培养温度对LPS诱导的离体大黄鱼头肾巨噬细胞抗氧化能力和炎性反应的影响

Effects of culture temperature on antioxidant capacity and inflammatory responses of LPS-induced macrophages from head kidney of large yellow croaker(Larimichthys crocea)

为研究温度对离体大黄鱼头肾巨噬细胞抗氧化能力和炎性反应的影响,从大黄鱼头肾组织中分离巨噬细胞结合贴壁筛选法得到细胞单层后,在不同温度(16、22和28°C)下培养备用。使用25μg/mL的脂多糖(LPS)孵育细胞2 h后,测定不同培养温度下离体细胞活力、呼吸爆发活性、抗氧化酶(SOD和CAT)活性以及相关基因(SOD、CAT、Hsp70和IL-1β)表达的情况。结果显示,体外培养细胞36 h后,16°C和22°C条件下培养的细胞活力显著高于28°C处理组;LPS处理组大黄鱼头肾巨噬细胞呼吸爆发活性显著升高,但SOD和CAT酶活性较对照组显著下降。高温(28°C)显著提高了细胞CAT酶的活性和基因表达水平,但SOD酶活性和基因表达变化差异不显著;LPS显著促进了大黄鱼头肾巨噬细胞IL-1β基因的表达,并且随培养温度升高细胞IL-1β基因的表达水平显著降低;但大黄鱼头肾巨噬细胞Nrf2和Hsp70的基因表达量随温度升高而显著增加,且LPS处理组细胞基因表达水平显著高于对照组。研究表明,温度显著影响了离体大黄鱼头肾巨噬细胞的抗氧化能力和LPS所诱导的促炎基因的表达,Nrf2和Hsp70在这一过程中可能发挥重要作用。

To study the effects of culture temperature on antioxidant capacity and inflammatory responses of Larimichthys crocea macrophages,macrophages were isolated from head kidney of L.crocea and resultant monolayer was maintained at 16℃,22℃and 28℃,respectively.After adding 25μg/mL LPS medium to macrophage monolayer for 2 h,cell viability,respiratory burst activity,antioxidant enzyme activities and gene expressions together with IL-1βand Hsp70 mRNA expressions of macrophage cultured at different temperatures were evaluated.Results showed that cell viability of macrophages placed at 16℃and 22℃was significantly higher than cells cultured at 28℃after incubation for 36 h.LPS could significantly promote the respiratory burst activity and inhibited the activities of SOD and CAT enzymes.Macrophage incubated at high temperature(28℃)showed significantly higher CAT enzyme activity and gene expression,whereas no significant difference was found in SOD enzyme activity and gene expression.LPS triggered potent pro-inflammatory response with IL-1βbeing the indicative parameter,which was inhibited by high culture temperature.However,transcript levels of Nrf2 and Hsp70 increased with the rise of temperature to a significant extent,and both gene expressions were markedly enhanced by LPS compared to control groups.Taken together,all the results in the present study indicated that temperature may regulate the antioxidant capacity and inflammatory responses of LPS-induced macrophages from head kidney of L.crocea through pathways involving Nrf2 and Hsp70.