The ovarian protective effect of pine pollen on cyclophosphamide induced premature ovarian failure in rats
Premature ovarian failure (POF) refers to the phenomenon of amenorrhea before the age of 40 caused by ovarian failure, characterized by an increase in gonadotropin levels and a decrease in estrogen levels. POF may also lead to clinical symptoms such as infertility. The incidence of POF is about 1% to 3%, and there is a gradually increasing and younger trend. Multiple factors can induce POF, such as environmental factors, genetic factors, and disease factors. Therefore, the prevention and treatment of POF have received widespread attention from the whole society.

At present, the commonly used treatment for POF is hormone replacement therapy (HRT), such as using natural estrogen estradiol or progesterone. Hormone analogs released by gonadotropins have also been shown to have good efficacy in preventing chemotherapy-induced POF in cancer patients. However, the use of hormone replacement therapy may lead to a higher risk of breast cancer, stroke and gallbladder disease. As is well known, Chinese herbal medicine, especially traditional medicinal and edible dual-use materials, have high safety. Studies have shown that traditional Chinese medicine treatment is more effective than HRT treatment in reducing FSH levels. Therefore, developing safer and more effective ovarian protection products based on the same origin of medicine and food is of great significance for maintaining people’s physical health.

Pine pollen is the male reproductive cell of several plants belonging to the same genus, such as Pinus massoniana or Pinus tabulaeformis. It contains more than 200 nutrients and active substances, including proteins, amino acids, enzymes, vitamins, minerals, nucleic acids, flavonoids, polysaccharides, choline, plant sterols, etc. It has been reported to have protective effects on the liver, enhance immunity and anti-tumor effects, as well as the ability to reduce inflammatory reactions and oxidative stress. In addition, pine pollen has been shown to have a protective effect on the prostate, indicating its potential ability to protect the reproductive system. The content of polysaccharides in pine pollen is relatively high, and its structure is similar to that of polysaccharides from wolfberry and Angelica sinensis, which have been reported to have ovarian protective properties. Pine pollen also contains a large amount of naringin, which is believed to have estrogen like effects and can bind to estrogen receptors to maintain physiological functions. Therefore, pine pollen has the potential for ovarian protection, but currently there is little research on the function of pine pollen that involves ovarian protection.

This study established a POF rat model induced by cyclophosphamide (CTX) and administered high, medium, and low doses of pine pollen suspension by gavage to investigate the effects and possible mechanisms of pine pollen on ovarian morphology, serum hormone levels, inflammatory factor concentrations, ovarian antioxidant enzyme activity, and anti granulosa cell apoptosis. The aim is to provide theoretical and experimental basis for the development of functional pine pollen products based on ovarian protection.

Pine pollen is a medicinal and edible raw material, and its nutritional components and functional properties have been widely studied. There are studies showing that pine pollen has prostate protection function, as well as antioxidant and anti-inflammatory effects. This article explores the protective function of pine pollen on the ovaries by inducing a rat POF model with cyclophosphamide.

Rats with POF often exhibit changes in serum hormone levels related to the reproductive system, such as a significant decrease in E2 and P levels. However, the results of this study showed a significant increase in the concentration of P in the model mice compared to the blank group. In POF rats, both ovarian cysts and endocrine disorders may lead to an increase in P levels. LH and FSH are both secreted by the pituitary gland and can regulate the synthesis of sex hormones, promoting follicular maturation and corpus luteum formation. CTX can disrupt hormone secretion related to the ovaries, and the results of this study are consistent with other studies. Ovarian reserve refers to the reserve of primordial follicles contained in the ovarian cortex, with AMH and INHB as the main diagnostic indicators. The pathological results of ovarian tissue showed a significant decrease in the number of follicles in the model group, consistent with the decrease in AMH. INHB is a hormone secreted by germ cells, closely related to reproductive ability, and its main function is to regulate the production of FSH. Studies have shown that there is negative feedback regulation between FSH and INHB, while the results of this study are opposite, possibly due to the high secretion of INHB indicating the presence of tumors in granulosa cells, as described by Carballo et al. The results of this study indicate that after oral administration of pine pollen, the secretion of serum hormones in rats tends to normal levels, and pine pollen has the function of regulating hormones with a dose effect relationship.

The main mechanism of CTX induced POF is to induce apoptosis of granulosa cells. The massive apoptosis of granulosa cells can directly affect the growth of follicles, ultimately leading to the development of blocked follicles and a significant reduction or even depletion of the number of follicles in the ovary, resulting in a decrease in ovarian reserve capacity. Caspase-3 only appears in granulosa cells of blocked follicles and not in granulosa cells of healthy follicles. Bcl-2 related pro apoptotic and anti apoptotic proteins play important roles in the decision-making steps of intracellular death programs upstream of Caspase protease. In this study, pine pollen can regulate the expression of apoptotic proteins, increase the ratio of Bcl-2 and Bax, reduce the expression of Cleaved Caspase-3, thereby inhibiting granulosa cell apoptosis and promoting normal follicular development.

Ovarian injury can be caused by oxidative stress induced by free radicals, such as reactive oxygen species and hydrogen peroxide. Increased levels of oxidative stress can ultimately lead to DNA damage and apoptosis of granulosa cells. For the female reproductive system, antioxidants are considered a key factor in ovarian physiological metabolism, which can effectively reduce the levels of these free radicals and maintain ovarian health. However, their exact molecular mechanisms and functions have not been fully elucidated. CAT, SOD, and GSH Px all play key roles in sex hormone regulation. The results of this study also indicate that high-dose pine pollen can significantly enhance the antioxidant enzyme activity of the ovary, which is of great significance for ovarian function.

Inflammation is also one of the possible pathways for ovarian aging, and long-term inflammation can lead to pathological changes in the ovaries. Tumor necrosis factor and interleukin are both major inflammatory factors, and they are highly expressed in rats with ovarian dysfunction. Some studies suggest that the inflammatory aging of the ovaries is essentially related to the level of oxidative stress, and oxidative stress and inflammatory response are mutually causal. The results of this study also indicate that the concentrations of TNF – α and IL-4 in the serum of POF rats increase, leading to a strong inflammatory response, while pine pollen can reduce the degree of inflammatory response.

This article confirms that pine pollen has a protective effect on the ovaries of CTX induced POF rats, such as regulating the levels of estrogen E2 and P, gonadotropins FSH and LH, anti granulosa cell apoptosis, and regulating apoptosis factors Bax, Bcl-2, and Cleaved Caspase-3, improving antioxidant capacity and increasing the activity of antioxidant enzymes GSH Px, SOD, CAT, and reducing the levels of inflammatory factors TNF – α and IL-4. The function of pine pollen is dose-dependent. Pine pollen contains a large amount of polysaccharides and flavonoids, which may be related to its ability to protect the ovaries and require further research. This study has certain theoretical and practical application value for developing healthy products with ovarian protection function using pine pollen as raw material.