The effect of spirulina polysaccharide on serum indicators and PI3K/Akt/mTOR signaling pathway gene expression in rats with endometriosis
In recent years, the incidence rate of endometriosis (EMs) has increased significantly. EMs refer to active endometrial tissue that appears outside the mucous membrane covering the uterine cavity. EMs are more common in women of childbearing age and may be related to excessive secretion of estrogen. Although they are benign lesions, they show local infiltration and growth, are easily invasive, and have obvious gynecological symptoms. After treatment, they are prone to multiple relapses, posing great harm to women’s physical and mental health. However, the methods for treating EMs are very limited. Apart from surgery, the commonly used internal medicine treatment is the long-term use of progesterone drugs. As the treatment time prolongs, patients may experience various side effects, such as bone loss, immune system dysfunction, and endocrine disorders. Therefore, it is urgent to find a new side effect free EMs treatment drug.

Natural products have abundant biological activity and the advantage of acting on different biological targets, thus making great progress in their development and utilization. Artemisinin, derived from Artemisia annua, is currently an effective treatment for malaria worldwide and has been used as a lead chemical in immune regulation, anti-inflammatory, and some cancer drugs. Taxus extract has also been used to treat breast cancer and ovarian cancer, and has become the research focus of chemical anti-tumor drugs. In numerous studies on the treatment of diseases with natural products, we have found that other extracts from terrestrial and aquatic plants, such as catechins, resveratrol, astaxanthin, and other seaweed extracts, exhibit various therapeutic effects, such as anti-inflammatory, antibacterial, and anti-tumor effects. Therefore, the idea of finding natural macromolecular compounds that are beneficial for treating endometriosis and providing new theoretical basis and drug resources for pharmaceutical libraries has become feasible. Tang et al. found that purple grass extract can effectively treat EMs and significantly affect the activity of inflammatory factors and apoptotic genes in ectopic endometrial tissue; Zhang et al. found that Juantongyin can effectively alleviate the symptoms of EMs in rats; Liu et al. found that saffron can improve EMs by inhibiting cell proliferation and inflammatory factors.

Spirulina is an early developed microalgae product with high market recognition. Polysaccharide from Spirulina (PSP) is a natural biomacromolecule derived from the prokaryotic organism Spirulina, which has multiple effects such as antioxidant, anti-inflammatory, antibacterial, anti-tumor, and antiviral. In recent years, PSP has been widely studied due to its unique characteristics that differ from commonly used anti-tumor drugs. At present, many studies have found that PSP can regulate the expression of downstream genes by acting on molecules in some tumor signal pathways. Xu et al found that PSP can inhibit the proliferation of human breast cancer cells. Yu et al. studied the effect of PSP on cervical cancer cells and found that PSP can inhibit the proliferation of Hela cells through periodic blockade. Sheng et al. demonstrated through in vitro and in vivo experiments that sulfated PSP has a significant inhibitory effect on mouse S180 sarcoma. Chen et al. found that PSP enhances the body’s ability to resist H22 cells by increasing immune cell activity. In addition, a large number of in vitro studies have shown that PSP can not only directly act on certain tumor cells, but also prevent cell damage in cancer patients during chemotherapy and radiotherapy. Therefore, we speculate that PSP can mediate some pathways to alleviate the production and proliferation of EMs, but there are no research reports on the treatment of EMs with natural polysaccharides. Therefore, this study aims to explore the therapeutic effect of PSP on endometriosis rats based on the PI3K/Akt/mTOR signaling pathway, providing new options for the development of novel EMs treatment plans and subsequent therapeutic drugs.

This experiment first extracted PSP by water extraction, and the purity was measured to be 89% by column chromatography; Using SD rats as model animals, a rat model of EMs was successfully prepared by autologous transplantation. The therapeutic effect on EMs was observed by feeding different doses of PSP; Western blot and RT-PCR were used to detect the protein and mRNA content of the PI3K/Akt/mTOR pathway.

The rate is increasing year by year, but its treatment methods are very limited, often requiring long-term internal medicine drug intervention, which not only affects patients’ confidence in treatment, but also leads to significant side effects. In addition, the development of new and effective EMs therapeutic drugs is urgent. With the development of the functional food industry, new natural products with pharmacological functions have attracted widespread attention. PSP is a natural biomacromolecule with multiple functions. Many studies have found that PSP can regulate downstream gene expression by acting on molecules in certain tumor signaling pathways. The formation and development of EMs are similar to the signaling pathways of most tumor cells. Therefore, we speculate that PSP can mediate some pathways to alleviate the production and proliferation of EMs.

This study first extracted and purified PSP, and the extraction rate of polysaccharides reached 1.95%. After column chromatography analysis, the purity was high (89%), which can be used for activity identification experiments; We used SD rats for EMs modeling, as the organs and tissues of rats are relatively large, making them easy to perform procedures such as dissection and implantation. EMs modeling can be divided into autologous transplantation and allogeneic transplantation. In order to avoid immune rejection, we choose autologous transplantation for modeling, and the success rate of modeling can reach over 80%. After PSP intervention, long-term dynamic observations were conducted on rats, and it was found that there was no significant change in their body weight, indicating that PSP does not have a negative impact on the growth of normal rats and EMs rats. By measuring the height and area of the abdominal transplant, it was found that PSP intervention significantly slowed down the development of EMs, suggesting that PSP may be useful for EMs treatment.

We speculate that PSP may produce therapeutic effects by regulating cytokines related to the formation and development of EMs. The regulation of cell adhesion and angiogenesis by cytokines is an important link in the development of EMs. For example, MMP-2 can regulate information exchange between cells and between cells and matrix. Studies have also shown that MMP2 can enhance cell adhesion, making endometrial cells more prone to ectopic implantation. In this study, the level of MMP2 in EMs rats was higher, which is consistent with Yuan et al.’s findings; PSP can reduce the activity of MMP2, indicating that MMP2 may be a target of PSP. TNF – α plays an important role in the process of cell proliferation and invasion, with many characteristics such as promoting cell proliferation and differentiation, and affecting vascular function. In this study, PSP significantly reduced the levels of TNF – α in the serum of EMs rats, and further reduced the downstream regulation of VEGF synthesis by TNF – α, thereby inhibiting the proliferation of EMs cells and the formation of blood vessels in diseased tissues. Blood vessel formation is an important reason for the easy metastasis and immune escape of EMs local lesions. The level of VEGF is also regulated by the PI3K/Akt pathway. The PI3K/Akt/mTOR pathway integrates a series of extracellular signals and regulates various cellular functions. This pathway mediates the adhesion of endometrial cells by integrating adhesins/intercellular adhesion factor-1, which is a necessary pathophysiological condition for endometriosis; Indirectly regulating metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) to mediate the invasion and metastasis of endometrial cells; Regulating VEGF to form neovascularization is an important step in the formation of EMs; In addition, the PI3K/Akt/mTOR signaling pathway can also regulate cell apoptosis and estrogen secretion, both of which are essential factors in the formation and development of EMs. We found that PI3K/Akt/mTOR was significantly upregulated in the model group mice, and the activation of this pathway implies cell growth, proliferation, and angiogenesis, which undoubtedly exacerbates the development of EMs. In this study, we found that PSP can inhibit the activation of the PI3K/Akt/mTOR signaling pathway, which may be an important reason for the reduced synthesis of cytokines such as TNF – α and VEGF. However, there have been few papers reporting the regulatory effect of PSP on this pathway so far. What pathway does PSP use to regulate PI3K/Akt? This will be our next research focus.

On the basis of PSP preparation and purification, this study completed the composition of PSP and prepared a rat model of endometriosis that meets the requirements based on autologous ectopic implantation. Based on the model, the improvement effect of PSP on rat endometriosis was explored, which is manifested as effectively reducing the levels of cytokines such as TNF – α, VEGF, MMP-2 in serum, and achieving the effect of inhibiting the development of EMs; Further exploration of the therapeutic mechanism of PSP on rat endometriosis based on the PI3K/Akt/mTOR signaling pathway showed that compared with the model group, the gene expression levels of PI3K, AKT, and mTOR in the ectopic endometrial tissue of the treatment group rats were significantly reduced. Protein expression detection confirmed that PSP could inhibit the phosphorylation of AKT, thereby inhibiting the protein expression of PI3K and mTOR. This indicates that PSP can improve the symptoms of EMs in rats by inhibiting the PI3K/Akt/mTOR signaling pathway. This article provides a reference for discovering new pharmacological functions of PSP and elucidating related mechanisms, laying a scientific foundation for further research and development.