Experimental Study Of Parthenolide Inhibiting Peritoneal Dialysis-related Peritoneal Fibrosis

Background:Peritoneal dialysis(PD)is a safe,effective,simple,convenient and home-based renal replacement therapy.Peritoneal fibrosis is the main cause of peritoneal failure,but there is no definite and effective treatment at present.Parthenolide(PTL),NF-κB inhibitor,is a sesquiterpene lactone extracted from Tanacetum balsamita,which can reduce pulmonary fibrosis by inhibiting inflammation,but its effect on peritoneal fibrosis and related mechanisms have not been reported.Chapter Ⅰ Inhibitory effect of PTL on mouse peritoneal fibrosisObjective:To investigate the effect of PTL on peritoneal fibrosis in vivo.Methods:A mouse model of peritoneal fibrosis associated with PD was established by daily intraperitoneal injection of 3 ml of 4.25%glucose dialysis solution for 28 days.To investigate the effect of PTL on peritoneal fibrosis,the mice were treated with daily intragastric administration of three different concentrations of PTL(12.5 mg/kg,25 mg/kg and 50 mg/kg),followed by daily PDF infusion.Delayed PTL(50 mg/kg)treatment groups were given PTL after daily PDF infusion for 14 days.PD mice with vehicle treatment(normal saline)were killed at day 14 and day 28 as untreated control groups.Peritoneal tissues,including the anterior abdominal wall and omentum,were collected.The thickness of parietal peritoneum was detected by Masson staining.Modified peritoneal equilibration tests(PET)were used to detect peritoneal function.Western Blot and immunohistochemistry(IHC)were used to detect the expression levels of peritoneal Fibronectin,Collagen I and E-cadherin.Results:1.Masson’s trichrome staining showed that the peritoneal thickness in the day 14 model group and day 28 model group increased significantly,and the day 28 model group was the most significant.At the same time,the transport of urea nitrogen and glucose increased.Western blot showed that compared with the normal control group,the expression of Fibronectin and Collagen I in the model group increased while the expression of E-cadherin decreased.2.Masson’s trichrome staining showed that compared with model group,the thickness of Parietal peritoneum in PTL treatment group with different concentrations and delayed intervention group were significantly reduced.Meanwhile,urea nitrogen and glucose transport decreased.Western Blot and IHC showed that compared with the model group,the expression levels of peritoneal Fibronectin,Collagen Ⅰ in PTL treatment group with different concentrations and delayed intervention group decreased,while the expression level of E-cadherin increased.Conclusion:PTL can effectively prevent and treat PD-related peritoneal fibrosis in vivo.Chapter Ⅱ Mechanism of PTL Inhibiting Peritoneal FibrosisObjective:The effect of PTL on the expression of pSmad2/3 in mouse peritoneum was investigated in vivo.Experiments were conducted to investigate the effect of PTL in TGF-β1-induced epithelial-mesenchymal transition(EMT)in HMrSV5 cells and related mechanisms,to clarify the relationship between peritoneal fibrosis and Smad2/3,and to explain the changes in p65 expression levels in vitro.Methods:In vivo experiment:IHC was used to detect the expression levels of psmad2/3 in peritoneum.In vitro experiments:First to examine the anti-fibrotic effect of PTL in TGF-β1-induced epithelial-mesenchymal transition(EMT)in HMrSV5 cells.Cells were exposed to TGF-β1(10 ng/ml)for 48 h and 24h in the presence or absence of different concentrations of PTL(1.25,2.5 and 5μM).Western Blot detected the expression levels of Fibronectin,Collagen I,E-cadherin,p65.Then to test the effect of PTL on TGF-β/Smad signalling,HMrSV5 cells were treated with the indicated amount of PTL for 12 h,followed by incubation with TGF-β1 for 3 h.Western Blot detected the expression levels of psmd2,Smad2,pSmad3,Smad3,psmd 1/5/9,Smad 1/5/9,p-AKT,AKT,p-ERK,ERK,p-p38 and p38 proteins.An immunofluorescence staining test and Western blot analysis of Smad2 and Smad3 expression in the cytoplasm and nucleus.In addition,HMrSV5 cells were preincubated with different concentrations of SIS3(0,1,3μm)for 1 h before TGF-β1(10ng/ml)treatment.Cells were collected 1 h and 48h after TGF-β1 stimulation.Western Blot detected the expression levels of pSmad3,Smad3,Fibronectin,Collagen I and E-cadherin.Results:1.Compared with the model group,the expression of pSmad2/3 in the peritoneum of the PTL treatment group and the delayed intervention group decreased.2.Western blot analysis of fibronectin,collagen I and E-cadherin expression after 48 h treatment in vitro.Results showed that all three concentrations of PTL could reduce EMT induced by TGF-β1 in HMrSV5 cells,and PTL 5μM group was the most obvious,showing the decrease of Fibronectin and Collagen I expression level and the increase of E-cadherin expression level.3.HMrSV5 cells were preincubated with PTL(5μM)for 12 h before TGF-β1(10 ng/ml)treatment.Cells were collected 3 h after TGF-β1 stimulation,Western blot analysis suggested that PTL selectively inhibited TGF-β1-induced Smad2 and Smad3 phosphorylation,did not affect TGF-β1-induced phosphorylation of Smad1/5/9,AKT,ERK or p38.4.Immunofluorescence staining test and Western blot analysis confirmed PTL further inhibited Smad2 and Smad3 nuclear translocation.5.HMrSV5 cells were preincubated with SIS3 for 1 h before TGF-β1(10ng/ml)treatment.Cells were collected 1 h after TGF-β1 stimulation Western Blot analysis showed SIS3 could reduce TGF-β1-induced phosphorylation of Smad3 in a dose-dependent manner.6.HMrSV5 cells were co-incubated with TGF-β1 and SIS3 at different concentrations(0,1,3μm).Western blot analysis of fibronectin,collagen Ⅰ and E-cadherin expression after 48 h treatment.Compared with the group stimulated by TGF-β1 alone,the expression level of Fibronectin and Collagen Ⅰ was lower,while the expression level of E-cadherin in SIS3 groups were higher,especially in SIS3 3μM group.7.PTL inhibits p65 nuclear translocation.Western blot analysis of p65 expression in the cytoplasm and nucleus at 24 h,compared with TGF-β1 stimulation alone,the cytoplasmic p65 increased gradually while the nuclear p65 decreased obviously in Three concentrations of PTL groups,the PTL 5μM group was the most obvious.Conclusion:PTL selectively inhibits TGF-β1-induced EMT of HMrSV5 cells through TGF-β/Smad pathway.Inhibition of Smad3 can effectively inhibit TGF-β1-induced EMT in HMrSV5 cells.