Effects Of Myo-inositol On The Antioxidant Ability In The Intestine Of Juveniles Jian Carp (Cyprinus Carpio Var. Jian) And The Mechanism Studies

Intestine is important to nutrient digestion and absorption, especially to stomachless fish, but it is susceptible to oxidative injury. myo-inositol (Ml) may improve the antioxidant capacity of the fish intestine, and thus maintain its integrity and function. CuZnSOD is the enzyme known that remove toxic superoxide anions and act as important endogenous antioxidants for protection against oxidative stress. We will study the antioxidant responsive element (ARE) that may locate in the promoter of CuZnSOD gene, and the expression of Nrf2et al. in the intestine of Jian carp. After that, we will investigate whether MI regulates CuZnSOD gene transcription through enhacing Nrf2nuclei translocation, increasing the binding of Nrf2and sMaf, and finally activating ARE-mediated CuZnSOD gene transcription. Meanwhile, we will investigate the roles of PKC8and Keapl in MI-mediated Nrf2nuclei translocation. Summarily, this study will reveal the mechanism of MI-regulated CuZnSOD gene transcription via Nrf2pathway. This study consists of five parts of experiments as follows.1Effects of MI on oxidative status, antioxidant ability and antioxidant enzyme gene expression in the intestine of juvenile Jian carpLipid peroxidation, protein oxidant, antioxidant status and gene expression of the intestine in juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of myo-inositol (MI)(163.5,232.7,384.2,535.8,687.3,838.8and990.3mg/kg diet) for60 days were investigated. MDA and PC contents significantly decreased with increasing dietary MI levels up to535.8and838.8mg/kg diet (P<0.05). respectively, and leveled off thereafter(P>0.05). Both ASA and AHR capacity were higher for fish fed MI-supplemented diets than MI-unsupplemented diet(P<0.05). SOD activity in the intestine was higher for fish fed MI-supplemented diets than Ml-unsupplemented diet (P <0.05). Intestinal CAT activity significantly increased with increasing dietary MI levels up to535.8mg/kg diet (P<0.05), and plateaued thereafter (P>0.05). GST activity in the intestine was lower for fish fed diets containing MI≤535.8mg/kg diet than fish fed diets containing MI≥687.3mg/kg diet (P<0.05). GPx activity was the lowest for fish fed Ml-unsupplemented diet, followed by232.7mg MI/kg diet, and the highest for fish fed diets containing MI>687.3mg/kg diet (P<0.05). GR was the highest for fish fed diets containing>687.3mg/kg diet, and the lowest for fish fed the232.7and163.5mg MI/kg diet (P<0.05). GSH content significantly increased with increasing dietary MI levels up to384.2mg/kg diet (P<0.05), and plateaued thereafter (P>0.05). MI significantly increased the gene expression of CuZnSOD, MnSOD, CAT and GPx1b in the proximal intestine, CuZnSOD, CAT, GPx1a and GPx1b in the mid intestine of Jian carp (P<0.05). MI decreased the mid intestinal MnSOD and proximal, mid and distal intestinal GR gene expression of Jian carp (P<0.05). MI did not change the proximal intestinal MnSOD, diatal CuZnSOD、MnSOD、CAT、GPx1a and GPx1b gene expression.2Effects of MI on the integrity of enterocytes of juvenile Jian carp and the potential action pathway studies2.1Effects of MI on the integrity of enterocytes of juvenile Jian carpThis study investigated the effects of myo-inositol (MI) on the growth and antioxidant capacity of carp enterocytes. The enterocytes were incubated in media containing0,15,30,45,60and75mg MI/L for96h. The results indicated that MI could increase cell viability. In addition, the activities of cellular alkaline phosphatase (AKP), gamma-glutamyl transpeptidase (γ-GT), Na+, K+-adenosine trisphosphatase (Na+ K+-ATPase) and creatinkinase (CK) increased with MI supplementation at levels ranging from15to60mg MI/L medium, indicating an improvement in cell differentiation and function. Further, enzymatic antioxidant ability, as measured by total-superoxide dismutase (T-SOD), CuZnSOD, MnSOD, catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activities, improved with Ml supplementation. Finally, cell damage, as indicated by lactic acid dehydrogenase (LDH) activity, malondialdehyde (MDA) content of the medium and cellular protein carbonyls (PC), were all depressed by MI. Correlation analyses showed that cell viability (MTT) was positively related to the antioxidant enzyme activities, but negatively related to cell damage (LDH, MDA and PC). In summary, the data showed that Ml could improve the growth of fish enterocytes.2.2Cu-induced oxidative stress in enterocytesThe second trial determined the effects of Cu exposure (0-6.0mg Cu/L of medium for24hours) on enterocytes in vitro. The results indicated that the LDH release gradually increased with increasing levels of copper exposure, suggesting that the cell injury was induced by Cu. MDA content in the medium also gradually increased when Cu exposure was increased to2.4mg Cu/L of medium or higher (P<0.05). In contrast, cell viability, as indicated by MTT OD, gradually decreased with increasing levels of copper exposure (P<0.05). Protein retention was lower in every copper-exposure group than it was in the Ctrl group (P<0.05). Every concentration of Cu exposure significantly decreased the cellular AKP activity (P<0.05). The T-SOD activity in enterocytes was significantly decreased by Cu exposure (P<0.05). The cellular CuZnSOD activity also significantly decreased with increasing Cu exposure, up to1.2mg/L of medium (P<0.05), but there was no significant difference among groups with higher Cu concentrations (P>0.05). The cellular CAT and GPx activities all significantly increased with increasing Cu levels up to0.6mg/L of medium (P<0.05) and remained nearly constant thereafter (P>0.05). The stress dose was established to be6mg/L Cu.2.3Pretreatment with MI reduced Cu-induced oxidative stress in enterocytesEnterocytes were pre-incubated with graded levels of MI (0-75mg MI/L of medium) for72h and exposed to6.0mg Cu/L of medium for24h. Pre-treatment with15-75mg/L of MI prior to exposure to Cu completely blocked the LDH release that was shown in the Ctrl/Cu group. The MDA content in the media was higher in the Ctrl+Cu group than that of the Ctrl+Ctrl group, indicating that Cu exposure caused severe peroxidation of the cellular membranes. As expected, pre-treatment of the cells with graded levels of MI partially prevented the MDA generation induced by Cu, but not completely (P<0.05). Also, MI pre-supplementation completely prevented the PC formation induced by Cu (P<0.05). Exposure to Cu was shown to cause a significant decrease in AKP activity compared with that of the Ctrl (P<0.05). However, pre-treatment of cells with30-75mg/L of MI completely prevented the decrease in AKP activity induced by Cu (P<0.05). Interestingly, pre-supplementation with30mg/L of Ml completely prevented the decrease of ASA activity induced by Cu, and pre-treatment with45-75mg MI/L of media significantly increased the ASA activities when compared with the Ctrl+Ctrl group (P<0.05). Exposure of cells to Cu was shown to cause a significant decrease in the AHR activity compared with the Ctrl group (P<0.05). However, pre-treatment with≥45mg MI/L completely prevented the decrease of AHR activity induced by Cu (P<0.05). As expected, pre-treatment with MI partially prevented the decrease in T-SOD and CuZnSOD activities induced by Cu (P<0.05). In contrast, exposure to Cu significantly increased the CAT activity compared with that of the unexposed Ctrl group (P<0.05). However, pre-treatment with increasing levels of MI partially prevented the increase in CAT activity induced by Cu, and75mg/L of MI completely prevented the CAT activity (P<0.05). Interestingly, cells pre-incubated with15-75mg/L of MI and further exposed to Cu (MI+Cu) also had high activities of GPx and GST (P<0.05). Compared with the Ctrl+Ctrl group, the GR activity and GSH content were significantly decreased in cells exposed to Cu (Ctrl+Cu)(P<0.05). Interestingly, pre-treatment with MI partially prevented the decrease of GR activities and GSH content induced by Cu exposure (P<0.05). Those results showed that pre-treatment IECs with MI can improve its integrity by enhacing the antioxidant ability.2.4Co-treatment with MI inhibited Cu-induced oxidative stress in enterocytesTo investigate the hypothesis that MI protects enterocytes against Cu toxicity via the intercept pathway, enterocytes were treated with different concentrations of MI (0-75mg/L medium) in the presence of6mg/L of Cu for24hours. The results of experiment1indicated that cells exposed to Cu alone for24h exhibited increases in lactate dehydrogenase release (LDH), malondialdehyde (MDA) formation and protein oxidation (P<0.05). Notably, a dose-dependent inhibitory effect on LDH release was observed with all doses of MI. Moreover, co-treatment with MI completely inhibited Cu-induced PC formation. However, Cu-induced lipid peroxidation was not altered by MI co-treatment. Additionally, Cu exposure suppressed total-superoxide dismutase (T-SOD), CuZnSOD and catalase (CAT) activities, and these changes were completely blocked by co-treatment with sufficient MI concentrations (P<0.05). In contrast, cells exposed to Cu exhibited adaptive increases in glutathione (GSH) content and the activities of anti-hydroxyl radical (AHR), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR). Interestingly, the Cu-stimulated increases in these antioxidants were blocked by co-treatment with sufficient MI concentrations. In conclusion, we demonstrated for the first time that MI protected enterocytes from Cu-induced oxidative damage.2.5Subsequent treatment with MI repaired Cu-induced oxidative damage in enterocytesWe investigated the potential reparative role of MI after a Cu challenge. The results indicated that cell injury (LDH release), lipid peroxidation (MDA formation) and protein oxidation induced by Cu were reversed by subsequent MI treatment. Meanwhile, Cu-induced decreases in alkaline phosphatase (AKP), anti-superoxide anion (ASA), T-SOD and CuZnSOD activities were completely restored by subsequent MI treatment, while the reduced CAT activity was partially restored (P<0.05). However, MI rescues partially restored the adaptive increase in GPx activity induced by Cu, whereas the adaptive increase in GSH content was completely reversed by75mg/L of MI (P<0.05). However, subsequent Ml treatments did not alter the induction of GST activity by Cu. In conclusion, we demonstrated for the first time that MI not only protected enterocytes from Cu-induced oxidative damage but also increased the repair activity in primary enterocytes after challenge with Cu. Moreover, MI-mediated increases in antioxidant enzyme activities contributed to lipid and protein oxidant repair.3The promoter of CuZnSOD, and Nrf2et al. genes cDNA cloning, characterization, and intestinal expressionThis study used molecular biology techniques to clone carp CuZnSOD gene promoter, Nrf2and PKC5cDNA sequences as well as Keapl and MafG etc. gene, and studied their expression patterns in fish intestine. The results showed that the carp CuZnSOD gene promoter was cloned to be1675bp, which contained an antioxidant response element ARE; Nrf2and PKC5cDNA sequence were2557bp and2862bp, respectively, the open reading frame of1761bp and2061bp, encoding586and686amino acid residues, protein molecular weight of66071.7Da and78498.36Da and the isoelectric point of4.49and7.53, respectively; Keap1a, Keap1b MafG1and MafG2part cDNA were843,521,394and344bp, coding240,173,130and114amino acid residues, respectively; gene expression patterns of those genes showed that Nrf2in the first segment of the intestine was the highest and was lowest in the7th segment (P <0.05), the PKC5in the1st and4th segments were the highest and was the lowest in the6th segment (P<0.05), Keap1a expression level in the5th,6th and7th intestinal segments were higher than that in the1st,2nd.3rd and4th intestinal segments (P<0.05), Keap1b in1st,2nd and7th segments were the highest and was the lowest in the5th segment (P<0.05), MafG1in the1st,6th, and7th segments was higher than that in the2nd,3rd,4th and5th segments (P<0.05), MafG2is the highest in the6th segment. followed by the4th segment, and the lowest in2nd segment (P<0.05). Those results indicated that there was an ARE located in the promoter of carp CuZnSOD, genes including PKCδ, Keap1a, Keap1b. MafG1and MafG2express in the intestine, those results play a foundational role to further explore mechanism of effects of MI on the CuZnSOD gene transcription.4Effects of MI on the Nrf2nucleus translocation and Nrf2-regulated gene transcriptions in the intestine of carpThis test is divided into two treatments, each treatment with three replicates; each repeated50fish, fed semi-purified experimental diets without MI and addtional inositol for60days, respectively. We investigated effects of MI on the nucleus Nrf2amounts, MafG gene and protein expression in the proxiamal intestine, mid intestine and distal intestine of carp. The results showed that inositol significantly increased Nrf2nuclear translocation, and also increased MafG gene and protein expression in the proxiamal and mid intestine of carp (P<0.05). The electromigration test showed that MI promoted the amount of the nucleus Nrf2combined the ARE (CuZnSOD) in the proxiamal and mid intestine of carp (P <0.05). These results demonstrate that MI promotes Nrf2nuclear translocation, and also promoting the nuclear translocation of Nrf2binding MafG, thus enhancing Nrf2combined the ARE, and finally activation of s the CuZnSOD gene transcription in the proximal and mid intestine. However, there had no significant influence on the Nrf2nuclear translocation, MafG gene and protein expression in the distal intestine by MI.5The mechanism of Nrf2nucleus translocation induced by MI in the intestines of carpResults indicated that inositol promoted the proximal intestinal Nrf2nuclear translocation was related to its promoting of the proxiamal intestinal Nrf2gene and protein expression and PKCδ phosphorylation of Nrf2(P<0.05). Secondly, inositol promoted the mid intestinal Nrf2nuclear translocation was related to that inositol improved the ratio between Nrf2gene expression and Keap1a/b gene expression as well as PKCδ phosphorylation of Nrf2(P<0.05). Inositol has no effect on the distal intestinal Nrf2nuclear translocation, and no effect on the Nrf2gene and protein expression, but reduced Keapla/b gene expression. Its mechanism needs further research.Summarily, MI improved the ability of anti-lipid peroxidation and anti-protein oxidation in the intestine of juvenile Jian carp through improving the enzymatic antioxidative capacity and non-enzymatic antioxidative capacity; MI improved the enzymatic antioxidant capacity was associated with the enhanced CuZnSOD, CAT and GPxlb gene expression in the proximal and mid intestine of juvenile Jian carp; MI could be benefit for the anti-lipid peroxidation, anti-protein oxidation, and thus maintaining the normal structural integrity and functionality of the IECs through protection, interception, and repair pathways; the carp intestinal CuZnSOD gene promoter contains antioxidant response element (ARE) and the Nrf2, MafG, PKCδ and Keapl express in the intestine of carp; MI can increase the CuZnSOD gene transcription by enhancing the Nrf2nuclear translocation and increasing the MafG gene and protein expression as well as improving the Nrf2binding ARE (CuZnSOD) in the proximal and mid intestine; MI increased the Nrf2nuclear translocation is accociated with the Nrf2gene and protein expression as well as increasing Nrf2and PKCδ phosphorylation; however, MI can not influence the Nrf2nuclear translocation, MafG gene and protein expression, Nrf2gene and protein expression, Nrf2and PKCδ phosphorylation, but indeed decressed the gene expression of Keap1a/b.