Antiaging Effect of Pine Pollen in Human Diploid Fibroblasts and in a Mouse Model Induced by D-Galactose
Zhejiang Provincial Key Lab of Geriatrics, Zhejiang Hosptial
Gen-Xiang Mao, 1 Ling-Di Zheng, 1 Yong-Bao Cao, 1 Zhuo-Mei Chen, 2 Yuan-Dong Lv, 1 Ya-Zhen Wang, 1 Xi-Lian Hu, 1 Guo-Fu Wang, 1 ,* and Jing Yan 1
The present paper was designed to investigate the effect of pine pollen against aging in human diploid fibroblast 2BS cells and in an accelerated aging model, which was established by subcutaneous injections with D-galactose daily for 8 weeks in C57BL/6J mice. Pine pollen (1 mg/mL and 2 mg/mL) is proved to delay the replicative senescence of 2BS cells as evidenced by enhanced cell proliferation, decreased SA-β-Gal activity, and reversed expression of senescence-associated molecular markers, such as p53, p21Waf1, p16INK4a, PTEN, and p27Kip1 in late PD cells. Besides, pine pollen reversed D-galactose-induced aging effects in neural activity and inflammatory cytokine levels, as indicated by improved memory latency time and reduced error rate in step-down test and decreased concentrations of IL-6 and TNF-α in model mice. Similar to the role of AGEs (advanced glycation endproducts) formation inhibitor aminoguanidine (AG), pine pollen inhibited D-galactose-induced increment of AGEs levels thus reversed the aging phenotypes in model mice. Furthermore, the declined antioxidant activity was obviously reversed upon pine pollen treatment, which may account for its inhibitory effect on nonenzymatic glycation (NEG) in vivo. Our finding presents pine pollen as an attractive agent with potential to retard aging and attenuate age-related diseases in humans.
Rodent chronically injected with D-galactose has been widely used as an animal aging model for brain aging or antiaging pharmacology research [1–4]. An increased level of advanced glycation endproducts (AGEs) is thought to account at least partially for the underlying mechanism as the AGEs inhibitor aminoguanidine (AG) could block most of the aging phenotypes in the D-galactose-induced mouse model . AGEs are a heterogeneous group of reaction products that form between a protein's primary amino group and a carbohydrate-derived aldehyde group by reducing sugars, such as D-glucose and D-galactose, by nonenzymatic glycation (NEG) in vitro and in vivo . Accumulating evidence indicates that AGEs exacerbate and accelerate aging process and contribute to the early phases of age-related diseases, including neurodegenerative disease, cataract, renal failure, arthritis, and age-related macular degeneration [7–9]. Moreover, AGEs and their precursors usually contain reactive carbonyl groups, which can be generated by the actions of reactive oxygen species (ROS) [10, 11].
As a kind of Chinese traditional medicine, pine pollen, which is the male spore of pine tree, has been used as a drug and food for thousands of years. Pine pollen has an effect in the treatment of different kinds of diseases such as colds, disease of the prostate, anemia, diabetes, hypertension, asthma, and rhinitis [12–14]. Pine pollen is collected artificially from Pinus massoniana Lamb., Pinus tabulaeformis Carr., and it has the characteristics of a single pollen source, pure quality, and is a stable component. Pine pollen powder, called “natural micronutrient storeroom,” is rich in many kinds of body-demanding amino acid, minerals, vitamin, enzyme, and flavonoids . Although it is well proposed that pine pollen may have antiaging effect due to its various benefits on human health, the direct supportive experimental evidence linking the drug with aging has rarely been reported so far. So, it is interesting to investigate whether pine pollen possesses any antiaging effect in vitro and in vivo.
Here, the antiaging effect of pine pollen in vitro was firstly investigated by using the human diploid fibroblasts (2BS) cell line, which has been well characterized and widely used as a cellular senescence model [15–17]. Then, the accelerate aging model in mice induced by D-galactose was used to evaluate the effect of pine pollen against aging in vivo [1, 5, 18]. We treated a group of 5-month-old C57BL/6J mice daily with D-galactose, D-galactose combined with various dosages of pine pollen (500, 1000, 1500 mg/kg, resp.), D-galactose combined with AGEs formation inhibitor AG, and control buffer for 8 weeks. At the end of the treatment, learning and memory abilities, serum and cerebral AGEs levels, indicators for antioxidant activity, and proinflammatory cytokines levels were determined. Our results demonstrated that pine pollen could retard the aging process in cells and mice thus presents pine pollen as an attractive agent with potential to retard aging and attenuate age-related diseases in humans.
Taken together, pine pollen is proved to delay the replicative senescence of human diploid fibroblasts and block D-galactose-induced increase of serum and cerebral AGEs level in model mouse, which may result in the reversal of D-galactose-induced aging effects in both neural and inflammation system. It is possible that pine pollen exerts its antiaging effects at least partially by its NEG-inhibiting effect in vivo. Our work first directly proves the antiaging efficacy of the natural agent both in vivo and in vitro and thus inspires the new application for this drug in gerontological area.