Mechanism Of Melatonin Pretreatement Attenuates Acute Lung Injury Induced By Ischemia-reperfusion In Mice

?Objective?To study the protective effect and mechanism of melatonin pretreatment on lung ischemia-reperfusion injury in mice.?Method?(1)30 C57BL/6J mice were divided into 5 groups randomly(n=6):(1)sham group;(2)lung ischemia reperfusion group(IR);(3)lung ischemia reperfusion +10mg/kg melatonin pretreatment group(IR+10MT);(4)lung ischemia reperfusion +20mg/kg melatonin pretreatment group(IR+20MT);(5)lung ischemia reperfusion+30mg/kg melatonin pretreatment group(IR+30MT);The IR group and the IR+5MT group,IR+10MT group,IR+15MT group(2,3,4,5 groups)via thoracotomy and clamping the left hilum for 1 hour,followed by reperfusion for 2 hours.Meanwhile,group 3,group 4,and group 5 received an intraperitoneal injection of melatonin at varied doses(5,10,and 15mg/kg)30 min before lung ischemia administration and 10 min before reperfusion administration respectively.The left lung in each mouse was harvested after IR administration for testing.In sham group,thoracotomy was performed to cut the left lower pulmonary ligament and free the pulmonary hilum.After 60 min,the thoracic cavity was closed and the mice were sacrificed after 120 min,the left lung was harvested for testing.(2)Histological assessment of lung injury.(3)Lung Wet/dry(w/d)weight ratio analysis.(4)Lung SOD activity.(5)Lung MDA content.(6)Detection of apoptosis in lung tissue by TUNEL.(7)Western blot was used to detect the changes of protein expression of TNF-?,NF-?B,IL-1?,IKK-?,caspase-3,BAX,bcl-2in different groups.(8)The expression of antioxidant indexes(GPX,GR)was detected by immunohistochemistry(IHC).?Results?(1)The pathological changes of lung tissue were observed under light microscope,the injury of IR group was the most serious,the injury of the sham group was the most lighter,the injury of the IR+10MT group,IR+20MT group and IR+30MT group were lighter than the IR group,and the injury of the IR+20MT group was lighter compared with the IR + 10 MT group and IR+30MT group,the difference was statistically significant (P<0.05).(2)The W/D ratio of the lung tissues was signifcantly higher in IR group compared with the sham group,the IR+10MT group,IR+20MT group,IR+30MT group was significantly lower than IR group,among them,IR+20MT group decreased the most obvious,the difference was statistically significant(P<0.05).(3)The content of MDA in IR group was significantly higher than that in sham group,the IR+10MT group,IR+20MT group,IR+30MT group was significantly lower than IR group,among them,IR+20MT group decreased the most obvious,the difference was statistically significant(P<0.05).(4)The content of SOD in IR group was significantly lower than that in sham group,the IR+10MT group,IR+20MT group,IR+30MT group was significantly higher than IR group,among them,the IR+20MT group rised the most obvious,the difference was statistically significant(P<0.05).(5)TUNEL detection of apoptosis showed that IR group increased apoptosis compared with sham group,the IR+10MT group,IR+20MT group,IR+30MT group decreased apoptosiscompared with the IR group,the IR+20MT group decreased apoptosis more obvious compared with IR 10 MT and IR 30 MT group,the difference was statistically significant(P<0.05).(6)The expression of TNF-?,IL-1?,NF-?B?IKK-? were detected by Western blot in each group,the experimental results showed that: compared with IR group,the IR+10MT group,IR+20MT group,IR+30MT group significantly decreased the levels of TNF-?,IL-1?,NF-?B?IKK-?,the difference was statistically significant(P<0.05).(7)Western blot detected the expression of caspase-3 and BAX in each group,the IR+10MT group,IR+20MT group,IR+30MT group was significantly lower than that of IR group(p<0.05),the difference was statistically significant(P<0.05).In contrast,protein expression of Bcl-2,an indicator of antiapoptosis,exhibited a reversed pattern compared with that of apoptotic protein expression of caspase 3 among each of the five groups.(8)The results of immunohistochemistry(IHC)showed that the GPX and GR(two indicators of antioxidants)gradually increased in sham group,IR group,IR + 10 MT group,IR + 30 MT group,IR +20MT group,the difference was statistically significant(P<0.05).?Conclusion?First,Melatonin pretreatment significantly attenuated lung ischemia reperfusion injury,20 mg/kg is a relatively more effective dose to relieve ischemia-reperfusion injury for mice.Second,the mechanism of melatonin pretreatment attenuated lung ischemia reperfusion injury may be related to the reduction of oxidative stress and the inhibition of TNF-?/ NF-??/ apoptosis pathway.