Investigation Of The Inhibitory Effect And Mechanism Of Dimethylaminomicheliolide Treatment With Peritoneal Fibrosis

Background:Peritoneal fibrosis(PF)is one of the leading complications of long-term peritoneal dialysis(PD),The pathogenesis of PF is complex and there are no effective treatinent strategies.Dimethylaminomicheliolide(DMAMCL)is a new lead compound with the advantages of high stability,wide therapeutic window and obvious anti-inflammatoryy and anti-fibrotic effects.However,the pharmacodynamic effect of DMAMCL and its related mechanism remain unknown in long-term PD related PF.(?)The effect of DMAMCL on PFObjective:To investigate the effect of DMAMCL on PF in a mouse model of PD and in cultured cells.Methods:In vivo:?We extablished a mouse PF model and randomly divided into several groups:normal mice,PD day 14 mice,PD day 28 mice(i.e.,PF model mice),early DMAMCL-treated mice,dalayed DMAMCL-treated mice.Mice were sacrificed at day 28.The parietal peritoneum and omentum were collected.?Masson's trichrome staining was performed to detect the thickness of the submesothelial tissue;Western Blot,QPCR and immunohistochemical technology(IHC)were used to detect the expression levels of Fibronectin,Collagen I,E-cadherin and a-SMA.(2)In vitro:Human peritoneal mesothelial cell line HMrSV5 cells were exposed to TGF-?1 for 48 h in the presence or absence of different concentrations of MCL.Western Blot technology was used to detect the expression levels of Fibronectin,Collagen I,E-cadherin and a-SMA.Results:(1)In a mouse model of PD,masson's trichrome staining revealed that compared with mice exposed to PDF plus vehicle treatment,both early DMAMCL-treated mice and dalayed DMAMCL-treated mice showed decreased thickening of the peritoneal membrance within the anterior abdominal wall.Western Blot,QPCR and IHC technology showed that DMAMCL alleviated extracellular matrix(ECM)accumulation at both the mRNA and protein levels,as evidenced by the downregulation of Fibronectin,Collagen I,and ?-SMA expression and the restoration of E-cadherin.(2)In vitro we found that cotreatment with TGF-?1 and MCL at three different concentrations reduced ECM deposition and reversed epithelial-mesenchymal transition(EMT),as evidenced by reduced levels of Collagen I,Fibronectin and a-SMA and upregulated E-cadherin expression.Conclusion:DMAMCL has a protective effect against PD-related PF in both experimental PF animal models and cultured cells.(2)The related mechanism of DMAMCL's inhibitory effect against PFObjective:To investigate the related mechanism of DMAMCL's inhibitory effect against PF.Methods:(l)In vivo:?We extablished a mouse PF model and randomly divided into several groups:normal mice,PF model mice,dimethyl sulfoxide(DMSO)-treated mice,rapamycin(RAPA)-treated mice,3-Methyladenine(3MA)-treated mice,DMAMCL-treated mice,MDAMCL plus 3MA-treated mice.Mice were sacrificed at day 28.The parietal peritoneum and omentum were collected.?Masson's trichrome staining was performed to detect the thickness of the submesothelial tissue;transmission electron microscopy technology was used to investigate the autophagosomes;Western Blot,QPCR and IHC technology were used to detect the expression levels of Fibronectin,Collagen I and LC3.(2)In vitro:?The cells were pre.treated with RAPA and then stimulated with TGF-?1.Westem Blot technology was used to detect the expression levels of Fibronectin,Collagen I,E-cadherin and LC3.?The cells were downregulated the expression of ATG7 by siRNA ATG7 technology and then stimulated with MCL.Western Blot technology was used to detect the expression levels of Fibronectin,Collagen I and LC3.Results:(1)In vivo RAPA treatment significantly increased the expression levels of LC3-?,alleviated PDF-induced aggravation of submesothelial thickening and decreased the levels of Collagen I and Fibronectin compared yith those of DMSO-treated mice;Compared with PF model mice,the DMAMCL treatment groups were found to have more autophagosomes via transmission electron microscopy technology;DMAMCL's protective effect against PF was lessened compared With co-administration of DMAMCL and 3MA,as evidenced by increased the submesothelial thickening and upregulated levels of Fibronectin and Collagen I.(2)In vitro pre-incubation with RAPA subsequent cotreatment with TGF-?1 appeared to increase LC3-? expression compared with that of the groups that were not subjected to RAPA pre-incubation.In addition,the levels of Collagen I and Fibronectin were significantly downregulated,and E-cadherin expression was upregulated;MCL upregulated the expression of LC3-? and ATG7,accompanied by reducing the level of Fibronectin and Collagen I induced by TGF-?1 treatment.However,this effect disappeared when ATG7 was knocked down by ATG7 siRNA technology.Conclusion:Activating autophagy plays a protective role against PF in a mouse PD model and in cultured cells.DMAMCL can effectively induce autophagy.The protective role of DMAMCL in PF can be attributed to the activation of autophagy.