The Role Of Shh Signaling Pathway In Chronic Cold Exposure Induced Lung Injury In Mice

The cold environment is a common stressor in north.Cold stress may limit the immunity,growth and fertility of animals,which hinder the development of animal husbandry.There are100 millions of alveoli in a lung,which directly contect to the cold air as the place of air exchange.It could turn the lung in a stress status.Previous studies have shown that Shh signaling pathway play an important role in the development of embryonic lung.It also take a part in regulating of lung diseases.So what is the mechanism of the lung injury under cold exposure?We model the mouse lung injury under induced by chronic cold exposure,aiming to research how chronic cold exposure affect in mouse lung injury and how Shh signaling pathway affect in mouse lung injury under chronic cold exposure.To research the effect of chronic cold exposure on mouse lung injury,the 5-year-old male C57BL/6 mice were divided into the cold exposure group(CE)or the room temperature group(RT)randomly.The mice in the CE group were exposed at 4℃ for 3 h per day,lasting 3 weeks.The mice in the RT group lived at 24±2℃.To research the effect of chronic cold exposure on oxidative stress of mouse lung,malondialdehyde(MDA),glutathione(GSH),reactive oxygen species(ROS),heat shock protein 60(HSP60),70,90,catalase(CAT),glutathione S transferase(GST),superoxide dismutase 1(SOD-1),Nuclear factor erythoid-2 related factor 2(Nrf-2),Kelch-like Ech-associated protein-1(Keap1)and heme oxygenase 1(HO-1)were detected by enzyme linked immunosorbent assay(Elisa),fluorescence probe and Western Blotting.The results showed that compared with RT group,the levels of ROS,MDA,HSP60,70,90 and SOD-1 were increased,while the activity of GSH and the levels of CAT,GST,Nrf-2,Keap1 and HO-1 were decreased in mouse lungs in CE group.It indicated that chronic cold exposure induced injury of protein and lipid in mouse lung,while decreasing the antioxidative ablity.To research the effect of chronic cold exposure on inflammation and apoptosis in mouse lung,HE staining and TUNEL staining were used for observing inflammation and apoptosis in mouse lung.The results showd that chronic cold exposure induced inflammation and apoptosis in mouse lung.Interleukin-1β(IL-1β),IL-6,tumor necrosis factor-α(TNF-α),B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax)and cysteinyl aspartate specific proteinase 3(Caspase3)were detected by Western Blotting.The results showed that compared with RT group,the levels of IL-1β,IL-6,TNF-α,Bax and Caspase3 were increased while the level of Bcl-2 was decreased.It proved the inflammation and apoptosis induced by chronic cold exposure,and also indicated that chronic cold exposure decreased the anti-apoptosis capacity in mouse lung.To research the effect of chronic cold exposure on Shh signaling pathway,sonic hedgehog(Shh),smoothened(Smo),zinc finger transcription factor glioma-associated oncogene homolog1(Gli1),2 and 3 were detected by Western Blotting and IHC.The results showed that compared with RT group,the levels of Shh,Smo,Gli1,Gli2 and Gli3 were decreased in mouse lung in CE group,indicating that chronic cold inhabited Shh signaling pathway.To research the effect of Shh signaling pathway on lung injury induced by chronic cold exposure,MLE12 cells was exposed at 32℃ for 3 h,treated with r Shh(activating Shh signaling pathway),Cyc(inhibiting Shh signaling pathway)or GANT58(inhibiting function of downstream transcription factor Gli1).The indictors of oxidative stress(Nrf-2,Keap1 and HO-1),inflammation(IL-1β,IL-6 and TNF-α)and apoptosis(Bcl-2,Bax and Caspase3)were detected by Western Blotting.The results showed that r Shh treatment increased the levels of Nrf-2,HO-1and Bcl-2,while decreased the levels of Keap1,IL-1β,IL-6,TNF-α,Bax and Caspase3 in MLE12 cells under cold exposure.The Cyc treatment or the GANT58 treatment weakened this effect on oxidative stress,inflammation and apoptosis.It means that Shh signaling pathway could increase antioxidative ability and decrease the level of inflammation and apoptosis in MLE12 cells.Conclusion: Chronic cold exposure induced oxidative stress,inflammation and apoptosis in mouse lung,while inhabiting Shh signaling pathway.Shh signaling pathway protect MLE cells from the oxidative stress,inflammation and apoptosis induced by cold exposure.