抗炎免疫调节活性益生菌的体外筛选及其相关组分的初步鉴定

In vitro screening of anti-inflammatory immunomodulatory active probiotics and preliminary identification of relevant components

为筛选出具有抗炎、抗感染免疫调节活性的益生菌并探究益生菌免疫调节活性的主要成分,本研究分别以10株益生菌Lac.1~Lac.10作为候选菌株,将益生菌的活菌体、灭活菌体、菌体裂解物分别作用于由脂多糖+尼日利亚菌素(LPS+Nigericin)诱导的急性炎症巨噬细胞模型及NF-κB和IRF信号通路报告细胞系J774-DualTM巨噬细胞,经荧光定量PCR检测细胞因子Il-1β和Il-10 mRNA的转录水平,通过检测报告基因SEAP和Lucia荧光素酶的活性分析NF-κB和干扰素调节因子(IRF)信号通路的激活情况。结果显示,在排除发酵液中MRS培养基干扰的情况下,益生菌株Lac.5活菌体能够极显著下调巨噬细胞炎症模型下Il-1β mRNA的转录水平(P<0.0001)、极显著上调Il-10 m RNA的转录水平(P<0.0001),并且能够极显著激活巨噬细胞的NF-κB和IRF信号通路(P<0.0001),进一步将益生菌Lac.5活菌体热灭活和超声破碎后显示,益生菌Lac.5灭活菌体和菌体裂解物的免疫调节活性均显著降低,仅益生菌Lac.5活菌体组分起到抗炎、抗感染的免疫调节活性。综上所述,本研究筛选出一株具有抗炎、抗感染的免疫调节活性的益生菌株Lac.5,并确认了其活菌体为免疫调节组分,对功能益生菌的筛选和新型益生菌治疗制剂的开发具有重要意义。

In order to screen probiotics with anti-inflammatory and anti-infective immunomodulatory activities and to explore the main components of probiotic immunomodulatory activity, 10 probiotic strains(Lac.1-Lac.10) were used as candidate strains,and living bacteria cells, inactivated bacteria cells and bacterial cell lysates of probiotics were used as candidate to conduct in vitro interaction experiments with the acute inflammatory macrophage model HD11 induced by LPS and Nigericin and the J774-DualTMmacrophages cell line respectively. Transcriptional levels of cytokines Il-1β and Il-10 m RNA were detected by RT-q PCR, and activation of the NF-κB and IRF signaling pathways was detected by measuring the activities of the reporter genes SEAP and Lucia luciferase. The results showed that under the condition of excluding the interference of medium MRS in the fermentation broth, the probiotic strain Lac.5 live bacteria cells could significantly down-regulate the transcriptional levels of Il-1β m RNA(P<0.0001) and significantly up-regulate the transcriptional levels of Il-10 m RNA(P<0.0001) in the macrophage inflammation model, and could significantly activate the NF-κB and IRF signaling pathways(P<0.0001) of macrophages. Further, the live bacteria of probiotic Lac.5 were subjected to thermal inactivation and ultrasonic fragmentation, and the results showed that the immunoregulatory activity of inactivated bacteria and cell lysates of probiotics Lac.5 was significantly decreased, only the live cells of probiotics Lac.5 had anti-inflammatory and anti-infective immunomodulatory activities. In conclusion, this study screened a probiotic strain Lac.5 with anti-inflammatory and anti-infective immunomodulatory activity, and confirmed that the living bacteria cell was an immunomodulatory component, which is of great significance for the screening of functional probiotics and the development of novel probiotic therapeutic agents.