IL-6 Promotes Epithelial-to-mesenchymal Transition Of Human Peritoneal Mesothelial Cells Possibly Through JAK2/STAT3 Signaling Pathway

Background and PurposePD has several advantages compared with hemodialysis,including a simpler and less invasive procedure and the retention of residual renal function.However,long-term PD therapy is known to result in functional and structural alterations in the peritoneal membrane.Peritoneal injuries resulting in failure can be caused by several physical stresses inherent in PD therapy,including exposure to PD fluid,catheter trauma and peritonitis.These factors increase the rate of peritoneal fibrosis and ultrafiltration failure occurrence.It was found that an increase in plasma IL-6 concentration during the acute phase reaction,which is also associated with the mortality in patients with PD.During PD,the local peritoneal cavity can produce IL-6 because the IL-6concentration in PD is more than its concentration in plasma.Some studies show that PD treatment in the first year has an increase in abdominal and systemic inflammatory response,and the IL-6 concentration in peritoneal fluid of PD patients is correlated positively with the transport status from the peritoneal equilibration test.The concentration of IL-6 in the solution is a sign of local inflammation of the abdominal cavity,and it increased significantly during peritonitis.The the Janus Kinase/Signal Transducer and Activator of Transcription(JAK/STAT)pathway is a pleiotropic cascade essential to signal transduction ofcytokines from the plasma membrane to the nucleus,which has been reported to mediate various cellular functions,including gene activation,cell differentiation,cell survival,and proliferation.In the study of cancer disease,JAK/STAT signaling pathway has become a target for cancer therapy.There is activation of STAT3 in a variety of human tumors,such as multiple myeloma,prostate cancer,colorectal cancer,breast cancer.JAK2/STAT3 signaling pathway is involved in the regulation of epithelial cell adhesion,and it may be related with EMT process of malignant lesion.IL-6 binding to the cell membrane binding receptor activates JAK/STAT signaling pathway to induce the expression of IL-6 gene.In the present study,It was also investigated that the effect of IL-6 on EMT of cultured human peritoneal mesothelial cells(HPMCs),involving E-cadherin,?-SMA and VEGF.Thereafter,we studied IL-6-induced alteration in the expression of signaling pathway proteins,JAK2 and STAT3.We also aimed to investigate the effect of WP1066 on the mechanism of HPMCs EMT and the expression of JAK2/STAT3 pathway proteins which were induced by IL-6.Methods1 The cells were divided into four groups:(I)control;(II)30 ng/mL IL-6 group;(III)50 ng/mL IL-6 group;(IV)100 ng/mL IL-6 group.After 5 days,Cell morphology was observed by inverted phase contrast microscope and photographed.2 Stimulated by 50 ng/mL IL-6 for 24 h,48 h and 72 h,cell proliferation was detected by MTT.The absorbance value of the wells was read at 490 nm using a microplate reader.3 At 24 h following exposure to IL-6,the mesenchymal markers(E-cadherin,?-SMA and)were measured.The concentration of IL-6 were 0(control),50 and 100ng/mL.The protein and mRNA expression measured by Western blot and Real-time fluorescence quantitative PCR.4 We set up 50 ng/mL as the stimulus concentration and observed EMT relative genes expression in different working times(0(control),24,48 and 72 h).The protein and m RNA expression measured by Western blot and Real-time fluorescencequantitative PCR.5 It was divided different groups as 3,we investigated the activation of the JAK2/STAT3 signaling pathway in 24 h.6 It was divided different groups as 4,it was observed that the P-JAK2(Tyr1007+1008)/JAK2 and P-STAT3(Ser727)/STAT3 relative protein expression in 0(control),24,48 and 72 h at exposure of 50 ng/m L IL-6.7 To investigate the effective concentration of WP1066 in HPMCs,we set up 0,2,4,6,8 and 10 ?M WP1066 to observe P-STAT3/STAT3 expression.8 Blockade of JAK2/STAT3 signaling pathway by WP1066,The proteins expression of JAK2/STAT3 pathway were observed by Western blot.9 Blockade of JAK2/STAT3 signaling pathway by WP1066,the EMT relative gens expression were investigated by Real-time fluorescence quantitative PCR and Western blot,including of E-cadherin,?-SMA and VEGF.10 Blockade of JAK2/STAT3 signaling pathway by WP1066,cell morphology was observed by inverted phase contrast microscope and cell proliferation was detected by MTT.Results1 After continuous stimulation with IL-6(50 ng/mL and 100 ng/mL)for 5 days,HPMCs converted into a fibroblast-like morphology.The intercellular space widened and the connection was loose.2 Stimulated by 50 ng/mL IL-6 for 24 h,48 h and 72 h,compared with the control group,an increase in HPMC viability was observed in the experimental groups,and this increase showed statistical significance.3 At 24 h following exposure to IL-6,the mesenchymal markers were measured.The concentration of IL-6 were 0(control),50 and 100 ng/mL.it was observed that the protein and mRNA expression of ?-SMA and VEGF dose-dependently increased(P<0.05),and the expression of E-cadherin dose-dependently decreased(P<0.05).4 Exposure of HPMCs to IL-6(50 ng/mL)for 0(control),24,48 and 72 h,the expression of ?-SMA and VEGF increased(P<0.05),the expression of E-cadherindecreased(P<0.05).5 At 24 h following exposure to IL-6,the concentration of IL-6 were 0(control),50 and 100 ng/mL.Compared with control group,IL-6 induced the increasing expression of phosphorylation of JAK2 and STAT3 in HPMCs(P<0.05).6 It was also observed the P-JAK2(Tyr1007+1008)/JAK2 and P-STAT3(Ser727)/STAT3 relative protein expression in 0(control),24,48 and 72 h at exposure of 50 ng/mL IL-6.The relative expression of P-JAK2(Tyr1007+1008)/JAK2 and P-STAT3(Ser727)/STAT3 significantly increased(P<0.05).7 The P-STAT3/STAT3 expression decreased significantly in 10 ?M WP1066.Less than 10 ?M,WP1066 couldn't prevent P-STAT3 in HPMCs.8 The proteins expression of JAK2/STAT3 pathway were observed.Treated with10 ?M WP1066 for 24 h,the p-JAK2 and p-STAT3 was significantly suppressed(P<0.05),the total JAK2 and total STAT3 were no change.9 Blocking the activation of JAK2/STAT3 pathway in HPMCs cells,the expression of EMT related protein and mRNA were not changed.10 WP1066 could prevent the morphologic and viability changes which was induced by IL-6 in HPMCs.Conclusions1 IL-6 could induce EMT process in HPMCs.The expression of EMT markers was dose-dependent and time-dependent.2 The activation of JAK2/STAT3 pathway was dose-dependent and time-dependent in IL-6.3 IL-6 induces EMT of HPMCs via JAK2/STAT3 signaling pathway.