| Objective:To study the anti-inflammatory effects of essential oils of Schizonepeta tenuifolia Briq.（EOST）and its main component pulegone on the mechanisms of NLRP3 inflammasome activation in THP-1 cell,to further improve the anti-inflammatory mechanisms of EOST related to pattern recognition rceptors（PRRs）.Methods:The chemical constituents of EOST were determined by GC-MS.Toxicity of EOST was detected by MTS.THP-1 cells were differentiated into macrophages with PMA,then treated with LPS and activators（ATP,Nigericin,CPPD or CaCl2）to activate NLRP3 inflammasome.To investigate the effects of EOST on NLRP3inflammasome activation.（1）The level of IL-1β,IL-18,Caspase-1（p20）and Pro-IL-1βin supernatants were measured by ELISA.（2）RT-PCR was performed to observe the mRNA expression of NLRP3,Caspase-1,IL-1β,IL-1αand IL-6.（3）The NLRP3,ASC,Pro-Caspase-1 and Pro-IL-1βprotein expression in cell lysate were detected by Western Blot.（4）The level of ROS in cell was determined by DCFH-DA.The protein expression of Cathepsin B,NLRP3 and ASC were detected by immunofluorescence.Results:（1）The relative content of pulegone in EOST was 33.40%.（2）The maximum non-toxic concentration of EOST and pulegone were 0.4mg/m L.（3）0.2mg/m L EOST and 0.2mg/m L pulegone could reduce the level of IL-1βinduced by ATP,Nigericin,CPPD or CaCl2（P<0.05 or P<0.01）,decrease the expression of Pro-IL-1βin supernatants induced by ATP or Nigericin（P<0.05 or P<0.01）.0.2mg/mL EOST could also reduce the level of IL-18 in supernatants induced by ATP or Nigericin（P<0.05 or P<0.01）.（4）0.2mg/m L EOST and 0.2mg/m L pulegone could inhibit the mRNA expression of NLRP3,Caspase-1,IL-1β,IL-1αand IL-6 in THP-1macrophage-like cells induced by ATP（P<0.05 or P<0.01）,reduce mRNA expression of Caspase-1,IL-1β,IL-1αand IL-6 induced by Nigericin（P<0.05 or P<0.01）.0.2mg/m L EOST could also down-regulate the mRNA expression of NLRP3 induced by Nigericin（P<0.05）.（5）The serection of Caspase-1（p20）induced by Nigericin in supernatant of THP-1 macrophage-like cells was decreased by 0.2mg/m L EOST and0.2mg/m L pulegone（P<0.05 or P<0.01）,.2mg/mL pulegone could also suppress the level of Caspase-1（p20）induced by ATP in supernatant of THP-1 macrophage-like cells（P<0.05）.0.2mg/mL EOST and 0.2mg/mL pulegone could reduce the expression of Pro-IL-1βin cell lysates induced by ATP or Nigericin（P<0.05）.（6）0.2mg/mL EOST and 0.2mg/mL pulegone could decrease the relative co-localization area of NLRP3 and ASC protein induced by ATP（P<0.01）,down-regulate the level of ROS induced by ATP or Nigericin（P<0.05 or P<0.01）,as well as the expression of Cathepsin B protein induced by CPPD（P<0.01）.Conclusion:EOST and pulegone could reduce the level of IL-1βin supernatant of THP-1 macrophage-like cells induced by ATP,Nigericin,CPPD or CaCl2.Its anti-inflammatory mechanism was probably related to inhibit the activation of NLRP3inflammasome.Pulegone had a similar effect of EOST,it could be considered as one of the effective components of EOST.The results of the study further enriched the modern pharmacological research of anti-inflammatory effect of EOST,and provided a more scientific basis for the clinical application of Schizonepeta tenuifolia to treat"exterior syndromes". |
