The accelerated pace of life and the recent increase in environmental pollution, unreasonable dietary habits, poor living habits, and mental factors often accompany modern women, causing a series of adverse reactions such as Mammary gland hyperplasia (MGH). MGH is a degenerative disease and progressive connective tissue growth caused by hyperplasia of mammary fiber and epithelial tissue (1). Modem medical research has shown that the development of MGH is closely linked to endocrine disorders. An imbalance in the ratio of estradiol to progesterone leads to excessive proliferation and regression of the breast parenchyma (2–4). It was reported that MGH was the disease with the highest incidence among female breast diseases, and its incidence rate is as high as 75%, and the incidence rate is still rising year by year. The age of onset is also getting lower and lower, and even some breast hyperplasia will have canceration, which has become a major health problem for women around the world (5–9). At present, for the examination and treatment of MGH, pathological biopsy or surgical resection of suspicious nodules revealed by some imaging examinations were typically utilized. Oral drugs mainly include oral sex hormone drugs (such as bromocriptine, tamoxifen, danazol, etc.) and non-sex hormone drugs (thyroxine, iodine, evening primrose oil, etc.). However, surgical resection has a large wound surface, which brings irreversible psychological and physiological damage to patients; drugs easily cause multiple toxic side effects such as menstrual blood loss, menstrual disorder, dizziness, nausea, vomiting, etc., and some drugs can also cause certain damage to the human gastrointestinal tract and central nervous system (10–12). Therefore, the search for a noninvasive, efficient treatment of MGH drugs has become a research hotspot at home and abroad.

Studies have demonstrated that endocrine imbalance can affect the composition of the intestinal microbiota and can directly or indirectly alter bacterial physiology and independent gene expression (13–15). MGH is caused by endocrine imbalance, in which the imbalance of estrogen secretion is an important signal of MGH. The abundance of intestinal microbiota is not simply related to the level of estrogen but also can affect the level of estrogen secretion. In endocrine balance, a variety of bacteria in the intestinal microbiota is involved in estrogen metabolism. For example, intestinal bacteria with structures such as β-glucuronidase and β-glucosidase can facilitate the reabsorption of estrogen through the intestine and re-enter the hepatic and intestinal circulation, eventually reaching the target organs it regulates to act (16–18). When there is an endocrine imbalance, the intestinal microbiota is altered and the β-glucuronidase content increases, raising the level of estrogen in the mammary glands. Breast epithelial cells that are chronically exposed to high levels of estrogen and become lesions occur, leading to their excessive proliferation, causing MGH (19, 20). Therefore, it was hypothesized that the treatment of MGH can be obtained by regulating the intestinal microbiota of homeostasis and balancing the level of estrogen secretion.

The earliest understanding of the mammary gland in Chinese medicine can be traced back to the Nei Jing, where it is written that “a woman’s breast belongs to the liver, while the nipple belongs to the stomach” and that “Chong and Ren meridians can regulate the Qi and blood of the 12 meridians and they stop at the breast upward and form menstruation downward”, which explains the close relationship between the breast and the spleen and stomach in terms of physiological function and meridian affiliation. Chinese herbal medicine has a very beneficial effect on the balance of the intestinal micro-ecolozy, and the intestinal microbiota coincides with the theory of “spleen and stomach” in Chinese Medicine (21–23). Atractylodes Macrocephala Rhizoma is the dry rhizome of the Asteraceae plant Atractylodes macrocephala Koidz, which has the effect of strengthening the spleen and stomach, drying and dampening water. Modern research shows that Atractylodes Macrocephala Rhizoma contains a variety of volatile components, polysaccharides, and amino acids, which have various pharmacological activities such as improving the function of the gastrointestinal tract and regulating the intestinal microbiota (24, 25).Therefore, this project will use AMP to treat MGH rats and analyze the effect of AMP on the intestinal microbiota of MGH rats through 16S RNA high-throughput sequencing, to provide theoretical support for the treatment of MGH from the perspective of intestinal microecology, as well as to provide an experimental basis for fully exploiting the medicinal value of AMP. The research idea is illustrated in Figure 1 .

The human intestinal microbiome is a diverse and complex ecosystem and is home to thousands of microorganisms that co-evolve with their hosts and play important roles in health and disease. The important role of intestinal microbial in health and disease has become a research focus (28–31). Intestinal microbial diversity is tightly associated with levels of estrogen and its metabolites (32–34). A reduction in intestinal microbial diversity can reduce the activity of β-glucuronidase that can reduce the circulation form of estrogen in the body and break it down into active ingredients, resulting in a large estrogen accumulation in the mammary gland, and leading to endocrine disorders (35–37). Modern medicine also believes that the occurrence of MGH is linked to endocrine disorders, in particular, it is related to the imbalance of balance hypothalamic-pituitary-ovarian-mammary gland endocrine axis, in which the secretion of estrogen increases and the secretion of progesterone decreases. The imbalance of female/progesterone ratio is the main cause of mammary gland hyperplasia (38–41). The mammary gland is an important target organ for the effect of estrogen, and when estrogen secretion in the female organism is too high, it will cause the female breast tissue to be in a state of over-stimulation, and the poor physical condition will have an impact on the mammary gland, leading to MGH (42, 43). Metabolic enzymes changed regulated by the intestinal microbiota will affect the estrogen level change, which leads to endocrine dysregulation, eventually causing MGH (36, 44). Thus, estrogen can be participated in the development of MGH through changes in the metabolic levels of intestinal microbiota.

In this experiment, E₂, P, and PRL were significant indicators to determine whether the rats had MGH. Compared with the Con group, the Mod group E₂ and PRL levels were increased significantly and P levels were decreased significantly, compared with the Mod group; the AMP group E₂ and PRL levels were reduced and P levels were increased, indicating that AMP could alleviate the symptoms on rats MGH effectively. By 16S RNA amplified sequencing, in alpha-diversity analysis, the Mod group Chao1, Pielou, Shannon, and Simpson indices were decreased in the intestinal tract of rats, indicating that MGH had an inhibitory effect on the richness and diversity of intestinal microbiota in rats. Compared with the Mod group, the AMP group Chao1, Pielou, Shannon, and Simpson indexes in the intestinal tract of rats were significantly increased, which was similar to that in the Con group, indicating that AMP had a certain improvement effect on the intestinal microbiota of rats with MGH. In the beta-diversity analysis, AMP improved the chances of microbial community structure in intestinal contents of MGH rats and gradually restored it to the normal level. However, there were still discrete individual samples in the Mod group, which may be due to the inter-individual differences.

Under normal conditions, Firmicutes and Bacteroidetes are in dynamic balance in the intestinal tract and the ratio of relative abundance is seen as an important marker of intestinal microbiota disorders, and an increase in F/B value represents an endocrine disorder on the other hand (45–47). In this experiment, MGH changed the structure and composition of the microbiota in the intestinal contents of rats, and through the phylum level analysis on the changes of the microbiota relative abundance, we can further understand how AMP treats the MGH by regulating the intestinal microbiota. In the Mod group, the relative abundance of Proteobacteres and the F/B value also increased, suggesting that MGH can lead to the imbalance of the intestinal micro-ecological environment; after AMP treatment, the relative abundance of Proteobacteres decreased, and the value was similar to the Con group; the F/B value also decreased but was still higher than that of the Con group. Therefore, it can be deduced that AMP is beneficial to restore the major intestinal microbiota. Actinobacteres are a gram-positive bacterium, which can protect the host and nutrient from attack by pathogens in the intestine (48–50). In this experiment, compared with Con group, the relative abundance of Actinobacteres in the Mod group increased, indicating that the MGH caused resistance of intestinal microbiota. Compared with Mod group, the relative abundance of Actinobacteres in the AMP group was reduced, which indicated that the administration of AMP could alleviate the intestinal microbiota disorder. According to the analysis of genus level, Clostridiales is generally considered to be related to body inflammation, Clostridium contains 20α-hydroxy steroid dehydrogenase, which converts glucocorticoids into androgens, when Clostridium is unbalanced, a large number of androgens will be synthesized, which will cause intestinal microorganisms dysregulation and eventually lead to endocrine disorders and pathological lesions in women (51, 52). Combined with the results of this experiment, compared to the Con group, the relative abundance of Allobaculum, Clostridiales, and Lachnospiraceae decreased and the Clostridium increased in the Mod group; compared to the Mod group, the relative abundance of Lachnospiraceae, Clostridium, Clostridiales increased, and Allobaculum decreased in the AMP group. Further studies are needed to investigate this difference. Based on the above experiments, AMP can maintain the endocrine balance by regulating the homeostasis of intestinal microbiota, so as to affect the occurrence and progress of MGH.

In general, MGH caused by endocrine dysregulation breaks the intestinal microbiota balance in rats, and the diversity and richness of intestinal microbiota are destroyed to varying degrees. Through the study of AMP in the treatment of MGH and its effect on intestinal microbiota, this experiment proved that AMP had a certain therapeutic effect on MGH and also had a definite recovery effect on the diversity, richness, and community structure of intestinal microbiota in MGH rats. However, the therapeutic effect of a single polysaccharide component on MGH by regulating intestinal microbiota has not been excellent. Therefore, the therapeutic effect of the AMP of MGH through intestinal microbiota needs further study.

The original contributions presented in the study are publicly available. This data can be found here: https://doi.org/10.5061/dryad.gf1vhhmtc.

The animal study was reviewed and approved by the Animal Ethics Committee of the Jiamusi University.

YP prepared the drafting of manuscript and interpretation of data. CL performed the experiment and analyzed in the analyzing of part of the data. LW and HZ designed the study and revised the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.