| Background:In recent years,the prevalence of chronic kidney disease(CKD)has been increasing year by year.According to incomplete statistics,the prevalence of CKD in the global adult population has reached 14.3%,while the prevalence of CKD in China is about 11%,in which the population with end stage renal disease(ESRD)reaches up to 3 million and the population undergoing dialysis is about 600 thousand.Peritoneal dialysis(PD),an effective alternative treatment for patients with end-stage of renal disease(ESRD),is currently accepted by about 11%of total dialysis patients.Compared with hemodialysis(HD),PD has the following advantages in the appropriate treatment population:stable hemodynamics,good protection of residual renal function,convenient home-based operation and low medical care costs.PD mainly relies on peritoneum as a biological semi-permeable membrane for ultrafiltration and solute diffusion.However,long-term peritoneal dialysis leads to peritoneal dysfunction and failure of ultrafiltration(UF).Although,epithelial-mesenchymal transition(EMT)of peritoneal mesothelium cell is the key step of peritoneal fibrosis,at the same time,the biocompatibility of dialysis fluid(hyperglycemia,high glucose degradation product(GDPs)and advanced glycation end-products(AGEs),hyperosmotic,low PH)was also involved in the changes of peritoneal mesothelium at function and biological characteristics,most detailed mechanisms are still unknown.Therefore,it is of great significance to further explore the mechanism of the occurrence and development of peritoneal fibrosis and seek for new therapeutic targets.Generalized peritoneal dialysis related peritoneal fibrosis has two core components:fibrosis itself and inflammatory response.These two factors influence each other and induce mutually.Firstly,the main pathological features of peritoneal fibrosis are the loss of peritoneal mesothelial cells,the activation of myofibroblasts,the massive accumulation of extracellular matrix(ECM)and angiogenesis.In traditional peritoneal dialysis,the most important components that affect the function and biological characteristics of peritoneal mesodermal cells are hyperglycemia and AGEs.In recent years,although many related preclinical studies have made significant breakthroughs,there is still a lack of therapeutic targets to relieve and intervene in peritoneal dialysis related peritoneal fibrosis.Secondly,peritoneal inflammatory reaction is still another common complication in patients with PD,and common causes of it include microbial infection,uremia toxins and non-biocompatible dialysis fluids.In the same way,repeated inflammatory reactions can lead to peritoneal skin cell damage,numerous inflammatory cells infiltration,a large number of inflammatory mediators released,and at last peritoneal fibrosis was developed.Rho is a member of the subfamily of small G protein superfamily and has the activity of GTPase,so it is also called Rho GTPase.Rho is essential in many cytological functions.They cycle between an active GTP-binding form and an inactivated GDP binding form through their internal hydrolytic enzymes,thus acting as molecular switches to regulate many signal transduction pathways downstream.At present,more than 20 Rho family members have been found,and the most in-depth and extensive studies are Racl,Cdc42 and RhoA.According to current studies,RhoA can participate in intercellular adhesion,cytoskeletal rearrangement,cell invasion,migration,and induction of apoptosis and gene expression by activating transcription factor AP-1 which contains serum response element(SRE).In many disease models,the RhoA/Rho-kinase signaling pathway is involved in the occurrence and development of multiple diseases such as multiple-organ fibrosis,inflammation,oncology and neuropathy,and also fasudil,the inhibitor of this signaling pathway showed obvious therapeutic effects in many disease models.But the mechanism involved in peritoneal fibrosis and inflammatory reaction related to peritoneal dialysis has not been clarified.Therefore,the purpose of this study was to explore the mechanism of RhoA/Rho-kinase signaling pathway in peritoneal dialysis related peritoneal EMT and inflammatory response,providing a new target for clinical treatment.Objective:1.Explore whether RhoA/Rho-kinase is activated in peritoneal dialysis related peritoneal fibrosis,and determine the role of RhoA/Rho-kinase in the occurrence and development of EMT and inflammation.2.To explore the specific role and molecular mechanism of RhoA/Rho-kinase in the development of EMT and inflammation.3.Select appropriate drugs to further block related signaling pathway,thereby inhibiting EMT and inflammatory reaction,and providing new targets for clinical treatment.Methods:Part 1:Preliminary study on the role and mechanism of RhoA/Rho-kinase signaling pathway in peritoneal fibrosis rat model1.Effect of fasudil on peritoneal thickness,fibrosis,inflammatory reaction and ultrafiltration function of ratsTwenty-four male SD rats at 6-8 weeks were selected and randomly divided into three groups:control group(control group),model group(PDF group)and intervention group(PDF+Fasudil group),with 8 rats in each group.The control group was given intraperitoneal injection of 25mL normal saline daily.In the PDF group and the PDF+Fasudil group,4.25%of the high-glucose peritoneal dialysis solution(1 00mL/kg)was administered intraperitoneally daily,and lipopolysaccharide(LPS)(0.6mg/kg)was administered simultaneously on the first 1/3/5/7 days of the study.One week after the start of the experiment,i.e.,after LPS peritoneal injection was completed,fasudil(30 mg/kg)was administered in the PDF+FAS group by gavage once a day.Peritoneal injection was stopped after 4 weeks.Peritoneal balance test was conducted on alternate days and peritoneal tissue was obtained to conduct HE staining,MASSON staining to evaluate and compare the changes in peritoneal thickness,fibrosis,inflammatory reaction and ultrafiltration ability in these three groups.2.Preliminary study about the effect and role of fasudil on rat peritoneal fibrosis and inflammatory reaction.Using the above rat peritoneal tissue specimens,WB and IHC were used for the detection of fibrosis-related indexes,including fibronectin(FN),transforming growth factor-?(TGF-?)and a-smooth muscle actin(?-SMA),and the changes were compared among these three groups.At the same time,changes about EMT related molecules and inflammatory molecules in peritoneal tissues were detected by WB,including changes in epithelial marker protein E-cadherin,mesenchymal marker protein N-cadherin and Vimentin,inflammatory molecules MCP-1 and NF-?B.ELISA was used to detected the content of IL-6 and TNF-? in peritoneal tissue homogenates.To further explore the relevant mechanisms,the changes in p-MYPT-Thr696,RhoA/Rho-kinase signaling pathway effector molecule,were detected by WB method in these three groups,and semi-quantitative analysis was performed.Summing up the above to investigate the important role of RhoA/Rho-kinase signaling pathway in PD related PF and inflammatory response.Part 2:Study on the role and mechanism of RhoA/Rho-kinase signaling pathway in AGEs-induced EMT in peritoneal mesothelial cells1.The expression of RhoA/Rho-kinase signaling pathway in AGEs-induced peritoneal mesothelial cellsHuman peritoneal mesothelium cell(HPMCs)line(HMrSV5)strain was continuously cultured in vitro and stimulated with AGEs.The concentration gradient and time gradient stimulated were set up respectively.RhoA activation was detected by the method of pull down assay.Simultaneously the expression of Rho kinase and its downstream molecular p-MYPT-Thr696 was detected by WB to explore the activation of this signaling pathway in our study.According to the experimental results,appropriate AGEs concentration and stimulation time were selected for the follow-up study.2.The mechanism of RhoA/Rho-kinase signaling pathway involved in AGEs-induced EMT in peritoneal mesothelial cellsAppropriate AGEs concentration and stimulation time were selected according to the above experiment to detect expression level of EMT-related indexes E-cadherin,N-cadherin,Vimentin and fibrosis-related molecules FN,TGF-?,and ?-SMA.Further,specific inhibitor Fasudil/HA-1077 and Y-27632 were added previously to detect whether the expression of E-cadherin,N-cadherin,Vimentin,FN,TGF-?,and?-SMA could restore.For specific role of RhoA,small interfering RNA(small interfering RNA,siRNA)was constructed.Firstly silence efficiency of RhoA in peritoneal mesothelium cell was detected by the WB.After transfected and silenced successfully,cells were stimulated with AGEs.The expression of downstream Rho-kinase and its substrate molecule p-MYPT-Thr696 was detected by WB,and also the related indicators E-cadherin,N-cadherin,Vimentin,FN,TGF-?,and ?-SM.In order to demonstrate the effect of AGEs on morphology and migration capacity of peritoneal mesothelial cells more intuitively,scratch test and transwell test were used to verify the above effect and mechanism.3.Mechanism of transcription factor AP-1 mediated by RhoA/ROCK signaling pathway in EMT of peritoneal mesothelial cells induced by AGEsIn order to further explore the mechanism of RhoA/Rho-kinase signaling pathway involved in EMT and fibrosis related indexes,transcription factor transcription activator protein-1(AP-1)activation was detected by EMSA.At first DNA probes specific combination for AP-1 was designed.Nucleoprotein was collected after cells were processed as mentioned to detect AP-1 activation.Further AP-1 specific inhibitor of curcumin was added before cells were stimulated by AGEs,Activation of AP-1 was detected by EMSA,and changes in expression of E-cadherin,N-cadherin,Vimentin,FN,TGF-?,and ?-SMA were detected by method of WB.According to these to speculate the role of AP-1 in AGEs induced EMT.Part 3:Study on the role and mechanism of RhoA/Rho-kinase signaling pathway in LPS-induced inflammatory response in peritoneal mesothelial cells1.Role and mechanism of RhoA in LPS-induced inflammatory reaction in peritoneal mesothelial cells.In vitro,continuously cultured cells HPMCs line HMrSV5 strain was treated with lipopolysaccharide(LPS).Concentration gradient and time gradient stimulated by LPS were set up respectively.RhoA activation was detected by RhoA pull down assay.Nucleus protein and plasma protein were extracted to detect the expression level of NF-?B respectively at the same time,so as to conform the activation of NF-?B in our study.According to the experimental results,appropriate LPS concentration and stimulation time were selected as the observation points of the follow-up study.After transfected with si-RhoA and verified its silencing efficiency,HPMCs were stimulated with LPS.Expression level of inflammatory factors interleukin-6(IL-6)and tumor necrosis factors-?(TNF-?)were determined by enzyme linked immunosorbent assay(ELISA)and expression changes of ?-SMA,monocyte chemo-attractant protein-1(MCP-1)were detected by WB to identify the role of RhoA in LPS induced inflammation.To further speculate the mechanism,Toll like receptor-4(TLR-4),p-I?B?,and NF-?B in cytoplasmic and nucleus were determined by WB.Above all we can speculate the role and mechanism of RhoA/Rho-kinase signaling pathway in LPS induced inflammation.2.Study on the role and mechanism of thymol on inflammatory reaction to LPS induced peritoneal mesenchymal cellsThymol is a phenolic derivative extracted from thyme,which has a strong anti-inflammatory effect.To further explore the effect of thymol on LPS-induced inflammatory reaction and whether it has cross-talk with RhoA/Rho-kinase signaling pathway,cells were stimulated by LPS after pretreated with thymol for 1 hour,.The expression changes of inflammatory factors IL-6 and TNF-a were measured by ELISA and the expression of a-SMA?MCP-1 were detected by WB.In order to further explore the related mechanism,WB was used to determine the expression changes of TLR-4?p-I?B??p-IKK and NF-?B in cytoplasm and nucleus.The activity of RhoA was detected by RhoA pull down assay,and the expression change of its downstream effector Rho-kinase was detected by WB so as to identify the role of thymol and related mechanism.Results:Part 1:Preliminary study the role and mechanism of RhoA/Rho-kinase signaling pathway in peritoneal fibrosis rat model1.Fasudil can reduce peritoneal thickness,inflammatory response and fibrosis,improve ultrafiltration function in peritoneal fibrosis rats 4.25%PDF was injected intraperitoneally for 4 weeks with SD male rats daily.After peritoneal dialysis related to peritoneal fibrosis model was constructed successfully,rats were stopped processing for 24h.Body weight and peritoneal ultrafiltration function were tested.The results showed that there was no statistical difference of weight among these three groups.Ultrafiltration function in PDF group was significantly reduced compared with the control group,and fasudil can obviously reverse ultrafiltration function compared with PDF group.HE staining showed that the peritoneal thickness and inflammatory cell infiltration of the PDF group were significantly increased compared with control group,while these changes could be significantly reduced after fasudil treatment.MASSON staining showed the same results,and further revealed that fasudil could reverse peritoneal fibrosis degree in PDF group.2.Fasudil alleviates peritoneal fibrosis including EMT and inflammatory response in rats through RhoA/Rho-kinase signaling pathwayThe results of peritoneal IHC showed that the fibrosis indexes,such as FN,TGF-?and ?-SMA were significantly up-regulated in PDF group compared with the control group,while fasudil treatment can significantly relieve these.The results of WB showed similar results as IHC.The EMT indexes in PDF group detected by WB showed that the expression level of E-cadherin was significantly down-regulated,N-cadherin and Vimentin were significantly up-regulated.All these indexes in fasudil group were significantly restored.Results of WB and ELISA showed that the expressions of inflammatory markers MCP-1,p-NF-?B,IL-6,TNF-? were significantly up-regulated in the dialysis group and decreased in fasudil group with statistically significant differences.Meanwhile,the expression of p-MYPT-Thr696 showed significantly upregulated in the PDF group,while fasudil can significantly relieve it.The above results show that fasudil can effectively alleviate peritoneal fibrosis including peritoneal EMT and inflammatory response induced by dialysis.Indirectly,we can conclude that RhoA/Rho-kinase signaling pathway plays an important role in peritoneal dialysis related peritoneal fibrosisPart 2:Study on the role and mechanism of RhoA/Rho-kinase signaling pathway involved in EMT of HPMCs induced by AGEs.1.RhoA/RhoA-kinase signaling pathway was significantly activated in HPMCs stimulated by AGEsContinuously cultured HMrSV5 was stimulated by AGEs to set concentration gradient and time gradient.RhoA pull down assay showed 50?g/mL AGEs can significantly activated RhoA in a time dependent manner within 6hours.WB detecting Rho-kinase,its downstream molecular p-MYPT-Thr696 and FN showed at 50?g/mL of AGEs,with extended time,the express level of these molecules increased gradually.According to the results,50?g/mL was chosen as the final concentration and 48h was chosen as the treatment time period with no cytotoxicity.2.RhoA/Rho-kinase signaling pathway mediates the appearance of EMT induced by AGEs in HPMCsIt was found that after cells were processed as detailed above,the expression levels of EMT related indicators,including N-cadherin,Vimentin and fibrosis-related molecules FN,TGF-?,and ?-SMA were significantly up-regulated,while E-cadherin was significantly decreased.When the specific inhibitors Fasudil/HA-1077 and Y-27632 were added,the expression of Rho-kinase signal pathway and EMT-related indicators(E-cadherin,N-cadherin,Vimentin,FN,TGF-?,and ?-SMA)was significantly recovered.To further clarify the specific role of RhoA,si-RhoA was transfected.When cells were stimulated with AGEs additional after RhoA was silenced,the activity of Rho-kinase and its downstream p-MYPT-Thr696 was obviously inhibited.And also,the indexes detailed previous were also restored.Intuitively,normal cell morphology is regular,with a cobblestone appearance and tight intercellular junction.AGEs treatment leads to long and narrow cells morphology with irregular intercellular junction while Fasudil and Y-27632 can significantly restore cell morphology.Wound healing assay and transwell assay further support the above results.3.RhoA/Rho-kinase signaling pathway mediates the induction of EMT via transcription factor AP-1By designing DNA probe specific for transcription factor AP-1,we detect the activity of it with method of EMSA.The results showed that the activity of AP-1 was significantly enhanced after AGEs stimulation,while fasudil and Y-27632 could inhibit its activity.Further the results of EMSA showed that the activity of AP-1 can also be inhibited by AP-1 specific inhibitor curcumin.WB was used to detect the activation of RhoA/Rho-kinase signal pathway and the expressions of fibrosis related indicators E-cadherin,N-cadherin,Vimentin,FN,TGF-?,and ?-SMA.The results showed that curcumin could also restore the downstream EMT indexes and alleviate the fibrosis state,but had no effect on the upstream Rho-kinase pathway.Part 3:Study on the role and mechanism of RhoA/Rho-kinase signaling pathway involved in inflammation of HPMCs induced by LPS1.The expression of RhoA mediated NF-?B was upregulated in LPS-induced inflammatory reaction of HPMCs.Continuously cultured HPMCs line HMrSV5 strain was stimulated with LPS to establish concentration gradient and time gradient.RhoA pull down assay showed 3?g/mL LPS incubation for 0.5h can activated RhoA maximum.At the same time,the expression of NF-?B in cytoplasm decreased and nucleus increased significantly.That is to say,?pg/mL LPS incubation for 0.5h can exhibit the greatest degree to activate the NF?kB to translocate from cytoplasm to nucleus by phosphorylation.With si-RhoA transfection,cells were stimulated with LPS again.The results of ELISA and WB showed that LPS can promote the expression of IL-6,TNF-a and a-SMA,MCP-1.When RhoA was silenced,the infammatory indexes were restored.Simultaneously,in order to further explore the mechanism,we found that when RhoA was silenced,the expression of p-I?B? and the translocation of NF-?B induced by LPS could be recovered,but had no effect on the expression of TLR-4.Therefore,we concluded LPS activated TLR-4 to participate in the inflammatory response of peritoneal mesothelial cells through the RhoA-mediated NF-?B signaling pathway.2.Role and mechanism of thymol on inflammatory reaction of PMCs induced by LPSAfter cells were pretreated with thymol for 1 hour in different concentrations,cells were stimulated with LPS.The results of ELISA and WB showed that thymol significantly recovered the inflammation indexes IL-6,TNF-?,?-SMA and MCP-1 induced by LPS in a concentration dependent manner.To further explore the relevant mechanism and verify if thymol acts overlap with RhoA,the expression of TLR-4,Rho-kinase,p-I?B?,p-IKK and NF-?B were detected by WB.The results showed that thymol could restore the changes of these molecules caused by LPS.RhoA pull down assay revealed that thymol could inhibit the activity of RhoA also in a concentration dependent manner.Immunofluorescence(IF)showed more intuitively that thymol and si-RhoA could restore NF-?B nuclear translocation caused by LPS.In conclusion,thymol can inhibit LPS-induced inflammatory response in HPMCs by blocking the TLR-4-mediated RhoA/NF-KB signaling pathway.Conclusions1.RhoA/Rho-kinase signaling pathway is involved in the occurrence and development of peritoneal dialysis related peritoneal fibrosis,and its inhibitor fasudil can effectively reduce peritoneal EMT and inflammatory responses.2.By activating the RhoA/Rho-kinase signaling pathway mediated transcription factor AP-1 high expression,AGEs can induce the occurrence of EMT in peritoneal mesothelial cell.Fasudil can effectively alleviate the occurrence of EMT in peritoneal mesothelial cell via this pathway.3.LPS can induce inflammatory response of peritoneal mesothelial cells by activating RhoA-mediated NF-?B signaling pathway,and thymol can inhibit the inflammatory response by down-regulating the TLR4-mediated RhoA/NF-?B signaling pathway.Innovations:In this study,for the first time,we found that the inhibitor of RhoA/Rho-kinase signaling pathway can alleviate EMT and inflammatory reaction induced by PD,and played an important role in the development of peritoneal dialysis related peritoneal fibrosis.Fasudil can restore ultrafiltration capacity of rats with PF.Meanwhile,in vitro studies we revealed the important role of RhoA in EMT and inflammatory responses of peritoneal mesothelial cells,and further elaborated its internal mechanism.Fasudil and thymol were found to have significant therapeutic effects on the treatment of peritoneal dialysis related complications,providing a new target for the clinical treatment of peritoneal dialysis related ultrafiltration failure. |
PDF Full Text Request