| Objective: To investigate the effect of corydalis saxicola bunting total alkaloid(CSBTA)on macrophage scorching and its possible mechanism of action,in order to provide some theoretical basis for the treatment of periodontitis with the use of traditional Chinese medicine.Methods: 1.THP-1 cells were induced to differentiate into macrophages by phorbol ester for 48 hours.Morphological change of the cells was observed using inverted microscope.Flow cytometry was applied to detect cell markers CD11 b and CD14,as well as the major marker of macrophages,which identify whether THP-1 cells are successfully induced into macrophages.2.1 μg/m L Pg-LPS was added to macrophages for 24 h and 5 m M adenosine triphosphate for 30 min,so as to establish the macrophages pyroptosis model.(1)Control group;(2)Pyroptosis model group(1 μg/m L Pg–LPS+5 m M ATP).Morphological change of the cells was observed using scanning electron microscope.The Pyroptosis rate of macrophages cell were evaluated by Inverted fluorescence Microscope.The m RNA expression of NLRP3,caspase-1,GSDMD was detected by RT-q PCR.The protein expression level of NLRP3,caspase-1,GSDMD was analyzed by western blotting.3.The CCK8 kit was used to detect the relative survival rate of macrophages under the intervention of CSBTA at a concentration of 2.5 mg/L,5mg/L,and 10 mg/L,and screen out the best inhibition of macrophage pyroptosis.4.The corydalis saxicola bunting total alkaloid was added to the macrophage pyrodeath model for intervention.(1)Pyroptosis Model group(1μg/m L Pg–LPS+5 m M ATP);(2)Negative control group(DMSO+1 μg/m L Pg–LPS+5 m M ATP);(3)CSBTA group(CSBTA+1 μg/m L Pg–LPS+5 m M ATP);(4)Positive group(Ac-YVAD-cmk+1 μg/m L Pg–LPS+5 m M ATP).The effects of CSBTA on pyroptosis in macrophages cell line were evaluated by Inverted fluorescence Microscope.The m RNA expression of NLRP3,caspase-1,GSDMD was detected by RT-q PCR.The protein expression level of NLRP3,cleave-caspase-1、cleave-GSDMD was analyzed by western blotting.Results: 1.After induction of THP-1 cells by 50 ng/m L PMA,the cells changed from a suspended state to an adherent state,and morphology changed from a spherical shape to a spindle shape.The cell surface was smooth with clear borders and no obvious tentacles.Meanwhile,the expression levels of CD14 and CD11 b in macrophages cells significantly increase than THP-1 cells(P < 0.05).2.The macrophages in the control group were mostly spindle-shaped and relatively small in size,while the macrophages in the pyroptosis model group were significantly swollen,enlarged,with ruptured cell membranes and cytoplasmic protrusions on the cell surface by Inverted fluorescence Microscope.The cell pyroptosis rate of the pyroptosis model group was significantly higher than that of the control group(P < 0.05).NLRP3,caspase-1,GSDMD m RNA in the pyroptosis model group was significantly higher than that in the control group(P < 0.05).The secretion of IL-1β and IL-18 in the supernatant in pyroptosis model group was significantly higher than that in the control group(P < 0.05).The protein expression of NLRP3,cleave-caspase-1 and cleave-GSDMD was significantly higher than that in the control group(P <0.05).3.The relative survival rate of macrophages was significantly higher than that of the pyroptosis model group after 24 h of intervention at concentrations of2.5 mg/L,5 mg/L and 10 mg/L CSBTA,and the relative survival rate of the cells was the highest when the concentration of CSBTA was 5 mg/L by CCK8 kit.4.After pretreated with 5 mg/L CSBTA for 24 h,(1)Compared with the pyroptosis model group,the cell pyroptosis rate of macrophages was significantly reduced in the CSBTA group and the positive control group(P <0.05),but there was no significant change in the negative control group(P >0.05).Compared with the CSBTA group,the cell pyroptosis rate was no significant change in the positive control group(P > 0.05),but was significantly lower than that the negative control group(P < 0.05).(2)Compared with the pyroptosis model group,the m RNA and protein expression levels of NLRP3,caspase-1 and GSDMD of macrophages was significantly reduced in the CSBTA group and the positive control group(P < 0.05),but there was no significant change in the negative control group(P > 0.05).Compared with the CSBTA group,the m RNA and protein expression levels of NLRP3,caspase-1 and GSDMD of macrophages was no significant change in the positive control group(P > 0.05),but was significantly lower than that of the negative control group(P < 0.05).(3)Compared with the pyroptosis model group,the secretion of inflammatory factors IL-1β and IL-18 was no significant change in the pyroptosis model than that the negative control group(P > 0.05),while the CSBTA group and the positive control group was significantly reduced(P <0.05).Compared with the CSBTA group,the expression of IL-1β and IL-18 was no significant change positive control group(P > 0.05),but were significantly lower than those in the negative control group(P < 0.05).Conclusion: 1.THP-1 cells could be successfully induced into macrophages after induced by 50 ng/m L PMA.2.After induced by 1 μg/m L Pg-LPS for 24 h,macrophages could be induced pyroptosis,and the model of macrophage pyroptosis was successfully established.3.CSBTA can significantly inhibit the pyroptosis of macrophages,which exhibits a pyroptosis inhibitory effect similar to that of the inhibitor of NLRP3/Caspase-1 inflammasome. |
PDF Full Text Request