Patients with POF usually have symptoms such as amenorrhea for more than four months, hot flashes, night sweats, low libido and infertility . Cisplatin as one of the most common-seen drugs for chemotherapy and considered to activate PTEN through the PI3K-AKT signaling pathway, which might potentially lead to follicular atresia . Herein, the POF mice were established by cisplatin in this study. The pathological morphology of ovary and vaginal exfoliated cells could reflect the ovarian status of mice. According to the above results, we found that EA could repair the injured of ovary and restore estrous cycle in cisplatin-induced POF mice. On the contrary, EN had little effect on the regulation of ovarian pathological morphology and estrus cycle.
FSH, LH, E2 and AMH are the important sex hormones that can reflect ovarian function , while both FSH and LH are thought to be secreted by hypophysis, while E2 is mainly secreted by granulosa cells in ovary. Serum AMH is applied to detect ovarian reserve function in clinic as reported . The decrease of AMH level indicated premature depletion of primordial follicles. E2 can affect the levels of FSH and LH through the mechanism of negative feedback in HPO axis, which makes the sex hormones maintain a dynamic balance . Follicular atresia causes the decrease of granulosa cells in follicles when POF occurs . Therefore, POF can cause E2 and AMH to maintain at a low level. The long-term maintenance of low serum E2 level will break the negative feedback mechanism between ovary and pituitary, and lead to the levels increased of FSH and LH . Therefore, both the high levels of FSH and LH in patients with POF, while low levels of E2 and AMH. According to the results, it was found that EA could effectively restore the levels of serum FSH, LH, E2 and AMH in POF mice. However, EN did not restore the levels of FSH, LH, E2 and AMH. These results might be related to EA increasing the number of antral follicles in ovary. It showed that acupoints could improve ovarian function more effectively than non-acupoints.
PI3K-AKT signaling pathway is involved in the process of oocyte growth, primordial follicle development and granulosa cell proliferation . FSH is a glycoprotein hormone synthesized by hypophysis. FSH can combine with the receptor on the membrane of ovarian granulosa cells to activate PI3K-AKT pathway to promote the maturation of ovarian granulosa cells . On the other hand, E2 can combine with the estrogen receptor α (ER-α) and estrogen receptor β (ER-β) in ovary . PI3K has subunits of p85 and P110. ER-α combines with the subunit p85 in PI3K to activate AKT [19, 20]. Meanwhile, mTOR is activated by AKT, which leads to proliferation and development of follicle . In this study, we found that the PI3K-AKT signaling pathway in ovary of cisplatin-induced POF mice was inhibited. The relative expression of PI3K, AKT, and mTOR were reversed to normal in POF mice by EA. The result indicated that PI3K-AKT signaling pathway could be activated by electro-acupuncture at acupoints to promote the proliferation of ovarian cells. The results were consistent with the previous research results of other subjects.
There are a large number of microbiomes in human intestine, including probiotics, pathogenic bacteria and conditioned pathogen . Gut microbiome is considered to be closely related to human health . Growing evidence shows that the disorder of gut microbiome can lead to ovarian diseases, endocrine diseases and cardiovascular diseases [24, 25]. It had been reported that the disorder of gut microbiome would break the barrier of intestinal mucosa . Pathogenic bacteria and their metabolites reached the ovary through the circulation of blood to reduce ovarian function . On the other hand, the enterohepatic circulation of estrogen was an important way to maintain the stability level of human serum estrogen . Serum free estrogen is converted into conjugated estrogen by the liver. Then, the conjugated estrogen is excreted into the intestine with the bile . β-glucuronidase was considered to be one of the metabolites of gut microbiome. Conjugated estrogen was converted into free estrogen by β-glucuronidase . Free estrogen enters the circulatory system of blood through intestinal resorption to promote growth of follicle and relieve the symptoms of POF.
In the control group, the three highest relative expression dominant microbiomes were Lactobacillus, Muribaculaceae and Monoglobus. Lactobacillus plays an important role in maintaining human health and is considered as one of the most important probiotics in human intestinal . It had been reported that Lactobacillus could inhibit the growth and reproduction of pathogenic bacteria . Muribaculaceae is widely distributed in the intestines of mice. Muribaculaceae can reduce the colonization of Clostridium difficile in the intestine . Meanwhile, Muribaculaceae can degrade carbohydrates. In addition, Monoglobus can degrade pectin and maintain intestinal health . According to the above results, we found that the three highest relative expression dominant microbiomes were probiotics in control group. They are important to maintain the health physiological function of intestine.
Lachnospiraceae, Eubacterium coprostanoligenes and Blautia were the dominant microbiomes in the POF group. Lachnospiraceae was considered to protect the intestinal mucosa . On the contrary, Lachnospiraceae also could damage the pathway of glucose metabolism and promote the process of inflammation . Therefore, Lachnospiraceae was considered as a conditional pathogen. Eubacterium coprostanogenes was considered to be pathogenic bacteria. It had been reported that Eubacterium coprostanogenes could reduce the intestinal absorption of cholesterol [37, 38]. Blautia could prevent inflammation and promote the production of short chain fatty acids (SCFAs) to maintain intestinal homeostasis, so it was considered to be a potential probiotic [39, 40]. The existence of beneficial bacteria in the first three dominant bacteria of POF mice whether be related to the self-healing function of mice, and it still needed to be further researched.
In the EA group, Anaeroplasma, Ruminococcaceae, and Eubacterium ventriosum were the three microbiomes with the highest relative expression. Anaeroplasma was an agent of anti-inflammatory. In addition, Anaeroplasma could maintain the homeostasis of immune in intestinal mucosal . Similarly, Ruminococcaceae and Eubacterium ventriosum had the function of anti-inflammatory, and both were the common beneficial bacteria in the intestine [42, 43]. As a member of SCFAs producer, Ruminocaceae was considered to maintain intestinal immune homeostasis. All the first three dominant microbiomes in POF mice were probiotics after treating by EA. The result indicated that EA could increase the abundance and diversity of probiotics.
The dominant microbiomes of three highest relative expressions in the EN group were Tannerellaceae, Parabacteroides, and Streptococcaceae. Several studies had found that Tannerellaceae promoted the occurrence of intestinal inflammation . Parabacteroides were considered to be beneficial bacteria . Moreover, it had been reported that Parabacteroides could alleviate liver injury and reduce the expression of inflammatory genes in liver . Streptococceae is a conditioned pathogen, which was a common microbiome in human oral cavity, skin, intestinal, and upper respiratory tract . Although the content of probiotics could be increased by EN, the dominant bacteria were pathogenic bacteria.
According to the results, EA could significantly reduce the F/B ratio, which indicated that EA had a good effect on improving the structure of gut microbiome. The abundance and diversity of gut microbiome in POF mice could be regulated by EA. According to the results of KEGG pathway analysis, the gut microbiome which related to estrogen signaling pathway, oocyte nutrition and PI3K-AKT signaling pathway were regulated by EA. These findings indicated that EA might play a role in the treatment of POF by regulating the abundance and diversity of gut microbiome.
In the future, antibiotic cocktail mice would be selected to further research the therapeutic mechanism of EA on POF by fecal microbial transplantation and macrogene sequencing.