Peng Yin

Effect of heat stress on the porcine small intestine: a morphological and gene expression study.

With the presence of global warming, the occurrence of extreme heat is becoming more common, especially during the summer, increasing pig susceptibility to severe heat stress. The aim of the current study was to investigate changes in morphology and gene expression in the pig small intestine in response to heat stress. Forty eight Chinese experimental mini pigs (Sus scrofa) were subjected to 40 degrees C for 5h each day for 10 successive days. Pigs were euthanized at 1, 3, 6, and 10 days after heat treatment and sections of the small intestine epithelial tissue were excised for morphological examination and microarray analyses. After heat treatment, the pig rectal temperature, the body surface temperature and serum cortisol levels were all significantly increased. The duodenum and jejunum displayed significant damage, most severe after 3 days of treatment. Microarray analysis found 93 genes to be up-regulated and 110 genes to be down-regulated in response to heat stress. Subsequent bioinformatic analysis (including gene ontology and KEGG pathway analysis) revealed the genes altered in response to heat stress related to unfolded protein, regulation of translation initiation, regulation of cell proliferation, cell migration and antioxidant regulation. Heat stress caused significant damage to the pig small intestine and altered gene expression in the pig jejunum. The results of the bioinformatic analysis from the present study will be beneficial to further investigate the underlying mechanisms involved in heat stress-induced damage in the pig small intestine.

Punicalagin Induces Nrf2/HO-1 Expression via Upregulation of PI3K/AKT Pathway and Inhibits LPS-Induced Oxidative Stress in RAW264.7 Macrophages

Reactive oxygen species (ROS) and oxidative stress are thought to play a central role in potentiating macrophage activation, causing excessive inflammation, tissue damage, and sepsis. Recently, we have shown that punicalagin (PUN) exhibits anti-inflammatory activity in LPS-stimulated macrophages. However, the potential antioxidant effects of PUN in macrophages remain unclear. Revealing these effects will help understand the mechanism underlying its ability to inhibit excessive macrophage activation. Hemeoxygenase-1 (HO-1) exhibits antioxidant activity in macrophages. Therefore, we hypothesized that HO-1 is a potential target of PUN and tried to reveal its antioxidant mechanism. Here, PUN treatment increased HO-1 expression together with its upstream mediator nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). However, specific inhibition of Nrf2 by brusatol (a specific Nrf2 inhibitor) dramatically blocked PUN-induced HO-1 expression. Previous research has demonstrated that the PI3K/Akt pathway plays a critical role in modulating Nrf2/HO-1 protein expression as an upstream signaling molecule. Here, LY294002, a specific PI3K/Akt inhibitor, suppressed PUN-induced HO-1 expression and led to ROS accumulation in macrophages. Furthermore, PUN inhibited LPS-induced oxidative stress in macrophages by reducing ROS and NO generation and increasing superoxide dismutase (SOD) 1 mRNA expression. These findings provide new perspectives for novel therapeutic approaches using antioxidant medicines and compounds against oxidative stress and excessive inflammatory diseases including tissue damage, sepsis, and endotoxemic shock.

Involvement of ERK1/2 signalling and growth-related molecules' expression in response to heat stress-induced damage in rat jejunum and IEC-6 cells

Our previous studies found small intestine epithelial tissues from several different animals (including rats, pigs and chickens) became significantly damaged following exposure to extreme heat. However, damaged tissue was rapidly repaired or regenerated in the following few days. Growth-related molecules are critical for cellular survival and promote endothelial cell proliferation and migration. The ERK1/2 signalling pathway is reported to regulate the growth and adaptation of endothelial cells to both physiological and pathological stimuli. However, little information is available concerning both growth-related molecules and ERK1/2 in response to heat stress. Herein, we employed both live rats and rat IEC-6 cells to investigate growth-related molecule expression and ERK1/2 activation in heat stress. Heat stress caused significant morphological damage to rat intestinal tissue and IEC-6 cells, reduced cell growth and proliferation, induced apoptosis, altered growth-related molecule mRNA expression and increased ERK1/2 phosphorylation. Addition of U0126 (a selective inhibitor of MEK kinase responsible for ERK phosphorylation) combined with heat stress exacerbated the morphological damage and apoptosis. With the addition of U0126, further up- or down-regulation of Egfr, Ctgf, Tgif, Vegfa, Okl38 and Gdf15 in response to heat stress was observed. In conclusion, extreme heat stress caused obvious damage to rat jejunum and IEC-6 cells. Both growth-related molecule expression and ERK1/2 phosphorylation were involved in response to heat stress. ERK1/2 inhibition exacerbated apoptosis and affected growth factor mRNA expression in heat stress.

Involvement of oxidative stress and mitogen-activated protein kinase signaling pathways in heat stress-induced injury in the rat small intestine

Extreme heat stress-induced gastrointestinal injury and dysfunction may occur during summer. We investigated possible mechanisms of heat stress-induced damage in the small intestine using male Sprague-Dawley rats subjected to 2 h of heat stress (40°C, 60% relative humidity) daily for 10 consecutive days. Rats were killed at specific times immediately following heat treatment to determine: morphological changes by optical and electron microscopy; intestinal permeability using fluorescein isothiocyanate–dextran; production of reactive oxygen species (ROS), malondialdehyde (MDA), and activities of superoxide-dismutase and glutathione-peroxidase by specific assays; phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) by immunocytochemistry and western-blot analysis. The rat intestinal epithelial cell line (IEC-6) and specific MAPK inhibitors were used for in vitro investigation of effects of activation of MAPKs by heat stress. Heat stress caused marked morphological damage to the small intestine and significantly increased intestinal permeability. Heat stress increased ROS and MDA production, and significantly reduced anti-oxidase activity. MAPK activity in small intestine was increased by heat stress. In vitro, heat stress caused damage and apoptosis in IEC-6 cells; inhibition of ERK1/2 activation (by U0126) exacerbated these effects, which were attenuated by inhibition of JNK (by SP600125) and p38 (by SB203580) activation. Hence, heat stress caused severe small intestine injury, increased oxidative stress, and activated MAPK signaling pathways. The in vitro studies indicated that ERK1/2 activation is anti-apoptotic, and JNK and p38 activation are pro-apoptotic in heat stressed intestinal epithelial cells.

The protective effect of caffeic acid against inflammation injury of primary bovine mammary epithelial cells induced by lipopolysaccharide

Caffeic acid possesses multiple biological effects, such as antibacterial, antioxidant, antiinflammatory, and anticancer growth; however, what effects it has on bovine mastitis have not been investigated. The aim of this study was to verify the antiinflammatory properties of caffeic acid on the inflammatory response of primary bovine mammary epithelial cells (bMEC) induced by lipopolysaccharide (LPS), and to clarify the possible underlying mechanism. Bovine mammary epithelial cells were treated with various concentrations (10, 50, 100, and 200 μg/mL) of LPS for 3, 6, 12, and 18 h; the results showed that LPS significantly inhibited cell viability in a time- and dose-dependent manner. When cells were treated with LPS (50 μg/mL) for 12h, the cell membrane permeability significantly increased, which promoted cell apoptosis. Various concentrations (10, 25, and 50 μg/mL) of caffeic acid could weaken the inflammation injury of bMEC induced by LPS without cytotoxicity. Proinflammatory cytokines (IL-8, IL-1β, IL-6, and tumor necrosis factor α) from bMEC were decreased. Nuclear transcription factor κB activity was weakened via blocking κB inhibitor α degradation and p65 phosphorylation. All these showed that the protective effect of caffeic acid on LPS-induced inflammation injury in bMEC was at least partly achieved by the decreased production of proinflammatory cytokines mediated by the effect of reducing the κB inhibitor α degradation and p65 phosphorylation in the nuclear transcription factor κB pathway. The use of caffeic acid would be beneficial in dairy cows during Escherichia coli mastitis as a safe and natural antiinflammatory drug.

Active components of common traditional Chinese medicine decoctions have antioxidant functions.

Many traditional Chinese medicine (TCM) decoctions are proven to have multiple functions in animal production. These decoctions are seldom recognized by the international scientific community because the mechanisms of action are not clearly elucidated. According to TCM theory, Cortex Phellodendri (COP), Rhizoma Atractylodes (RA), Agastache Rugosa (AR), and Gypsum Fibrosum (GF) can be used to formulate a medicinal compound that prevents or cures animal disease caused by heat stress. The aim of this research was to study the regulatory functions of the active components of TCM and to elucidate the effects of different TCM decoctions on antioxidant activity and lipid peroxide content, using in vitro and in vivo models of heat stress. For in vitro experiments, intestinal crypt-like epithelial cell line-6 (IEC-6) cells were employed to evaluate the effects of the active components of COP, RA, AR, and GF. For in vivo experiments, forty-eight 2-mo-old Chinese experimental mini-pigs (7.20 ± 0.02 kg) were randomly assigned to 4 groups: a normal-temperature group (NTG); a high-temperature group (HTG); HTG treated with COP, RA, AR, and GF (1:1:1:1, TCM1); and HTG treated with COP, RA, AR, and GF (1:1:1:0.5, TCM2). Results showed that the active components of the COP, RA, AR, and GF increased (P < 0.05) the proliferation and viability of heat-stressed IEC-6 cells and that the most effective treatment doses of COP alkaloid, RA Aetherolea, Herba Agastachis Aetherolea, and GF water extract were 200, 100, 100, and 200 µg/mL, respectively. All 4 active components increased (P < 0.05) superoxide dismutase, glutathione peroxidase activities, and glutathione content, and decreased (P < 0.05) malondialdehyde content with respect to the heat-stressed group to concentrations similar to those seen in NTG. In vivo experiments demonstrated that TCM1 and TCM2 improved (P < 0.05) the poor growth performance seen in HTG pigs. The superoxide dismutase, glutathione peroxidase activities, and malondialdehyde content in porcine jejunum treated with TCM1 and TCM2 were not different (P > 0.05) from those seen in the NTG and were better (P < 0.05) than results seen in the HTG. Overall, it appeared that TCM2 was more effective than TCM1 in ameliorating the effects of heat stress in pigs. In conclusion, this study revealed that the active components of common TCM decoctions have antioxidant functions.

Protective Effects of Ferulic Acid against Heat Stress-Induced Intestinal Epithelial Barrier Dysfunction In Vitro and In Vivo.

Heat stress is important in the pathogenesis of intestinal epithelial barrier dysfunction. Ferulic acid (FA), a phenolic acid widely found in fruits and vegetables, can scavenge free radicals and activate cell stress responses. This study is aimed at investigating protective effects of FA on heat stress-induced dysfunction of the intestinal epithelial barrier in vitro and in vivo. Intestinal epithelial (IEC-6) cells were pretreated with FA for 4 h and then exposed to heat stress. Heat stress caused decreased transepithelial electrical resistance (TER) and increased permeability to 4-kDa fluorescein isothiocyanate (FITC)-dextran (FD4). Both effects were inhibited by FA in a dose-dependent manner. FA significantly attenuated the decrease in occludin, ZO-1 and E-cadherin expression observed with heat stress. The distortion and redistribution of occludin, ZO-1 and E-cadherin proteins were also effectively prevented by FA pretreatment. Moreover, heat stress diminished electron-dense material detected in tight junctions (TJs), an effect also alleviated by FA in a dose-dependent manner. In an in vivo heat stress model, FA (50 mg/kg) was administered to male Sprague–Dawley rats for 7 consecutive days prior to exposure to heat stress. FA pretreatment significantly attenuated the effects of heat stress on the small intestine, including the increased FD4 permeability, disrupted tight junctions and microvilli structure, and reduced occludin, ZO-1 and E-cadherin expression. Taken together, our results demonstrate that FA pretreatment is potentially protective against heat stress-induced intestinal epithelial barrier dysfunction.

Effect of simulated transport stress on the rat small intestine: A morphological and gene expression study.

The present study investigated the effects of simulated transport stress on morphology and gene expression in the small intestine of laboratory rats. Sprague Dawley rats were subjected to 35°C and 0.1×g on a constant temperature shaker for physiological, biochemical, morphological and microarray analysis before and after treatment. The treatment induced obvious stress responses with significant decreases in body weight (P<0.01), increases in rectal temperature, serum corticosterone (CORT), serum glucose (GLU), creatine kinase (CK) and lactate dehydrogenase (LDH) levels (P<0.01), as well as expression of Hsp27/70/90 mRNA (P<0.05; P<0.01). The rat jejunum was severely damaged and apoptotic after mimicking transport stress, which may mainly be related to cell death, oxidation reduction and hormone imbalance determined by microarray analysis. The bioinformatics analysis from the present study would provide insight into the potential mechanisms underlying transport stress-induced injury in the rat small intestine.

Punicalagin induces Nrf2 translocation and HO-1 expression via PI3K/Akt, protecting rat intestinal epithelial cells from oxidative stress

Abstract Purpose: Oxidative stress plays a central role in heat stress-induced gastrointestinal injury. Punicalagin (PUN), a major polyphenol abundant in pomegranate fruit, husk and juice, exhibits antioxidative effects. In this study we used a heat stress model to investigate the intestinal protection effect of PUN and the underlying mechanisms. Materials and methods: IEC-6 cells were pretreated with PUN for 6 h and exposed to 42 °C for 6 h. Intracellular reactive oxygen species levels, malondialdehyde, nitrogen oxide, and superoxide dismutase activity were measured. IEC-6 cells were treated with PUN at different times and doses, the protein levels of haeme oxygenase-1 (HO-1) were evaluated. The nuclear translocation of the transcription factor NF-erythroid 2-related factor (Nrf2) and the phosphorylation level of PI3K/Akt were also investigated. Results: PUN significantly decreased the heat stress-induced cell death and apoptosis. Heat stress increased reactive oxygen species, malondialdehyde and nitrogen oxide production, while it decreased superoxide dismutase activity. These effects were markedly inversed when the cells were pretreated with PUN. Furthermore, PUN treatment induced the expression of HO-1 and increased Nrf2 nuclear translocation in a time- and dose-dependent manner. The Nrf2-related cytoprotective effects of PUN were via the PI3K/Akt signalling pathway, as LY294002, a specific PI3K/Akt inhibitor, suppressed the PUN-induced nuclear translocation of Nrf2, the HO-1 up-regulation and the protective effect of PUN against oxidative stress. Conclusions: PUN protects IEC-6 cells against oxidative stress by up-regulating the expression of HO-1 via a mechanism that involves PI3K/Akt activation and Nrf2 translocation.

Endoplasmic Reticulum Stress in Heat- and Shake-Induced Injury in the Rat Small Intestine

We investigated the mechanisms underlying damage to rat small intestine in heat- and shake-induced stress. Eighteen Sprague-Dawley rats were randomly divided into a control group and a 3-day stressed group treated 2 h daily for 3 days on a rotary platform at 35°C and 60 r/min. Hematoxylin and eosin-stained paraffin sections of the jejunum following stress revealed shedding of the villus tip epithelial cells and lamina propria exposure. Apoptosis increased at the villus tip and extended to the basement membrane. Photomicrographs revealed that the microvilli were shorter and sparser; the nuclear envelope invaginated and gaps in the karyolemma increased; and the endoplasmic reticulum (ER) swelled significantly. Gene microarray analysis assessed 93 differentially expressed genes associated with apoptosis, ER stress, and autophagy. Relevant genes were compiled from the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Forty-one genes were involved in the regulation of apoptosis, fifteen were related to autophagy, and eleven responded to ER stress. According to KEGG, the apoptosis pathways, mitogen-activated protein kinase(MAPK) signaling pathway, the mammalian target of rapamycin (mTOR) signaling pathway, and regulation of autophagy were involved. Caspase3 (Casp3), caspase12 (Casp12), and microtubule-associate proteins 1 light chain 3(LC3) increased significantly at the villus tip while mTOR decreased; phosphorylated-AKT (P-AKT) decreased. ER stress was involved and induced autophagy and apoptosis in rat intestinal damage following heat and shake stress. Bioinformatic analysis will help determine the underlying mechanisms in stress-induced damage in the small intestine.