Jujuboside A attenuates norepinephrine-induced apoptosis of H9c2 cardiomyocytes by modulating MAPK and AKT signaling pathways.

Cardiomyocyte apoptosis is closely associated with the pathogenesis of heart failure. Jujuboside A (JUA) is a type of saponin isolated from the seeds of Zizyphus jujuba. In traditional Chinese medicine, it is believed that JUA possesses multiple biological effects, including antianxiety, antioxidant and anti­inflammatory activities. The present study aimed to evaluate the effects of JUA on norepinephrine (NE)­induced apoptosis of H9c2 cells and to investigate its underlying mechanisms. Rat H9c2 cardiomyocytes were pretreated with JUA and were then exposed to NE as an in vitro model of myocardial apoptosis. A cell viability assay, scanning electron microscopy, transmission electron microscopy, flow cytometry assay, acridine orange/ethidium bromide staining, reverse transcription­quantitative polymerase chain reaction and western blotting, all revealed that NE induced H9c2 cell apoptosis. The results demonstrated that NE inhibited cell viability, and enhanced cell damage and apoptosis of H9c2 cells. Conversely, pretreatment with JUA was able to reverse NE­induced decreased cell viability and increased apoptosis. Furthermore, JUA suppressed upregulation of the B­cell lymphoma 2 (Bcl­2)­associated X protein/Bcl­2 ratio, and inhibited the increased protein expression levels of cleaved caspase­3 and cleaved caspase­9 following NE exposure. However, the protein expression levels of cleaved caspase­12 and cleaved caspase­8 were not significantly altered following exposure to NE or JUA pretreatment. In addition, in JUA­pretreated cells, the protein expression levels of phosphorylated (p)­p38 and p­c­Jun N­terminal kinase were downregulated compared with in NE­treated cells. Furthermore, JUA regulated the activation of extracellular signal­regulated kinase (ERK) in NE­treated cells and significantly increased the expression levels of p­AKT. Taken together, these data suggested that JUA may protect against NE­induced apoptosis of cardiomyocytes via modulation of the mitogen­activated protein kinase and AKT signaling pathways. Therefore, JUA may be considered a potential therapeutic strategy for the treatment of heart disease.

Jujuboside A Protects H9C2 Cells from Isoproterenol-Induced Injury via Activating PI3K/Akt/mTOR Signaling Pathway.

Jujuboside A is a kind of the saponins isolated from the seeds of Ziziphus jujuba, which possesses multiple biological effects, such as antianxiety, antioxidant, and anti-inflammatory effects; however, its mediatory effect on isoproterenol-stimulated cardiomyocytes has not been investigated yet. In this study, we tried to detect the protective effect and potential mechanism of JUA on ISO-induced cardiomyocytes injury. H9C2 cells were treated with ISO to induce cell damage. Cells were pretreated with JUA to investigate the effects on the cell viability, morphological changes, light chain 3 conversion, and the activation of PI3K/Akt/mTOR signaling pathway. Results showed that ISO significantly inhibited the cell viability in a time- and dose-dependent manner. JUA pretreatment could reverse the reduction of cell viability and better the injury of H9C2 cells induced by ISO. Western blot analysis showed that JUA could accelerate the phosphorylation of PI3K, Akt, and mTOR. Results also indicated that JUA could significantly decrease the ratio of microtubule-associated protein LC3-II/I in H9C2 cells. Taken together, our research showed that JUA could notably reduce the damage cause by ISO via promoting the phosphorylation of PI3K, Akt, and mTOR and inhibiting LC3 conversion, which may be a potential choice for the treatment of heart diseases.

Transport stress induces apoptosis in rat myocardial tissue via activation of the mitogen-activated protein kinase signaling pathways.

The present study aimed to elucidate the mechanism of myocardial damage induced by simulated transport stress. Sprague-Dawley rats were subjected to 35 °C and 60 rpm (0.1×g rcf) on a constant temperature shaker. The blood samples were prepared for detection of epinephrine (E), norepinephrine (NE), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and serum cardiac troponin T (cTNT); myocardium samples were prepared for morphological examination and signaling protein quantitative. The result showed that plasma norepinephrine (NE) and epinephrine (E) concentrations increased in all stressed groups (P < 0.01). Levels of serum cardiac troponin T (cTNT) were elevated in both the S2d (P < 0.05) and S3d groups (P < 0.01). The concentration of plasma BNP was increased significantly in S3d group (P < 0.05); the difference in ANP was not remarkable. Morphological observation demonstrated obvious microstructure and ultrastructure damage after simulated transport stress. There was also a significant increase in the number of TUNEL-positive cardiomyocytes in stressed hearts. Western blot analysis found that the mitogen-activated protein kinase (MAPK) pathways were activated by strengthening phosphorylation of ASK-1, JNK, P38 and ERK in rat myocardial tissue after simulated transport stress (P < 0.05, P < 0.01). In addition, the ratio of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins was increased in stressed rats (P < 0.01), and the amount of cleaved-caspase3 increased in all stressed rats (P < 0.01). The expression of cleaved-caspase9 protein was also elevated in S2d and S3d groups (P < 0.01). Consequently simulated transport stress induced obvious myocardial damage, which may be attributed to the activation of caspase 9-mediated mitochondrial apoptotic pathway and MAPK pathways.

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.

Activation of transcription factor AP-1 in response to thermal injury in rat small intestine and IEC-6 cells.

BACKGROUND: Our previous studies indicated that heat stress can cause significant damage to the intestinal epithelium and induce differential expression of many genes in rat small intestine. The transcription factors AP-1 and NF-κB, which act as important mediators by binding to specific DNA sequences within gene promoters, regulate the transcription of genes associated with immune regulation, stress response and cell fate. METHODS: To determine whether AP-1 and NF-κB are involved in hyperthermia-induced injury in rat small intestine and IEC-6 cells, we investigated their activity, and the expression of related proteins, by electrophoretic mobility shift assays and western blotting, respectively. RESULTS: Heat stress resulted in severe damage to the epithelium of the small intestine. The cell morphology and viability were obviously altered when IEC-6 cell was exposed to hyperthermia. AP-1 was activated in the small intestine of heat-stressed rats, as was phosphorylation of the JNK signaling pathway. In IEC-6 cell line, AP-1 activation in groups exposed to 42 °C for 1 h, 2 h and 4 h was significantly increased. In contrast, NF-κB was not activated in both in vivo and in vitro models. CONCLUSION: These results reveal that AP-1 is likely to play an important role in regulating gene transcription in rat small intestine and IEC-6 cells during exposure to heat stress.

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.

Quantitative proteomic analysis reveals heat stress-induced injury in rat small intestine via activation of the MAPK and NF-κB signaling pathways.

The intestinal epithelium plays a critical role in absorbing nutrients and maintaining the integrity of the gut barrier. Extreme heat stress induces damage to the intestinal epithelium. However, the protein expression changes and the mechanism behind this damage remain poorly understood. In this study, morphological observation showed that heat stress induced desquamation of intestinal epithelial cells, and destruction of intestinal microvilli and mitochondria. Heat stress-induced changes in the intestinal proteome were quantified using the iTRAQ method followed by mass spectrometry and software analysis. A total of 1689 proteins were identified in rat intestine tissue, of which 41 showed significantly altered expression between the heat stressed and control groups. However, these proteins with significant alterations were involved in biological processes such as cellular assembly and organization, developmental disorder, organismal injury and abnormalities, and inflammation. We found that members of the MAPK and NF-κB signaling pathways act as hub proteins in the network interaction analysis. Furthermore, western blot analysis verified that the MAPK and NF-κB signaling pathways were activated by heat stress as expected. This study suggests that heat stress induces cell cytoskeleton reorganization and an inflammatory response, and the activation of the MAPK and NF-κB signaling pathways, which may ultimately contribute to intestinal injury.

Microarray analysis of intestinal immune-related gene expression in heat-stressed rats.

PURPOSE: This study aimed to investigate immune-related gene expression in rat small intestine after heat stress. MATERIALS AND METHODS: Twelve Sprague Dawley (SD) rats were randomly divided into control and heat-stressed groups. Rats in both groups were housed at 25 °C with 60% relative humidity. The heat-stressed group was subjected to 40 °C for 2 h/day for 3 days. After heat stress, the mRNA expression profile of small intestine epithelial tissue was evaluated by microarray analysis. RESULTS: A total of 23 genes related to immune responses were significantly altered, of which 12 genes were up-regulated and 11 genes were down-regulated. CONCLUSIONS: Microarray analysis demonstrated the JAK-STAT pathway had a potentially important role in the regulation of inflammation in the small intestine, and changes in antigen presentation might reduce intestinal immune responses after heat stress.

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-1ß, 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.

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 inhibits inflammation in LPS-induced RAW264.7 macrophages via the suppression of TLR4-mediated MAPKs and NF-κB activation.

Punicalagin (2,3,hexahydroxydiphenoyl-gallagyl-D-glucose and referred to as PUN) is a bioactive ellagitannin isolated from pomegranate, which is widely used for the treatment of inflammatory bowel disease (IBD), diarrhea, and ulcers in Chinese traditional medicine. In this study, we detected the anti-inflammation potentials of PUN in lipopolysaccharide (LPS)-induced macrophages and tried to uncover the underlying mechanism. Results demonstrated that PUN (25, 50, or 100 µM) treatment could significantly decrease the LPS-induced production of nitric oxide), prostaglandin E2 (PGE2), interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in RAW264.7 cells. Molecular research showed that PUN inhibited the activation of upstream mediator nuclear factor-κB by suppressing the phosphorylation of IκBα and p65. Results also indicated that PUN could suppress the phosphorylation of mitogen-activated protein kinase including p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase. In conclusion, we observed that PUN could inhibit LPS-induced inflammation, and it may be a potential choice for the treatment of inflammation diseases.

Inhibition of heat-induced apoptosis in rat small intestine and IEC-6 cells through the AKT signaling pathway.

BACKGROUND: As the world warms up, heat stress is becoming a major cause of economic loss in the livestock industry. Long-time exposure of animals to hyperthermia causes extensive cell apoptosis, which is harmful to them. AKT and AKT-related serine-threonine kinases are known to be involved in signaling cascades that regulate cell survival, but the mechanism remains elusive. In the present study, we demonstrate that phosphoinositide 3-kinase (PI3K) /AKT signal pathway provides protection against apoptosis induced by heat stress to ascertain the key point for treatment. RESULTS: Under heat stress, rats showed increased shedding of intestinal epithelial cells. These rats also had elevated levels of serum cortisol and improved expression of heat shock proteins (Hsp27, Hsp70 and Hsp90) in response to heat stress. Apoptosis analysis by TUNEL assay revealed a higher number of villi epithelial cells that were undergoing apoptosis in heat-treated rats than in the normal control. This is supported by gene expression analysis, which showed an increased ratio of Bax/Bcl-2 (p < 0.05), an important indicator of apoptosis. During heat-induced apoptosis, more AKTs were activated, showing increased phosphorylation. An increase of BAD phosphorylation, which is an inhibitory modification, ensued. In rat IEC-6 cell line, a significant higher level of AKT phosphorylation was observed at 2 h after heat exposure. This coincided with a marked reduction of apoptosis. CONCLUSION: Together, these results suggest that heat stress caused damages to rat jejunum and induced apoptosis to a greater degree. HSPs and pro-survival factors were involved in response to heat stress. Among them, AKT played a key role in inhibiting heat-induced apoptosis.