Preliminary study on the protective effect of icariin activating Nrf2 signaling pathway against D-galactose-induced injury in mouse testicular Sertoli 15P-1 cells
With the continuous development of the economy and society and the increasing pressure on people’s survival, infertility has become a global problem. Research has found that about 15% of couples worldwide suffer from infertility, with male factors accounting for about 50%, and the mechanism is not yet fully understood. Testicular Sertoli Cells, as the only somatic cells connected to spermatogenic cells, structurally surround spermatogonial stem cells, spermatogonia, and various levels of spermatogenic cells, forming a blood testis barrier through various tight junction proteins such as Occludin and Claudin-1. Research has shown that testicular supporting cells secrete various growth factors such as glial cell line derived neurotrophic factor (GDNF), bone morphogenetic protein-4 (BMP4), and stem cell factor (SCF), which regulate the proliferation and differentiation of spermatogonial stem cells and promote sperm formation. Another study found that male rats exposed to hexavalent chromium during pregnancy showed downregulation of the expression of tight junction proteins Occludin and Claudin-1 in the supporting cells after adulthood, leading to increased apoptosis of spermatogenic cells and impaired sperm production. Under heat stress stimulation, bovine testicular Sertoli cells are damaged, and GDNF and SCF mRNA levels are downregulated, leading to sperm dysfunction. Therefore, improving the structure and function of supporting cells may be a new direction for treating male infertility.

Epimedium is a traditional Chinese medicinal herb with nourishing properties, first recorded in the “Shennong Bencao Jing”. It has the effects of tonifying kidney yang, strengthening muscles and bones, and dispelling wind and dampness. Modern pharmacological research has shown that Epimedium has significant effects in delaying aging and enhancing male reproductive function. Its main active ingredient is icariin (ICA). Studies have found that ICA can promote the proliferation of primary Sertoli cells in rats and enhance the function of male Sertoli cells. Research has shown that ICA can enhance the reproductive function of rats by increasing the mRNA expression levels of follicle stimulating hormone receptors and tight junction protein Claudin-11 in supporting cells, thereby improving the antioxidant stress resistance of testicular tissue. Nuclear factor erythroid like 2 (Nrf2), as the main regulator of the body’s cellular defense system, regulates the expression of a series of genes encoding antioxidant proteins and oxidative stress response proteins by binding to enhancer sequences in the promoter regulatory region of antioxidant response elements (ARE), coordinating fine-tuning responses to various stress conditions and harmful attacks, and achieving cellular microenvironment homeostasis. Research has found that Epimedium can alleviate oxidative stress-induced liver damage by activating Nrf2. Our previous research found that ICA can alleviate DNA damage in testicular germ cells of naturally aging rats by upregulating Nrf2. ICA can upregulate the protein expression of the tight junction related proteins Occludin and Claudin-1 in the mouse testicular support cell line TM4 induced by D-Gal. However, there is currently no research report on whether ICA can improve D-Gal-induced 15P-1 cell damage through the Nrf2 signaling pathway. Previous studies have shown that D-Gal can induce tight junction damage in mouse testicular supporting cell line TM4 cells. Therefore, this experiment used D-galactose (D-Gal) – induced 15P-1 cells as a damage model to explore the protective effect and mechanism of ICA on Sertoli cell damage from the Nrf2 signaling pathway.

In recent years, infertility has been recognized as a public health problem by WHO. The incidence rate of infertility in the general population has increased year by year, affecting about 15% of couples of childbearing age in the world. Research shows that in the United States and other Western countries, sperm count decreases by 1.5% annually, and it is estimated that nearly 70 million couples worldwide have become victims of infertility or sterility. A large-scale epidemiological survey conducted by WHO found that about half of couples’ infertility is caused by male factors, and the mechanism is not yet fully understood.

Research has found that D-Gal can reduce the number and function of mouse supporting cell line TM4 cells. Therefore, we chose the D-Gal model to evaluate whether ICA can alleviate 15P-1 cell damage. There are literature reports that after treatment with 35, 70, and 140 μ M ICA on endothelial cells ECV-304, the cell inhibition rate was 1.2% and there was no significant cytotoxicity. The MTT results of this experiment showed that compared with the control group, when ICA was used alone to treat 15P-1 cells, the IC50 was greater than 50% at a concentration of 40 μ M, indicating that ICA had no significant cytotoxicity. Our preliminary research found that ICA has a protective effect on TM4 cell damage treated with D-Gal, and has a significant proliferative effect at concentrations of 0.5 and 1 μ M. The MTT results of this experiment also showed that compared with the D-Gal treatment group, ICA increased cell survival rate in a concentration dependent manner at 0.125-1 μ M, and had a significant promoting effect on cell proliferation at concentrations of 0.5 and 1 μ M. The promoting effect of ICA was weakened when the concentration was greater than 1 μ M, which may be due to the longer stimulation time of D-Gal on 15P-1 cells in this experiment, more severe cell damage, and greatly increased sensitivity of damaged cells to drug stimulation, thus exhibiting a phenomenon of low concentration promotion and high concentration inhibition. Testicular Sertoli Cells are somatic cells located in the seminiferous tubules of the testes that are directly connected to spermatogenic cells. They regulate the proliferation and differentiation of spermatogonial stem cells and promote sperm formation by secreting various cytokines such as GDNF, BMP4, and SCF. GDNF is a member of the transforming growth factor beta superfamily and an important factor in regulating the proliferation of spermatogonial stem cells. It plays a crucial role in the development of male reproductive cells and early testicular development. BMP4 and SCF are important factors that promote the differentiation of spermatogonial stem cells. Previous research by our research group has shown that total flavonoids of Epimedium can upregulate the mRNA and protein levels of GDNF, BMP4, and SCF secreted by Sertoli cells in naturally aging rat testes, improving Sertoli cell secretion function. The results showed that compared with the control group, the mRNA and protein expression levels of Sertoli cell secreted factors were significantly decreased after D-Gal treatment, while the mRNA and protein expression levels of Sertoli cell secreted factors were significantly up-regulated in 15P-1 after ICA treatment. ICA can improve the secretion function of 15P-1 testicular Sertoli cells.

The tight junctions between supporting cells are the most important component of the blood testis barrier, mainly including the three transmembrane protein families of Occludin, Claudin, and adhesion proteins, as well as the cytoplasmic protein family of the blocking junction. The tight junctions between cells not only protect reproductive cells from immune system invasion and provide a good physiological environment for spermatogenesis, but also allow pre fine and post fine spermatocytes to pass through the blood testis barrier regularly for further development. Research has found that Occludin and Claudin are the earliest identified tight junction membrane proteins, and tight junction ability and epithelial barrier function are mainly mediated by Claudin. Another study has shown that injecting synthetic Occludin peptide into rat testes can reversibly ablate most spermatogonia after 27 days, while knocking out Occludin in vitro can also lead to a decrease in tight junction function of supporting cells. The results of this experiment showed that compared with the control group, the expression of tight junction protein in 15P-1 cells decreased after D-Gal treatment, while the expression of tight junction protein in 15P-1 supporting cells was significantly upregulated after ICA treatment. ICA has a protective effect on tight junction protein damage in 15P-1 supporting cells.

When the body is in a normal state, Kelch like ECH associated protein-1 (Keap1) recruits Nrf2 and forms a complex in the cytoplasm that is degraded by ubiquitin protease; When oxidative stress occurs, Nrf2 dissociates from Keap1, and Nrf2 is transferred from the cytoplasm to the nucleus to bind to ARE, activating the transcription of downstream protective genes such as HO-1 and NQO1, thereby resisting oxidative stress damage caused by various stimuli to the body. Our previous research has shown that ICA can alleviate testicular DNA damage in naturally aging rats and improve reproductive function decline caused by aging by activating the Nrf2/HO-1 signaling pathway. This suggests that ICA’s improvement of spermatogenic dysfunction may be related to the Nrf2 signaling pathway. Previous studies have shown that ICA can improve D-Gal-induced testicular support cell line TM4 cell damage. Therefore, this study used Western blot to detect Nrf2 pathway proteins. The results showed that compared with the control group, the expression levels of important Nrf2 pathway proteins Nrf2, HO-1, and NQO-1 in 15P-1 cells were significantly downregulated after D-Gal stimulation, while they were significantly increased after ICA administration. Subsequently, we further detected the expression and localization of Nrf2 using immunofluorescence technology. Compared with the normal control group, the fluorescence expression of Nrf2 in the nucleus of 15P-1 cells in the D-Gal treatment group was weakened, but improved after ICA treatment. The above results suggest that the functional and structural damage of 15P-1 cells is related to the downregulation of the Nrf2 signaling pathway, which leads to a decrease in cellular antioxidant capacity. ICA may improve the damage of 15P-1 cells by enhancing their antioxidant capacity.

In summary, ICA can upregulate the expression levels of secretion related proteins GDNF, BMP4, SCF, as well as tight junction related proteins Occludin and Claudin-1 in 15P-1 cells by activating the Nrf2 signaling pathway, thereby improving D-Gal-induced damage in 15P-1 cells. This experiment has laid a theoretical foundation for our subsequent research on the protective effect and mechanism of ICA on germ cells, in order to better explore the application value of Chinese herbal medicine Epimedium in the reproductive system, and provide new directions for the clinical treatment of male infertility.