| Objective: Paraquat(PQ)is a highly effective and inexpensive herbicide.It can be rapidly deactivated after contact with soil,and there are few residues left in plants.If used appropriately,PQ has high safety and less environmental pollution.The above advantages are conducive to the sustainable agriculture.However,PQ poisoning caused by accidental ingestion or deliberate suicide is common in clinical setting.It is a common one of oral pesticide suicide accidents worldwide,with a high fatality rate more than 50% after severe poisoning.As the mechanism of PQ poisoning is still unknown and there is no specific antidote,its harm to people after poisoning is not negligible.PQ poisoning can lead to multiple organ dysfunction,and the lungs are the most serious.Acute lung injury(ALI)and acute respiratory distress syndrome(ARDS)are the main manifestations of PQ poisoning in the early stage,which are also the main cause of death in the early stage of poisoning.It is believed that oxidative damage,immune response and inflammation play a pivotal role in PQ-induced ALI,but the specific mechanism is unknown.PQ can cause DNA damage.Normally,DNA in eukaryotic cells only stays in the nucleus and mitochondria.DNA is the carrier of genetic information and is crucial to the vital life activities.After cell damage,the leakage of genetic material leads to the increase of “mislocated” self-DNA.self-DNA,as a kind of DAMPs,is an important initiating factor of immune and inflammatory response,and can lead to pulmonary inflammatory injury.It is recognized that mt DNA,a kind of self-DNA,played a vital role in the pathogenesis of PQ poisoning.The c GAS-STING signaling pathway,as a self-DNA sensing,mediates immune and inflammatory diseases,and has recently attracted the attention of scholars.Ursolic acid(UA),a pentacyclic triterpenoid,has the effects of anti-inflammatory,antioxidant,protecting mitochondrial function,DNA repairing,and et al,and can reduce LPS-induced mice ALI and septic lung injury in rats.Our study aimed to investigate whether UA could alleviate PQ-induced ALI,and to provide the evidence that UA alleviates PQ-induced ALI by inhibiting the c GAS-STING pathway.Methods: 1.Establishment of animal model C57BL/6J mice(SPF grade,male,weight 18-22g)was intraperitoneally injected PQ 30mg/kg to establish an ALI model.2.Establishment of cell model RAW264.7 cells were cultured in high-glucose DMEM containing 10% heat-inactivated fetal bovine serum.Cells were treated with PQ with different concentrations,and the optimal concentration of PQ used for experiment was screened according to the results of CCK8 assay.3.The STING-IRF3 pathway mediates PQ-induced ALI 3.1.Mouse samples were collected at 2,12,24 and 48 h after PQ administration.MDA and SOD assay were used to assess pulmonary oxidative damages.The m RNA or protein expression levels of STING,IRF3 and IFN? in lung tissues or serum were detected by RT-q PCR,western blot and ELISA.3.2.Mice were injected with dexamethasone(5 mg/kg,i.p.)at 1 h after PQ administration,and samples were collected at 48 h after PQ administration.Histopathological changes of lung tissues were observed by HE staining,and the W/D ratio of lung tissues was calculated to evaluate the degree of pulmonary edema.The expression of STING and IRF3 in lung tissues was observed by immunohistochemistry.The expression levels of Sting,Irf3 and Ifn? m RNA or protein in lung tissue or alveolar lavage fluid were detected by RT-q PCR,western blot and ELISA.3.3.si RNA was targeted to silence the expression of Sting and Irf3 in RAW264.7 cells.Immunofluorescence,RT-q PCR,western blot and ELISA were used to observe the m RNA or protein expression levels of STING,IRF3 and IFN? in RAW264.7 cells or culture supernatant.4.TBK1 plays an important role in the STING-IRF3 pathway mediating PQ-induced ALI 4.1.Mice were administered with amlexanox(100 mg/kg,once a day)by gavage,beginning at three days prior to the PQ injection,and samples were collected at 72 h after PQ administration.HE staining and alveolar lavage fluid protein concentration were used to assess lung injury.ROS detection was used to assess lung oxidative damage.Immunohistochemical method was used to observe the expression of NF-?Bp65 and RF3 in lung tissue.Western blot was used to detect the expression of STING,TBK1,p TBK1 in cytoplasmic proteins and NF-?Bp65 and IRF3 in nucleus proteins of lung tissues.RT-q PCR and ELISA were used to detect Il-1? and Ifn? m RNA or protein level of lung tissues or alveolar lavage fluid.4.2.CCK8 assay was used to screen the optimal concentration of amlexanox on RAW264.7 cells.The oxidative damage of RAW264.7 cells was assessed by ROS detection.The expression of NF-?Bp65 and IRF3 in RAW264.7 cells was observed by immunohistochemical method.The expression of STING,TBK1,p TBK1 in cytoplasmic proteins and NF-?Bp65 and IRF3 in nucleus proteins of RAW264.7 cells were detected by western blot.The level of Il-1? and Ifn? m RNA or protein in RAW264.7 cells or culture supernatant were detected by RT-q PCR and ELISA.5.UA alleviates PQ-induced ALI by inhibiting the c GAS-STING pathway 5.1.Mice were administered with UA(50 mg/kg,once a day)by gavage beginning at one day prior to the PQ injection,and samples were collected at 72 h after PQ administration.Mouse weights were measured.Lung tissue pathology was assessed by HE staining.Pulmonary edema was evaluated by W/D ratio calculation.Lung oxidative damage was observed by DHE staining.The levels of ds DNA in alveolar lavage fluid and serum was detected.The source of ds DNA was identified by real-time PCR.The expression levels of c GAS,STING,TBK1,p TBK1,IRF3,and p IRF3 of lung tissues were detected by western blot.The secretion level of IFN? in alveolar lavage fluid and serum was detected by ELISA.5.2.The optimal concentration of UA on RAW264.7 cells was screened by CCK8 assay.The oxidative damage of RAW264.7 cells was observed by DHE staining.The levels of ds DNA in cell culture supernatant was detected.The source of ds DNA was identified by real-time PCR.The expression levels of c GAS,STING,TBK1,p TBK1,IRF3,and p IRF3 of RAW264.7 cells were detected by western blot.The secretion level of IFN? in cell culture supernatant was detected by ELISA.Results: 1.PQ induced lung histopathological changes and increased W/D ratio in mice,increased expression levels of STING,IRF3 and IFN? in lung tissue and RAW264.7 cells,and induced the translocation of STING and IRF3.Dexamethasone reduced the expression evels of STING,IRF3 and IFN? in lung tissues of PQ-poisoned mice,and inhibited the translocation of STING and IRF3.Targeted silencing Sting of RAW264.7 cells can inhibit the activation of IRF3 and reduce IFN? secretion.Targeted silencing Irf3 of RAW264.7 cells had no significant effect on STING,but could inhibit IFN? secretion.2.After PQ administration,the level of p TBK1/TBK1 increased in mouse lung tissues and RAW264.7 cells,occurring with the activation of the STING-IRF3 pathway.Amlexanox,a TBK1 inhibitor,inhibited the activation of TBK1,NF-?Bp65 and IRF3,reduced the secretion of IL-1? and IFN?,reduced the pathological damage of PQ-poisoned mouse lungs,and reduced the protein concentration in alveolar lavage fluid,but had no significant effect on the activation of STING.3.After PQ administration,the expression of ds DNA(including n DNA and mt DNA)increased in alveolar lavage fluid,serum and cell culture supernatant,the expression levels of c GAS,STING,p TBK1/TBK1,p IRF3/IRF3 in lung tissue and RAW264.7 cells increased,and the secretion level of IFN? in alveolar lavage fluid,serum and cell culture supernatant increased.UA effectively alleviated the above conditions,reduced the pathological damage of PQ-poisoned mouse lungs,and alleviated the weight loss in mice.Conclusion: 1.The STING-IRF3 pathway mediated PQ-induced ALI.2.TBK1 played an important role in the activation of the STING-IRF3 pathway in PQ-induced ALI.Amlexanox,a TBK1 inhibitor,can alleviate PQ-induced ALI.3.ds DNA(including mt DNA and n DNA)played a vital role in PQ-induced ALI.The c GAS-STING signaling pathway contributed to PQ-induced ALI.UA could reduce ds DNA secretion,inhibit activation of the c GAS-STING signaling pathway,and alleviate PQ-induced ALI. |
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