Dysmenorrhea refers to a kind of uterine cramping pain that occurs in women before or after the menstrual period (Shirvani et al., 2015). In recent years, with the increasement of social pressure, the incidence rate of dysmenorrhea is gradually growing. According to epidemiological statistics, about 50% of women suffered from dysmenorrhea, 10% of which had endured more severe menstrual cramps. This kind of pain not only affects individual’s quality of life, but also lower the level of happiness (Lefebvre et al., 2005). The treatment of dysmenorrhea in western medicine is mainly the application of non-steroidal anti-inflammatory drugs, which could merely provide temporary pain relief instead of fundamental treatment and additionally cause drug resistance, gastrointestinal adverse as well as liver and kidney damage. On the contrast, traditional Chinese medication focus on curing dysmenorrhea itself, where patients are diagnosed and treated based on the overall analysis of the disease and physical condition (Xiu-Hong et al., 2015).

Generally, dysmenorrhea could be categorized as primary dysmenorrhea and secondary dysmenorrhea, in which the primary one accounts for more than 90% (Meirong et al., 2020). Caused by excessive pathological uterine contraction, primary dysmenorrhea mainly occurs in women during their teenage years without any organic pelvic lesions, which pathogenesis is not clear. Syndrome of cold coagulation and blood stasis is one of the most common clinical classification of it. There are a variety of methods to establish the cold coagulation syndrome dysmenorrhea model: whole body freezing method (Shujing et al., 2018; Shujing et al., 2020), local frostbite method (Wang and Feng, 2000), wind cold environment method (Tang et al., 2000), ice water immersion method (Cheng et al., 2005), ice water immersion joint drug method (Yan et al., 2014; Xihong et al., 2017; Meng, 2018) and surgery plus drug method (Lu et al., 2010). However, the ice-water immersion method uses short-term overstimulation to build the model, and the local frostbite method ignores the overall concept, which both cannot fit well with the disease. Therefore, the cold coagulation dysmenorrhea model used in this experiment is established on the basis of the traditional dysmenorrhea model using the method of whole body freezing. This model comes from the dysmenorrhea animal model preparation specifications issued by the Chinese Society of Traditional Chinese Medicine and the Professional Committee of Experimental Pharmacology of Traditional Chinese Medicine in 2018 (medicine and Medicine., 2018). It conforms to the modeling ideas and characteristics of the TCM syndrome model and has been adopted by a large number of researchers.

Guizhi Fuling Capsules evolved from Guizhi Fuling Pills in “The Synopsis of the Golden Chamber” by Zhang Zhongjing in the Eastern Han Dynasty (Yunxi et al., 2016). It is mainly composed of Chinese medicinal materials such as Guizhi, Poria, White Peony, Peony Bark, and Peach Kernel. Jiangsu Kangyuan Pharmaceutical Co., Ltd. (Nanjing, Jiangsu, China) has continuously improves the production process of Guizhi Fuling Capsules, and now its production of Guizhi Fuling Capsules has successfully overcomes the unstable characteristics of traditional Chinese medicine preparations (Qingli et al., 2019). Guizhi Fuling Capsules have a variety of pharmacological effects such as anti-inflammatory, analgesic, anti-tumor, smooth muscle regulation, endocrine regulation and immunity enhancement. It is mainly used for gynecological indications, such as the treatment of dysmenorrhea, uterine fibroids, dysfunctional uterine bleeding, menorrhagia, amenorrhea, endometriosis, and some symptoms related to ovarian cysts (Yan et al., 2014). Meanwhile, studies have shown that the extract of Guizhi Fuling Capsules can inhibit the activity of protein tyrosine kinases and exert anti-tumor effects (Lianhua et al., 2012). Guizhi Fuling Capsules has definite curative effect, less side effects and adverse reactions, and is the preferred drug for the treatment of gynecological related diseases. However, the main active ingredients of Guizhi Fuling Capsules for anti-dysmenorrhea, the metabolic pathways involved and the target of the drug effect are not clear. Due to the complexity of the traditional Chinese medicine compound, it has not carried out in-depth modern pharmacology and the mechanism of action.

The objects of metabolomics research are small molecular compounds in biological samples, systems, tissues or cells in the body. Metabolomics is an emerging discipline that uses modern analytical methods such as chromatography and mass spectrometry to determine qualitative/quantitative information of endogenous compounds. The changes from these endogenous substances can reflect the interaction between the inside and outside of the organ or system, which is to reflect the physiological changes in a certain pathological process as a whole. In recent years, with the advancement of analytical methods such as HPLC and GC-MS, metabolomics research has developed rapidly, especially in the interpretation of traditional Chinese medicine prescriptions, pharmacological effects of traditional Chinese medicine and pharmacological effects of active ingredients in natural medicine. Metabolomics is also an important method on the road of Chinese medicine going abroad and being recognized and accepted by the world. Metabolomics can reflect the changes of small molecule metabolites in the body at an overall level. Therefore, this research will explore the possible mechanism of Guizhi Fuling Capsules’s anti-dysmenorrhea effect by using metabolomics technology.

We choose female rats to establish primary dysmenorrhea model, because female rats have a short estrus cycle, leading them suitable to study the changes in the reproductive cycle (Marcondes et al., 2002). Then, dysmenorrhea itself is a physiological phenomenon that mostly occurs within a certain age group of human beings, especially primary dysmenorrhea, which is caused by non-pelvic organic diseases. Primary dysmenorrhea is mostly caused by mental factors, and most of them occur in menarche. The average age at which Eastern women experience menarche is 12–16 years old. The three-month-old rat is equivalent to a human being 12–14 years old. Therefore, we finally chose three-month-old female rats as our model animals.

The model of experimental dysmenorrhea could be established by using estradiol benzoate and oxytocin, in order to synchronize the uterine cycle and cause artificial estrus, animals were given estrogen for 10 consecutive days to increase uterine sensitivity. After artificial estrus, oxytocin was injected to the animals to induce contraction of uterine smooth muscle and cause pain, which was manifested as writhing response. Writhing response is the most direct expression of dysmenorrhea in rats, so it is the most widely used behavioral index for the overall evaluation of visceral pain response. Studies have shown that the occurrence of dysmenorrhea is mainly related to the abnormal synthesis and release of prostaglandins in the endometrium of the menstrual period. The increased ratio of PGF2α/PGE2 leads to enhanced spastic contraction of uterine smooth muscle and dysmenorrhea (Yoshino and Ellis, 1987). PGE2 is one of the most important endogenous substances for inhibiting the inflammatory response. It has the effects of dilating blood vessels, increasing blood flow to organs throughout the body, reducing peripheral resistance of blood vessels, immunosuppression and anti-inflammatory. In the pharmacodynamics experiment, PGF2α/PGE2 in the serum of the model group rats was significantly increased, and significantly decreased after administration. β-EP has a powerful endogenous analgesic effect. It is affected by sex hormones, and participates in the regulation of reproductive endocrine. In the rat pharmacodynamics experiment, β-EP and PGE2 showed the same trend. Compared with the control group, the concentration of β-EP in the serum of the model group was significantly reduced; compared with the model group, that of the Guizhi Fuling Capsules group was significantly higher. This is the same as the results of previous studies (Sun et al., 2014).

The uterine tissue samples of dysmenorrhea rats were analyzed by metabolomics. It was found that many fatty acids had significant changes in the dysmenorrhea model, but Guizhi Fuling Capsules can reverse this change to varying degrees after intervention. Among them, arachidonic acid metabolic pathway is in a pivotal position. Arachidonic acid has many metabolic pathways, one of which is to generate prostaglandin and thromboxane through cyclooxygenase metabolism (Wang et al., 2014). Prostaglandin endoperoxidase (PTGS), also known as cyclooxygenase (COX), is the key enzyme that regulates the release of prostaglandins, including two isoforms: structural cyclooxygenase-1 (COX-1) and inducible cyclooxygenase-2 (COX-2), in which COX-2 is an inducible form only existing in inflammation. After being stimulated by inflammatory mediators, endotoxin, hypoxia factor, epidermal growth factor (EGF), COX-2 gene is induced to be highly expressed, resulting in the increase of PGI2, PGE1, PGE2 content, which could participate in inflammatory reaction. This is considered as the main source of inflammatory prostaglandins (Mitchell and Kirkby, 2019). In dysmenorrhea model rats, the expression of COX-2 in liver and uterus of rats increased significantly, and the expression of mRNA in rats from Guizhi Fuling group decreased significantly compared with the model group (Figure 10). The results suggested that Guizhi Fuling Capsules could reduce the production of prostaglandin by regulating the expression of COX-2.

Moreover, docosahexaenoic acid (DHA) is a type of n-3 unsaturated fatty acid. Recent studies have shown that when metabolized by cyclooxygenase and lipoxygenase, DHA is transformed into powerful anti-inflammatory molecules, and DHA may produce anti-inflammatory effects by inhibiting the arachidonic acid cascade (Tokuyama and Nakamoto, 2011). Lactic acid is closely related to the metabolism and polarization of macrophages. Lactic acid is a regulator of macrophage metabolism and can prevent excessive inflammation. Studies have revealed a new connection between lactic acid and the control of uterine inflammation, and found that high levels of lactic acid act on uterine GPR81 and down-regulate pro-inflammatory genes (Zhou et al., 2021). Taurine is an endogenous anti-injury substance in humans. Studies have shown that taurine has a protective effect on nonylphenol-induced uterine pathological damage in mice (Ding et al., 2018).

In summary, we speculate that Guizhi Fuling Capsules may partially reduce the synthesis of arachidonic acid through the arachidonic acid metabolic pathway, and regulate the expression of COX-2 to reduce the production of downstream prostaglandins, thereby reducing the production of inflammatory mediators release to achieve the purpose of alleviating dysmenorrhea.

In a word, we have proved that Guizhi Fuling Capsules has a good effect on cold coagulation dysmenorrhea model rats, can significantly reduce the number of writhing, regulate the level of serum PGE2, PGF2α, β-EP, improve endometrial hyperplasia and ovarian function. The uterine tissue samples of dysmenorrhea rats were analyzed by metabolomics. It was found that Guizhi Fuling Capsules could reduce the interference of estradiol benzoate and oxytocin on the metabolism products of rats. Guizhi Fuling Capsules mainly played an anti-dysmenorrhea role by regulating the metabolism of arachidonic acid. More results suggest that Guizhi Fuling Capsules may inhibit the conversion of arachidonic acid into prostaglandins by down-regulating the expression of COX-2. Further experiments are needed to study the effect of Guizhi Fuling Capsules on other substances in arachidonic acid metabolism pathway.