The occurrence of pediatric diarrhea is frequently associated with inflammatory responses, compromised barrier function, and dysbiosis in the gut. These conditions are commonly triggered by stressors, similar to postweaning diarrhea observed in piglets. Garlic-derived exosome-like nanoparticles (GELNs) hold the potential for ameliorating stress-induced diarrhea, yet supporting evidence remains scarce. Following the successful isolation of GELNs, this study employed weaned piglets as a model to evaluate the regulatory effects of GELNs on intestinal barrier integrity, mucosal inflammation, and the gut microbiota and its metabolites. Weaned Bama miniature piglets were orally administered phosphate buffer saline (PBS) or GELNs, and 1 week later, samples were collected following slaughter. Histological and molecular biological techniques were performed to examine intestinal structure, tight junction protein expression, mucin secretion, T lymphocyte infiltration, and the levels of pro-inflammatory cytokines. The composition of the gut microbiota was analyzed using 16S rRNA sequencing, while its derived metabolites were profiled via untargeted metabolomics. Subsequently, correlation analyses were performed to evaluate the associations between the microbiota and its derived metabolites, as well as between the microbiota and the key indicators of intestinal barrier function and cytokine levels in response to GELNs. The isolated GELNs exhibit typical exosome characteristics in size and morphology, alongside a rich content of proteins and RNAs. The incidence of diarrhea in weaned piglets was reduced with supplementation of GELNs at a dosage of 50 mg/kg body weight, compared to the control group. In addition, piglets receiving GELNs displayed an increase in mucin content within the tissues of the jejunum, ileum, and colon, a decrease in CD8+ T lymphocyte counts in the colon, and suppression of pro-inflammatory cytokines (IL-8 and TNF-α) levels in the mucosal layers of both the jejunum and ileum. Furthermore, 16S rRNA sequencing unveiled that GELNs reshaped the colonic microbiota in weaned piglets by augmenting beneficial bacteria, notably Lactobacillus and Lactobacillus reuteri, correlating strongly with diminished TNF-α protein levels and heightened mucin expression. Metabolite analysis demonstrated a significant increase in indole-3-propionic acid, derived from the gut microbiota, in piglets supplemented with GELNs. This increase was positively correlated with the abundance of Lactobacillus and Lactobacillus reuteri and negatively linked with the protein levels of IL-8 and TNF-α in the gut. In summary, our study demonstrates that GELNs mitigate stress-related intestinal mucosal inflammation and enhance mucin production in the gut of weaned piglets, which is potentially achieved through the optimization of gut microbiota composition, specifically by increasing the abundance of Lactobacillus and Lactobacillus reuteri, as well as via the induction of the anti-inflammatory microbial metabolite indole-3-propionic acid. The findings presented here provide essential groundwork for the future development of GELNs as a therapeutic strategy aimed at enhancing gut homeostasis disruption caused by stress in both weaned piglets and children.

Piglets receive far less hydroxyproline (Hyp) from a diet after weaning than they obtained from sow's milk prior to weaning, suggesting that Hyp may play a protective role in preserving intestinal mucosal homeostasis. This study aimed to evaluate the effect of Hyp on intestinal barrier function and its associated gut microbiota and metabolites in early-weaned piglets. Eighty weaned piglets were divided into four groups and fed diets containing different Hyp levels (0 %, 0.5 %, 1 %, or 2 %) for 21 days. Samples, including intestinal contents, tissues, and blood, were collected on day 7 for analysis of microbial composition, intestinal barrier function, and metabolites. We demonstrated that dietary supplementation with 2 % Hyp improved the feed conversion ratio and reduced the incidence of diarrhea in early-weaned piglets compared to the control group. Concurrently, Hyp enhanced intestinal barrier function by facilitating tight junction protein (zonula occludens (ZO)-1 and occludin) expression and mucin production in the jejunal, ileal, and colonic mucosas. It also improved mucosal immunity (by increasing the amount of secretory IgA (sIgA) and the ratio of CD4+/CD8+ T lymphocytes and decreasing NF-κB phosphorylation) and increased antioxidant capacity (by raising total antioxidant capacity (T-AOC) and glutathione levels) in the intestinal mucosa. In addition, Hyp supplementation resulted in an increase in the levels of glycine, glutathione, and glycine-conjugated bile acids, while decreasing the concentrations of cortisol and methionine sulfoxide in plasma. Intriguingly, piglets fed diet containing Hyp exhibited a remarkable increase in the abundance of probiotic Enterococcus faecium within their colonic contents. This elevation occurred alongside an attenuation of pro-inflammatory responses and an enhancement in intestinal barrier integrity. Further, these changes were accompanied by a rise in anti-inflammatory metabolites, specifically glycochenodeoxycholic acid and guanosine, along with a suppression of pro-inflammatory lipid peroxidation products, including (12Z)-9,10-dihydroxyoctadec-12-enoic acid (9,10-DHOME) and 13-L-hydroperoxylinoleic acid (13(S)-HPODE). In summary, Hyp holds the capacity to enhance the intestinal barrier function in weaned piglets; this effect is correlated with changes in the gut microbiota and metabolites. Our findings provide novel insights into the role of Hyp in maintaining gut homeostasis, highlighting its potential as a dietary supplement for promoting intestinal health in early-weaned piglets.

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.

Heat stress (HS) occurs when the sensible temperature of animals exceeds their thermoregulatory capacity, a condition that exerts a detrimental impact on health and growth. The intestinal tract, as a highly sensitive organ, has been shown to respond to HS by exhibiting mucosal injury, intestinal leakage, and disturbances in the gut microbiota. Oxidative stress and endoplasmic reticulum stress (ERS) are both potential outcomes of long-term exposure to high temperatures and have been linked to apoptosis, autophagy, and ferroptosis. In addition, HS alters the composition of the gut microbiota accompanied by changed levels of bacterial components and metabolites, rendering the gut more vulnerable to stress-related injury. In this review, we present recent advances in mechanisms of oxidative stress-associated ERS in response to HS, which is destructive to intestinal barrier integrity. The involvement of autophagy and ferroptosis in ERS was highlighted. Further, we summarize the relevant findings regarding the engagement of gut microbiota-derived components and metabolites in modulation of intestinal mucosal injury induced by HS.

Heat stress (HS) induced by high ambient temperatures compromises intestinal epithelial cell. However, the underlying mechanisms by which HS causes intestinal barrier dysfunction remain unclear. In this study, we established an in vitro acute-HS-induced intestinal damage using porcine small intestinal epithelial cell (IPEC-J2) that exposed to the high temperatures (43 degrees C) for 2 h. The cell proliferation, apoptosis, tight junction (TJ) barrier integrity and transcriptomic profiles were measured. The results showed that HS decreased cell viability while increased proapoptotic signaling evidenced by Bax/bcl2 ratio, cytochrome C release to cytosol and active-caspase 3 increases (p < 0.01). HS led to decreased transepithelial electrical resistance, increased cell permeability, and downregulated TJ proteins including ZO1, occludin, and claudin 3 (p < 0.05). Transcriptome sequencing and KEGG pathway analysis revealed HS-induced cell cycle arrest and activation of endoplasmic reticulum stress (ERS) response mediated by a critical transcript eif2 alpha and proapoptotic molecule DDIT3 (known as CHOP). Furthermore, inhibition of ERS by 4-phenylbutyrate (4-PBA) administration and knockdown of eif2 alpha and CHOP significantly attenuated IPEC-J2 cells apoptosis (p < 0.05). Transmission electron microscopy analysis suggested that 4-PBA inhibited HS-induced increase in ER lumen diameter, indicating ultrastructural sign of ERS. In addition, HS-induced impairment of TJs was significantly attenuated by 4-PBA (p < 0.05). Collectively, HS induces ERS and activates the p-eif2 alpha/CHOP signaling pathway to impair epithelial barrier integrity through triggering the intestinal epithelial cell apoptosis.

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.