| Alzheimer’s disease (AD), a primary neurodegenerative disorder, is the most important type of senile dementia. The P-amyloid peptide (Aβ) is produced from the enzymolysis of β-amyloid precursor protein (APP). Researches illustrated that Aβ oligomer is one of the main toxicants that lead to damage and apoptosis of neurons and can eventually promote the development of AD.Neprilysin (NEP), a type Ⅱ zinc-dependent metalloprotease, is considered as a major Aβ-degrading enzyme and normally involved in maintaining a low Aβ level in the brain. The down-expression of NEP can break the balance between the synthesis and clearance of Aβ and subsequently cause cerebral Aβ aggregation. Hence the expression of NEP is closely related with the pathological progress of AD.Ginsenoside Rb1, a monomer extracted from ginseng, is one of the main active constituents that ginseng acts on the nervous system and has neuroprotective effects [9,10,11]. Our previous studies showed that Rbl can obviously increase the expression and activity of NEP in human neuroblastoma cells.To further elucidate how Rbl promotes NEP expression, a series of NEP promoter deletion mutant-luciferase reporter plasmids were constructed, and human neuroblastoma SH-SY5Y cells were transfected with them. The transfected cells were treated with Rb1, and the dual-luciferase reporter assay was performed to explore sites in the NEP promoter region that were related to the regulation of the NEP promoter activity by Rb1. It was found that there were two positive and two negative regulatory regions in the NEP promoter, and Rbl might increase the NEP promoter activity through one of the above positive regulatory regions. Methods1. The NEP promoter-luciferase reporter plasmid pGL3-nep2.4was identified through dual-digestion of Xho I and BamH I and sequence analysis using the general primers RVprimer3and GLprimer2.2. Dual-luciferase reporter assay was conducted to SH-SY5Y cells transfected with the plasmid pGL3-nep2.4and treated with treated with Rbl.3. Erase-a-Base System was applied to construct a series of5’NEP promoter deletion mutant-luciferase reporter plasmids derived from pGL3-nep2.4. SH-SY5Y cells were transfected with the mutants identified by digestion and sequencing, and the NEP promoter activities in them were determined by dual-luciferase reporter assay.4. Mutants from deletion series corresponding to-894bp/-534bp and-247bp/-82bp regions were conformed through digestion by Xho I and BamH I and sequencing, dual-luciferase reporter assay was conducted to SH-SY5Y cells transfected with the validated mutants.5. SH-SY5Y cells transfected with mutants corresponding to-894bp/-857bp,-559bp/-534bp,-223bp/-179bp, and-100bp/-82bp regions were treated with50umol/L Rb1, and luciferase reporter assay was performed.Results1. Restriction digestion and sequencing indicated that the2.4kb DNA fragment was inserted to the right location of pGL3-basic at a correct direction.2. Dual-luciferase reporter assay showed that the luciferse activity in SH-SY5Y cells transfected with pGL3-nep2.4was13.1times of that in the cells transfected with pGL3-basic (P<0.01). After SH-SY5Y cells transfected with pGL3-nep2.4were treated with Rbl, the luciferase activity in them was2.9times of that in the control cells (P<0.01).3. Restriction digestion and agarose gel electrophoresis showed that the sizes of DNA fragments after digestion by BamH I and Kpn I coincided with the expectation and the inserted DNA fragments of the mutants were validated by sequence analysis.4. Dual-luciferase reporter assay also showed that the NEP promoter activity was decreased to29%(P<0.01) or25%(P<0.01) after the-894bp to-857bp (Region Ⅰ) or-100bp to-82bp (Region IV) region upstream of the NEP gene was deleted, whereas it was increased to5.12(P<0.01) or1.81times (P<0.01) after the-559bp to-534bp (Region Ⅱ) or-223bp to-179bp (Region Ⅲ) region upstream of the NEP gene was deleted.5. After Rbl treatment, the luciferase activity in SH-SY5Y cells transfected with the mutant deleting Region Ⅰ, Ⅱ or Ⅲ was no different from that in the control cells (P>0.05), and the luciferase activity in SH-SY5Y cells transfected with the mutant pGL3-244containing Region Ⅳ was1.61times of that in the control cells (P<0.01) while it in SH-SY5Y cells transfected with the mutant pGL3-226without Region Ⅳ was no different from that in the control cells (P>0.05).Conclusions1. The2.4kb DNA fragment upstream of the NEP gene had the strong promoter activity that could be increased by Rbl.2. NEP mutant series in whole or2defined area of the2.4kb fragment were successfully constructed.3. Two positive (Region Ⅰ and Ⅳ) and two negative (Region Ⅱ and Ⅲ) regulatory regions were identified in the NEP promoter, and the increase of the NEP promoter activity caused by Rbl was related to Region Ⅳ. |
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