| Lung is one of the preferred target organs of ischemia-reperfusion injury.Lung ischemiareperfusion injury(LIRI) usually occurs when the patients experienced lung transplantation. Inflammation is known to be a key mediator of LIRI.Reactive oxygen species(ROS),calcium overload,TNF-αand IL-1βare the mainly agents of LIRI which also induce apoptosis.Apoptosis is a distinct form of single-cell death in response to injury,and there are strong indications that apoptosis plays an important role in various disease processes. More and more evidences show that apoptosis plays a critical role in lung ischemia-reperfusion injury.Experimental studies indicated that TGF-β1 may attenuate ischemia-reperfusion injury. TGF-β1 is a kind of multifunction cytokine.The effect of TGF-βsignaling pathway was depended on activation of membrane receptor and downstream molecular Smads.Smad3 is the first player in TGF-βsignaling system.Phosphorylation of Smad3 translocates to the nucleus and interacts with transcription factors to regulate downstream gene expression. These findings raised the question of the role of Smads during the LIRI period.In this study,a stable mouse LIRI model was established.In order to evaluate the role of pSmad3 of LIRI apoptosis,TGF-β1 and LY-364947 was used.Then,the content of pSmad3 was tested with ELISA method and SOD,TNF-αand IL-1βwere tested.To discuss the the role of Smads during the LIRI period,apoptosis of cell was detected using TUNEL staining.This study can provide clues for studying the LIRI and looking for new therapeutic targets.Part 1:Role of phosphorylation of Smad3 to inflammatory mediators during the LIRI period.Objective:Inflammation is known to be a key mediator of LIRI.ROS,TNF-αand IL-1βare the mainly agents of LIRI.In this study we hypothesized that phosphorylation of Smad3 inhibit the expression of ROS,TNF-αand IL-1βduring LIRI. Methods:We used mouse model of single left lung of ischemia-reperfusion in situ as Sekido'S model.Forty Sprague-Dawle mouse as experimental were randomly divided into 4 groups:Shame operation group(SO group,n=10),ischemia-reperfusion group(IR group, n=10),TGF-β1 group(T group,n=10) and LY-364947 group(L group,n=10).TGF-β1 was administered intravenously at doses of l0ug/kg in T group and L group.LY-364947 was administered intravenously at doses of lmg/kg In L group.All mouse were ventilated with the assistance of a manual mechanical ventilator at 70-80 breaths/min and 1.5-2.0 ml tidal volum.The left lung of mouse was subjected to ischemia for 60 minutes,followed by reperfusion for 120 minutes,to induced LIRI.Lung tissue samples were obtained at the end of reperfusion.Tissue homogenate was used for test the pSmad3 with ELISA method. SOD was tested with spectrophotometric method.TNF-αand IL-1βwere tested with ELISA method.Results:Phosphorylation of Smad3 was activeted during the LIRI period(48.482±19.602). ROS were largely generated,while SOD was depleted(15.499±3.487).At the same time TNF-αand IL-1βwas expressed(2.492±0.690,2.557±0.542).TGF-β1 inhanced phosphorylation of Smad3(63.607±17.194).With the increasing of pSmad3,depletion of SOD was decreased(22.441±3.909),and expressed of TNF-αand IL-1βwere inhibited significantly(1.438±0.392,1.452±0.498).Additionally,LY-364947 inhibited the activation of Smad3(13.763±8.732),expression of TNF-αand IL-1βwere increased (1.921±0.417,2.029±0.599),and SOD was depleted largely(18.892±3.176).Conclusions:Reactive oxygen species(ROS),TNF-αand IL-1βare the mainly agents of LIRI.Phosphorylated Smad3 inhibits the expression of ROS,TNF-αand IL-1β. LY-364947 blocks the effect of PSmad3.Part 2:Role of phosphorylation of Smad3 to apoptosis during the LIRI period.Objective:Apoptosis plays a critical role in lung ischemia-reperfusion injury.We have demonstrated that phosphorylation of Smad3 inhibits the ewpression of ROS,TNF-αand IL-1βduring LIRI.The part of study tested the hypothesis that phosphorylation of Smad3 reduces apoptosis LIRI by inhibiting the expression of ROS,TNF-αand IL-1β.Methods:Stable mouse LIRI model was used.TGF-β1 was administered intravenously at doses of l0ug/kg in T group and L group.LY-364947 was administered intravenously at doses of lmg/kg In L group.Lung tissue samples were obtained at the end of reperfusion. Apoptosis ratio was determined by TUNEL staining.Tissue homogenate was used for test the pSmad3 with ELISA method.Results:TGF-β1 inhanced phosphorylation of Smad3(63.607±17.194).Apoptosis index in T group(10.500±4.127) was significantly lower than IR group(18.042±4.789). Additionally,LY-364947 inhibited the activation of Smad3(13.763±8.732),AI in L group was markly higher than T group(14.276±3.705 vs 10.500±4.127).Conclusion:Phosphorylation of Smad3 plays an important role in lung self-repair after ischemia-reperfusion injury via reducing apoptosis.
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