China Animal Husbandry and Veterinary Medicine ›› 2021, Vol. 48 ›› Issue (3): 846-854.doi: 10.16431/j.cnki.1671-7236.2021.03.008

• Physiology and Biochemistry • Previous Articles     Next Articles

Mechanism Study on Inactivated E. coli and Lipopolysaccharide Co-induced IPEC-JⅡ Cells Expressing RegⅢγ

MENG Jianghai1,2, XIANG Qionghao1, SONG Yunyun1,2, CAO Bin1, CHEN Tao1,2   

  1. 1. College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China;
    2. Hunan Veterinary Drug Engineering and Technology Research Center, Changsha 410128, China
  • Received:2020-07-02 Online:2021-03-20 Published:2021-03-18

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Abstract: This experiment was intended to explore the regulation mechanism of Gram-negative bacterium Escherichia coli (E.coli) and its surface molecules lipopolysaccharide (LPS) induced the pancreas regeneration protein Ⅲ gamma (RegⅢγ) expression.Firstly,different concentration E. coli (109,108,107,106,105 and 104 CFU/mL) and LPS (0.01,0.1,1,5,10,20,40 and 80 μg/mL) were used to induce intestinal epithelia cells of porcine (IPEC-JⅡ),and D490 nmvalue was determined by MTT method to judge the effect of E. coli and LPS on IPEC-JⅡ cell viability.Then,different concentration E. coli (107,106 and 105 CFU/mL) and LPS (0.01,0.1,1 and 5 μg/mL) were used to induce IPEC-JⅡ for 24 h,and Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression of RegⅢγ.Finally,1 μg/mL LPS was used to treat IPEC-JⅡ for 24 h,and Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression and phosphorylation level of p65,p38,JNK and ERK.The results showed that except 0.01 μg/mL LPS did not inhibit IPEC-JⅡ cell viability,all the other concentration of E. coli and LPS inhibited IPEC-JⅡ cell viability,moreover,the cell viability of 109 and 108 CFU/mL E. coli and 10,20,40,80 μg/mL LPS group were extremely significantly decreased (P<0.01).Compared with control group,107,106 and 105 CFU/mL E. coli could increase the expression of RegⅢγ in IPEC-JⅡ cells,and the RegⅢγ mRNA expression of 105 CFU/mL E. coli group was extremely significantly higher than that of control group (P<0.01),the quantity of protein expression was significantly higher than control group (P<0.05).0.01,0.1,1 and 10 μg/mL LPS could increase the expression of RegⅢγ in IPEC-JⅡ cells,moreover,the RegⅢγ mRNA expression of 0.1 and 1 μg/mL LPS group was extremely significantly higher than that of control group (P<0.01),although RegⅢγ protein expression had increasing trend,but had no significant difference (P>0.05).Compared with control group,the mRNA expression of p65 and p38 of 1 μg/mL LPS group were extremely significantly increased (P<0.01),and the mRNA expressions of JNK and ERK were significantly increased (P<0.05).Meanwhile,the protein expression levels of p38 and JNK and the phosphorylation level of p65 were significantly increased (P<0.01), and the phosphorylation level of p65 was significantly increased (P<0.05).Both ERK protein and phosphorylation levels increased,but the difference were not significant (P>0.05).The above results showed that E. coli and LPS could induce RegⅢγ expression,1 μg/mL LPS increased the phosphorylation levels of p65,p38 and JNK proteins.

Key words: RegⅢγ; IPEC-JⅡ; Escherichia coli; LPS; NF-κB; MAPK

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