| Objective: In order to study the protective effects of essential oils of Schizonepeta tenuifolia Briq.(EOST)and pulegone on endotoxin poisoning mice induced by LPS,and the anti-inflammatory mechanism of EOST and pulegone via NLRP3 inflammasome pathway was explored to improve the anti-inflammatory mechanism of EOST,and revealing its main material basis.Methods:(1)Extraction and analysis of the chemical constituents in EOST: EOST was extracted according to the fourth general rule 2204 of Chinese Pharmacopoeia(2015),and the relatively content of each compound in Schizonepeta essential oils was measured using GC-MS.(2)Screening the best optimal dose and detection time of endotoxin poisoning mice induced by LPS: Mice were intraperitoneally injected with different doses of LPS,then the changes of respiratory rate,pleen index,thymus index,lung index,the level of IL-1β in serum and NLRP3 protein expression in lung were observed at different time points to establish the best optimal dose and detection time of endotoxin poisoning mice induced by LPS.(3)The protective effect of drugs on endotoxin poisoning mice induced by LPS:(1)the effects of drugs on white blood cell(WBC)count and platelet count(PLT)in blood,organ index and pathological features of lung tissue in model mice were observed.(2)the level of IL-18,IL-1β,IL-5,TNF-α,IFN-γ,monocyte hemoattractant protein-1(MCP-1),macrophages and macrophages(MIP-1β),macrophage colony-stimulating factor(M-CSF),granulocyte-macrophage colony-stimulating factor(GM-CSF),and IL-10 were measured by ELISA and liquid protein microarray.(3)the level of NO in lung tissue was determined by Griess method.(4)Anti-inflammatory mechanism of drugs via NLRP3 inflammasome pathway:(1)the expression of NLRP3,ASC,caspase-1,IL-1β,p65,i NOS,TNF-α,STAT1,IFN-α,and IFN-β m RNA in lung tissue were detected by real-time fluorescent quantitative PCR.(2)The expression of P2X7 R and cathepsin B in lung tissue was detected by immunohistochemistry.(3)the expression of NLRP3,ASC,Pro-caspase-1,Caspase-12,COP1,POP1,Caspase-1(p20),and pro-IL-1β protein in lung tissue were detected by Western blotting.Results:(1)The results determined by GC-MS showed that the relative content of menthone and pulegone are 46.67% and 33.92%,respectively,and the content of the two compounds contributed the 80.59% of total essential oils.(2)LPS was intraperitoneally injected at 15 mg/kg.After LPS injection 12 h,the respiratory rate was accelerated,the content of IL-1β in serum,the WBC,the lung index,and spleen index were significantly increased.The expression of NLRP3 protein in lung tissue was higher,so the LPS dosage and detection time were used as the conditions to induce endotoxin poisoning mice.(3)EOST and pulegone have a trendency to suppress the significantly increased WBC,PLT,spleen index,and the decreased thymus index,but there were no statistically difference.Dexamethasone could significantly reduce spleen index and thymus index.(4)EOST could decrease the level of IL-5,TNF-α,MCP-1,MIP-1β,M-CSF,and IFN-γ in serum.In addition,the 0.452 g/kg EOST decreased the level of IL-18 and GM-CSF in serum.Pulegone could decrease the level of IL-18,IL-1β,IL-5,MIP-1β,and M-CSF in serum,and 0.19 g/kg pulegone decreased the levels of TNF-α,MCP-1,and GM-CSF in serum.0.226 g/kg EOST and 0.19 g/kg pulegone significantly reduced the number of neutrophils in lung,thus reducing the infiltration of acute inflammatory cells in lung tissue.0.452 g/kg EOST and 0.19 g/kg pulegone significantly reduced the level of NO in lung tissue.(5)EOST could down-regulate the expression of NLRP3,i NOS,p65,IL-1β,and STAT1 m RNA in lung tissue,and it also inhibited the expression of IFN-β and IFN-α m RNA in lung tissue.The expression of Cathepsin B and p20 protein in lung tissue can be down-regulated by EOST and pulegone,and they also increased the expression level of COP1 protein.EOST could inhibit the expression of P2X7 R and NLRP3 protein,and pulegone inhibited the expression of pro-IL-1β protein in lung tissue.Conclusion: EOST and pulegone exhibited good anti-inflammatory and protective effects on endotoxin poisoning mice induced by LPS,which could inhibit the release of inflammatory cytokines and reduce the infiltration of inflammatory cells.The anti-inflammatory mechanism was related to the intervention and regulation of the activation of NLRP3 inflammasome,and plugone was one of the active components with anti-inflammatory effects in EOST. |
