Effect of Sun's Bushen Huayu formula on M1/M2-associated markers and endometrial repair in a thin endometrium model

The presence of thin endometrium (TE) and defective embryo implantation, indicative of endometrial disorders, majorly contributes to sterility. The Sun's Bushen Huayu formula (BHD), a multi-herbal decoction prepared under standardized conditions, has shown potential in modulating M1/M2-associated inflammatory markers, but its efficacy in endometrial repair remains unclear. Female Sprague-Dawley rats with confirmed regular estrous cycles were first subjected to a thin endometrium (TE) model induced by hydroxyurea, high-molecular dextran, and intrauterine ethanol injury. After model establishment, rats received oral BHD or vehicle for 20 days, then were mated. On gestational day 8, uterine tissues were collected for analysis. Estrous cycle synchronization, body weight dynamics, embryo count, and histopathological evaluation were performed. Immunohistochemistry for M1/M2 macrophage markers, cytokine profiling by ELISA, and molecular analyses (qRT-PCR, flow cytometry) of M1/M2-associated markers and endometrial repair markers were conducted. In addition, western blotting was performed to examine STAT3 activation, apoptosis-related proteins, and nuclear/cytoplasmic NF-κB signaling. The administration of BHD notably lessened body weight loss and improved reproductive outcomes, as indicated by increased embryo implantation compared with the Model group, although implantation was not fully restored to Control levels. Representative histological analysis showed that BHD improved endometrial morphology. Treatment with BHD altered the expression of M1- and M2-associated markers, as evidenced by elevation of Arg-1 (an M2-related marker) and reduction of iNOS (an M1-related marker) in uterine tissue. Mechanistically, BHD was associated with a reduced p-STAT3/STAT3 ratio, decreased Cleaved-Caspase-3 and Bax expression, restored Bcl-2 levels, lower nuclear p-NF-κB/NF-κB, and higher cytoplasmic p-NF-κB/NF-κB, suggesting attenuation of inflammatory-apoptotic signaling and a shift in NF-κB subcellular distribution. Molecular analyses confirmed BHD's role in shifting the balance of M1/M2-associated markers, increasing CD206⁺ cell populations, and reducing CD86⁺ cells in uterine tissue. BHD was associated with improved endometrial repair in a TE model, accompanied by changes in M1/M2-related markers and cytokine balance. These findings suggest that BHD may serve as a potential therapeutic agent for restoring endometrial function and enhancing reproductive outcomes in conditions associated with endometrial dysfunction.