猫乳源乳酸片球菌的分离及对巨噬细胞极化的影响

Isolation of Pediococcus acidilactici from Feline Milk and Its Effects on Polarization of Macrophages

本试验旨在研究筛选出的猫乳源乳酸片球菌(YH-15)抑制脂多糖(LPS)诱导的巨噬细胞极化,以确定YH-15对机体免疫功能的调节作用。从猫乳中分离乳酸菌得到猫乳源益生菌YH-15,利用LPS建立小鼠巨噬细胞炎症模型。采用CCK-8法检测YH-15对RAW264.7细胞增殖的影响,采用酶联免疫吸附法(ELISA)检测细胞中的促炎因子肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)及抑炎因子白细胞介素-10(IL-10)的含量;采用Western blot法检测巨噬细胞极化相关指标诱导型一氧化氮合酶(iNOS)和精氨酸酶-1(Arg-1)的蛋白表达量,并检测乙酰化细胞核因子-κB p65(NF-κB p65)的蛋白表达以确定YH-15是否通过抑制NF-κB的核转位,从而达到对炎症的抵抗作用。结果表明:从猫乳中成功分离出乳酸片球菌菌株并命名为YH-15;高浓度(10^(9) CFU/mL)YH-15对小鼠巨噬细胞增殖无显著影响(P>0.05),但能够显著降低细胞促炎因子TNF-α、IL-1β、IL-6的含量,显著提高抑炎因子IL-10的含量(P<0.05);高浓度YH-15可抑制巨噬细胞向M1型极化,同时诱导巨噬细胞向M2型极化;YH-15显著降低NF-κB p65的蛋白表达(P<0.05)。综上可知,YH-15可诱导巨噬细胞向M2型极化,抵抗LPS诱导的M1型巨噬细胞的生成及NF-κB p65的蛋白表达,具有预防和抑制炎症的作用。

To study the inhibitory effects of Pediococcus acidilactici named YH-15 on lipopolysaccharide(LPS)-induced polarization of macrophages and to determine the regulatory effects of YH-15 on immune function,lactic acid bacteria were isolated from feline milk to obtain feline milk probiotic YH-15,and a mouse macrophage inflammation model was established by LPS.The effects of YH-15 on the proliferation of RAW264.7 cells was detected by the CCK-8 method,and the contents of pro-inflammatory factors tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6)and anti-inflammatory factor interleukin-10(IL-10)were detected by enzyme-linked immunosorbent assay(ELISA).The expression levels of inducible nitric oxide synthase(iNOS)and arginase-1(Arg-1)in macrophages related to polarization were detected by Western blot assay,and the expression of acetylated nuclear factor-κB p65(NF-κB p65)was detected to determine whether YH-15 could inhibit the inflammation by inhibiting the nuclear translocations of NF-κB.Results showed as follow:the strains of Pediococcus acidilactici were successfully isolated from feline milk and named YH-15.High concentrations of YH-15(10^(9) CFU/mL)had no significant effect on macrophage proliferation(P>0.05),but significantly decreased the contents of pro-inflammatory cytokines TNF-α,IL-1β,and IL-6 and significantly promoted the content of anti-inflammatory factor IL-10(P<0.05).High concentrations of YH-15 could inhibit the M1-typepolarization of macrophages and induce the M2-type polarization of macrophages.YH-15 significantly decreased the protein expression of NF-κB p65(P<0.05).In conclusion,YH-15 can induce M2-type polarization of macrophages,resist the formation of LPS-induced M1-type macrophages and the expression of NF-κB p65,and have the effects of preventing and inhibiting inflammation.