| Objectives In this study,lipopolysaccharide(LPS)-induced acute lung injury(ALI)model in mice was constructed to observe the impact of germacrone(GM)on the degree of lung injury,and to evaluate its effects on macrophage transformation and the activity of NOD-like receptor protein 3(NLRP3)inflammasome.Methods(1)The MTT experiment was used to determine the maximum non-toxic dose of germacrone for macrophage cell line ANA-1,which was used in the subsequent experiments.(2)The cultured ANA-1 cells were divided into control,LPS,IL-4 and LPS+GM groups.In order to observe the effect of germacrone on M1/M2transformation,the contents of IL-12 and IL-10 in different groups were detected by using ELISA,and mRNA expression levels of iNOS and CD206 were detected by real-time quantitative PCR.(3)ANA-1 cells were divided into control group,LPS+Nigericin group and GM+LPS+Nigericin group.In order to observe the effect of geminone on the activity of NLRP3 inflammasome,the levels of NLRP3,ASC(apotosis-associated speck-like protein containing a CARD),caspase-1 and IL-1?were measured by Western blot in three groups of cells.(4)32 C57BL/6 mice were randomly divided into control group,acute lung injury model group(ALI group),Germacrone group(GM Group)and NLRP3 overexpression group(pc-NLRP3group).HE staining was used to observe the success of ALI model.Western blot was used to verify the success of NLRP3 gene overexpression.(5)HE staining was used to observe the lung tissue damage in the Control group,ALI group and GM group.(6)The expression of CD16/32,CD206 and F4/80 in BALF of Control group,ALI group and GM group were detected by flow cytometry.the mRNA expression levels of iNOS and CD206 were detected by real-time quantitative PCR.(7)The MPO activity and wet/dry weight ratio of lung tissues of four groups of mice were determined.The levels of TNF-?,IL-1?and IL-6 in bronchoalveolar lavage fluid(BALF)of mice were detected by ELISA.Results(1)The MTT experiment determined that the maximum concentration of was2?M.(2)After the LPS-induced ANA-1 cells were treated with germacrone,the content of IL-12 and the m RNA expression of iNOS decreased(P<0.05,P<0.01),while the content of IL-10 and the mRNA expression of CD206 increased(P<0.01),suggesting that germacrone can accelerate the conversion of macrophages type from M1 to M2.(3)Western blot results showed that the expression levels of NLRP3 and ASC were increased in the LPS+nigericin group when compared to the control group(P<0.001),and the expression levels of mature Caspase-1 and IL-1?also increased(P<0.001).This indicates that the stimulation was effective and the cells had an inflammatory response.Compared to the LPS+nigericin group,the expression of the above-mentioned proteins was decreased in the GM+LPS+nigericin group(P<0.001,P<0.01,P<0.001,P<0.01),indicating that germacrone inhibited the expression of them.(4)HE staining showed that the damage of mice in the ALI group were more severe than the control group,indicating the modeling was successfully constructed.Compared with the ALI group,the GM group had less damage than the ALI group,which indicated that gestion had a therapeutic effect on acute lung injury.Western blot results showed that the expression of NLRP3 in lung tissue of pc-NLRP3 group was increased than control group(P<0.001),which verified the successful overexpression of NLRP3.(5)Flow cytometry results showed that the proportion of macrophage F4/80~+CD16/32~+cells in BALF of GM group was lower than ALI group(P<0.05),while the proportion of F4/80~+CD206~+cells in GM group was higher than ALI group(P<0.05).Real-time quantitative PCR results showed that the mRNA expression of macrophage iNOS in BALF of GM group was lower than that of ALI group(P<0.05),while the mRNA expression of CD206 was increased(P<0.001).This indicates that the proportion of M1 type macrophages decreased and the proportion of M2 type increased after treatment with germacrone.(6)The inflammatory index test results of the four groups of mice showed that the proportion of wet weight and MPO activity in lung tissue increased after LPS stimulation(P<0.05),and the level of TNF-?,IL-1?,and IL-6 in BALF was increased significantly(P<0.001),indicating that the mice had an inflammatory response after LPS stimulation;the above indicators were decreased(P<0.05)in ALI group when compared to the GM group,indicating that the treatment of germacrone is effective;pc-NLRP3 group had no significant differences in the above indicators compared to the ALI group(P>0.05),indicating that the therapeutic effect of germacrone was inhibited when NLRP3 was overexpressed.Conclusion Our experiments confirmed that germacrone can significantly reduce LPS-induced injury in ALI mice.This effect may be achieved by promoting the transformation of macrophage M1 to M2 typing and inhibiting the activation of NLRP3 inflammasome. |
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