| Objective:Paraquat(PQ)poisoning can cause multiple organ dysfunction syndrome mainly in acute lung injury(ALI)and acute respiratory distress syndrome,with a very high mortality rate,and there is no effective treatment.As an important mechanism for controlling mitochondrial mass,mitochondrial DNA(mtDNA)is cleared of mitochondrial DNA(mtDNA)as a pattern of damage-related molecules in this process,thereby improving the TLR9 inflammatory pathway activated by mtDNA.Melatonin(MT),as a common hormonal drug,can promote the expression of PINK 1 and BNIP3,which are key proteins inmitophagy,which is speculated to produce a certain protective effect in the ALI caused by PQ poisoning by upregulatingmitophagy.This study aims to explore the effect and mechanism of MT on ALI caused by PQ poisoning through in vitro and in vitro experiments,in order to provide new ideas for the clinical treatment of PQ poisoning.Methods:In this study,adult male C57BL/6J mice and A549 cells were studied as the research subjects.1.Animal model construction and grouping The SPF-grade C57BL/6J mice were divided into four groups:control group(group A),PQ group(group B),PQ+MT group(group C)and MT group(group D),among which the concentration of PQ intervention was 30mg/kg and the concentration of MT intervention was 20mg/kg.2.HE staining and lung wet weight/dry weight ratio to evaluate the severity of ALI,Western Blot(WB)and immunofluorescence(IF)to detect the expression changes of PINK1 and BNIP3,transmission electron microscope(TEM)to observe autophagic bodies in lung tissue,The levels of inflammatory factors TNF-α and IL-1β in the serum of four groups of mice were detected by ELISA,and the changes of mitophagy and inflammatory response in mouse lung tissues after MT intervention were explored.3.A549 cells were cultured in different concentrations(0,100,200,400,600,800,1000,1200μM)PQ and different concentrations(0,400,800,1200,1600,2000,2400,2800μM)MT,and CCK8 was screened for the best PQ and MT intervention concentrations and grouped:control group(Ⅰ.group),PQ group(Ⅱ.group),PQ+MT group(Ⅲ.group)and MT group(Ⅳ.group).4.Chromatin Immunoprecipitation(ChIP)-qPCR to explore whether mtDNA leakage can activate the TLR9 inflammatory pathway,qPCR detects four groups of mitochondrial DNA leakage levels to quantify MT-CO2 to measure mtDNA levels.WB and IF detected the expression levels of PINK1,BNIP3 and TLR9 proteins,the autophagic bodies of A549 cells were observed by TEM,and the inflammatory factors TNF-α and IL-1β levels were detected by ELISA in the Supernatant of A549 cells.5.After transfection inhibited the expression of PINK1 and BNIP3,MT intervention was performed on the PQ group,and the expression levels of PINK1,BNIP3 and p-P65 proteins were detected by WB,the leakage level of mtDNA was detected by qPCR,and the TNF-α and IL-1β levels were detected by ELISA.Results:1.In vivo experiments verified the protective effect of MT on PQ-induced ALI:(1)The results of HE staining and lung wet weight/dry weight ratio showed that compared with group A,group B had obvious hyperemia,edema and fibrosis,and no obvious change in group D.Group C had less lung injury than Group B.(2)WB and IF results showed that compared with group A,the expression of PINK1 and BNIP3 in group B and group D was increased;Compared with group B,group C had elevated expression of PINK1 and BNIP3.(3)TEM results showed that,compared with group A,group B cell structure was abnormal,mitochondria were swollen and deformed,the number of autophagsomes increased,the cell structure of group D did not change significantly,and the number of autophagsomes increased;Compared with group B,group C had a more complete and clear cell structure,and the number of autophagic bodies increased.(4)ELISA results showed that compared with group A,the level of inflammatory factors in group B was significantly increased,and there was no obvious change in group D;Group C had reduced levels of inflammatory factors compared to group B.2.In vitro experiments to explore the specific mechanism of melatonin to produce protective effect:(1)CCK8 results showed that after applying different concentrations of PQ to culture cells,the cell survival rate gradually decreased with the increase of paraquat concentration,and when the PQ concentration was 600μM,the cell survival rate was close to 50%,that is,half of the inhibitory concentration.The change of cell viability when different concentrations of MT were measured in the same method,and it was found that there was no significant difference in cell survival rate between different concentrations of MT,which indicated that MT had no obvious cytotoxicity.In addition,when we intervened simultaneously with different concentrations of MT and PQ(600μM),we found that cell survival was highest at 1200 μM.Therefore,we used 600 μM and 1200 μM as the optimal intervention concentrations for PQ and MT,respectively.(2)ChIP-qPCR results showed that after PQ intervention,compared with the control group,the content of mtDNA in the extracted and purified DNA increased significantly,which indicated that the activation of TLR9 was closely related to mtDNA leakage during PQ poisoning.(3)qPCR results showed that compared with group Ⅰ.,the MT-CO2 level of group Ⅱ was increased,and there was no significant change in group Ⅳ.Compared with group Ⅱ,group Ⅲ had a lower level of MT-CO2,which indicated that mtDNA leakage into the cytoplasm was reduced after MT intervention.(4)The results of TEM,WB,IF,ELISA showed that compared with group Ⅰ.,the cell structure of group Ⅱwas significantly destroyed,the number of autophagies increased,the expression of PINK1,BNIP3 and TLR9 increased,the level of inflammatory factors increased,there was no significant change in the cell structure of group Ⅳ.,the number of autophagoids increased,the expression of PINK 1 and BNIP3 increased,and the expression of TLR9 and inflammatory factors did not change significantly.Compared with group Ⅱ.,groupⅢ.had a complete cell structure,increased the number of autophagies,increased the expression of PINK1 and BNIP3,decreased the expression of TLR9,and decreased the level of inflammatory factors.(5)After transfection inhibited the expression of PINK1 and BNIP3,the results of WB,qPCR and ELISA showed that MT intervention in the PQ group increased mtDNA leakage,increased p-P65 expression,and increased the level of inflammatory factors,which indicated that the protective effect of MT disappeared.Conclusion:Melatonin can promote the expression of PINK1 and BNIP3 upregulatemitophagy,clear abnormal mitochondria,thereby reduce mtDNA leakage,and improve acute lung injury mediated by TLR9 inflammatory pathway in paraquat poisoning. |
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