Effects And Mechanism Of Pirfenidone On Radiation-induced Intestinal Fibrosis

Part Ⅰ Effects and underlying mechanism of pirfenidone on radiation-induced intestinal fibrosis in rats Objective: This study was aimed to explore whether pirfenidone could protect against radiation-induced intestinal fibrosis in rats,and to evaluate the underlying mechanism.Materials and methods: 40 Sprague-Dawley rats were randomly assigned into 4 groups: Control group,Radiation group(radiotherapy),PFD200 group(radiotherapy plus pirfenidone 200mg/kg/d),and PFD400 group(radiotherapy plus pirfenidone 400mg/kg/d).An animal model of radiation-induced intestinal fibrosis was induced by exposure of a single dose of 20 Gy to the pelvis.Rats were orally administered with pirfenidone(200,400md/kg/d)or sodium carboxymethyl cllulose for 12 weeks from the second day after radiotherapy.The rats were sacrificed 12 weeks after radiotherapy,gross and pathological damage score,the rectal submucosa thickness was measured under a microscope,fibrosis was assessed by using HE and VG staining for pathological examination and fibrosis score was evaluated.Real time PCR and Western Blot were performed to evaluate the expression of collagen I and α-SMA mRNA and protein in rectal tissues,as well as the protein expression of TGF-β1,pSmad2,pSmad3,Smad2,Smad3,and CTGF.Result: The rats in Radiation group experienced body weight loss after radiotherapy.Most rats developed radiation fibrosis,e.g.intestinal strictures of the irradiated segments intestinal stenosis,and even intestinal obstruction symptoms.Radiation induced remarkable fibrotic lesions with thickened submucosal layers,which were replaced by dense collagen depositions,as demonstrated by HE and VG staining.Such effects were attenuated by treatment with pirfenidone,and most rats exhibited slight and mild stenotic lesion in irradiated intestinal segments.Pirfenidone treatment attenuated fibrotic lesion and collagen deposition,together with a decrease in the fibrosis score and depth of thickened submucosa.By using qRT-PCR and western blot analysis,we showed that administration with pirfenidone down-regulated radiation-induced over-expressed collagen I and α-SMA mRNA and protein levels in a dose-dependent manner.Significant up-regulation of TGF-β1,pSmad2,pSmad3,and CTGF proteins were observed in irradiated intestines in rats.Additionally,the up-regulation of these molecules was suppressed dose-dependently by treatment with pirfenidone in vivo.Conclusion: Our findings suggested that pirfenidone attenuated radiation-induced intestinal fibrosis in rats through inhibition of the phosphorylation of Smad2 and Smad3 in the TGF-β1/Smad/CTGF signaling pathway.Part Ⅱ Effects and mechanism of pirfenidone on rat intestinal fibroblastsObjective: To observe the effect of pirfenidone on intestinal fibrosis in rats with radiation-induced fibrosis,and to explore the underlying mechanism.Materials and methods: Intestinal fibroblasts from rats(RIFs)were cultured in vitro after primary culture,and were identified by vimentin and keratin using flow cytometry.TGF-β1 was used to activate RIFs to mimic the “fibrotic” environment in vivo.LDH cytotoxicity experiment was used to detect cytotoxic effects of pirfenidone on RIFs.CCK-8 assay was utilized to evaluate the effects of pirfenidone on TGF-β1-induced RIFs proliferation.Real time PCR and Western Blot assay were performed to detect the effect of pirfenidone on TGF-β1-induced cell differentiation(α-SMA)and collagen synthesis(collagen I).Western Blot was carried out to examine the TGF-β1-related pathway proteins,and to explore the possible underlying mechanism.The phosphatase inhibitor,l-p-bromotetramisole,was added to further verify the corresponding effects of pirfenidone on the inhibition of phosphorylation of Smad2 and Smad3 on RIFs,and to detect the effects on cell differentiation,collagen synthesis and expression of TGF-β1/Smad pathway proteins.Results: According to the location,primary cell morphology,vimentin and keratin flow cytometry,the cultured cells were determined as RIFs.LDH cell toxicity test showed no obvious cytotoxic effects after 48 h of pirfenidone treatment.CCK-8 assay showed that pirfenidone(0.5,1mg/ml)exerted inhibiting effects on cell proliferation in RIFs.After stimulation with TGF-β1(5ng/ml),cell proliferation of RIFs was significantly enhanced.However,with the increased concentration(0.5,1mg/ml)of pirfenidone,TGF-β1-enhanced inhibitory effect on cell proliferation was reduced by pirfenidone.Real-time PCR and Western Blot assay demonstrated that TGF-β1 could induce increased expression of α-SMA and collagen I m RNA and protein in RIFs.After pirfenidone treatement,TGF-β1-induced up-regulated expression of α-SMA and collagen was reduced.Pirfenidone could obviously inhibit TGF-β1-induced elevated phosphorylation of Smad2 and Smad3.After treatment with l-p-bromotetramisole,TGF-β1-induced increased m RNA and protein levels of α-SMA and collagen I in RIFs were reduced.Inhibition of phosphatase activity could significantly inhibit the phosphorylation of Smad2 and Smad3 induced by TGF-β1.Additionally inhibition of phosphatase activity inhibited the protein expression of the downstream molecule,CTGF,which was induced by TGF-β1.Conclusion: Pirfenidone inhibited TGF-β1-induced enhancement of intestinal fibrosis related activities into vitro,including cell proliferation,myofibroblast differentiation and collagen synthesis.Effect of anti-fibrosis in vitro intestinal pirfenidone may be related to inhibition of Smad2 and Smad3 phosphorylation in the TGF-β1/Smad/ CTGF signaling pathway.Part Ⅲ Effects and mechanism of pirfenidone on human intestinal fibroblastsObjective: To observe the effect of pirfenidone on human intestinal fibroblasts(HIFs),and to explore the underlying mechanism.Materials and methods: TGF-β1 was used to activate HIFs to mimic the fibrotic environment in vivo.LDH cytotoxicity experiment was used to detect cytotoxic effect of pirfenidone on HIFs.CCK-8 assay and colony formation assay were utilized to evaluate the effect of pirfenidone on TGF-β1-induced HIFs proliferation.Flow cytometry and TUNEL assay were utilized to assess the effect of pirfenidone on TGF-β1-induced HIFs apoptosis.Western Blot assay was performed to determine the expression of apoptosis-related proteins.Real time PCR and Western Blot were used to examine pirfenidone effects on TGF-β1 induced cell differentiation(α-SMA)and collagen synthesis(collagen I and fibronectin).Western Blot assay was used to determine the protein expression of TGF-β1/Smad and PI3K/Akt pathways,and to explore the possible underlying mechanism.Results: LDH cell toxicity test showed no obvious cytotoxic effect after 48 h treatment of pirfenidone.CCK-8 and colony formation assay showed that pirfenidone exerted inhibiting effect of cell proliferation in TGF-β1-induced HIFs.In addition,pirfenidone enhanced HIFs cell apoptosis and increase the number of TUNEL positive cells,accompanied by increased protein expression of Bad and the decreased protein level of Bcl-2.Real time PCR and Western Blot showed that TGF-β1 could induce the increase expression of α-SMA,collagen I,and fibronectin m RNA and protein in HIFs.Pirfenidone inhibited TGF-β1 induced elevated m RNA and protein expression of α-SMA,collagen I and fibronectin.Pirfenidone can inhibit TGF-β1 protein phosphorylation,induced by elevated Smad2/3.Additionally,pirfenidone inhibited TGF-β1-induced increased protein expression of PI3 K and phosphorylation level of AKT protein.Conclusion: Pirfenidone inhibited TGF-β1-induced HIFs proliferation and reduced cell apoptosis.Pirfenidone inhibited TGF-β1-induced fibrosis-related activities into vitro,including myofibroblast differentiation and collagen synthesis.Effect of anti-fibrosis in vitro intestinal pirfenidone may be related to inhibition of phoshporylation of the TGF-β1/Smad and PI3K/Akt signaling pathways.