Effects of E.coli Infection on Fibroblast Injury and TLR4 Expression in Sheep Mammary Glands

doi:10.7668/hbnxb.20192742

Abstract

Abstract:

In order to study the damage of mammary fibroblasts in sheep E.coli mastitis and the mechanism of TLR4,primary sheep mammary fibroblasts was obtained through in vitro culture,and E.coli artificially infected cells to establish E.coli mastitis cell model.Analyze the effect of E.coli on cell injury and the role of TLR4 in E.coli mastitis.Enzymatic digestion combined with differential digestion to obtain primary sheep mammary fibroblasts;MTT method to detect cell viability and cytotoxicity of the TLR4 blocker CLI-095;LDH method detects the damage of E.coli to sheep mammary fibroblasts and determines the optimal MOI;qRT-PCR to detect the relative expression of caspase-3 and TLR4 mRNA and the blocking effect of CLI-095;colorimetric method to detect the enzymatic activity of pyroptosis protein caspase-4,5;WB to detect the relative expression of TLR4 protein;the plate viable bacteria count method was used to detect the effect of CLI-095 on the adhesion of E.coli to sheep mammary fibroblasts.Sheep mammary fibroblasts was isolated,and the results of MTT assay showed good cell viability.After sheep mammary fibroblasts were infected with E.coli MOI=4:1,the release of LDH was significantly increased in each time period;the relative expression of caspase-3 mRNA increased significantly within 1-3 h of infection,and decreased significantly after 3 h;caspase-4,5 enzyme activity increased within 1-3 h of infection,and decreased after 3 h.The mRNA and protein expression of TLR4 were significantly up-regulated,and the CLI-095 could inhibit the adhesion of E.coli to cells.After E.coli infects fibroblasts,the release of LDH was increased,the expression of caspase-3 gene was up-regulated,and the activity of caspase-4,5 was increased,which promotes the injury,apoptosis and pyroptosis of sheep mammary fibroblasts.E.coli infection promotes the expression of TLR4 mRNA and protein in sheep mammary fibroblasts.The TLR4 blocker CLI-095 may inhibit the adhesion of E.coli to cells by inhibiting the expression of TLR4.

Key words: Sheep, E.coli, Mastitis, Fibroblast, TLR4

MA Xiaojun, WANG Jiamian, LI Shiying, YAN Lijiao, MA Qiangsheng, ZHANG Xiaoli. Effects of E.coli Infection on Fibroblast Injury and TLR4 Expression in Sheep Mammary Glands[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(2): 231-238. doi: 10.7668/hbnxb.20192742.

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Figures/Tables 14

Tab.1 Sheep caspase-3 primer information

引物名称 Primer name	引物序列(5'—3') Primer sequence	产物大小/bp Produce size	退火温度/℃ Tm	登录号 Accession
caspase-3 F	GGCTCTGAGTGTTTGGGGAA	131	60	XM-015104559.1
caspase-3 R	CCTGGACAAAGTTCCGTGGT

Tab.2 Sheep TLR4 primer information

引物名称 Primer name	引物序列(5'—3') Primer sequence	产物大小/bp Produce size	退火温度/℃ Tm	登录号 Accession
TLR4 F	ACTGACGGGAAACCCTATCC	208	60	AY957615
TLR4 R	CAGGTTGGGAAGGTCAGAAA

Fig.1 Mammary fibroblast morphology

Fig.2 Giemsa staining of mammary fibroblasts

Fig.3 Growth curve of sheep mammary fibroblasts
Different lowercase letters show significant difference(P<0.05);The same lowercase letters indicate that the difference is not significant(P>0.05).The same as Fig.4,6—9,11.

Fig.4 Changes in LDH release

Fig.5 Inverted microscope observation of E.coli infected fibroblasts

Fig.6 Relative expression of caspase-3 mRNA

Fig.7 caspase-4,5 enzyme activity change

Fig.8 Effect of CLI-095 on cell proliferation rate

Fig.9 Relative expression of TLR4 mRNA

Fig.10 TLR4 protein expression

Fig.11 Relative expression of TLR4 protein

Tab.3 Bacterial adhesion number and adhesion blocking rate

组别 Group	黏附数/(cfu/mL) Adhesion number	黏附阻断率/% Adhesion blocking rate
TLR4非阻断组	40.3×10⁶ ± 2.3×10⁶	18.32
TLR4 non-blocking
TLR4阻断组	33.0×10⁶ ± 1.2×10⁶
TLR4 blocking

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