The Role Of Nrf2 Pathway In Numb-mediated Epithelial-mesenchymal Transition During Pulmonary Fibrosis And The Regulatory Effect Of Melatonin

Pulmonary fibrosis?PF?is a chronic interstitial lung disease.The current clinical treatments of PF are still glucocorticoid and immunosuppressive drugs,but the effect is not obvious and possesses side effects.Therefore,exploring the pathogenesis of PF and looking for new therapeutic drugs to treat PF are significant clinical problem to be solved immediately.Epithelial-mesenchymal transition?EMT?plays a key role in the development of PF.It is a phenotypic transformation of epithelial cell,which performed by the loss of epithelial adhesion protein E-cadherin?E-cad?and the acquisition of interstitial cell marker?-smooth muscle actin??-SMA?.It is one of the most important source of myofibroblasts?MFbs?.A large number of MFbs lead to the excessive deposition of extracellular matrix?ECM?,which eventually promotes the formation of PF.Recent studies have reported that a membrane-associated protein Numb has a close relationship with EMT and plays a crucial role in EMT in many oncological and fibrotic diseases.However,relevant studies of Numb in EMT-induced PF are still barren.Nuclear factor E2-related factor 2?Nrf2?,a key regulator of cellular defense against oxidative stress?OS?,protects cells and tissues from damages induced by OS.It has been found that Nrf2 can suppress EMT-induced PF in our previous studies.Whether Nrf2 and Numb have a potential relationship in the PF still need further study.Melatonin,an amine hormone produced by the pineal bodies of mammals.Recent researches have shown that it has effects on preventing OS,anti-aging,anti-tumor,improving the quality of sleeping and so on.The role of Melatonin in the treatment of PF has also been paid more and more attention in recent years,but the mechanism of Melatonin treating PF still need more studies.Whether Melatonin can inhibit Numb-mediated EMT in PF by activating the expression of Nrf2 has not been reported.Objective In this study,intratracheally injecting bleomycin?BLM?into both Nrf2-knockout(Nrf2^-/-)and wild-type?WT?mice to build PF models in vivo and using TGF-?1 to induce EMT models in vitro to detect expressions of Nrf2,HO-1,NQO1,EMT-related proteins and Numb and further explore the regulatory effect between Nrf2 and Numb,as well as the role of melatonin on PF and its possible mechanism.Methods1.Tracheal intratracheal instillation of BLM?4.5 mg/kg?in the Nrf2^-/-mice and WT mice,the control group of mice by intratracheal instillation of the same volume of0.9%normal saline?normal saline,NS?instead.Mice were anesthetized by intraperitoneal injection of 10%chloral hydrate on the 7th day,the 14th day and the28th day after modeling respectively.The lung tissues were removed and the mice were sacrificed.?1?Left lung tissue was used for hematoxylin-eosin?H&E?,Masson trichrome staining and IHC to observe histopathology,collagen expression and expressions of key proteins.?2?The expressions of Nrf2,HO-1,NQO1,Numb and EMT in lung tissue were detected by Western blot.2.In vitro,TGF-?1 induced EMT on RLE-6TN and A549 cells to explore the regulation of Nrf2 and Numb on EMT and the relationship between Nrf2 and Numb:?1?Numb was silenced by siRNA,then TGF-?1 was used to build EMT models on RLE-6TN and A549 cells.Western blot analysis was used to observe the expressions of E-cad,?-SMA,Nrf2,HO-1,NQO1 and Numb.?2?Activating Nrf2 by sulforaphane?SFN?before TGF-?1 stimulation on RLE-6TN and A549 cells,Western blot analysis and immunofluorescence?IF?were used to observe the expressions of E-cad,?-SMA,Numb,Nrf2,HO-1 and NQO1 in TGF-?1-induced EMT.?3?Silencing Nrf2 by siRNA before TGF-?1 stimulation on RLE-6TN and A549 cells,Western blot and IF analysis were used to observe the expressions of E-cad,?-SMA,Numb,Nrf2,HO-1 and NQO1 in TGF-?1-induced EMT.?4?Numb was silenced with siRNA and then treated with SFN and TGF-?1 on RLE-6TN and A549 cells.The expression of E-cad,?-SMA,Numb,Nrf2,HO-1 and NQO1 were observed by Western blot analysis.?5?MTT method was used to select the optimal concentration of Melatonin on RLE-6TN.Then,cells were treated with Melatonin and TGF-?1.The expression of E-cad,?-SMA,Numb,Nrf2,HO-1 and NQO1 were detected by Western blot analysis.Results1.After BLM treatment,the normal structure of alveolar were damaged,the infiltration of inflammatory cells and the accumulation of collagen became more serious in mice lung tissues,and the expressions of EMT-related proteins became more obvious,accompanied by further up-regulation of Numb protein expression in Nrf2^-/-group compared with WT BLM group.2.In vitro,Numb silenced by si RNA inhibited the EMT induced by TGF-?1,which showed by the down-regulation of the interstitial cell marker protein?-SMA and the up-regulation of E-cad.3.In vitro,activation of Nrf2 attenuated the progress of EMT induced by TGF-?1,which performed by the decrease of the interstitial cell marker of?-SMA and the up-regulation of E-cad,and the decrease of Numb expression.4.In contrast,silencing Nrf2 increased the EMT progression induced by TGF-?1,which was shown by the up-regulation of interstitial cell marker protein?-SMA and the down-regulation of E-cad accompanied by an increase in Numb protein expression.5.In vitro,fluorescence experiments further demonstrated that Numb transfer from the cell membrane to cytoplasm and nuclear after TGF-?1 treatment,but when treated cell with SFN,this phenomenon is inhibited.Moreover,Numb protein further expressed in the cytoplasm and the nucleus after Nrf2 is silenced by si RNA.6.In vitro,when Numb was silenced,the inhibitory effect of SFN on TGF-?1-induced EMT was relieved,which mainly manifested as?-SMA and E-cad were up-regulated and down-regulated respectively compared with SFN+TGF-?1 groups.7.In vitro,we selected a certain concentration of melatonin to stimulate RLE-6TN,expressions of Nrf2,HO-1,NQO1 and E-cad were increased accompanied by down-regulations of Numb and?-SMA detected by western blot.Conclusion1.Experiments in vivo suggest that Nrf2,HO-1,NQO1,Numb and EMT are involved in the pathological process of PF.2.Numb silenced in vitro can inhibit the EMT.In addition,activation of Nrf2attenuated TGF-?1-induced EMT,but silencing Nrf2 aggravated EMT accompanied by expression alterations of HO-1,NQO1 and Numb,and silencing Numb blocked the protection of SFN against EMT.These results show that Nrf2 can reduce the EMT induced PF by inhibiting the expression of Numb.In addition,melatonin can activate the Nrf2 anti-oxidant pathway to reduce Numb-mediated EMT.These studies provide some theoretical and experimental evidence for further exploring the pathogenesis of PF.