| Objective: Oral inflammation,including periodontitis and peri?implant disease,was caused by bacteria and accompanied by the destruction and absorption of alveolar bone.The principal etiological factor for these inflammatory diseases is plaque accumulation,and lipopolysaccharide(LPS)was the main toxic factor.LPS can induce a variety of cells to express cytokines,such as Interleukin 1β(IL-1β),Interleukin 6(IL-6),tumor necrosis factor-α(TNF-α)and nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome,which can significantly affect the development of periodontitis and peri-implantitis.NLRP3 inflammasome is the central link of chronic inflammation.Thioredoxin interacting protein(TXNIP)is involved in the activation of NLRP3 inflammasome,which play an important role in the pathogenesis of LPS-induced inflammation.N-acetyl cysteine(NAC)contains active sulfhydryl groups that control inflammation.The aim of this study was to investigate the regulatory effect of NAC on LPS-induced bone marrow mesenchymal stem cells(BMSCs)inflammatory response and to clarify the underlying molecular mechanism.Methods: BMSCs were cultured in DMEM medium containing 10% fetal bovine serum.Firstly,in order to investigate the effects of different concentrations of NAC on BMSCs viability,the cells were divided into five groups: blank control group,0.1 m M NAC,0.5 m M NAC,1 m M NAC and 2m M NAC(the 5 groups were treated with the right concentration of NAC,respectively).After 24 h,the viability of BMSCs cells was detected by CCK-8method.Subsequently,in order to investigate the protective effect of NAC on BMSCs,the cells were randomly divided into 6 groups: blank control group,LPS(1 μg/m L)group,the NAC(0.1 m M)+ LPS(1 μg/m L)group,the NAC(0.5 m M)+ LPS(1 μg/m L)group,the NAC(1 m M)+ LPS(1 μg/m L)group and the NAC(2 m M)+ LPS(1 μg/m L)group.Blank control group were cultured in DMEM medium containing 10% fetal bovine serum.LPS group were stimulated with LPS for 24 h,other groups respectively corresponding to the concentration of NAC pretreatment for 1 h before LPS stimulation for 24 h.Finally CCK 8 method was adopted to detect cell viability.Finally,in order to investigate the effect of NAC on the expression of cytokines in bone marrow mesenchymal stem cells induced by inflammation and the related mechanisms,BMSCs were randomly divided into 4 groups: blank control group,NAC(1m M)+LPS(1 μg/m L)group(NAC pretreatment for 1 h,LPS stimulation for 24h),resveratrol(Res)(50 μm)+LPS(1 μg/m L)group(Res pretreatment for 2 h,LPS stimulation for 24 h),LPS(1 μg/m L)group(only LPS stimulation for 24h).In order to investigate the effect of NAC on the secretion of inflammatory cytokines,the levels of IL-1β,IL-6 and TNF-α in BMSCs cells were detected by ELISA.In order to investigate the associated anti-inflammatory mechanism of NAC,the m RNA expression levels of ASC,NLRP3,caspase-1,TXNIP and TRX in BMSCs were detected by real-time fluorescence quantitative(RT?)PCR.The protein levels of ASC,NLRP3,caspase-1,TXNIP and TRX in BMSCs cells were detected by western blot.Results:1.To investigate the effects of different concentrations of NAC on the viability of BMSCs,the cells were pretreated with various concentrations of NAC.The results of CCK-8 showed that,compared with blank control group,there is no significant difference among groups under the stimulation of various concentrations of NAC(0.1 m M,0.5 m M,1 m M,2 m M)(P>0.05),which indicating that NAC had no toxic effect on BMSCs.2.To explore the protective effect of NAC on BMSCs,the results of CCK-8showed that,compared with the blank control group,the cell viability of LPS(1 μg/m L)group,NAC(0.1 m M)+LPS(1 μg/m L)group and NAC(0.5m M)+LPS(1 μg/m L)group decreased,and the difference was statistically significant(P<0.05).Compared with the LPS group,the cell viability of NAC(0.5 m M)+LPS(1 μg/m L)group,NAC(1 m M)+LPS(1 μg/m L)group and NAC(2 m M)+LPS(1 μg/m L)group increased.As the growth of NAC concentration from 0 to 1 m M,the proliferation rate increased;while a sharp increase of concentration cannot bring a significant difference correspondingly when it comes to 1 to 2 m M(P>0.05).The variations in the proliferation rate indicated that the NAC concentration of 1 m M played the strongest role in the proliferation rate of BMSCs.3.The inflammatory mediators in response to NAC were detected by ELISA kit,to investigate whether NAC could restrain the inflammation in BMSCs induced by LPS.BMSCs of LPS group showed a high expression of IL-1β,IL-6 and TNF-α,compared to the control group(P<0.05);however,the pretreatment of NAC for 1 h led to a significantly decrease of the inflammatory mediators(P<0.05);the Res + LPS group brought out the similar result with NAC + LPS group(P>0.05).4.To further identify the potential molecular mechanism of the anti-inflammatory effect of NAC on LPS-induced BMSCs,RT-q PCR were adopted to evaluate the expression levels of m RNA in LPS-mediated BMSCs.According to the results of RT-q PCR,it was noticed obviously that,compared with other groups,the LPS group(P<0.05)possessed the highest expression of m RNA,such as ASC,NLRP3,caspase-1 and TXNIP,but the lowest of TRX;however,NAC could reverse the effect of LPS on BMSCs(P<0.05).In addition,BMSCs were pretreated with the signaling pathway inhibitor(Res),got the same result with NAC(P>0.05).5.Western blotting analysis were adopted to evaluate the expression levels of protein in LPS-mediated BMSCs.Western blotting analysis of protein expression levels displayed a highly consistent with the m RNA results.Briefly,pretreatment with NAC or Res could decrease the expression of ASC,NLRP3,caspase-1 and TXNIP,but increase TRX,compared with cells only exposed to LPS.Conclusion:1.NAC could reduce the secretion of inflammatory factors,such as IL-1β,IL-6 and TNF-α,which induced by LPS.2.LPS could up-regulate the expression of ASC,NLRP3,caspase-1 and TXNIP,but down-regulate the expression of TRX.While NAC could down-regulate the expression of ASC,NLRP3,caspase-1 and TXNIP,and up-regulate the expression of TRX.The pretreatment of NAC could reverse the effect of LPS.3.NAC showed an anti-inflammatory effect on LPS-stimulated BMSCs,and associated with the TXNIP/NLRP3 signaling pathway. |
