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.
FA protected against heat stress-induced intestinal epithelial barrier dysfunction via activating the PI3K/Akt-mediated Nrf2/HO-1 signaling pathway in IEC-6 cells.
BackgroundAs 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.ResultsUnder 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.ConclusionTogether, 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.
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.
To investigate the potential applications and the molecular mechanisms of transcranial direct current stimulation (tDCS) on cognitive impairment in a vascular dementia (VD) animal model. Sprague-Dawley rats were used in this study. VD rat model was induced by modified permanent bilateral common carotid artery occlusion (2-VO) approach. Anodal tDCS was applied to the animals. Morris water maze was used to analyze spatial memory and navigation ability. The pathological changes in the hippocampal CA1 region and cerebral cortex were examined via Hematoxylin-Eosin staining. The rats were sacrificed for the measurement of the level of superoxide (SOD), glutathione (GSH), reactive oxidative species (ROS), malondialdehyd (MDA), Interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α level in the hippocampus. Western blot was carried out to measure the hippocampal expression of microtubule-associated protein 1 light chain 3 (LC-3) and p62. Rats with VD have decreased number of neurons in the hippocampus and cerebral cortex, as well as worse cognitive impairment. The proliferation of activated microglia and astroglia, accompanied with attenuation of myelination were observed in the white matter about 1 month after 2-VO operation. These abnormalities were significantly ameliorated by tDCS treatment. Further study revealed that anodal tDCS could suppress the MDA and ROS level, while enhance the SOD and GSH level to reduce the oxidative stress. Anodal tDCS could inhibit hypoperfusion-induced IL-1β, IL-6, and TNF-α expression to attenuate inflammatory response in hippocampus. Moreover, anodal tDCS treatment could alleviate autophagy level. The study has demonstrated a possible therapeutic role of tDCS in the treatment of cognitive impairment in VD.
Intestinal barrier dysfunction is an important clinical problem in various acute and chronic pathological conditions. Ferulic acid (FA) can attenuate the intestinal epithelial barrier dysfunction, however, the underlying mechanism remains unclear. The present study aimed to uncover the protective effect of FA on intestinal epithelial barrier dysfunction in a Caco-2 cell model of lipopolysaccharide (LPS) stimulation and the underlying mechanism. Caco-2 cells were pretreated with FA and then exposed to LPS stimulation. The barrier function of Caco-2 cells was evaluated by measuring trans-epithelial resistance (TER) and 4-kDa fluorescein isothiocyanate (FITC)-dextran (FD4) flux, and analyzing the tight junction protein expression and structure. The results showed that decreased TER and increased FITC-FD4 flux were observed in Caco-2 cells stimulated with LPS, but these effects were attenuated by FA pretreatment. FA pretreatment inhibited LPS-induced decrease in occludin and ZO-1 mRNA and protein expression. LPS stimulation decreased miR-200c-3p expression, whereas this decrease was inhibited by FA pretreatment. Furthermore, overexpression of miR-200c-3p strengthened the protective effects of FA on LPS-induced Caco-2 cell barrier dysfunction by decreasing epithelial permeability, increasing occludin and ZO-1 protein expression, and maintaining of ZO-1 protein distribution, while suppression of miR-200c-3p reversed the protective effects of FA. LPS treatment increased the expression of PTEN protein and decreased expression of phosphorylated PI3K and AKT proteins. However, pretreatment of FA inhibited expression of PTEN protein and promoted activation of PI3K/AKT signaling pathway in the LPS-treated Caco-2 cells, and this regulatory effect of FA on the PTEN/PI3K/AKT signaling pathway was strengthened or weakened by miR-200c-3p overexpression or suppression, respectively. Our findings suggested that in Caco-2 cells, FA promotes activation of PI3K/AKT pathway by miR-200c-3p-mediated suppression of the negative
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.
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