Chronic heat stress (HS) impairs intestinal barrier function and microbiota homeostasis. Taurine, a functional amino acid, is well characterized by anti-oxidation, anti-inflammation and cytoprotection. However, whether taurine can maintain intestinal barrier integrity and microbiota under chronic HS remains unknown. Mice (6-week-old; C57BL/6; Male) were randomly assigned one of three groups including a control group (CON, 23°C), a heat stress group (HS, 42°C), or a heat stress + taurine group (HT, received 0.5% taurine in drinking water under HS condition). This study investigated the effects of taurine supplementation on intestinal morphology, barrier function, endoplasmic reticulum stress (ERS), apoptosis, and gut microbiota in heat-stressed mice. Taurine supplementation contributed to decreased rectal temperature and body weight loss in heat-stressed mice. Taurine improved intestinal morphology as demonstrated by elevated villus height in HT group compared to HS group. HS-induced intestinal permeability indicative of increased d-lactate was inhibited by upregulating ZO-1 expression in mice administrated by Taurine. Supplementary taurine normalized the expression of endoplasmic reticulum (ER) stress proteins (BIP, IRE1α, XBP-1, CHOP) as well as proapoptotic proteins (BAX, Cytc, and Active-caspase 3) in HT mice. Taurine intervention resulted in expansion of taurine-utilizing Desulfovibrio in HT mice. Moreover, taurine supplementation enhanced enrichment of SCFA-producing microbiota such as Bifidobacterium, Dubosiella, and Faecalibaculum in heat-stressed mice. Collectively, these results suggested that taurine could attenuate HS-induced intestinal barrier dysfunction via reversing ER stress-mediated apoptosis and gut microbial dysbiosis, suggesting taurine as a promising dietary additive against the intestinal injury induced by HS in mice.

Heat stress (HS) compromises intestinal barrier integrity and microbiota homeostasis. Trehalose, a nonreducing disaccharide, is well known as a molecular chaperone to prevent protein misfolding under stress. However, whether trehalose supplementation can affect intestinal barrier integrity and microbiota under HS remains unknown. Male C57BL/6 mice (6-week-old) were randomly divided into 3 groups (7 mice/group): a normal control group (CON, 23℃), a heat stress group (HS, 42℃) and heat stress + trehalose group (HT, received 2.0% trehalose in drinking water under HS condition). Trehalose supplementation decreased rectal temperature and body weight loss in heat-stressed mice. HS-induced intestinal permeability characterized by increased d-lactate was inhibited by upregulating protein expression of tight junction proteins including occludin and Zo1 in mice administrated by trehalose. Supplementary trehalose significantly ameliorated HS-induced oxidative stress by elevating activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) as well as protein expression of catalase (CAT). Trehalose intervention reversed the up-regulation of endoplasmic reticulum (ER) stress proteins glucose-regulated protein 78 (GRP78), growth arrest and DNA damage-inducible protein 34 (GADD34), phosphorylated eukaryotic initiation factor 2α (p-eif2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) as well as proapoptotic proteins BAX, Cytc, and active-caspase3 in heat-stressed mice. Trehalose supplementation evidently elevated the abundance of beneficial bacteria Lachnospiraceae_NK4A136_group while reducing that of harmful bacteria Bacteroidetes in heat-stressed mice. Taken together, these results revealed that supplementary trehalose could attenuate HS-induced intestinal barrier dysfunction via reducing oxidative stress, reversing ER stress induced apoptosis and gut microbial dysbiosis, suggesting trehalose as a promising dietary additive to counteract the intestinal injury induced by HS in mice.

本试验旨在研究N-乙酰半胱氨酸(NAC)对热应激诱导的猪小肠上皮细胞IPEC-J2抗氧化能力、内质网应激通路蛋白表达和细胞凋亡的影响.采用单因子试验设计,试验分3组,分别为对照组(CON,37℃)、热应激组(HS,43℃)和NAC加热应激组(NAC+HS,43℃).其中NAC+HS组细胞在NAC(0.5 mmol/L)预处理12 h后,进行热应激处理4 h,测定细胞氧化损伤情况、活性氧自由基(ROS)含量、抗氧化酶的活性、线粒体膜电位变化、细胞内质网应激和凋亡相关蛋白的表达.结果表明:与CON组相比,热应激导致细胞中ROS和丙二醛(MDA)含量显著增加,铜锌超氧化物歧化酶(SOD1)、锰超氧化物歧化酶(SOD2)和过氧化氢酶(CAT)的表达量提高(P<0.05);与HS组相比,添加NAC降低了氧化损伤和抗氧化酶(SOD2、CAT)的表达(P<0.05).与CON组相比,热应激增加热休克蛋白A5(HSPA5)、磷酸化真核细胞翻译启始因子2α(p-eif2α)、转录激活因子4(ATF4)、C/EBP同源蛋白(CHOP)蛋白表达量(P<0.01);添加NAC降低HSPA5、p-eif2α、ATF4、CHOP蛋白表达量(P<0.01).NAC预处理降低了热应激诱导的凋亡标志蛋白细胞色素C(Cytc)和活化半胱氨酸天冬氨酸蛋白酶3(Active-caspase3)的表达(P<0.05).综上,添加NAC可以缓解热应激诱导的IPEC-J2细胞的氧化应激和内质网应激,减少细胞凋亡.

This study was carried out to investigate the effects of trehalose (Tre) on antioxidant capacity, endoplasmic reticulum stress (ERS) response and apoptosis of heat-stressed intestinal porcine epithelial cells (IPEC-J2). IPEC-J2 cells were cultured at 37 °C until the end of the experiment (control, CON); exposed to heat stress for 2 h (43 °C, HS); or pretreated with 0.1, 1, 5, 10, and 15 mM trehalose at 37 °C for 4 h prior to heat stress exposure for 2 h. The optimum level of trehalose for protecting against HS-induced cell injuries was determined to be 10 mM, as evidenced by the highest cellular viability and lowest malondialdehyde (MDA) content and lactate dehydrogenase (LDH) activity. Based on these, IPEC-J2 cells were divided into three groups: the first group was cultured at 37 °C until the end of the experiment (control, CON); the second group was exposed to heat stress for 2 h (43 °C, HS); the third group was pretreated with 10 mM trehalose for 4 h at 37 °C prior to heat stress exposure for 2 h (Tre + HS). The reactive oxygen species (ROS) content, superoxide dismutase (SOD) activity, mitochondrial membrane potential (MMP) changes, and expressions of the manganese superoxide dismutase (SOD2), ERS and apoptosis-related proteins were determined. Compared to the CON group, HS significantly increased ROS generation (p < 0.01), decreased SOD activity (p < 0.05), and downregulated protein expression of SOD2 (p < 0.01). Compared to the HS group, Tre supplementation reduced ROS levels and increased SOD activity and SOD2 expression to the levels that were comparable to the control (p < 0.05). The HS-induced ERS response was evidenced by the increased protein expressions of glucose-regulated protein 78 (GRP78) (p < 0.01), eukaryotic translation initiation factor 2α (p-eif2α) (p < 0.01), transcription activator 4 (ATF4) (p < 0.01), and the protein expression of C/EBP homologous protein (CHOP) (p < 0.01), which were the four hallmarks of ERS. The Tre + HS group showed lower expressions of GRP78 (p < 0.01), p-eif2α (p < 0.01), ATF4 (p < 0.01), and CHOP (p < 0.01) than that of the HS group. Tre pretreatment attenuated HS-induced mitochondrial apoptosis in IPEC-J2 cells, demonstrated by the increased MMP and decreased proapoptotic proteins active caspase 3, Bax, and cytochrome c (Cyt c). Taken together, trehalose can protect against HS-induced oxidative damage and endoplasmic reticulum stress-mediated apoptosis in IPEC-J2 cells. These data may provide a nutritional strategy for alleviating heat stress in pig production.

The intestinal epithelium is susceptible to heat stress (HS), which leads to gut leakage and inflammation. However, the mechanisms underlying HS-induced intestine dysfunction have yet to be elucidated. We established an in vitro chronic heat exposure-induced intestinal injury of intestinal porcine epithelial cells (IPEC-J2) exposed to high temperatures (43 °C) for 12 h. The results revealed that HS increased reactive oxygen species (ROS) generation and decreased superoxide dismutase 2 (SOD2) expression, leading to oxidative stress. Western blotting analysis demonstrated that HS induced apoptosis as evidenced by increased cytochrome c (Cyt c) release in the cytoplasm and caspase 3 activation. Transcriptome sequencing analysis revealed that HS activated the endoplasmic reticulum stress (ERS) response/unfolded protein response (UPR) but inhibited glutathione metabolism. Specifically, HS triggered the pro-apoptotic activating transcription factor 4 (ATF4)/CEBP-homologous protein (CHOP) branch of the UPR. Interestingly, glutathione-specific gamma-glutamylcyclotransferase1 (CHAC1) involved in glutathione degradation was upregulated due to heat exposure and was proved to be downstream of the ATF4-CHOP signal pathway. Knockdown of CHAC1 attenuated the HS-induced decrease in glutathione level and cell apoptosis. These studies suggest that crosstalk between ERS and oxidative stress in HS-induced apoptosis might be dependent on the ATF4-CHOP-CHAC1 signal pathway in IPEC-J2 cells.