated PBMCs can indirectly induce EMT in HPMCs.2.PBMCs secrete IL-8 and promote the EMT process of HPMCs.With the prolongation of IL-8 concentration and time,the expression of EMT-related markers increased gradually.3.IL-8 induces EMT in HPMCs via the NF-?B signaling pathway. |
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