高NEFA血症通过内质网应激加剧奶牛乳腺上皮细胞炎性通路的活化

High concentration of blood NEFA exacerbates the activation of inflammatory pathway in bovine mammary epithelial cells by activating the endoplasmic reticulum stress

酮病是高产奶牛常发的一种以高非酯化脂肪酸(non-esterified fatty acids,NEFA)为病理学特征的能量代谢障碍疾病。酮病奶牛脂解产生的大量NEFA被乳腺上皮细胞(bovine mammary epithelial cell,BMEC)所吸收,而高浓度的NEFA具有显著的细胞毒性,这可能与乳腺的非感染性炎性水平显著升高有关,但内在机制尚未明确。本研究利用BMEC进行体外试验,探讨高浓度NEFA对BMEC炎性通路的影响和作用机制。体外培养BMEC,添加不同浓度的NEFA和shIRE1α,通过免疫荧光法检测细胞角蛋白-18(CK-18)鉴定分离的BMEC,采用Western blot和qRT-PCR检测内质网应激关键分子和NF-κB炎性信号通路关键分子的基因转录水平和蛋白表达水平。结果显示,高NEFA可以显著增加IRE1α的磷酸化水平和葡萄糖调节蛋白(glucose regulated protein 78,GRP78)的表达,增加p65的磷酸化水平和下游炎性因子肿瘤坏死因子α(TNF-α)、白介素-6(IL-6)和白介素-1β(IL-1β)的mRNA表达水平。shIRE1α转染BMEC后,显著降低IRE1α的磷酸化水平,且沉默IRE1α能显著逆转高NEFA对BMEC的NF-κB信号通路的活化,使p65的磷酸化水平显著降低,且显著降低其下游炎性因子TNF-α、IL-6和IL-1β的mRNA表达水平。结果表明,NEFA能够诱导BMEC的内质网应激,并通过IRE通路激活NF-κB信号通路促进BMEC炎性因子的表达,增加乳腺非感染性炎性水平,本研究为进一步探究酮病奶牛乳腺损伤的分子机制奠定了基础。

Ketosis is a common disease of energy metabolism disorder characterized by high levels of non-esterified fatty acids(NEFA)in high-yielding cows.Large amounts of NEFA produced by lipolysis in ketosis cows are absorbed by bovine mammary epithelial cells(BMEC),whereas high concentrations of NEFA have significant cytotoxicity,which may be correlated with significantly increased levels of non-infectious inflammation in the mammary gland,however,the intrinsic mechanism has not been clarified.In this study,we investigated the effects and mechanisms of high concentrations of NEFA on inflammatory pathways in BMEC by in vitro experiments with BMEC.BMEC were cultured in vitro with different concentrations of NEFA and shIRE1α,and isolated BMEC were validated by identifying cytokeratin-18(CK-18)using immunofluorescence assay.Western blot and qRT-PCR were utilized to detect the expression levels of key genes and proteins in the endoplasmic reticulum stress and the NF-κB inflammatory signaling pathway.The results revealed that high NEFA significantly increased the phosphorylation level of IRE1αand the expression level of glucose regulated protein 78(GRP78),and also enhanced the phosphorylation level of p65and the mRNA expression level of downstream inflammatory factors such as tumor necrosis factorα(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β).After shIRE1αtransfection,the phosphorylation level of IRE1αwas significantly reduced,and silencing of IRE1αsignificantly reversed the activation of NF-κB signaling pathway in BMEC by high NEFA,resulting in significant reduction of p65phosphorylation level and the mRNA expression level of its downstream inflammatory factors TNF-α,IL-6and IL-1β.The data above suggest that NEFA can induce endoplasmic reticulum stress in BMEC and promote the expression of inflammatory factors in BMEC through the activation of NF-κB signaling pathway by IRE pathway,increasing the level of non-infectious inflammation in mammary gland,thus laying the foundation for further investigation into the molecular mechanisms of mammary gland injury in ketosis cows.