NF-E2-Related Factor 2 Suppresses Intestinal Fibrosis By Inhibiting TGF-β1/SMADs Pathway

Objective:Inflammatory bowel disease（IBD）is a chronic relapsing disease,including Crohn’s disease（CD）and ulcerative colitis（UC）.Intestinal fibrosis is a common complication of inflammatory bowel disease,especially Crohn’s disease;however,its mechanism of intestinal fibrosis is largely unclear.NF-E2-Related Factor 2（Nrf2）,a nuclear transcription factor,plays an indispensable role in cellular defense against oxidative stress.Nrf2 is released from Keap1 after stimulation by reactive oxygen species（ROS）or inflammatory cytokines,and translocates into the nucleus.It combines with antioxidant response element to initiate the expression of various antioxidant-associated genes expression including heme oxygenase 1（HO-1）.The downstream products of Nrf2 can clear ROS and reduce the accumulation of oxidative stress substances.The expression of Nrf2 was upregulated at inflammatory sites of IBD tissues.Nrf2pathway possessed the ability to alleviate acute colitis,chronic colitis,and associated colorectal cancer in the mouse model,but whether Nrf2 can protect against colitis-associated fibrosis is unexplored.Meanwhile,it has been confirmed that the Nrf2pathway is a negative regulator of fibrosis in other organs.Transforming growth factor-β1（TGF-β1）is the most important profibrogenic molecule in intestinal fibrosis.It mainly mediates through the SMADs family to activate the transcription of collagen and fibronectin,the primary component of ECM.The degradation of ECM is also mediated by the matrix metalloproteinases（MMPs）and tissue inhibitor of metalloproteinases（TIMPs）.TGF-β1 can promote the expression of TIMP1 and inhibit the expression of MMPs to prevent the degradation of ECM.ECM is mainly produced by intestinal myofibroblast cells,andα-smooth muscle actin（α-SMA）is an extracellular marker of myofibroblast.ROS is reported to induce fibrosis through the TGF-β1/SMADs pathway.Nrf2 and downstream products have the ability to scavenge ROS.This study aimed to evaluate the antifibrotic effects of NF-E2-Related Factor 2（Nrf2）on intestinal fibrosis.Methods:BALB/c mice received 2,4,6-trinitrobenzene sulfonic acid weekly via intrarectal injections to induce chronic fibrotic colitis.They also diet containing received 1%（w/w）tert-butylhydroquinone（tBHQ）,which is an agonist of Nrf2.The main fibrosis markers,inflammation markers and TGF-β1/SMADs signaling pathway were detected by Quantitative real-time RT-PCR,immunohistochemical analysis,and Western blot analysis.Human intestinal fibroblasts（CCD-18Co）cells were pretreated with tBHQ or siRNA-Nrf2 followed by stimulation with transforming growth factor-β1（TGF-β1）,which transformed the cells into myofibroblasts.The main fibrosis markers and TGF-β1/SMADs signaling pathway were detected by Quantitative real-time RT-PCR and Western blot analysis.CCD-18Co cells were pretreated with tBHQ and H₂O₂ which is the primary component of ROS,then treated with TGF-β1,TGF-β1/SMADs signaling pathway were detected by Western blot analysis.CCD-18Co cells were transferred with siRNA-Nrf2,then treated with N-acetyl cysteine which can scavenge ROS,finally treated with TGF-β1,TGF-β1/SMADs signaling pathway were detected by Western blot analysis.And levels of cellular ROS were detected by dichlorodihydrofluorescein diacetate.Results:Nrf2 agonist tBHQ was used to investigate the antifibrotic role of Nrf2 in intestinal fibrosis.H&E and Masson’s trichrome staining showed that the oral administration of tBHQ significantly alleviated high levels of inflammation and fibrosis in the colon of TNBS-induced chronic colitis mouse model.This study demonstrated that administration of tBHQ decreased the high expression of Myeloperoxidase（MPO）and proinflammatory cytokines,including Tumor necrosis factor（TNF-α）and Interleukin-1 beta（IL-1β）in TNBS-induced colitis.The high expression of collagen I,α-SMA and TIMP1 in TNBS-induced colitis and TGF-β1–stimulated CCD-18Co cells was attenuated by Nrf2.It was also found that the downregulation of Nrf2 by siRNA transfection could enhance TGF-β1-induced overexpression ofα-SMA,collagen I and TIMP1.tBHQ treatments in mice with TNBS-induced intestinal fibrosis downregulated the expression of TGF-β1,phosphorylation SMAD3,phosphorylation SMAD2/3 and SMAD2/3 in the Nrf2-dependent manner.The same result was obtained in CCD-18Co fibroblast cells stimulated with TGF-β1.Moreover,this study also demonstrated that Nrf2 knockdown increased the expression of TGF-β1/SMADs signaling pathway in CCD-18Co fibroblasts cells.ROS significantly increased in TGF-β1–stimulated CCD-18Co cells.Pretreatment with H₂O₂,the primary component of ROS,was demonstrated to block the effect of tBHQ on reducing the expression of TGF-β1.Moreover,scavenging ROS by N-acetyl cysteine could inhibit the increasing expression of TGF-β1 promoted by Nrf2knockdown.Conclusions:The results suggested that tBHQ suppressed the intestinal fibrosis through the TGF-β1/SMADs signaling pathway in TNBS-induced colitis and CCD-18Co cells.Moreover,Nrf2 knockdown enhanced the TGF-β1–induced differentiation of CCD-18Co cells.Nrf2 suppressed intestinal fibrosis by inhibiting ROS mediated TGF-β1/SMADs pathway in vivo and in vitro.