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A nutrient mixture reduces the expression of matrix metalloproteinases in an animal model of spinal cord injury by modulating matrix metalloproteinase-2 and matrix metalloproteinase-9 promoter activities.

Zhang H, Chu G, Pan C, Hu J, Guo C, Liu J, Wang Y, Wu J - Exp Ther Med (2014)

Bottom Line: The expression levels of MMP-2 in the NM-LD and NM-HD groups were decreased by ~50% compared with the saline group as indicated by western blotting results.These results suggest that NM significantly inhibits the expression of MMP-2 and MMP-9 proteins.Reverse transcription quantitative polymerase chain reaction results indicated that NM reduced the levels of MMP-2 and MMP-9 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Spine Surgery, Xiangya Spinal Surgery Center, Xiangya Hospital of Central South University, Changsha, Hunan 410008, P.R. China.

ABSTRACT
This study aimed to determine whether a novel nutrient mixture (NM), composed of lysine, ascorbic acid, proline, green tea extracts and other micronutrients, attenuates impairments induced by spinal cord injury (SCI) and to investigate the related molecular mechanisms. A mouse model of SCI was established. Thirty-two mice were divided into four groups. The sham group received vehicle only. The SCI groups were treated orally with saline (saline group), a low dose (500 μg 3 times/day) of NM (NM-LD group) or a high dose (2,000 μg 3 times/day) of NM (NM-HD group). The levels of mouse hindlimb movement were determined every day in the first week post-surgery. The protein expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 were determined by western blotting. Wild-type and mutant MMP-2- and MMP-9-directed luciferase constructs were generated and their luciferase activities were determined. NM significantly facilitated the recovery of hindlimb movement of the mice in comparison to that in the saline group. The expression levels of MMP-2 in the NM-LD and NM-HD groups were decreased by ~50% compared with the saline group as indicated by western blotting results. The expression levels of MMP-9 in the NM-LD and NM-HD groups were decreased to ~25 and ~10%, respectively. These results suggest that NM significantly inhibits the expression of MMP-2 and MMP-9 proteins. Reverse transcription quantitative polymerase chain reaction results indicated that NM reduced the levels of MMP-2 and MMP-9 mRNA. Furthermore, the luciferase results indicated that site-directed mutagenesis comprising a -1306 C to T (C/T) base change in the MMP-2 promoter and a -1562 C/T base change in the MMP-9 promoter abolished the inhibitory effects of NM on MMP-2 and MMP-9 promoters. These results suggest that NM attenuates SCI-induced impairments in mice movement by negatively affecting the promoter activity of MMP-2 and MMP-9 genes and thus decreasing the expression of MMP-2 and MMP-9 proteins.

No MeSH data available.


Related in: MedlinePlus

Site-directed mutations on human MMP-2 and MMP-9 promoters abolish the effect of NM. (A) The −1306 C/T and −1562 C/T mutations were generated on the luciferase constructs. (B) HeLa cells were co-transfected with 1 μg mutant pMMP-2-LUC or mutant pMMP-9-LUC construct in addition to 1 μg pCMV-β-galactosidase construct. After 4 h, cells were treated with vehicle only (sham group), saline (saline group), 100 μg/ml NM (NM-LD group) or 500 μg/ml NM (NM-HD group). Luciferase and β-galactosidase activity was determined and the luciferase activity of each sample was normalized to β-galactosidase activity. Data are the mean ± standard deviation from at least five experiments. NM, nutrient mixture; MMP, matrix metalloproteinase; LD, low dose; HD, high dose; C/T, C to T; LUC, firefly luciferase gene.
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f5-etm-08-06-1835: Site-directed mutations on human MMP-2 and MMP-9 promoters abolish the effect of NM. (A) The −1306 C/T and −1562 C/T mutations were generated on the luciferase constructs. (B) HeLa cells were co-transfected with 1 μg mutant pMMP-2-LUC or mutant pMMP-9-LUC construct in addition to 1 μg pCMV-β-galactosidase construct. After 4 h, cells were treated with vehicle only (sham group), saline (saline group), 100 μg/ml NM (NM-LD group) or 500 μg/ml NM (NM-HD group). Luciferase and β-galactosidase activity was determined and the luciferase activity of each sample was normalized to β-galactosidase activity. Data are the mean ± standard deviation from at least five experiments. NM, nutrient mixture; MMP, matrix metalloproteinase; LD, low dose; HD, high dose; C/T, C to T; LUC, firefly luciferase gene.

Mentions: Site-directed mutagenesis was performed to generate the −1306 C/T base change on the MMP-2 promoter and the −1562 C/T base change on the MMP-9 promoter in the luciferase constructs (Fig. 5A). The luciferase assay results (Fig. 5B) indicate that NM did not significantly inhibit the MMP-2 and MMP-9 promoter-directed luciferase activities when compared with those in the sham and saline groups. These results suggest that these loci are important for the inhibitory effect of NM on MMP-2 and MMP-9 gene expression.


A nutrient mixture reduces the expression of matrix metalloproteinases in an animal model of spinal cord injury by modulating matrix metalloproteinase-2 and matrix metalloproteinase-9 promoter activities.

Zhang H, Chu G, Pan C, Hu J, Guo C, Liu J, Wang Y, Wu J - Exp Ther Med (2014)

Site-directed mutations on human MMP-2 and MMP-9 promoters abolish the effect of NM. (A) The −1306 C/T and −1562 C/T mutations were generated on the luciferase constructs. (B) HeLa cells were co-transfected with 1 μg mutant pMMP-2-LUC or mutant pMMP-9-LUC construct in addition to 1 μg pCMV-β-galactosidase construct. After 4 h, cells were treated with vehicle only (sham group), saline (saline group), 100 μg/ml NM (NM-LD group) or 500 μg/ml NM (NM-HD group). Luciferase and β-galactosidase activity was determined and the luciferase activity of each sample was normalized to β-galactosidase activity. Data are the mean ± standard deviation from at least five experiments. NM, nutrient mixture; MMP, matrix metalloproteinase; LD, low dose; HD, high dose; C/T, C to T; LUC, firefly luciferase gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4218658&req=5

f5-etm-08-06-1835: Site-directed mutations on human MMP-2 and MMP-9 promoters abolish the effect of NM. (A) The −1306 C/T and −1562 C/T mutations were generated on the luciferase constructs. (B) HeLa cells were co-transfected with 1 μg mutant pMMP-2-LUC or mutant pMMP-9-LUC construct in addition to 1 μg pCMV-β-galactosidase construct. After 4 h, cells were treated with vehicle only (sham group), saline (saline group), 100 μg/ml NM (NM-LD group) or 500 μg/ml NM (NM-HD group). Luciferase and β-galactosidase activity was determined and the luciferase activity of each sample was normalized to β-galactosidase activity. Data are the mean ± standard deviation from at least five experiments. NM, nutrient mixture; MMP, matrix metalloproteinase; LD, low dose; HD, high dose; C/T, C to T; LUC, firefly luciferase gene.
Mentions: Site-directed mutagenesis was performed to generate the −1306 C/T base change on the MMP-2 promoter and the −1562 C/T base change on the MMP-9 promoter in the luciferase constructs (Fig. 5A). The luciferase assay results (Fig. 5B) indicate that NM did not significantly inhibit the MMP-2 and MMP-9 promoter-directed luciferase activities when compared with those in the sham and saline groups. These results suggest that these loci are important for the inhibitory effect of NM on MMP-2 and MMP-9 gene expression.

Bottom Line: The expression levels of MMP-2 in the NM-LD and NM-HD groups were decreased by ~50% compared with the saline group as indicated by western blotting results.These results suggest that NM significantly inhibits the expression of MMP-2 and MMP-9 proteins.Reverse transcription quantitative polymerase chain reaction results indicated that NM reduced the levels of MMP-2 and MMP-9 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Spine Surgery, Xiangya Spinal Surgery Center, Xiangya Hospital of Central South University, Changsha, Hunan 410008, P.R. China.

ABSTRACT
This study aimed to determine whether a novel nutrient mixture (NM), composed of lysine, ascorbic acid, proline, green tea extracts and other micronutrients, attenuates impairments induced by spinal cord injury (SCI) and to investigate the related molecular mechanisms. A mouse model of SCI was established. Thirty-two mice were divided into four groups. The sham group received vehicle only. The SCI groups were treated orally with saline (saline group), a low dose (500 μg 3 times/day) of NM (NM-LD group) or a high dose (2,000 μg 3 times/day) of NM (NM-HD group). The levels of mouse hindlimb movement were determined every day in the first week post-surgery. The protein expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 were determined by western blotting. Wild-type and mutant MMP-2- and MMP-9-directed luciferase constructs were generated and their luciferase activities were determined. NM significantly facilitated the recovery of hindlimb movement of the mice in comparison to that in the saline group. The expression levels of MMP-2 in the NM-LD and NM-HD groups were decreased by ~50% compared with the saline group as indicated by western blotting results. The expression levels of MMP-9 in the NM-LD and NM-HD groups were decreased to ~25 and ~10%, respectively. These results suggest that NM significantly inhibits the expression of MMP-2 and MMP-9 proteins. Reverse transcription quantitative polymerase chain reaction results indicated that NM reduced the levels of MMP-2 and MMP-9 mRNA. Furthermore, the luciferase results indicated that site-directed mutagenesis comprising a -1306 C to T (C/T) base change in the MMP-2 promoter and a -1562 C/T base change in the MMP-9 promoter abolished the inhibitory effects of NM on MMP-2 and MMP-9 promoters. These results suggest that NM attenuates SCI-induced impairments in mice movement by negatively affecting the promoter activity of MMP-2 and MMP-9 genes and thus decreasing the expression of MMP-2 and MMP-9 proteins.

No MeSH data available.


Related in: MedlinePlus