| [Backgrounds]With the improvement of people’s living standard, the aging of the population has gradually accelerated and the age-associated diseases 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. 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 act on the insulin signaling pathway and improve oxidative stress-inflammation in aging liver, as well as its role in insulin signaling pathway in aging liver cells. Insulin is an important growth factor in cells that can regulate cell oxidative stress-inflammation and aging through PI3K/Akt/FOXO signaling by binding to the insulin receptor substrates(IRs). Meanwhile, the inflammation and aging can affect insulin signaling pathway. Therefore, we assume that huperzine A might affect insulin thereby regulate PI3K/Akt/FOXO/NF-K B pathway through insulin, and participate in the regulation of inflammation in aging mice liver, which could contribute to liver protection and delaying aging.[Objective]1. Research the expression change of insulin, clarify the effect of huperzine A on insulin in aging liver.2. Research the expression changes of PI3K, Akt, p-Akt, FOXO3a and p-FOXO3a levels in liver. And clarify the influence of huperzine A on insulin signaling pathway in aging liver.3. Research expression changes of MnSOD, MDA and NF-K B p50. And clarify the influence of huperzine A on oxi-inflam in aging liver.[Methods]1. Thirty six 3-month male mice were randomly divided into three groups (n=12 per group):the huperzine A-treated group, the huperzine A+ inhibitor-treated group, and the control group. The huperzine A-treated group was injected subcutaneously with 0.1mg/kg huperzine A daily. In the huperzine A+ inhibitor-treated group, the mice were co-injected subcutaneously with 0.1mg/kg huperzine A and 5mg/Kg huperzine A inhibitor daily. The control group was injected with saline. After 12 months of treatment, all the mice were culled for tissue preparation.2. Specimens of blood and liver tissue were obtained and automatic biochemistry analyzer was used to assess insulin level.3. The expression of hepatic PI3K, Akt, p-Akt, FOXO3a and p-FOXO3a were determined by both immunohistochemical staining and Western Blot analysis. We used image analysis software for semi-quantitative determination of the results, and the data were statistically analyzed.4. Serum was obtained and automatic biochemistry analyzer was used to assess liver MnSOD. The expression of hepatic MDA and NF-κB were determined by both immunohistochemical staining and Western Blot analysis. We used image analysis software for semi-quantitative determination of the results, and the data were statistically analyzed.[Results]1. When compared with the control group, insulin level was lower in huperzine A+ inhibitor-treated group and huperzine A-treated group (huperzine A+ inhibitor-treated group:P<0.05; huperzine A-treated group:P<0.01). There were no significant differences in huperzine A+inhibitor-treated group and huperzine A-treated group (one-way ANOVA,F=3.086, P=0.154). These changes suggested that Huperzine A could reduce age-related insulin.2. Relative to the control group, a much stronger intracellular Akt and FOXO3a signal was detected in the huperzine A-treated group, while PI3K、p-Akt and p-FOXO3a were reduced. These results suggested that huperzine A could inhibit hepatic insulin signaling pathway. Huperzine A inhibitor can weaken the effect.3. Detected through Microplate reader, MnSOD level in the huperzine A-treated group was higher relative to the control group(F=32.018,P<0.01), MnSOD level in the huperzine A+ inhibitor-treated group was between the control group (F=15.399, P=0.002) and he huperzine A-treated group (F=6.262, P=0.023). These changes suggested that huperzine A could promote the expression of MnSOD, and promote the synthesis process, which may contribute to the enhance antioxidant capacity and the delay of hepatic aging. Huperzine A inhibitor can weaken the effect.4. Immunohistochemical staining showed the expression of MDA in huperzine A-treated group was significantly lower than the other two groups (P<0.05). The expression of NF-κB in huperzine A-treated group was significantly lower than the other two groups (P<0.01). Western Blot analysis showed MDA expression in huperzine A-treated group was significantly lower than that in the control group (P<0.001). Huperzine A could reduce intracellular oxidative stress and inflammation, thereby delay hepatic senescence. Huperzine A inhibitor can weaken the effect. We surmised that the adjustment of huperzine A on oxidative stress may be associated with insulin signaling pathway.[Conclusion]1. Huperzine A could reduce the insulin level in liver of aging mice, this effect is not affected by huperzine A inhibitor.2. Huperzine A could inhibit insulin signaling pathway in aging liver which involved in the regulation of aging. Huperzine A inhibitor may weaken this effect.3. Huperzine A could promote the expression of MnSOD and enhance antioxidant capacity of hepatocytes. Huperzine A inhibitor may weaken this effect.4. Huperzine A could accelerate the elimination of intracellular lipid peroxidation product MDA, which may contribute to inhibiting oxidative stress and delaying aging. Huperzine A inhibitor may weaken this effect.5. Huperzine A could inhibit the activation of NF-K B in aging liver, thereby inhibiting the expression of inflammatory cytokines and delaying inflame-aging. Huperzine A inhibitor may weaken this effect. |
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