Effect Of Aloperine On The Activation Of NLRP3 Inflammasomes In Mice With Acute Lung Injure And Its Mechanism

Objectives:Acute lung injury(ALI)is one of the critical illnesses with a high clinical fatality rate.The uncontrolled severe inflammation in the lung is one of the key pathological mechanisms leading to ALI.The study took ALI mice and lipopolysaccharide(LPS)-induced RAW264.7 cells as the research object to observe the effects of Alo on the activation of NLRP3 inflammasomes in the lung tissues of ALI mice and RAW264.7 cells,and preliminarily discussed its possible mechanism.Methods:In the in vivo experiment,the LPS(5 mg/kg)tracheal administration method was used to replicate the ALI mouse model.25 mice of C57BL/6 were randomly divided into 5 groups,namely Control group,ALI group,ALI+25 mg/kg Alo group,ALI+50 mg/kg Alo group,and ALI+100 mg/kg Alo group.Hematoxylin-eosin staining(HE)observe the morphological changes and inflammation pathological scores of lung tissue.Real-time quantitative polymerase chain reaction(RT-q PCR)discern the expression of NLRP3 m RNA and pro-caspase-1 m RNA in lung tissue.Western blotting discover the expression of NLRP3,pro-caspase-1 and caspase-1 p10 protein in lung tissue.Enzyme-linked immunosorbent assay(ELISA)perceive the content of IL-1β and IL-18 in the lung tissue to confirm that Alo can inhibit the activation of NLRP3 inflammasome in the lung tissue of mice with ALI.The in vitro experiment was carried out in two parts:In vitro experiment 1: we used LPS(100 ng/m L)and ATP(5 mmol/L)dual stimulation to induce RAW264.7 cells to establish NLRP3 inflammasome activation model.The experiment is divided into Control group,LPS+ATP group,LPS+ATP+50 μmol/L Alo group,LPS+ATP+100 μmol/L Alo group and LPS+ATP+200 μmol/L Alo group.MTT discern the viability of RAW264.7 cells.Colorimetric method discern the viability of LDH in the culture supernatant of RAW264.7 cells.RT-q PCR discern the expression of NLRP3 m RNA and procaspase-1 m RNA in RAW264.7 cells.Western Blot discern the expression of NLRP3,procaspase-1 caspase-1 p10,Nrf2 total protein and nuclear translocation in RAW264.7 cells.ELISA detects the content of IL-1β and IL-18 in the culture supernatant of RAW264.7 cells.It is clear that Alo can inhibit the activate the NLRP3 inflammasome of RAW264.7 cells.In vitro experiment 2: NLRP3 inflammasome activated cell model as the same as in vitro experiment 1,divided into Control group,LPS+ATP group,LPS+ATP+200 μmol/L Alo group,LPS+ATP+200 μmol/L Alo+ML385(5 μmol/L)group.RT-q PCR detects the expression of NLRP3 m RNA and pro-caspase-1 m RNA in RAW264.7 cells after adding Nrf2 blocker.Western Blot detected the expression of NLRP3,pro-caspase-1 and caspase-1 p10 protein in RAW264.7 cells after adding Nrf2 blocker.ELISA detected the content of IL-1β and IL-18 in the culture supernatant of RAW264.7 cells after adding Nrf2 blocker.It is clear that Alo can inhibit the activation of NLRP3 inflammasomes in RAW264.7 cells through the Nrf2 pathway.Results:In vivo experiments,HE results showed that Alo reduced significantly the lung tissue lesions of ALI mice.RT-q PCR results show that Alo can down-regulate the expression of NLRP3 m RNA and pro-caspase-1 m RNA in the lung tissue of ALI mice.Western Blot results show that Alo can down-regulate the expression of NLRP3,pro-caspase-1 and caspase-1 p10 protein in the lung tissue of ALI mice.ELISA results show that Alo can down-regulate the content of IL-1β and IL-18 in the lung tissue of ALI mice.In vitro experiment 1,RAW264.7 cells were used as the research object.MTT results showed that LPS can reduce the viability of RAW264.7 cells.Alo can reverse this effect,which is dose-dependent.The colorimetric results show that Alo can reduce the activity of LDH in the culture supernatant of RAW264.7 cells.RT-q PCR results showed that Alo can reduce the expression of NLRP3 m RNA and pro-caspase-1 m RNA in RAW264.7 cells.Western Blot results showed that Alo can reduce the expression of NLRP3,pro-caspase-1 and caspase-1 p10 protein in RAW264.7 cells.Alo can increase the expression of Nrf2 protein and promote nuclear translocation of Nrf2.ELISA results showed that Alo can reduce the expression of IL-1β and IL-18 in the culture supernatant of RAW264.7 cells.In vitro experiment 2,it can weaken the effect of Alo on NLRP3 m RNA and pro-caspase-1 m RNA after adding Nrf2 blocker ML385.And ML385 can weaken the effect of Alo on NLRP3,pro-caspase-1,and caspase-1 p10 protein.In addition,the ML385 can also weaken the effect of Alo which can down-regulation of IL-1β and IL-18.It shows that Alo can inhibit the activation of NLRP3 inflammasomes in RAW264.7 cells through the Nrf2 pathway.Conclusion:Alo inhibit the activation of NLRP3 inflammasomes to alleviate acute lung injury in mice,and its mechanism may be achieved through the Nrf2 pathway.