副干酪乳酪杆菌E6通过其代谢物改善长春瑞滨诱导的斑马鱼免疫抑制

doi:10.12122/j.issn.1673-4254.2025.02.14

摘要/Abstract

摘要：

目的通过斑马鱼模式生物探究副干酪乳酪杆菌E6改善长春瑞滨诱导免疫抑制的作用机制。方法用异硫氰酸荧光素（FITC）标记副干酪乳酪杆菌E6，在荧光显微镜下观察副干酪乳酪杆菌斑马鱼肠道的定植情况。运用长春瑞滨构建斑马鱼免疫低下模型，分别通过斑马鱼尾部造血组织（CHT）处巨噬细胞、中性粒细胞的生成，胸腺Ｔ细胞的生成，以及斑马鱼体内免疫基因白介素-12（IL-12）、干扰素-γ（IFN-γ）的表达来研究副干酪乳酪杆菌E6的免疫调节活性。运用液相色谱串联质谱（LC-MS/MS）靶向代谢组学技术分别检测副干酪乳酪杆菌E6发酵上清液和副干酪乳酪杆菌E6在斑马鱼体内代谢物短链脂肪酸（SCFAs）的含量。通过斑马鱼免疫低下模型评价SCFAs乙酸钠、丙酸钠、丁酸钠的免疫调节作用。结果与正常组相比，斑马鱼肠球、中肠和后肠部位均清晰可见FITC标记副干酪乳酪杆菌E6的绿色荧光。与模型组相比，副干酪乳酪杆菌E6缓解长春瑞滨诱导斑马鱼CHT巨噬细胞和中性粒细胞的数量减少（P<0.05），增加斑马鱼体内胸腺Ｔ细胞的荧光强度（P<0.05）。同时副干酪乳酪杆菌E6可促进斑马鱼免疫低下模型免疫因子IL-12和IFN-γ基因的表达（P<0.01）。与MRS培养基相比，副干酪乳酪杆菌E6发酵上清液中代谢物SCFAs乙酸、丙酸、丁酸的含量增加（P<0.001）。副干酪乳酪杆菌E6在斑马鱼体内的代谢物SCFAs乙酸、丙酸、丁酸的含量也增加（P<0.05）。SCFAs乙酸钠、丙酸钠均能够增加斑马鱼免疫低下模型CHT处巨噬细胞和中性粒细胞的数量（P<0.001），抑制长春瑞滨诱导斑马鱼胸腺T细胞荧光强度的降低（P<0.01）。结论副干酪乳酪杆菌E6通过其代谢物SCFAs乙酸、丙酸改善长春瑞滨诱导斑马鱼免疫抑制。

关键词: 副干酪乳酪杆菌E6, 代谢物, 短链脂肪酸, 长春瑞滨, 免疫抑制

Abstract:

Objective To explore the mechanism of Lacticaseibacillus paracasei E6 for improving vinorelbine-induced immunosuppression in zebrafish. Methods The intestinal colonization of L. paracasei E6 labeled by fluorescein isothiocyanate (FITC) in zebrafish was observed under fluorescence microscope. In a zebrafish model of vinorelbine-induced immunosuppression, the immunomodulatory activity of L. paracasei E6 was assessed by analyzing macrophage and neutrophil counts in the caudal hematopoietic tissue (CHT), the number of T-lymphocyte, and the expressions of interleukin-12 (IL-12) and interferon-γ (IFN-γ). The contents of short-chain fatty acids (SCFAs) in L. paracasei E6 fermentation supernatant and the metabolites of L. paracasei E6 in zebrafish were detected by LC-MS/MS-based targeted metabolomics. The immunomodulatory effects of the SCFAs including sodium acetate, sodium propionate and sodium butyrate were evaluated in the zebrafish model of immunosuppression. Results After inoculation, green fluorescence of FITC-labeled L. paracasei E6 was clearly observed in the intestinal ball, midgut and posterior gut regions of zebrafish. In the immunocompromised zebrafish model, L. paracasei E6 significantly alleviated the reduction of macrophage and neutrophil counts in the CHT, increased the fluorescence intensity of T-lymphocytes, and promoted the expressions of IL-12 and IFN-γ. Compared with MRS medium, L. paracasei E6 fermentation supernatant showed significantly higher levels of acetic acid, propionic acid and butyric acid, which were also detected in immunocompromised zebrafish following treatment with L. paracasei E6. Treatment of the zebrafish model with sodium acetate and sodium propionate significantly increased macrophage and neutrophil counts in the CHT and effectively inhibited vinorelbine-induced reduction of thymus T cells. Conclusion L. paracasei E6 can improve vinorelbine-induced immunosuppression in zebrafish through its SCFA metabolites acetic acid and propionic acid.

Key words: Lactobacillus paracei E6, metabolites, short-chain fatty acids, vinorelbine, immunosuppression

许欣筑, 郭丽娜, 郑康帝, 马燕, 林淑娴, 何盈犀, 盛雯, 许素哗, 邱峰. 副干酪乳酪杆菌E6通过其代谢物改善长春瑞滨诱导的斑马鱼免疫抑制[J]. 南方医科大学学报, 2025, 45(2): 331-339.

XU Xinzhu, Lina GUO, Kangdi ZHENG, Yan MA, Shuxian LIN, Yingxi HE, Wen SHENG, Suhua XU, Feng QIU. Lacticaseibacillus paracasei E6 improves vinorelbine-induced immunosuppression in zebrafish through its metabolites acetic acid and propionic acid[J]. Journal of Southern Medical University, 2025, 45(2): 331-339.

图/表 7

表1 qRT-PCR引物序列

Tab.1 Sequences of the primers for qRT-PCR

Gene	Forward (5'—3')	Reverse (5'—3')
β-actin	TCGAGCAGGAGATGGGAACC	CTCGTGGATACCGCAAGATTC
IL-2	AACTCCTACAAGCCCAGCAC	ACACTCGGTCGTCAAACGAA
INF-γ	GCCTGGGGAGTATGTTTGCT	CAGGAAGATGGGGTGTGCAT

图1 副干酪乳酪杆菌E6在斑马鱼肠道定植情况

Fig.1 Colonization of L. paracasei E6 in the intestinal tract of zebrafish.

图2 副干酪乳酪杆菌E6对长春瑞滨诱导的斑马鱼免疫抑制的改善作用

Fig.2 L. paracasei E6 improves vinorelbine-induced immunosuppression in zebrafish. A: L. paracasei E6 inhibits vinorelbine-induced reduction of macrophage count in the caudal hematopoietic tissue (CHT; dashed box) of zebrafish (left). Macrophage counts are shown in the diagram on the right. B: L. paracasei E6 inhibits vinorelbine-induced reduction of neutrophil counts in the CHT (dashed box) in zebrafish. *P<0.05, ***P<0.001.

图4 副干酪乳酪杆菌E6对斑马鱼体内IL-12和IFN-γ mRNA表达水平的影响

Fig.4 Effect ofL. paracasei E6 on mRNA expression levels of IL-12 and IFN-γ in zebrafish. **P<0.01, ***P<0.001

表2 副干酪乳酪杆菌E6发酵清液中SCFAs

Tab.2 Short-chain fatty acids (SCFAs) detected in culture supernatant of L. paracasei E6 (Mean±SD, n=3)

SCFAs	Concentration(ng/mL)
SCFAs	MRS	Culture supernatant
Acetic acid	354.70±5.03	33576.01±200.71***
Propionic acid	-	74.65±1.70***
Isobutyric acid	-	-
Butyric acid	-	33.40±2.29***
2-Methylbutyric acid	-	-
Isovaleric acid	-	-
Valeric acid	-	-
2, 2-Dimethylbutanoic acid	-	-
2-Ethyl butyric acid	-	-
3, 3-Dimethylbutanoic acid	-	-
2-Methylvaleric acid	-	-
3-Methylvaleric acid	-	-
4-Methylvaleric acid	-	-
Caproic acid	-	-

图5 副干酪乳酪杆菌E6在斑马鱼体内代谢产生的SCFAs含量变化

Fig.5 SCFAs produced by the metabolism of L. paracasei E6 in zebrafish. *P<0.05, **P<0.01, ***P<0.001.

图6 SCFAs乙酸钠、丙酸钠、丁酸钠对长春瑞滨诱导斑马鱼免疫抑制的影响

Fig.6 Effects of SCFAs (sodium acetate, sodium propionate, and sodium butyrate) on vinorelbine-induced immunosuppression in zebrafish. A: Effects of sodium acetate, sodium propionate, and sodium butyrate on vinorelbine-induced reduction of macrophage number in the CHT (dashed box) of zebrafish. B: Effects of sodium acetate, sodium propionate, and sodium butyrate on vinorelbine-induced reduction of neutrophil number in the CHT (dashed box) of zebrafish. ***P<0.001.

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