Research on the Anti-breast Hyperplasia Mechanism of Liqi Sanjie Granules Based on Network Pharmacological Analysis and ExperimentalVerification

Background: Breast hyperplasia has become a major public health challenge due to changes in dietary patterns. However, there is still a lack of effective pharmacotherapy for Breast hyperplasia. Methods: The chemical components of Liqi Sanjie Granules (LSG) were quantified through HPLC. Three key components were screened out by combining network pharmacology methods and experimental determination methods, whereas key components in sera were quantified through LC-MS. The potential molecular mechanisms of LSG and these three blood-entering chemicals for the treatment of breast hyperplasia were predicted by network pharmacology. Estradiol benzoate and progesterone were injected intramuscularly to establish a breast hyperplasia model. Serum estradiol (E2), progesterone (P), testosterone (T), and prolactin (PRT) were detected by ELISA. H Li et al., 2022b). Breast hyperplasia and breast cancer pathogenesis are caused by endocrine disorders and associated with high estrogen levels. In general, the development mechanism of breast cancer is normal → hyperplasia → atypical hyperplasia → carcinoma in situ → invasive carcinoma(Schindler, 2009). Atypical hyperplasia is a necessary stage in carcinogenesis. Currently, many factors, such as Wnt-2, c-myc, ER, and PR are related to the growth, development, and metastasis of breast cancer(Yip and Rhodes, 2014; Satriyo et al., 2019), and the continuous activation of secretory cells in the lumen of the mammary gland leads to the development of breast cancer(Sopel, 2010). It has been reported that the mammary gland is a target organ for estrogen and progesterone, and the ER and PR can bind specifically to estrogen and progesterone, and the occurrence of breast hyperplasia is closely related to ER and PR disorders(Li et al., 2017). An HPLC method was used in comparing the chromatographic profile and data obtained from the standards and samples. The following parameters were considered: retention time, analysis time for samples, separation, and peak shapes. To optimise the chromatographic separation conditions of the seven components, we used different percentages of phosphoric acid water mixed with acetonitrile were as the separation system at different wavelengths (230, 240, 250, 260, and 270 nm), temperatures (25 °C, 30 °C, and 35 °C), and flow rates (0.6, 0.8, and 1.0 mL·min−1).The best animal model for collecting blood samples was determined by investigating the dosages of different animal models; blood collection times (30, 60, 90, and 120 min); different protein precipitants (methanol, acetonitrile, ethyl acetate, 25% ethyl acetate acetonitrile, 50% ethyl acetate acetonitrile, and 75% ethyl acetate acetonitrile); precipitant volumes (800, 1000, and 1200 μL); and multiple protein precipitants. Solvent (methanol and acetonitrile) determined the most appropriate method of serum treatment. Seven chemical components of LSG were quantitatively analysed, and the amounts of the three components of LSG after entering the blood of rats were determined through HPLC-MS, which laid the foundation for the study of the pharmacodynamic material basis and pharmacokinetics of LSG. In this study, a rat mammary gland hyperplasia model was established through the intramuscular injection of estradiol benzoate for 25 days, followed by progesterone intramuscular injection for 5 days. The specific manifestations were as follows: mammary tissue hyperplasia was obvious, that is, the number of mammary lobules and alveoli increased significantly, and the glandular epithelial cells showed vacuolar degeneration, hyperplasia, detachment, and necrosis; part of the mesenchyme was widened because of fibrous tissue hyperplasia, the alveoli were of different sizes, and the luminal walls thinned; and the content of PRL in the sera considerably increased. LSG exert a therapeutic effect by reducing the number of mammary lobules and alveoli and inhibiting changes in vacuolar degeneration, hyperplasia, and detachment of glandular epithelial cells. LSG may also exert a therapeutic effect on rat mammary hyperplasia by decreasing the expression levels of ER ESR1 mRNA and PGR mRNA in the mammary tissues. These effects in turn may decrease the expression level of PR protein in the mammary tissues. We used network pharmacology to explore the potential targets and mechanisms of LSG for the treatment of breast hyperplasia and screened a total of 63 potential targets and 36 signaling pathways. Estrogen, PRT, oxytocin, and other signaling pathways play an essential role in mammary gland development and pathological changes(Yan et al., 2022). The mammary gland is one of the target organs of estrogen and progesterone. ER and PR bind specifically to estrogen and progesterone. The overexpression of ER and PR aggravates the development of breast hyperplasia and cancer. When E2 levels are elevated and progesterone levels are reduced, estrogen stimulates breast tissue for a prolonged period of time, leading to ductal and lobular proliferation, which promotes tissue sodium retention and further causes interstitial edema. Moreover, no proliferation and recovery of the mammary gland occur because of the lack of regulation and protection against progesterone, and thus ductal epithelium and fibrous tissues of the mammary gland proliferate(Yi et al., 1994). PRT can enter the cell and bind to nuclear receptors, leading to a series of subsequent transcription and synthesis and increased proliferation. Other abundant relevant biological processes also suggest LSG exert inhibitory effects on breast proliferation by regulating signaling pathways, such as breast cell proliferation, inflammatory response, angiogenesis, and apoptosis(Shao et al., 2021). Network pharmacology studies have shown that the LSG multitargeted treatment of breast hyperplasia confirms the synergistic effect of LSG. The results of the network pharmacological study of the three chemical components entering the blood screened out 18 potential targets and five signaling pathways. NF-κB can regulate the expression levels of many genes and then participate in various biological processes, such as immune response, inflammatory response, apoptosis, and tumorigenesis. The cGMP-PKG signaling pathway is an intracellular signaling pathway in Chinese medicines and participates in a variety of physiological or pathological processes. These three chemical components enter the bloodstream and are potentially effective components of LSG for the treatment of breast hyperplasia. These three components can be separated, and their effects on breast hyperplasia can be studied separately. The mechanism of action was not comprehensively studied in this study, and other pathways were not investigated. Western blot and other advanced m