| Oxidative stress plays a central role in heat stress-induced gastrointestinal injury. Punicalagin (PUN), a major polyphenol abundant in pomegranate husk, exhibits antioxidative effects. In the present study, we used a heat stress model to investigate the intestinal protection effect of PUN and the underlying mechanisms.Experiment oneThe heat stress model of IEC-6 was established, cell viability was determined by MTT, cell morphology was observed by inverted microscope, the level of HSPs mRNA were detected by RT-PCR, cell apoptosis was investigated by AO/EB. Results showed that 42℃ 6 h was screened out as the thermal stress model and the punicalagin concentrations were 2.5,5,10 μmol/L respectively. Also, the injury of IEC-6 cells significantly reduced, mRNA expressions of HSP27、HSP70、HSP90 significantly decreased after pretreatment with punicalagin.Experiment twoROS production was detected by DCF assessment method, SOD activity was determined by hydroxylamine assay method and MDA content were detected by thiobarbituric acid method. Results showed that heat stress increased reactive oxygen species, malondialdehyde production and meanwhile it decreased superoxide dismutase activity. These findings revealed that PUN significantly inhibits heat stress-induced oxidative stress.Experiment threeThe protein level of HO-1, Nrf2 and P-AKT were detected by western blot, the nucleu level of Nrf2 was detected by immunofluorescence, cell viability was determined by MTT assay, cell apoptosis was investigated by flow cytometry. Results showed that PUN treatment induced the expression of heme oxygenase-1 (HO-1), increased Nrf2 nuclear translocation and phosphorylation of AKT 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 upregulation and the protective effect of PUN against oxidative stress. |
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