In Vitro Study on Protective Effect of Chitosan against Inflammation Injury of Bovine Mammary Epithelial Cells Induced by Streptococcus agalactiae

SUN Mingwei; TONG Jinjin; JIANG Linshu; XIONG Benhai; MAO Shengyong

doi:10.3969/j.issn.1006-267x.2020.03.028

2020 , Vol. 32 >Issue 3: 1204 - 1215

DOI: https://doi.org/10.3969/j.issn.1006-267x.2020.03.028

In Vitro Study on Protective Effect of Chitosan against Inflammation Injury of Bovine Mammary Epithelial Cells Induced by Streptococcus agalactiae

SUN Mingwei ,
TONG Jinjin ,
JIANG Linshu ,
XIONG Benhai ,
MAO Shengyong

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1. Key Laboratory for Dairy Cow Nutrition, College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206, China;
2. Institute of Animal Science and Veterinary, Chinese Academy of Agricultural Science, Beijing 100193, China;
3. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210014, China

Received date: 2019-09-07

Online published: 2020-03-13

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Abstract

The aim of this study was to investigate the protective effect of chitosan (CTS) against Streptococcus agalactiae (S. agalactiae)-induced inflammatory injury of bovine mammary epithelial cells (BMECs) using an in vitro method. Firstly, the BMECs were treated with different concentrations (0, 15.625, 31.25, 62.5, 125, 250, 500 and 1 000 mg/mL) of CTS for 12 and 24 hours, respectively. The BMECs viability was detected by methyl thiazoyl terazolium (MTT) assay, and the appropriate action time and action concentrations (31.25, 125 and 250 mg/mL) were screened out according to BMECs viability for follow-up experiment. Then, a two-factor experiment design was adopted and divided into four groups with 4 replicates in each group. BMECs in control group were not treated with CTS and not stimulated by S. agalactiae, BMECs in S. agalactiae group were stimulated by S. agalactiae for 6 hours, BMECs in CTS groups were treated with CTS at different concentrations (31.25, 125 and 250 mg/mL) for 24 hours, and BMECs in CTS(-)+sgc group were treated with CTS at different concentrations (31.25, 125 and 250 mg/mL) for 24 hours, and then stimulated by S. agalactiae for 6 hours. The mRNA expression levels of interleukin (IL)-6, IL-1β, tumor necrosis factor-α (TNF-α), IL-8, Toll-like receptor 2 (TLR2), myeloid differentiation factor 88 (MyD88)、IL-1 receptor associated kinase 4 (IRAK4), tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β activated kinase 1 (TAK1) in BMECs were detected by quantitative real-time PCR method, and the protein expression levels of nuclear transcription factor-κB (NF-κB) inhibitor-α (IκB-α), phosphorylated NF-κB-p65 (p-NF-κB-p65), phosphorylated p38 (p-p38), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated c-Jun N-terminal kinase (p-JNK) in BMECs were detected by Western blot method. The results showed as follows:1) CTS at 31.25 and 125 mg/mL significantly or extremely significantly decreased the mRNA expression levels of IL-6, IL-8, TLR2, MyD88, IRAK4 and TRAF6 in BMECs (P<0.05 or P<0.01); CTS at 31.25, 125 and 250 mg/mL extremely significantly decreased the mRNA expression levels of IL-6, IL-1β, TNF-α, IL-8, TLR2, MyD88, IRAK4, TRAF6 and TAK1 in BMECs induced by S. agalactiae (P<0.01). 2) CTS at 31.25, 125 and 250 mg/mL significantly or extremely significantly reduced the protein levels of p-NF-κB-p65, p-p38, p-ERK1/2 and p-JNK in BMECs and BMECs induced by S. agalactiae (P<0.05 or P<0.01). It can be concluded that CTS can effectively protect cells and reduce inflammatory injury by inhibiting the NF-κB and mitogen activated protein kinase (MAPK) signaling pathways of BMECs and inhibiting S. agalactiae-induced inflammatory response.

Key words： chitosan; Streptococcus agalactiae; bovine mammary epithelial cells; inflammation injury; protective effect

Cite this article

SUN Mingwei , TONG Jinjin , JIANG Linshu , XIONG Benhai , MAO Shengyong . In Vitro Study on Protective Effect of Chitosan against Inflammation Injury of Bovine Mammary Epithelial Cells Induced by Streptococcus agalactiae[J]. Chinese Journal of Animal Nutrition, 2020 , 32(3) : 1204 -1215 . DOI: 10.3969/j.issn.1006-267x.2020.03.028

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