Effect of Enterococcus faecalis lipoteichoic acid on macrophage autophagy

doi:10.3877/cma.j.issn.1674-1366.2018.02.001

Abstract

Abstract:

Objective

By investigating the effect of Enterococcus faecalis lipoteichoic acid (LTA) on macrophages autophagy, this study aimed to provide experimental evidence for further exploration of the pathogenesis of persistent periapical periodontitis.

Methods

RAW 264.7 was cultured and cell counting kit-8 (CCK-8) was used to detect the cytotoxicity of LTA. Western blot was used to detect the expression level of autophagy-related protein LC3-Ⅱ/LC3-Ⅰ and Beclin1 and qPCR was applied to detect the expression level of LC3 and Beclin1. RAW 264.7 cell strain stably expressing HBLV-mRFP-GFP-LC3-PURO was established, and laser confocal microscopy was used to observe the formation of autophagosome. SPSS 20.0 statistical software for data analysis.

Results

The result of CCK-8 showed no obvious toxicity to macrophage when stimulated with 20 μg/mL and the lower concentration of LTA for 24 hours (t = 2.102, P = 0.1106) . After LTA stimulated, Western blot suggested that the expression of LC3-Ⅱ/LC3-Ⅰ (0.42 ± 0.04) and Beclin1 (0.56 ± 0.11) significantly increased with dose-dependent effect (F = 14.25, P<0.001; F = 3.459, P = 0.0258) . In the meanwhile, qPCR results showed that the expression of LC3 (5.94 ± 0.85) and Beclin1 (2.22 ± 0.21) significantly increased with dose-dependent effect (F_LC3 = 12.93, P_LC3<0.001; F_Beclin1 = 6.036, P_Beclin1<0.001) after LTA stimulated. Laser confocal microscopy detected that LTA group (58 ± 6) had significant difference (F = 12.36, P = 0.0021) compared with control group (18 ± 8) .

Conclusion

Enterococcus faecalis LTA may regulate macrophages autophagy with dose-dependent effect.

Key words: Enterococcus faecalis, Lipoteichoic acid, Macrophages, Autophagy

Donghjia Lin, Yanhuo Chen, Zhixiang Peng, Yan Gao. Effect of Enterococcus faecalis lipoteichoic acid on macrophage autophagy[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2018, 12(02): 69-75.

/ / Recommend

Add to citation manager EndNote|Ris|BibTeX

URL: https://zhkqyxyjzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1674-1366.2018.02.001

https://zhkqyxyjzz.cma-cmc.com.cn/EN/Y2018/V12/I02/69

Figures/Tables 6

References 21

[1]	Mg S. Contemporary Oral Microbiology and Immunology[M]. 1st edn ed. St. Louis，Missouri，USA：Mosby，1992：59-62.
[2]	凌均棨.难治性根尖周炎的感染控制与显微治疗策略[Z]//全国第三次牙体牙髓病学临床技术研讨会论文汇编，重庆，2009.重庆：中华口腔医学会牙体牙髓病学专业委员会，2009.
[3]	Zhang C，Du J，Peng Z. Correlation between Enterococcus faecalis and Persistent Intraradicular Infection Compared with Primary Intraradicular Infection：A Systematic Review[J]. J Endod，2015，41（8）：1207-1213.
[4]	Stuart CH，Schwartz SA，Beeson TJ，et al. Enterococcus faecalis：its role in root canal treatment failure and current concepts in retreatment[J]. J Endod，2006，32（2）：93-98.
[5]	Mizushima N. Autophagy：process and function[J]. Genes Dev，2007，21（22）：2861-2873.
[6]	Cadwell K. Crosstalk between autophagy and inflammatory signalling pathways：balancing defence and homeostasis[J]. Nat Rev Immunol，2016，16（11）：661-675.
[7]	中华人民共和国国家质量监督检验检疫总局. GB/T 16886.5-2017/ISO 10993-5：2009医疗器械生物学评价第5部分：体外细胞毒性试验[S].北京：中国标准出版社，2017.
[8]	Nair PN. On the causes of persistent apical periodontitis：a review[J]. Int Endod J，2006，39（4）：249-281.
[9]	Zhao L，Chen J，Cheng L，et al. Effects of Enterococcus faecalis lipoteichoic acid on receptor activator of nuclear factor-κB ligand and osteoprotegerin expression in periodontal ligament fibroblasts[J]. International Endodontic Journal，2014，47（2）：163-172.
[10]	Wan J，Ma J，Anand V，et al. Morphine potentiates LPS-induced autophagy initiation but inhibits autophagosomal maturation through distinct TLR4-dependent and independent pathways[J]. Acta Physiologica，2015，214（2）：189-199.
[11]	Abada A，Elazar Z. Getting ready for building：signaling and autophagosome biogenesis[J]. EMBO Rep，2014，15（8）：839-852.
[12]	Zou J，Shankar N. The opportunistic pathogen Enterococcus faecalis resists phagosome acidification and autophagy to promote intracellular survival in macrophages[J]. Cellular Microbiology，2016，18（6）：831-843.
[13]	Gomes LC，Dikic I. Autophagy in antimicrobial immunity[J]. Mol Cell，2014，54（2）：224-233.
[14]	Yuan K，Huang C，Fox J，et al. Autophagy plays an essential role in the clearance of Pseudomonas aeruginosa by alveolar macrophages[J]. J Cell Sci，2012，125（Pt 2）：507-515.
[15]	Li Y，Lu L，Luo N，et al. Inhibition of PI3K/AKt/mTOR signaling pathway protects against d-galactosamine/lipopolysaccharide-induced acute liver failure by chaperone-mediated autophagy in rats[J]. Biomed Pharmacother，2017（92）：544-553.
[16]	Liu X，Wang Y，Cao Z，et al. Staphylococcal lipoteichoic acid promotes osteogenic differentiation of mouse mesenchymal stem cells by increasing autophagic activity[J]. Biochem Biophys Res Commun，2017，485（2）：421-426.
[17]	Shi CS，Kehrl JH. TRAF6 and A20 regulate lysine 63-linked ubiquitination of Beclin-1 to control TLR4-induced autophagy[J]. Sci Signal，2010，3（123）：ra42.
[18]	Glick D，Barth S，Macleod KF. Autophagy：cellular and molecular mechanisms[J]. J Pathol，2010，221（1）：3-12.
[19]	Ni H，Bockus A，Wozniak AL，et al. Dissecting the dynamic turnover of GFP-LC3 in the autolysosome[J]. Autophagy，2014，7（2）：188-204.
[20]	Kimura S，Fujita N，Noda T，et al. Monitoring autophagy in mammalian cultured cells through the dynamics of LC3[J]. Methods Enzymol，2009（452）：1-12.
[21]	He J，Zhang G，Pang Q，et al. SIRT6 reduces macrophage foam cell formation by inducing autophagy and cholesterol efflux under ox-LDL condition[J]. FEBS J，2017，284（9）：1324-1337.

RGR(%)	毒级
≥100	0
75 ~ 99	1
50 ~ 74	2
25 ~ 49	3
1 ~ 24	4
0	5

RGR(%)	毒级
≥100	0
75 ~ 99	1
50 ~ 74	2
25 ~ 49	3
1 ~ 24	4
0	5

基因	引物序列
LC3	F:5′-GAGCGAGTTGGTCAAGAT-3′
?	R:5′-TCATCCTTCTCCTGTTCATAG-3′
Beclin1	F:5′-AGTTGGGTGATGTGGGGAAAG-3′
?	R:5′-CGTCGCACACAGTATCAAACA-3′
GADPH	F:5′-GGGAAATCGTGCGTGAC-3′
?	R:5′-AGGCTGGAAAAGAGCCT-3′

基因	引物序列
LC3	F:5′-GAGCGAGTTGGTCAAGAT-3′
?	R:5′-TCATCCTTCTCCTGTTCATAG-3′
Beclin1	F:5′-AGTTGGGTGATGTGGGGAAAG-3′
?	R:5′-CGTCGCACACAGTATCAAACA-3′
GADPH	F:5′-GGGAAATCGTGCGTGAC-3′
?	R:5′-AGGCTGGAAAAGAGCCT-3′

Please choose a citation manager

Content to export