关键词: 藏猪, LPS, 促炎因子, 血液生理指标, SNP

Abstract: This study, using Yorkshire pigs as controls, aimed to explore the differences in blood physiological indicators and the expression profiles of immune-related organ tissues between Tibetan pigs and Yorkshire pigs. Additionally, the expression levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in Tibetan pig peripheral blood lymphocytes stimulated with LPS was investigated, to understand their impact on the immune characteristics of Tibetan pigs. Forty Tibetan pigs and forty Yorkshire pigs aged 180 days were divided into two experimental groups based on breed. Fresh blood samples were collected for detecting blood physiological indicators, and peripheral blood lymphocytes were cultured for further analysis. Moreover, 8 Tibetan pigs and 8 Yorkshire pigs from each breed were randomly selected and divided into two experimental groups. Fresh blood was collected from the anterior vena cava, and liver, spleen, and submandibular lymph node tissues were collected post-slaughter for RNA extraction using the Trizol method. Each individual sample was subjected to 3 replicates for RT-qPCR and next-generation sequencing analysis to assess the expression levels and genetic polymorphisms of IL-1β, IL-6, and TNF-α genes. Peripheral blood lymphocytes were stimulated with 4 concentrations of LPS (0, 1, 10, 100 μg·mL^-1), with 3 replicates per group. Cells were collected at 0, 24, 36, 48, and 72 h, and RT-qPCR was performed to assess the expression of pro-inflammatory cytokine mRNA. Results indicate that: 1) Tibetan pigs exhibited significantly higher levels of WBC, RBC, HGB, HCT, and PLT compared to Yorkshire pigs (P<0.01). Additionally, the average MCV and PCT were significantly higher in Tibetan pigs than those in Yorkshire pigs (P<0.05). 2) RT-qPCR results revealed significantly higher mRNA expression levels of IL-1β, IL-6, and TNF-α genes in the blood, spleen, and liver tissues of Tibetan pigs compared to Yorkshire pigs (P<0.01). In the submandibular lymph nodes of Tibetan pigs, the expression levels of IL-1β and TNF-α genes were significantly higher (P<0.05), while the mRNA expression level of the IL-6 gene was extremely significantly higher (P<0.01). 3) SNP site screening identified 5 mutation sites in the 3' flanking region of the IL-1β gene. Among them, G⁶⁹⁰T, C¹³⁸³G, C¹⁴⁸⁰T, and A¹⁴⁹⁷G sites showed extremely significant differences between Tibetan pigs and Yorkshire pigs (P<0.01), and the C¹⁴⁵⁴T site exhibited significant differences (P<0.05). The IL-6 gene’s 3' flanking region contained a non-synonymous mutation site C²⁶⁵T, which showed significant differences (P<0.05), and a non-synonymous mutation site A-72G in the 5' flanking region exhibited highly significant differences (P<0.01). The TNF-α gene had a non-synonymous mutation site in the 5' flanking region, but the difference between the two breeds was not significant (P>0.05). 4) Different LPS concentrations stimulated Tibetan peripheral blood lymphocytes, with 10 μg·mL^-1 LPS showing the most significant proliferative effect. At concentrations of 1 and 10 μg·mL^-1 LPS stimulation, the IL-1β gene responded significantly, and at a concentration of 100 μg·mL^-1 LPS, the IL-6 gene exhibited a strong response. In summary, compared to Yorkshire pigs, Tibetan pigs demonstrated stronger resistance in blood physiological indicators, immune organ cytokine expression, and genetic polymorphism. The A-72G site in the IL-6 gene may be closely related to immunological and disease resistance capabilities. The significant responses of IL-1β and IL-6 genes to LPS in Tibetan pig peripheral blood lymphocyte culture experiments highlight their critical role in immune regulation.

Key words: Tibetan pigs, LPS, pro-inflammatory factors, blood physiological parameters, SNP