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Activation of NLRP1 and NLRP3 inflammasomes contributed to cyclic stretch-induced pyroptosis and release of IL-1β in human periodontal ligament cells - PubMed

  • ️Fri Jan 01 2016

Activation of NLRP1 and NLRP3 inflammasomes contributed to cyclic stretch-induced pyroptosis and release of IL-1β in human periodontal ligament cells

Dan Zhao et al. Oncotarget. 2016.

Abstract

Inflammasomes have been reported to be present in periodontal inflammatory tissue, but the exact role of inflammasomes in periodontal inflammatory reactions especially those related to mechanical stimulations has not been clarified. In this study, it was shown that cyclic stretch activated the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 1 and 3 (NLRP1 and NLRP3) inflammasomes and induced the release of IL-1β and pyroptosis via a caspase-1-related mechanism in human periodontal ligament cells (HPDLCs). This study firstly demonstrated that activation of NLRP inflammasomes contributed to the stretch-induced inflammatory response in HPDLCs. As inflammasomes have been reported to be involved in both programmed cell death and inflammation, further studies are required to elucidate the exact roles and signaling pathway of inflammasomes in stretch-induced periodontal inflammation.

Keywords: IL-1β; cyclic stretch; human periodontal ligament cell; inflammasome; pyroptosis.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. NLRP1 and NLRP3 expressed in HPDLCs in response to cyclic stretch

(A) Real-time PCR result of NLRP1 in HPDLCs showed no significant difference among different groups, while the results of NLRP3 showed that its mRNA increased in response to 6 h cyclic stretch significantly and decreased to the control level after 24 h stretch. (B) Western blot analysis revealed that the expressions of NLRP1 and NLRP3 increased in response to 6 h cyclic stretch. Expression of NLRP3 further increased in response to 24 h cyclic stretch. The expression of NLRP1 decreased to the control level after 24 h cyclic stretch. GAPDH was used as a loading control. The results were quantified from at least 3 independent experiments and expressed as mean ± S.E. Statistical significance was calculated using one-way ANOVA with multiple comparisons. *P < 0.05 versus control group; #P < 0.05 versus 6 h group.

Figure 2
Figure 2. Cyclic stretch induced the activation and release of IL-1β in HPDLCs

(A) Real-time PCR result revealed that the expression of IL-1β mRNA increased in response to 6 h cyclic stretch and was reduced after 24 h cyclic stretch. (B) Western blot analyses showed that 6 h cyclic stretch increased the expressions of pro-IL-1β and mature IL-1β proteins. GAPDH was used as a loading control. (C) ELISA assay verified the appearance of IL-1β in the cell-culture medium of HPDLCs in response to 1, 2, 4, 6, 12 and 24 h cyclic stretches and showed that both 4 and 6 h stretches stimulated the release of IL-1β. The appearance of IL-1β in the cell-culture medium of HPDLCs reached a peak after 6 h stretch and then decreased to the control level after 12 h stretch. The results were quantified from at least 3 independent experiments and expressed as mean ± S.E. Statistical significance was calculated using one-way ANOVA with multiple comparisons. *P < 0.05 versus control group; #P< 0.05 versus 6 h group.

Figure 3
Figure 3. Cyclic stretch activated caspase-1 and -5 in HPDLCs

(A) Real-time PCR result showed that the expressions of caspase-1 and -5 mRNA in human PDLCs increased in response to 6 h cyclic stretch and decreased after 24 h cyclic stretch. (B) Western blot analysis showed that 6 h cyclic stretch increased the expressions of pro-caspase-1, caspase-1 (p20), pro-caspase-5 and caspase-5 (p20). The expressions decreased to the control level after 24 h cyclic stretch. GAPDH was used as a loading control. (C) The activities of caspase-1 and -5 increased substantially after 6 h cyclic stretch. The results were quantified from at least 3 independent experiments and expressed as mean ± S.E. Statistical significance was calculated using one-way ANOVA with multiple comparisons. *P < 0.05 versus control group; #P < 0.05 versus 6 h group.

Figure 4
Figure 4. Cyclic stretch promoted the formation and activation of inflammasome in HPDLCs

(A) Real-time PCR result showed that ASC mRNA did not change significantly in response to cyclic stretch. (B) Western blot analysis showed that 6 h cyclic stretch increased the expression of ASC, and 24 h cyclic stretch reduced its expression. GAPDH was used as a loading control. (C) The assembly of inflammasome was detected using immunoprecipitation (IP) with anti-ASC Ab followed by immunoblotting (IB) for caspase-1. The results were quantified from at least 3 independent experiments and expressed as mean ± S.E. Statistical significance was calculated using one-way ANOVA with multiple comparisons. *P < 0.05 versus control group; #P < 0.05 versus 6 h group.

Figure 5
Figure 5. Cyclic stretch-induced expression of IL-1β and pyroptosis were caspase-1 dependent

(A) The addition of the caspase-1 inhibitor significantly inhibited the appearance of IL-1β in the culture medium of the cyclic stretched cells, compared with the non-inhibited 6 h stretched cells. (B) The pyroptotic rate of HPDLCs in response to 6 h cyclic stretch increased significantly. While the addition of the caspase-1 inhibitor significantly inhibited the pyroptotic rate in response to 6 h cyclic stretch, compared with the non-inhibited 6 h stretched cells. (C) Western blot analysis showed that 6 h cyclic stretch increased the expressions of pro-IL-1β and mature IL-1β proteins. While the addition of the caspase-1 inhibitor significantly inhibited the expressions of both pro-IL-1β and mature IL-1β, compared with the non-inhibited 6 h stretched cells. The GAPDH was used as a loading control. results were quantified from at least 3 independent experiments and expressed as mean ± S.E. Statistical significance was calculated using one-way ANOVA with multiple comparisons. *P < 0.05 versus control group; #P < 0.05 versus 6 h group.

Figure 6
Figure 6. A proposed model illustrating the mechanism of inflammation and pyroptosis via NLRP3 and NLRP1 inflammasomes in HPDLCs.

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