Study on the role of NALP3 inflammasome in Porphyromonas gingivalis lipopolysaccharide induced RAW264.7
10.3760/cma.j.issn.1002-0098.2017.05.006
- VernacularTitle: NALP3炎性体在牙龈卟啉单胞菌脂多糖刺激RAW264.7细胞中的作用
- Author:
Fang YANG
1
;
Mingyue CHEN
2
;
Yingying HU
1
;
Changning WANG
3
Author Information
1. The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, School of Stomatology, Wuhan University, Wuhan 430079, China
2. The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, School of Stomatology, Wuhan University, Wuhan 430079, China [Present address: Department of Stomatology, Taihe Hospital, Shiyan Hubei 442000, China]
3. Department of Periodontology, School of Stomatology, Wuhan University, Wuhan 430079, China
- Publication Type:Journal Article
- Keywords:
Periodontitis;
Porphyromonas gingivalis;
Lipopolysaccharides
- From:
Chinese Journal of Stomatology
2017;52(5):289-293
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To illuminate the effect of NALP3 inflammasome on regulating the expression of cytokines of macrophages in periodontitis.
Methods:RAW264.7 cells were cultured and divided into three groups. The first group stayed normal as control, the second group was stimulated by 1 mg/L Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS), the third group was pretreated with AC-YVAD-CMK (caspase-1 inhibitor) before stimulated with 1 mg/L Pg LPS. RAW264.7 cells pretreated with various concentrations (0, 5, 10, 25, 50, 75, 100, 200 μmol/L) of AC-YVAD-CMK for 2 h, and stimulated by 1 mg/L Pg LPS for 24 h in the third group. After that, cell survival rate were detected by cell counting kit-8. Every group cells gene transcription of NALP3 and interleukin-1β (IL-1β) were detected by quantitative real-time PCR (qPCR) after 6 h, protein expression of NALP3 and IL-1β were separately detected by Western blotting and enzyme linked immunosorbent assay (ELISA) after 24 h, respectively.
Results:It is observed that treatment with 5, 10, 25, 50, 75, 100, 200 μmol/L AC-YVAD-CMK did not significantly affect the viability of RAW264.7 cells. qPCR showed that mRNA expression of IL-1β level (1.03±0.08, 5.48±0.22, 4.31±0.20) and NALP3 level (0.96±0.05, 2.62±0.44, 1.73±0.09). Western blotting showed that protein expression of NALP3 level (1.00±0.10, 2.34±0.04, 1.64±0.04), ELISA showed protein secretion of IL-1β level ([40.20±0.25], [61.50±1.81], [52.40±1.91] ng/L). After stimulated by Pg LPS, mRNA and protein expression of IL-1β (P<0.01, P<0.01) and NALP3 (P<0.01, P<0.01) significantly increased; but the expression of IL-1β (P=0.002, P=0.027) and NALP3 (P<0.01, P<0.01) were decreased when pretreated with AC-YVAD-CMK.
Conclusions:NALP3 inflammasome signal pathway can be activated by Pg LPS in RAW264.7. Block of the pathway can inhibit Pg LPS-induced secretion of cytokines.
