| [Backgrounds]With the improvement of people’s living standard, the aging of the population has gradually accelerated and become a major challenge of the society nowadays. Therefore, it becomes more and more important to study on the mechanism of aging and age-associated diseases. At present, the exact mechanism of aging is not clear yet. The chronic administration of D-galactose (D-gal) could cause oxidative damage and induce changes that resemble natural aging in animals, and is widely used as aging animal model in today’s studies and researches. Huperzine A is a reversible and selective acetylcholinesterase inhibitor (AChEI) and has been used to treat neurodegenerative disorders such as Alzheimer’s disease. However, it has not been well-understood whether huperzine A can reduce D-gal-induced aging in liver, as well as its role in apoptosis and autophagy in aging liver cells. Vascular endothelial growth factor (VEGF) is an important cytokines that can regulate cell apoptosis through PI3K/Akt/GSK-3β signaling by binding to the vascular endothelial growth factor receptor-2(VEGFR-2/Flk-1). Meanwhile, the PI3K/Akt/mTOR pathway is involved in autophagy, and GSK-30also participates in the regulation of mTOR activity. Obviously, both pathways could share those common cytokines, by which the two physiological processes contact with each other closely. Therefore, we assume that huperzine A might activate PI3K/Akt/GSK-3β7mTOR pathway through Flk-1, and participate in the regulation of apoptosis and autophagy in D-gal-treated rat liver, which could contribute to liver protection and delaying aging.[Aims]1. To establish the D-gal aging rat model, and to evaluate hepatic impairment and senescence caused by D-gal as well as the effects of huperzine A on it.2. To investigate the influence of huperzine A on apoptosis in aging liver, and to further explore the pathway by which it regulate cell apoptosis.3. To investigate the effect of huperzine A on autophagy in aging liver, and to further explore the possible mechanism of it. [Methods]1. Thirty six two-month-old male Sprague Dawley rats were randomly divided into three groups (n=12per group):the D-gal-treated group, the D-gal+huperzine A-treated group, and the control group. The D-gal-treated group was injected subcutaneously with300mg/kg D-gal daily. In the D-gal+huperzine A-treated group, the rats were co-injected subcutaneously with300mg/kg D-gal and O.lmg/kg huperzine A daily. The control group was injected with saline. After8weeks of treatment,3rats in each group were labeled with BrdU. All the rats were culled for tissue preparation.2. Serum was obtained and automatic biochemistry analyzer was used to assess liver function. The expression of hepatic SA-β-Gal and BrdU in three groups was measured via immunohistochemical staining. Hepatic cell microstructure was observed under electron microscope.3. TUNEL staining was performed to evaluate cell apoptosis in liver. Apoptotic index (AI) was then calculated and analyzed statistically. The expression of hepatic Flk-1(A-3), p-Flk-1(Tyr996)-R and p-GSK-3β(Ser9) were measured via immunohistochemical staining. Western Blot analysis was performed for caspase3expression. We used image analysis software for semi-quantitative determination of the results, and the data were statistically analyzed.4. The expression of hepatic p-mTOR(S2448) was determined by both immunohistochemical staining and Western Blot analysis, while LC3B and Atg7were only measured via immunohistochemical staining. The results were semi-quantitated and analyzed statistically.[Results]1. After8weeks of D-gal injection, serum AST, TBIL, DBIL and ALP levels were significantly higher than those in the control group (AST, DBIL:P<0.01; TBIL, ALP:P<0.001). Huperzine A significantly suppressed the D-gal-induced increases in AST and TBIL (P<0.05), but not for ALP or DBIL. When compared with the control group, levels of AST, TBIL and ALP were higher in D-gal+huperzine A-treated group(AST, ALP:P<0.05; TBIL:P<0.01). There were no significant differences in ALT values among the three groups (one-way ANOVA, F=0.0227, P=0.978).Huperzine A could not completely reverse D-gal-induced hepatic injury. 2. Relative to the control group, a much stronger intracellular SA-β-Gal signal was detected in the D-gal-treated group, while the percentage of BrdU positive hepatic cells was dramatically reduced. In the D-gal+huperzine A-treated group, a similar SA-β-Gal staining pattern with the control group was observed, and the BrdU-positive cell count were higher than the D-gal-treated group but lower than the control group. These results suggested that huperzine A could recover D-gal-induced hepatic senescence and loss of replicative potential.3. Detected through electron microscope, a large number of lipid droplets and swelling mitochondria with fewer cristae were shown in hepatic cells in D-gal-treated group, as well as some lysosomes with lipofuscin deposition. Huperzine A could significantly inhibit the accumulation of lipid droplets and impaired mitochondria, and increase the amount of rough endoplasmic reticulum. These changes suggested that huperzine A could accelerate the elimination of lipid drops and damaged mitochondria, and promote the synthesis process, which may contribute to the delay of hepatic aging.4. Hepatic cell apoptosis assay by TUNEL indicated a full screen of positive cells with brown nuclear shrinkage and fragmentation in D-gal-treated group, while only a few were seen in the control group. Huperzine A treatment could reduce these apoptotic cells. Apoptotic index (AI) in D-gal-treated group[(73.150±2.635)%] was higher than that in the control group[(2.117±0.651)%]and D-gal+huperzine A-treated group[(13.884±1.547)%](P<0.05). AI in D-gal+huperzine A-treated group is still higher than that in the control group (P<0.05). The expression of caspase3in D-gal-treated group was significantly higher than that in the control group (P<0.001). Huperzine A could inhibit the D-gal-induced increase of caspase3effectively (P<0.01), but the level was still higher than that in the control group (P<0.01). Huperzine A could partly reverse D-gal-induced hepatic cell apoptosis.5. Immunohistochemical staining showed the expression of Flk-1(A-3) and p-Flk-1(Tyr996)-R in D-gal+huperzine A-treated group was significantly higher than the other two groups (P<0.05).There were no significant differences between the control group and the D-gal-treated group [Flk-1(A-3):P=0.999; p-Flk-1(Tyr996)-R:P=0.122].P-GSK-3β(Ser9) expression were significantly different among the three groups (one-way ANOVA, F=1026.676, P<0.001), which was highest in D-gal+huperzine A-treated group and lowest in D-gal-treated group(Pairwise Multiple Comparison Procedures using the Holm-Sidak method, P<0.001).These results suggested that huperzine A might inhibit hepatic cell apoptosis through Flk-1/Akt/GSK-3β signaling pathway.6. Immunohistochemical staining showed the expression of p-mTOR (S2448) in D-gal-treated group was significantly higher than that in the control group (P<0.01). Huperzine A could inhibit the D-gal-induced increase of p-mTOR (S2448) effectively (.P<0.05), and the level was similar to that in the control group (P=0.731). LC3B expression was significantly different among the three groups (one-way ANOVA, F=38.4, P<0.001), which is lower in D-gal-treated group than that in the control group (P<0.001) and D-gal+huperzine A-treated group (P<0.01). Huperzine A could promote the expression of LC3B, but still lower than that in the control group(P<0.01). There were no significant differences in the expression of Atg7among the three groups (one-way ANOVA, F=2.747, P=0.117), but the average optical density value displayed highest in the control group, and lowest in the D-gal-treated group. Western Blot analysis showed p-mTOR (S2448) expression in D-gal-treated group was significantly higher than that in the control group (P<0.001).Huperzine A could effectively inhibit the expression of p-mTOR (S2448)(P<0.01), but still higher than that in the control group (P<0.01). Huperzine A could reverse D-gal-induced inhibition of autophagy.7. After comprehensive analysis of hepatic p-GSK-3β(Ser9) and p-mTOR(S2448) expression in the three groups, we surmised that the adjustment processes of huperzine A on mTOR signaling pathway and autophagy may be extremely complex. It may not only perform through the Flk-1/Akt/GSK-3β pathway, but also be associated with other pathways. These pathways cooperate with each other and promote hepatic autophagy finally.[Conclusion]1. Eight-week administration of D-gal could induce hepatic impairment, senescence and loss of replicative potential. Huperzine A intervention can reverse D-gal-induced hepatic injury, and delay hepatic aging.2. D-gal could promote cell apoptosis in aging liver. Huperzine A could partly reverse D-gal-induced hepatic cell apoptosis.3. Huperzine A might regulate cell apoptosis in aging liver through Flk-1/Akt/GSK-3β signaling pathway.4. D-gal could inhibit autophagy in aging liver. Huperzine A could reverse D-gal-induced inhibition of autophagy.5. Huperzine A could accelerate the elimination of lipid drops and damaged mitochondria, and promote the synthesis process, which may be associated with its effect on autophagy.6. The adjustment processes of huperzine A on mTOR signaling pathway and autophagy may be extremely complex. It may not only perform through the Flk-1/Akt/GSK-3β pathway, but also be associated with other pathways. |
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