枯草芽孢杆菌肽聚糖对β-伴大豆球蛋白诱导的鲤幼鱼肠上皮细胞损伤的保护作用

摘要

为观察不同浓度枯草芽孢杆菌肽聚糖对β-伴大豆球蛋白(β-conglycinin)诱导的鲤幼鱼肠上皮细胞炎性损伤的保护作用,将原代培养72 h (26 °C、6%的CO2)的24孔鲤幼鱼IECs培养板随机设为6组,每组4个重复.其中1组为阴性对照,其余5组用浓度为1.0 mg/mL的枯草芽孢杆菌肽聚糖诱导24 h后,分别更换浓度为 0(阳性对照)、0.15、0.30、0.45 和0.60 mg/mL枯草芽孢杆菌肽聚糖培养液,相同条件培养12、24和36 h,分别检测细胞的抗超氧阴离子(ASA)和抗羟自由基(AHR)能力、蛋白羰基(PC)含量、抗氧化酶(SOD、CAT和GPx)活性和细胞因子IL-1β、IL-8、IL-10、TNF-α1、TGF-β基因表达.结果显示, β-conglycinin上调细胞炎性因子表达,降低细胞抗氧化性能.添加枯草芽孢杆菌肽聚糖后,随PG浓度增加和处理时间延长,细胞抗氧化性能增加,呈显著的量效关系,且36 h呈二次曲线相关.IL-1β、IL-8、TNF-α1 mRNA表达呈二次曲线递减,IL-10、TGF-β mRNA表达呈线性递增.研究表明,不同浓度枯草芽孢杆菌肽聚糖均可保护由β-congly-cinin诱导引起的鲤幼鱼IECs氧化损伤,提高IECs抗氧化能力;高浓度PG通过抑制致炎因子和促进抗炎细胞因子的基因表达,提高IECs的抗炎能力.%This experiment was conducted to investigate the protective effects of different concentration of Bacil-lus subtilis peptidoglycan on the β-conglycinin-induced inflammatory lesions in intestinal epithelial cells of juven-ile carp(Cyprinus carpio).In 24-cell microplates,the intestinal epithelial cells(IECs)of juvenile carp were primarily cultured for 72 h at 26 °C and 6% CO2, and then the IECs were randomly divided into 6 groups with 4 replicates per group. One of the 6 groups was set as negative control group, and the other groups were all supple-mented with 1.0 mg/mL β-conglycinin in culture medium in order to establish inflammatory lesions. After 24 h further culture in the same conditions,the culture medium was changed into the B.subtilis peptidoglycan culture medium with 0 (positive control group), 0.15, 0.30, 0.45 and 0.60 mg/mL, respectively. The samples were collec-ted to measure the antioxidant and anti-inflammatory indices after 12,24 and 36 h B.subtilis peptidoglycan cul-ture. The results showed as follows: β-conglycinin exposure significantly decreased the activity of ASA, AHR, SOD, CAT, GPx, and increased the content of PC and the mRNA expression of inflammatory cytokines (IL-1β, IL-8, TNF-α1, IL-10 and TGF-β). The 12, 24 and 36 hours after the peptidoglycan were given, the activity of ASA, AHR, SOD, CAT, GPx and the content of PC in the cells were decreased in a dose-dependent manner; and the mRNA levels of IL-1β, IL-8 and TNF-α1 were down-regulated and IL-10 and TGF-β were up-regulated. Different concentration of B.subtilis peptidoglycan could significantly improve the anti-oxidative activity,and increase the mRNA levels of anti-inflammation cytokine, and decrease the pro-inflammation cytokine, and inhibit the inflam-matory response of the carp IECs induced by β-conglycinin.