Mechanism Study On The Involvement Of SIRT1-PGC-1α Signaling Pathway In Acute Lung Injury Induced By Paraquat

Objective: Paraquat(PQ)poisoning is a toxic disease that seriously endangers health of human.Patients can develop multiple organ dysfunction within several days,especially lung and kidney injury.The severity of early acute lung injury(ALI)is closely related to the survival rate and long-term prognosis of patients.In common cases of suicide,the effects of paraquat poisoning are serious and difficult to reverse.Patients who have passed the acute stage often have different degrees of irreversible pulmonary fibrosis,which brings serious burden to patients and their families.The core pathological process of PQ poisoning lies in severe oxidative stress and inflammation.Due to the polyamine uptake system in the lung,the concentration of PQ in the lung even reaches 6-10 times of that in the serum.Serious oxidative stress and inflammation destroy the structure and function of the alveoli,which are the basis of lung lesions.Mitochondria,as the main sites of reactive oxygen species(ROS)generation,are also severely damaged in structure and function.Meanwhile,the protein network that regulates and maintains mitochondrial homeostasis and biogenesis also plays an important role in the pathogenesis of PQ poisoning.PGC-1α is one of the important molecules in regulating mitochondrial biogenesis and maintaining its functional homeostasis.PGC-1α is also widely involved in the metabolic regulations of various tissues,and regulated by upstream factors through posttranslational modification.In recent years,PGC-1α has attracted much attention due to its extensive regulatory functions and close relationship with mitochondrial functions.SIRT1,as its main upstream regulator,controls PGC-1α activity by regulating its acetylation status,and is involved in mitochondrial function maintenance and other metabolic processes.High-mobility group protein box 1(HMGB1)has been shown to be involved in the development of multiple inflammatory disease models.When inhibited,it can reduce the activation of the inflammasome and reduce the production of inflammatory chemokines.Its inhibitor glycyrrhizin has been confirmed to play a therapeutic role in PQ poisoning,but the mechanism of PGC-1α and HMGB1 signaling regulation in PQ poison has not been further studied.EX527 is a selective inhibitor of SIRT1,which can significantly reduce its deacetylation activity and thus inhibit its downstream regulatory network.The purposes of this study were to explore the role of PGC-1α signaling pathway in acute lung injury caused by PQ poisoning,and to explore its upstream and downstream regulatory mechanisms.The treatment of PQ poisoning model was carried out by means of signal inhibitors and transgene to confirm the therapeutic effect was related to the regulation of PGC-1α signaling.Methods:Ⅰ.Establishment of animal models and collection of specimens1.Experimental animals: wild-type C57BL/6J mice(SPF grade,male,weight 18-22g)were purchased from Beijing Huafukang Biotechnology Co.,LTD.,and raised in the Animal Experimental Center of Research and Development Center of Shengjing Hospital Affiliated of China Medical University.Feeding conditions: temperature 20-25℃,humidity 40-70%,12 hours of day and night alternation.The mice were fed and drank freely and were fed for 7 days before the experiment.PQ was injected intraperitoneally at a dose of 30mg/kg,and the acute lung injury model was established after 72 hours of feeding.2.Sample collection: Experimental mice were anesthetized by isoflurane inhalation,the chest was fully exposed after disinfection,and 0.5ml of blood was taken from the ventricle.The pulmonary circulation was irrigated with normal saline through the right ventricle,and the systemic circulation through the left ventricle until the perfusion fluid was clear and blood-free.The left lung was fixed at 4℃ overnight with 4%paraformaldehyde,and the right lung was frozen at-80℃ for subsequent experiments.After standing for half an hour,the blood was centrifuged at 4℃ and 4000 rpm for 25 min,and the supernatant was taken and frozen in the refrigerator at-80℃.Ⅱ.Establishment of the cell modelA549 human alveolar epithelial cells were cultured in a special medium for A549 cell containing 10% fetal bovine serum.The cells were planted in 96-well plates with a density of 5000/ well.After cells adhering,the medium was changed once,and 100μl PQ(0,100,200,300,400,500,600,750μM)of different concentrations were added.After24 hours,The original liquid was discarded,and CCK8 reagent was diluted 10 times with the medium,then was add to 96-well plates,100μl per well,and continued to culturing in37℃ incubator.After 2 h,the absorbance at wavelength 450 nm was measured by microplate reader.The survival rate of cells in each group was calculated according to the formula.The concentration of PQ with the survival rate closest to 50% was selected as the concentration for subsequent experimental treatment.According to different treatment intervention plans,they were divided into normal control group,poison group,transfection group,transfection poison group,inhibitor group and inhibitor treatment group.Ⅲ.Study on the differences of expression of SIRT1,PGC-1α and HMGB1 proteins in PQ poisoning model1.The mice were divided into control group and paraquat group with 6 mice in each group.Samples were collected 72 h after PQ administration.Pathological changes of lung tissue were observed by HE staining and lung injury score was given.The expression of SIRT1 in lung tissue was observed by immunohistochemistry.m RNA or protein expression levels of SIRT1,PGC-1α and HMGB1 in lung tissue of mice were detected by RT-q PCR and western blot.The distribution characteristics of PGC-1α and HMGB1 in lung tissues were observed by immunofluorescence method.2.The protein expression levels of SIRT1,PGC-1α and HMGB1 in A549 cells were observed 24 h after PQ exposuring by western blot method,and the expression changes and distribution of PGC-1α in A549 cells were observed by immunofluorescence method after 6h,12 h and 24 h PQ treatment.The reactive oxygen species(ROS)levels of A549 cells in normal control group and infected group were observed by DHE staining.Ⅳ.Study on the effect of PGC-1α-HMGB1 signal on acute lung injury caused by PQ poisoning1.The mice were divided into control group,paraquat group,SR-18292 control group and SR-18292 treatment group,with 6 mice in each group.SR-18292,a selective inhibitor of PGC-1α,was injected intraperitoneally once,and samples were collected after 72 hours of observation of PQ poisoning.Pathological changes of lung tissues were observed by HE staining and lung injury score was given.m RNA or protein expression levels of SIRT1,PGC-1α and HMGB1 in lung tissues of mice were detected by RT-q PCR and western blot.The distribution characters of SIRT1,PGC-1α and HMGB1 in lung tissues were observed by immunofluorescence method.Tissue homogenates were used to detect MDA level and SOD activity.Levels of IL-1β and IL-6 in serum were determined by ELISA.2.The reactive oxygen species(ROS)levels of A549 cells were observed by DHE staining.Flow cytometry FITC-PI staining was used to observe the apoptosis rate of poisoning A549 cells after PGC-1α knockout.The interaction between PGC-1α and HMGB1 was detected by immunoprecipitation.Ⅴ.The involvement of SIRT1 in PGC-1α-HMGB1 signaling mediated acute lung injury caused by PQ poisoning.1.The mice were divided into control group,paraquat group,EX527 control group and EX527 treatment group,with 6 mice in each group.SIRT1 selective inhibitor EX527 was injected intraperitoneally every 24 hours,pre-treated for 72 hours,and samples were collected after 72 hours of PQ poisoning observation.Pathological changes of lung tissues were observed by HE staining and lung injury score was given.The distribution characters of SIRT1,PGC-1α and HMGB1 in lung tissues were observed by immunofluorescence method.After knockdown PGC-1α by si RNA,the protein levels of SIRT1,PGC-1α and HMGB1 in A549 cells were observed 24 h after PQ exposure by western blot method.2.The protein expression levels of SIRT1,PGC-1α and HMGB1 in A549 cells were observed by western blot after 24 h pretreatment with EX527 and 24 h exposure to PQ.The expression and distribution of PGC-1α and HMGB1 in A549 cells treated with PQ for 24 h were observed by immunofluorescence method.The reactive oxygen species(ROS)levels of A549 cells were observed by DHE staining.Flow cytometry FITC-PI staining was used to observe the apoptosis rate of infected A549 cells after SIRT1 overexpression or PGC-1α knockout.Results: 1.HE staining was performed to observe the pathological manifestations of lung tissue injury in PQ group mice under the microscope,and the lesion score was performed.Compared with the Control group,the pulmonary alveolar structure and bleeding were significantly damaged in the PQ group.SIRT1 protein levels in lung tissue decreased,PGC-1α and HMGB1 protein levels increased.The m RNA levels of SIRT1 and PGC-1α in A549 cells decreased significantly at 6,12 and 24 h after paraquat exposuring,while the m RNA levels of HMGB1 increased with time.The protein levels of SIRT1,PGC-1α and HMGB1 increased significantly after poisoning.2.In the PQ group,lung tissue of mice showed alveolar collapse,intra-alveolar bleeding,thickening of alveolar wall,infiltration of a large number of inflammatory cells,and increased lung tissue injury score.However,the paraquat group treated with PGC-1αinhibitor reduced lung injury and injury score.By detecting MDA,the oxidative stress level of paraquat group was significantly increased,while the level in paraquat group treated with PGC-1α inhibitor was decreased.The levels of IL-1β and IL-6 in serum of mice were detected by ELISA.Compared with the normal group,their levels in the PQ group were significantly increased,while the levels in the poisoning group treated with PGC-1α inhibitor were decreased.DHE staining was used to detect ROS levels in A549 cells of each treatment group.Fluorescence microscopy showed that oxidative stress levels in the PQ group were significantly enhanced,while ROS levels in PGC-1αinhibitor treatment group and PGC-1α knockdown group were significantly lower than those in the PQ group.Flow cytometry showed that the apoptosis rate of the PQ group was significantly higher than that of PGC-1α knockdown followed by PQ poison.The immunoprecipitation experiment showed that there was an interaction between PGC-1αand HMGB1.3.Compared with paraquat exposured group,the mice treated with SIRT1 inhibitor EX527 had less lung injury,less alveolar hemorrhage and congestion,and lower injury score.The protein expression levels of SIRT1,PGC-1α and HMGB1 in lung tissue decreased after treatment with inhibitor,and the expression of PGC-1α in perinuclear decreased compared with the PQ group.In A549 cells,oxidative stress level in the PQ group was significantly enhanced,the level of ROS in EX527 treating group and PGC-1αknockdown group was significantly lower than that in the PQ group,and apoptosis rate was significantly lower than that in the PQ group.Conclusion:1.PGC-1α-HMGB1/ROS signaling pathway mediates acute lung injury and apoptosis of pulmonary epithelial cells induced by PQ poisoning in mice.2.Inhibition of SIRT1 can regulate PGC-1α-HMGB1/ROS signaling to improve acute lung injury and apoptosis of pulmonary epithelial cells induced by PQ.