| [Backgrounds]The nuclear lamina is a10nm thick proteinaceous layer underlying the inner nuclear membrane.It is juxtaposed to the inner nuclear membrane and combinded with the specific area of chromatin, providing shape and structural integrity to the nucleus. They are major determinants of nuclear size and shape and are involved in essential functions such as DNA replication and transcription. The lamins are type V intermediate filament (IF) proteins comprising the major structural components of the nuclear lamina. Right amounts of mature lamins are essential for cell normal functions. There are large amounts of lamin A and lamin C in many tissues such as heart、liver and kidney. The amount of lamin A is a little more than lamin C. Overexpression of lamin A in osteoarthritis chondrocytes can promote aging and even lead to apoptosis. Accumulation of prelamin A could accelerate vascular smooth muscle cell senescence. Prelamin A accumulation is triggered by downregulation of FACE1. However, researchs on the level changes of lamin A/C, prelamin A and FACE1in the aged liver are rare. Huperzine A is a potent, selective and reversible inhibitor of acetylcholinesterase (AChE). It has the roles of anti-oxidation, anti-apoptosis, reducing the active oxygen free radicals, increase activity of key enzymes in respiratory chain or tricarboxylic acid cycle and so on. It has been widely used in China for the treatment of Alzheimer’s disease (AD). But its role for ameliorating liver aging is still unclear.[Aims]1. To build aging rats model via D-galactose. Research the expression changes of lamin A/C, prelamin A and FACE1, then clarify the relationships between liver aging and lamin A/C, prelamin A, FACE1levels.2. Research the effects of huperzine A on lamin A/C, prelamin A and FACE1levels in liver. And clarify the protective role of huperzine A for liver aging.3. Discuss the mechanisms of liver aging induced by lamins and mechanisms of the protective role huperzine A play on liver aging.[Methods]1. Two-month-old thirty-six healthy male SD rats were randomly assigned into three groups (n=12per group):D-galactose treatment group, D-galactose combined with huperzine A treatment group and normal group. D-galactose group were injected subcutaneously with D-galactose300mg/Kg/d for eight weeks. D-galactose combined with huperzine A treatment group were injected subcutaneously with D-galactose300mg/Kg/d and huperzine A O.lmg/Kg/d for eight weeks. The normal group were injected with equal amount of normal saline every day. For5-bromo-2-deoxyUridine (BrdU, Sigma, USA) labeling, the rats (n=3per each treatment group) received3intraperitoneal injections of BrdU (1injection every6h for18h at a dose of100mg/kg b.w.).6h after the last BrdU injection, the rats were culled for tissue preparation.2. Using immunohistochemistry to measure liver cells express senescence-associated β-Galactosidase (SA-β-Gal), proliferation liver cells labeled by Brdu and liver cells with the presence of8-Oxoguanine (8-oxoG), which is a common marker of oxidative DNA damage. We use quantitative real-time PCR to measure p16mRNA, TNF-a mRNA and IL-6mRNA. Use dichlorofluorescein (DCF) as a probe to (measure ROS levels. Semi-quantitative western blot analysis were used to measure p65, phospho-p65, IκBa, TNF-a and IL-6levels.3. Immunohistochemistry and Western blot were used to measure the expression of lamin A/C, prelamin A and FACE1in liver.4. Spectrophotometry was used to measure the activity of acetylcholinesterase (AChE).[Results]1. In the normal, D-galactose combined with huperzine A and D-galactose treated groups, liver cells expressing SA-β-Gal, labeled by Brdu and with the presence of8-oxoG were gradually increased; the levels of p16mRNA and ROS were increased gradually. In contrast to normal group, the level of TNF-a mRNA was increased significantly in D-galactose treated group, while there was no significant changes in the D-galactose combined with huperzine A treated group. Levels of IL-6mRNA were gradually increased. Western blot analysis indicated that the TNF-α level was highest in the D-galactose treated group and lowest in the D-galactose combined with huperzine A treated group. The levels of IL-6were gradually increased. Nuclear p65and phospho-p65levels were increased gradually; cytoplasmic p65level was lowest in the D-galactose treated group and highest in the D-galactose combined with huperzine A treated group. The levels of IκBα were gradually decreased in the three groups.2. Immunohistochemistry results indicated that liver cells labeled by lamin A/C, lamin A antibody were gradually increased in normal, D-galactose combined with huperzine A and D-galactose group. While, cells labeled by FACE1were gradually decreased in the three groups. Western blot analysis indicated that lamin A/C, prelamin A were gradually increased, FACE1was gradually decreased in the three groups.3. In the normal, D-galactose combined with huperzine A and D-galactose treated groups, AChE activity were0.103±0.00790,0.0702±0.00521and0.0926±0.00447, respectively.[Conclusions]Higher levels of lamin A/C, prelamin A and lower levels of FACE1indicate liver aging in D-galactose treatment rats. Huperzine A can remarkably ameliorate these proteins changes indicating its anti-aging role for liver. |
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