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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

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Front. Med.    2021, Vol. 15 Issue (2) : 292-301    https://doi.org/10.1007/s11684-020-0806-5
RESEARCH ARTICLE
Bioactive hyaluronic acid fragments inhibit lipopolysaccharide- induced inflammatory responses via the Toll-like receptor 4 signaling pathway
Na You1, Sasa Chu2, Binggang Cai3, Youfang Gao1, Mizhou Hui4, Jin Zhu5(), Maorong Wang6()
1. Department of Infectious Disease, The People’s Hospital of Bozhou, Bozhou 236800, China
2. Department of Infectious Disease, The People’s Hospital of Linyi, Linyi 276000, China
3. Department of Infectious Disease, The People’s Hospital of Yancheng, Yancheng 224000, China
4. AnRuipu Biological Products Research Co., Ltd., Hangzhou 310019, China
5. Huadong Medical Institute of Biotechniques, Nanjing 210002, China
6. Institute of Liver Disease, Jinling Hospital, Nanjing 210002, China
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Abstract

The high- and the low-molecular weight hyaluronic acids (HMW-HA and LMW-HA, respectively) showed different biological activities in inflammation. However, the role of LMW-HA in inflammatory response is controversial. In this study, we aimed to investigate the effect of bioactive hyaluronan (B-HA) on lipopolysaccharide (LPS)-induced inflammatory responses in human macrophages and mice. B-HA was produced from HA treated with glycosylated recombinant human hyaluronidase PH20. Human THP-1 cells were induced to differentiate into macrophages. THP-1-derived macrophages were treated with B-HA, LPS, or B-HA+LPS. The mRNA expression and the production of inflammatory cytokines were determined using quantitative real-time PCR and enzyme-linked immunosorbent assay. The phosphorylation levels of proteins in the nuclear factor-kB (NF-kB), mitogen-activated protein kinase (MAPK), and IRF-3 signaling pathways were measured using Western blot. The in vivo efficacy of B-HA was assessed in a mouse model of LPS-induced inflammation. Results showed that B-HA inhibited the expression of TNF-α, IL-6, IL-1, and IFN-β, and enhanced the expression of the anti-inflammatory cytokine IL-10 in LPS-induced inflammatory responses in THP-1-derived macrophages and in vivo. B-HA significantly suppressed the phosphorylation of the TLR4 signaling pathway proteins p65, IKKα/β, IkBα, JNK1/2, ERK1/2, p38, and IRF-3. In conclusion, our results demonstrated that the B-HA attenuated the LPS-stimulated inflammatory response by inhibiting the activation of the TLR4 signaling pathway. B-HA could be a potential anti-inflammatory drug in the treatment of inflammatory disease.
Keywords bioactive hyaluronan      lipopolysaccharide      inflammatory cytokines      TLR4      human macrophages     
Corresponding Author(s): Jin Zhu,Maorong Wang   
Just Accepted Date: 17 August 2020   Online First Date: 17 September 2020    Issue Date: 23 April 2021
 Cite this article:   
Na You,Sasa Chu,Binggang Cai, et al. Bioactive hyaluronic acid fragments inhibit lipopolysaccharide- induced inflammatory responses via the Toll-like receptor 4 signaling pathway[J]. Front. Med., 2021, 15(2): 292-301.
 URL:  
https://academic.hep.com.cn/fmd/EN/10.1007/s11684-020-0806-5
https://academic.hep.com.cn/fmd/EN/Y2021/V15/I2/292
Fig.1  Effect of B-HA concentration on LPS-induced inflammatory response. THP-1 cell-derived macrophages were pretreated with 0.5%, 1%, or 2% B-HA 2 h before LPS (100 ng/mL) treatment. The mRNA levels of TNF (A), IL-1 (B), IL-6 (C), IFNB1 (D), and IL-10 (E) were measured using qRT-PCR 4 h after LPS treatment. L, LPS; B-HA, bioactive hyaluronan. *P<0.05, **P<0.01 compared with LPS treatment alone.
Fig.2  Effect of B-HA on cytokine transcription in LPS-treated THP-1 cells. The B-HA pretreatment suppresses the transcription of TNF (A), IL-1 (B), IL-6 (C), and IFNB1 (D) and enhances the transcription of IL-10 (E) in LPS-treated THP-1 cells. Cells were pretreated with HA for 2 h and treated with 100 ng/mL LPS for 2, 4, or 8 h. Transcript levels were determined using qRT-PCR. L, LPS; B-HA, bioactive hyaluronan. * P<0.05, ** P<0.01, **** P<0.001 compared with the LPS group.
Fig.3  Effect of B-HA on the secretion of inflammatory cytokines in LPS-induced inflammatory response. The levels of TNF-α (A), IL-1 (B), IL-6 (C), IFN-β (D), and IL-10 (E) in the supernatants of the treated cells were measured using ELISA. L, LPS; B-HA, 2% bioactive hyaluronan. * P<0.05, ** P<0.01, **** P<0.000 compared with LPS group.
Fig.4  Inhibition of NF-kB activation by B-HA in LPS-induced inflammatory response. The phosphorylation levels of IKK, IkB, and p65 proteins were detected in THP-1 cell-derived macrophages treated with B-HA and LPS by Western blot. L, LPS; B-HA, bioactive hyaluronan. * P<0.05, ** P<0.01 compared with the LPS group.
Fig.5  Inhibition of MAPK activation by B-HA in LPS-induced inflammatory response. The phosphorylation levels of p-JNK1/2, p-ERK1/2, and p38 proteins were detected in THP-1 cell-derived macrophages treated with B-HA and LPS by Western blot. L, LPS; B-HA, bioactive hyaluronan. * P<0.05, ** P<0.01 compared with the LPS group.
Fig.6  Inhibition of IRF-3 activation by B-HA in LPS-induced inflammatory response. The phosphorylation level of the IRF-3 protein was detected in THP-1 cell-derived macrophages treated with B-HA and LPS by Western blot. L, LPS; B-HA, bioactive hyaluronan. * P<0.05, ** P<0.01 compared with the LPS group.
Fig.7  Effect of B-HA on the production of inflammatory cytokines in vivo. The effect of B-HA on the production of inflammatory cytokines in vivo was assessed in a mouse model of LPS-induced inflammation. Animals were treated with LPS, B-HA, or LPS+ B-HA. The serum levels of TNF-α (A), IL-1 (B), IL-6 (C), and IL-10 (D) were measured using ELISA. L, LPS; B-HA, bioactive hyaluronan. * P<0.05, ** P<0.01 compared with the LPS group.
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