| Based on the previous studies on the MnSOD expression in the broiler cadiocytes stimulated by Manganese, the current study was applied to further investigate the different Manganese sources in the regulation of MnSOD gene expression and its potential mechanism in cadiocytes by the means of primary culture broiler. Wherefore three experiments were conducted as follows.Experiment I According to the preliminary studies of the project, the present experiment was designed as a two factors completely random test by 5 X 2. That was 5 manganese sources X 2 coincubating times, in which of 5 manganese sources with 0.5mM supplementation were MnC12, a inorganic form, three organic forms with complex strenght of weak (Availa-Mn), moderate (MnAAB) and strong (Bioplex Mn) as well as a Mn blank control; two times were Mn coincubation for 12h and 48h. There were ten treatment groups with 6 repeats. The results showed that manganese and the coincubating time exerted an interaction (p<0.0001) on cellular MnSOD mRNA. Compared with control, there was no significant differ for 12h coincubation after Mn supplementation and for the different manganese sources. However, by 48h, Mn supplementation increased cellular MnSOD mRNA (p＜0.0001) and significant differ was showed between manganese sources, in which of MnC12 was the highest induction for MnSOD mRNA, higher than MnAAB (P=0.0049) and AvailaMn (p＜0.0001) without significant difference from Bioplex Mn. Bioplex Mn was higher than MnAAB (P=0.0111) and AvailaMn (p＜0.0001), MnAAB was higher than AvailaMn (P=0.0002) too. Manganese source and coincubating time had no interaction for MnSODp (p=0.6241). Mn supplementation induced the MnSODp (p=0.0157), especially at 12h, MnCl2 (p=0.0062) and MnAAB (p=0.0028) increased the cellular MnSODp, AvailaMn and Bioplex Mn also increased MnSODp (by 4.2%and 4.9%, respectively) without significant difference. However, there were no significant difference for Mn supplementation and between manganese sources, so the coincubating time was also a significant effect on the MnSODp (p=0.0427). Manganese source and coincubating time had no interaction for MnSOD activity (p=0.05720). MnSOD activity at 12h was higher than that at 48h (50.596 versus 33.160,p＜0.0001). MnC12 increased the MnSOD activity at 12h (P=0.0047), the others also increased the activity in some extent despite no significance. There was no significant difference between manganese sources in MnSOD activity. The results suggested that Mn was the reliable inducer of MnSOD expression in broiler cadiocytes. MnSOD expression induced by Mn was time-dependent, and regulated at transcriptional level (48h) as well as translational and/or post-translational levels (12h). MnSOD mRNA was the sensitive indicator for distinguishing different manganese sources. MnCl2 and MnAAB were similar induction for MnSOD expression. MnCl2 and MnAAB were selceted as the representatives of inorganic manganese and organic manganese, respectively, to further investigate the effects of different manganese sources on MnSOD gene expression at trancriptional (48h) and translational (12h) levels.ExperimentⅡAccording to the previous studies, the experiment was designed as a mono factor test, in which of treatments 1-3 were the inhibitor balnk control including 0.5 mM supplementaion of MnCl2 and MnAAB and Mn blank control, 10μM SB203580,30μM PD98059,50nM JNKI1 and 500μM NAC were introduced into the control groups and designed the treatment groups 4-6,7-9,10-12 and 13-15, respectively. There were 15 treatment groups, each of 6 repeats. The results showed that Mn activated the p38MAPK, JNK, ERK pathways and the activation were inhibited by special inhibitors and NAC, a ROS scavenger; MnCl2 (p=0.0079) and MnAAB (p=0.0009) increased the cellular MnSOD mRNA level with a significant difference (p=0.0071); SB203580, a special inhibitor of p38MAPK, depressed the induction of MnCl2 and MnAAB for MnSOD mRNA (by 28.1% versus 22.7%, p=0.0267 versus p=0.0245, respectively); PD98059, a special inhibitor of ERK, depressed the MnSOD mRNA induced by MnCl2 and MnAAB 1.2% and 6.2%, respectively, with no significant difference; JNKI1, a special inhibitor of JNK, depressed the MnSOD mRNA induced by MnAAB 20.4 % with no differ significance (p=0.2588), and did not affect the MnCl2 induction of MnSOD mRNA; NAC did not inhibit the MnCl2 induction of MnSOD mRNA and depressed 11.4% for MnAAB without differ significane (p=0.3030). Both MnCl2 (P=0.0044) and MnAAB (P=0.0006) increased cellular ROS levels with no significant differ between the two manganese sources. NAC declined the cellular ROS levels (p=0.0118 and p=0.0442 versus MnC12 and MnAAB). The results suggested that Mn regulated MnSOD expression at transcriptional level, both manganese sources remarkably increased cellular MnSOD mRNA; Mn-activated p38MAPK, ERK and JNK pathways were mediated by ROS; Mn-induced MnSOD mRNA expression was mediated by ROS, in which of MnC12 induction was through p38MAPK and MnAAB may be through both p38MAPK and JNK. EERK pathway was not involved in Mn-induced MnSOD mRNA expression; Mn may directly affect transcription factors related with MnSOD gene expression and then induce MnSOD mRNA.Experiment III To investigate whether the singal pathways of Mn-induced MnSOD gene expression at translational level were mediated by PTK and/or PKC and its potential mechanism, a experiment was conducted as follows. The experiment was a monofactor test design, in which of treatments 1-3 were the inhibitor blank control including 0.5mM supplementation of MnC12 and MnAAB and Mn blank control, 50μM genistein, 1μM calphostin C and 500μM NAC were introduced into the control groups and designed the treatment groups 4-6,7-9 and 10-12, respectively. There were 12 treatments groups, each of 6 repeats. The results showed that Mn activated the signal pathways mediated by PTK or PKC and the activation were inhibited by special inhibitors and NAC; Cellular ROS was not affected by Mn coincubation for 12h (p=0.4045). NAC depressed the cellular ROS level (6.88 vs 8.42, P=0.0003), and the two manganese sources were no significant differ in the profile. Both MnC12 and MnAAB increased the cellular MnSODp (p=0.0074 and p＜0.0001, respectively) and MnSOD activity (p＜0.0001 and p<0.0001, respectively). MnC12 was lower MnAAB in induction of MnSODp (p=0.0187), and higher in MnSOD activity induction (p=0.0332). Genistein, calphostin C and NAC depressed the MnSODp induced by MnC12 and MnAAB (p=0.0115 and p=0.0061, p=0.0399 and p＜0.0001, p=0.0238 and P=0.1253, respectively), Cellular MnSODp was no significant differ between MnC12 and MnAAB groups after inhibitor introduction. Genistein inhibited the acivation of MnSOD acitivty induced by MnC12 (p=0.3299) and MnAAB (p=0.1875) without statistic significance. Calphostin C and NAC depressed the MnSOD activities induced by MnCl2 and MnAAB (p=0.0031 and p＜0.0001, p=0.0003 and p=0.0039, respectively), Cellular MnSODp was no significant differ between MnCl2 and MnAAB groups after inhibitor introduction. The results suggested that Mn remarkably increased cellular MnSOD protein and activity, and MnSOD activity was a sensitive indicator for distinguishing different manganese sources. MnCl2 was lower MnAAB in the induction of MnSODp and higher in MnSOD activity induction; Mn activated PTK and PKC pathways mediated by ROS; MnSOD protein and activity induced by MnCl2 and MnAAB were through PKC, may through PTK too, which was mediated by ROS.It was proved that Mn induced MnSOD gene expression and regulated at transcriptional as well as translational and/or post-translational levels in primary culture broiler cadiocytes; MnSOD mRNA was the most sensitive indicator distinguishing different manganese sources; The moderate complex strength organic (eg. MnAAB) was more inducible than inorganic manganese (eg. MnCl2) for MnSOD gene expression; The signaling pathways of MnSOD expression induced by Mn were through p38MAPK, potential through JNK at trancriptinal level, and through PKC, potential through PTK at translational and/or post-translational levels, which was mediated by ROS. Mn activated the signaling pathways mediated by p38MAPK, ERK, JNK, PTK and PKC through ROS. |
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