1 Introduction
Primary dysmenorrhea (PDM) refers to the pain and distention of the lower abdomen before, during, and/or after menstruation caused by non-organic lesions of the reproductive organs. PDM is often accompanied by low back pain or other discomforts, all belonging to the category of “menstrual abdominal pain” (Chai et al., 2020). Epidemiological studies show that primary dysmenorrhea is the most common disease in gynecology, affecting 25–95% of women around the world, with 10% having serious symptoms. This is one of the most common and frequent causes for disruptions in women’s normal work and deterioration of their quality of life (De Sanctis et al., 2015; Nguyen et al., 2015; Sahin et al., 2018). According to TCM, dysmenorrhea is mainly caused by “pain when Qi and Blood are blocked” or “pain when the uterus is not well-nourished.” It is also often caused by damp heat accumulation, kidney qi deficiency, cold coagulation and blood stasis, qi stagnation and blood stasis, and Qi and Blood weakness.
According to modern research, the occurrence of primary dysmenorrhea is mainly related to increased proportion of prostaglandins (PGs), mainly PGF2 a/PGE2, in the endometrium during menstruation (Chan 1983; Maia et al., 2005). PGF2 a promotes contraction of the uterine smooth muscle and vasospasm, which can cause pain due to local ischemia and hypoxia, while PGE2 has the opposite reaction (Ju et al., 2014; Iacovides et al., 2015). In addition, related studies have shown that primary dysmenorrhea is also related to tumor necrosis factor (TNF - α), interleukin (IL), plasma thromboxane B2 (TXB2), 6-keto-PGF1 α, NO, Ca2+, and beta-endorphin (β - EP) (Dawood and Khan-Dawood, 2007a; Pu et al., 2014; Xie et al., 2014; Huang et al., 2016). At present, the treatment of primary dysmenorrhea in Western medicine is not yet satisfactory: non-steroidal anti-inflammatory drugs and oral contraceptives have limited clinical use due to their serious adverse reactions (Oladosu et al., 2018). TCM has many effective methods in the treatment of primary dysmenorrhea and has unique advantages in the improvement of dysmenorrhea symptoms and long-term curative effects (Xu et al., 2019). In the treatment of primary dysmenorrhea, ancient and modern doctors started with blood stasis, which is the main syndrome type. The main treatment methods revolve around promoting blood circulation and removing blood stasis. TCM treatments for promoting blood circulation and removing blood stasis can improve microcirculation, dilate blood vessels, and change hemorheology, all of which are significant and effective ways to relieve pain.
The dried rhizomes of CAS, a ginger plant, are spicy, bitter, and warm in nature. It promotes Qi and breaking blood, eliminating accumulation and relieving pain. It is used for treating syndromes such as lump, blood stasis and amenorrhea, chest arthralgia and heartache, food accumulation, and flatulence. According to the 2020 edition of Chinese Pharmacopoeia (Chinese Pharmacopoeia, 2020), through different processing methods of steaming and vinegar, two processed products of CAS, RCAS, and VCAS, were prepared. Both RCAS and VCAS have the effect of removing blood stasis and pain, but their clinical applications have differences despite their similarity (Liu et al., 2017; Xie et al., 2020). CAS can relieve pain by promoting Qi, breaking blood, and removing blood stasis, and these effects are enhanced after vinegar processing. VCAS has a very high frequency in the clinical application of primary dysmenorrhea. For example, there are 18 prescriptions containing VCAS in the Chinese Pharmacopoeia 2020 edition, which mainly treat blood stasis-related diseases (Hao et al., 2018; Chinese Pharmacopoeia, 2020). In addition, Fufang Ezhusan uses VCAS as the principal medicine for treating dysmenorrhea of blood stasis type. Moreover, clinical and animal studies of formulae containing VCAS in the treatment of primary dysmenorrhea are also more common than those of RCAS (Gu et al., 2018; Hao et al., 2018). However, few reports can be found on the mechanism of enhancing stasis and relieving pain and relieving dysmenorrhea after vinegar processing. As a result, VCAS can enhance the treatment of primary dysmenorrhea which deserves further study.
Ultra-performance liquid chromatography–quadrupole–time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) has the characteristics of high resolution, sensitivity, simplicity, and high-throughput. It has been used to successfully study metabolic changes caused by diseases and drug toxicity (Su et al., 2019). In this study, metabolomics technology based on UHPLC-Q/TOF-MS and multivariate statistics were used to screen out potential biomarkers in blood, urine, and fecal metabolites related to primary dysmenorrhea, analyze related metabolic pathways, and compare the efficacy and mechanism of RCAS and VCAS in the treatment of primary dysmenorrhea.
This study provides a basis for further research on the mechanism of VCAS in the treatment of primary dysmenorrhea. Through the in-depth analysis of dysmenorrhea-related pain factors, related metabolites, and metabolic pathways, it is found that metabolomics can comprehensively demonstrate the impact of the disease on the whole body, and the selected indicators can holistically reflect the state of the disease and provide a new approach for the study of mechanism, screening, or clinical drug treatment of diseases related to blood stasis syndrome.
2 Materials and methods
2.1 Preparation of RCAS and VCAS extracts
The fresh rhizome of CAS was purchased from Hebei Anguo juyaotang Pharmaceutical Co., Ltd. (#1810003), Hebei, China in December 2018. The samples were identified as the rhizome of Curcuma aromatica Salisb. [Zingiberaceae] by Professor Jianwei Cheng at Nanjing University of Chinese Medicine.
RCAS slices: RCAS was steamed for 2 h, cut into 3-mm thickness, and dried in the oven.
VCAS slices: RCAS was boiled with vinegar (per 100 kg samples with 20 kg vinegar and percentile of vinegar is 20% approximately), and dried in the oven at 40°C (Chinese Pharmacopoeia, 2020).
The RCAS and VCAS slices were refluxed for 1.5 h with 90% ethanol 15 times and extracted twice. The residues were extracted for 1.5 h with water 10 times. The aforementioned extracts of RCAS and VCAS were combined. After filtrating and concentrating, per 1 ml RCAS and VCAS extracts containing 1 g of raw medicines were prepared (Xie et al., 2020).
2.2 Chemicals and reagents
LC-MS-grade acetonitrile, LC-MS-grade methyl alcohol, HPLC-grade methanoic acid (Merck. Co. Inc., Darmstadt, Germany), and ultra-pure-grade water were obtained from a Milli-Q system (Millipore, Bedford, MA, United States). The other solvents were of analytical grade.
Epinephrine hydrochloride injection (#10180505) was purchased from Harvest Pharmaceutical Co., Ltd. (Shanghai, China). Estradiol benzoate (#20190301) injection was purchased from Jinke Pharmaceutical Co., Ltd. (Sichuan, China). Oxytocin injection (#190207) was purchased from Hongye Pharmaceutical Co., Ltd. (Anhui, China). Tong Jing Bao Ke Li (#181203) was purchased from Zhongjingwanxi Pharmaceutical Co., Ltd. (Henan, China). The ELISA kits of interleukin-6 (IL-6, #EK306-01), tumor necrosis factor-a (TNF-α, #EK382HS-01), 6-keto-prostaglandin F1a (6-keto-PGF1a, #E-EL-0054c), prostaglandin E2 (PGE2, #E-EL-0034c), prostaglandin F2α (PG F2α, #E-EL-R0795c), thromboxane B2 (TXB2, #E-EL-R0965c), and beta-endorphin (β-EP, #E-EL-R0105c) were purchased from Nanjing Kaiji Technology Co., Ltd., Nanjing, China. The colorimetric assay kits of calcium (Ca2+, #C004-2-1) and nitric oxide (NO, #A013-2-1) were provided by Jiangcheng Bioengineering Institute, Nanjing, China.
2.3 Chemical analysis of RCAS and VCAS extracts
RCAS and VCAS extracts were prepared with a reverse phase-solid phase extract column (RP-SPE) by gradient elution, and the analysis determination performance by UHPLC-Q/TOF-MS coupled with chemometric analysis according to literature and compared the differences in the chemical profiles of RCAS and VCAS (Hao et al., 2018). At the same time, the volatile oil content in RCAS and VCAS was analyzed by GC-MS. However, due to the thermal instability of very few sesquiterpenoids, this part of the research was just a supplement to UHPLC-Q/TOF-MS (Yang et al., 2007). The results of the chemical composition analysis are detailed in the Supplementary Materials.
2.4 Animals and drug administration
A total of 70 specific pathogen-free (SPF) degree female Sprague–Dawley (SD) rats (180 ± 20 g) were obtained from the animal breeding farm of Qinglong Mountain in Jiangning District, Nanjing, China (license approval number: SCXK (SU) 2019-0001). Before the experiment, the rats were fed in an environment-controlled breeding room. A 12-h light/dark cycle was set. Room temperature and relative humidity were regulated at 25 ± 2°C and 60 ± 5%, respectively. In this study, all experimental animals were allowed free access to food and tap water. All rats were adapted for 7 days before the experiment. The study protocol was in accordance with the Guide for the Care and Use of Laboratory Animals and was approved by the Animal Experimental Ethics Committee of the Nanjing University of Chinese Medicine.
A total of 40 rats were randomly divided into five groups with each group having eight rats. Five groups were set as below: the normal control group (NC), model group (M), RCAS, VCAS, and Tong Jingbao group (TJB, a Chinese patent medicine granule), which was the positive control group. TJB, RCAS, and VCAS were intragastrically administered to the TJB group (2.1 g/kg/day), RCAS group (3.8 g/kg/day), and VCAS group (3.8 g/kg/day), respectively, for the last 7 days. The dose of administration was changed according to the clinical equivalent dose of rats. NC and M groups were intragastrically administered normal saline. The models in M, TJB, RCAS, and VCAS groups were established on the 10th day after the dose according to the references: subcutaneous injected 0.1% adrenaline (0.9 mg/kg/d) for 10 days, and 4 h later, the rats were given comprehensive stimulation [A: sound stimulation (60 dB, (10 ± 5) Hz] for 10 min; B: light stimulation [(2 ± 1) Hz) for 10 min; C: binding for 10 min; D: nipping tail for 10 min; E: swimming in ice water (0–4°C) for 5 min]. The models were subcutaneously injected with 0.2% estradiol benzoate for 10 days, given a dose (2.5 mg/kg/d) on the 1st and 10th day, and another dose (1.25 mg/kg/d) from the 2nd to the 9th day. After the last estradiol benzoate injection, 1 h later, the rats were intraperitoneally injected with oxytocin (10 U/kg), while NC groups were intragastrically administered normal saline. After that, all rats were intraperitoneally injected with 1% pentobarbital sodium (50 mg/kg, i.p). Blood samples were collected by the abdominal aortic method.
2.5 Sample collection
The experiment continued for 10 days. At the end of treatment, the rats were euthanized, and blood samples were collected by heparin sodium blood collection tubes. The samples were centrifuged at 3000 r/min for 10 min, and then the supernatants were stored at −80°C for related function detection. Urine and feces samples were collected in the 12 h after drug administration and stored at −80°C for UHPLC-Q/TOF-MS analysis.
2.6 Sample preparation
Before mass spectrometric detection, the urine and feces samples were unfrozen at ambient temperature. A total of 100-mg feces samples were added to 70% chromatographic pure methanol (2 ml). Eight times the volume of methanol was added to the 200-µl plasma or urine sample. The mixed samples were shaken violently for 30 s by a vortex mixer. Then, the mixture was centrifuged at 12,000 r/min at 4°C for 10 min. The supernate was prepared for mass detection.
2.7 Histopathology and writhing response
The uterus of rats was fixed in 10% formalin solution and processed routinely for paraffin embedding. Sections (5 μm thick) were deparaffinized and then stained with hematoxylin and eosin solutions (H&E). Then, the histopathology samples were examined under light microscopy (Leica, DFC-259, and Germany). According to the degree of endometrial hyperplasia, the morphological changes of all rats’ uterus were graded as 0∼ 4: 0 as no pathological phenomenon and 4 as the most serious pathological damage. The specific scoring criteria are as follows: 0: the uterine structure is clear, with a three-layer structure, including the endometrium, myometrium, and adventitia from inside to outside. The endometrial epithelial cells are complete, the distribution of glands in the lamina propria is normal, and the thickness of the endometrium and myometrium is normal. Four points: the uterine structure is disordered, the uterine cavity is not smooth, gland distribution in lamina propria is abnormal, gland hypertrophy, endometrial hyperplasia is obvious, myometrial hyperplasia is obvious, and inflammatory cell infiltration is obvious (Thaina et al., 2009).
On the 10th day of the experiment, after oxytocin was injected into the abdominal cavity of rats in each group, the writhing times and writhing latency of rats within 0–30 min were immediately observed: abdominal concave contraction, trunk and hind limb extension, and hip and one side limb internal rotation. Writhing latency: the time from the beginning of the intraperitoneal injection of oxytocin to the first writhing reaction in rats.
2.8 Biochemical assays
The rat uterine sample was washed with normal saline, dried, and weighed. Then, 10% of the tissue homogenate was prepared with pre-cooled normal saline at a ratio of 1:9 (weight: volume). The homogenate was centrifuged at 3000 rpm for 5 min, and the supernatant was stored at −80°C for HYP detection, which was determined by ELISA or colorimetric assay method. PGF2α, PGE2, β-EP, TXB2, 6-keto-PGF1α, TNF-α, IL-6, NO, and Ca2+ were carried out in perfect accordance with the product’s instructions (Hao et al., 2018).
2.9 Mass spectrum analysis and verification of methodology
The plasma, urine, and feces sample analysis was managed by Shimadzu UPLC (Kyoto, Japan) which consisted of an LC-30AD binary liquid pump, SIL-30SD auto sampler and DGU-20A5R on-line solvent degasser, a CTO-30 A column oven, an AB SCIEX Triple TOF 5600+ system, and an ESI source (Hao et al., 2018). Chromatographic conditions were as follows: Agilent C18 reversed phase column (2.1 mm × 100 mm, 1.8 µm, Palo Alto, CA, United States), mobile phase A (0.1% formic acid aqueous solution)-B (acetonitrile), gradient elution program: 0–1 min, 5–25% B; 1–3 min, 25–30% B; 3–13 min, 30–55% B; 13–15 min, 55–70% B; 15–25 min, 70–100% B; 25–28 min, 5–100% B; flow rate: 0.3 ml/min; column temperature: 35°C; injection volume: 1 µl. Mass spectrometer condition: ESI source and data collection in positive and negative ion mode. The source parameters were set as follows: ion spray voltage floating: +4500/− 4500; decluttering potential: +60/− 60 V; source temperature: 550°C; the atomizing gas is N2, curtain gas: 35 psi; gas1 (nebulizer gas): 55 psi; gas 2 (heater gas): 55 psi; collision energy: +35/− 35e V; using MS/MS secondary mass spectrometry mode: the MS spectrometer ion scanning range was m/z 100–2000. The MS/MS spectrometer ion scanning range was m/z 50–1,000, and dynamic background subtraction was turned on. The quality control (QC) sample was obtained by even mixing of all groups of each kind of sample to ensure system suitability. Before the samples started testing, the QC sample was continuously detected six times. Moreover, system consistency was verified by QC samples after every five detected samples.
2.10 Multivariate statistical analysis
Analyst TF 1.6 software (AB Sciex, Boston, MA, United States) was used to extract the original metabolic fingerprint profiles. MarkerView1.2.1 (AB Sciex, United States) software was used for peak detection and calibration of the original samples of each group. The parameters were set as follows: retention time range: 0.5∼30 min; quality range: 100–1,200 Da; retention time error limit: 0.01 min; mass error limit: 10 ppm; peak intensity threshold: 10; minimum peak width: 25 ppm; noise threshold: 100; retention time error range: 0.5 min. After the original map of each sample was calibrated and normalized by software, they were converted into a three-dimensional matrix containing the peak name (Rt-m/z) sample number and normalized peak area. The three-dimensional matrices of each group were imported into SIMCA-P 14.1 software (Umetrics AB, Sweden). The data were pretreated with Pareto-scaling, followed by PCA and OPLS-DA multivariate statistical analysis. The model predictors are R2X (cum) and Q2X (cum), with R2X (cum) representing the ability of the variable to interpret the model and Q2X (cum) signifying the ability to predict the model. The applicability of the model was good when R2X (cum) and Q2X (cum) both approached 1.0. In the OPLS-DA model, the ions with VIP values greater than 1.0 were identified as potential differential ions. At the same time, t-test single-dimensional statistical analysis was carried out on the three-dimensional matrices among different groups. p < 0.05 was determined as the ion with a significant difference. Potential differential ions with VIP >1.0 and p < 0.05 were confirmed as the final differential ions after taking the intersection, whose structures were to be identified later (Yao et al., 2014).
2.11 Biomarker identification and metabolic pathway analysis
The online database One-Map (http://www.5omics.com/) was used for identification of the differential metabolites of plasma, urine, and feces. Excel data (including sample ID, mass/charge ratio/retention time, and ion strength) of all samples after the original chromatogram dimensionality reduction were imported into the One-MAP online cloud platform, and the differential metabolite search and metabolic pathway analysis were automatically carried out. According to the variable importance for the project (VIP) of the OPLS-DA model in each group, the differential ions were selected. Referring to the literature on metabolomics, this study set VIP > 1 as the differential ions between the two groups with independent sample t-test, excluding data with p > 0.05 (Xing et al., 2017; Su et al., 2019). Finally, the p < 0.05 differential ions were input into the HMDB database, the structure of the compounds according to the first-order mass spectrometry information was predicted, the predicted exogenous compounds were eliminated, and the final selected differential compounds for structure identification were obtained, which are compared with the HMDB database (http://www.hmdb.ca/), METLIN database (https://www.sisweb.com/software/ms/wiley-metlin.htm), and KEGG database (http://www.genome.jp/kegg/). The standard secondary mass spectrometry data and related literature were compared, and finally the endogenous differential metabolites were identified and the related metabolic pathways were analyzed.
4 Discussion
The endometrium is an important part of prostaglandin synthesis. It is generally believed that the occurrence of primary dysmenorrhea is mainly related to the increase of prostaglandin synthesis and release in the endometrium during menstruation. Prostaglandin (PG) can be converted into different pGs in the body, such as PGF2 a (PGF2 α), PGE2 (PGE2), and TXA2 (thromboxane A2) (Guimarães and Povoa, 2020). PGF2 a stimulates uterine contraction, increases uterine tension, and decreases blood flow; on the contrary, PGE2 inhibits uterine contraction, inhibits the spontaneous activity of smooth muscle, and relaxes the uterus (Ruoff and Lema, 2003; Harel, 2004). Therefore, the concentration of PGF2 a/PGE2 in the endometrium and blood of dysmenorrhea patients 48 h before menstruation was significantly higher than that of normal people: the more severe the dysmenorrhea, the higher the level of PGF2 a (Richter et al., 2006; Dawood and Khan-Dawood, 2007a). In order to reduce the contractility of uterine smooth muscle and achieve the purpose of treating primary dysmenorrhea, it is necessary to inhibit the content of PGF2 a and increase the content of PGE2 (Dawood and Khan-Dawood, 2007b; Shi et al., 2011).
Thromboxane B 2 (TXB 2) and 6-keto-prostaglandin F 1 a (6-Keto-PGF 1 α) are relatively stable products transformed from two unstable bioactive substances, thromboxane A 2 (TXA 2) and prostacyclin I 2 (PGI 2) (Dawood and Khan-Dawood, 2007b). According to relevant research, TXA 2 is one of the most powerful vasoconstrictors reported in vivo, which can enhance platelet activation and aggregation, promote thrombosis, and cause Ca2+ influx, thus inducing dysmenorrhea (Das, 2008). Prostacyclin (PGI2) is mainly produced in vascular endothelial cells. By stimulating adenylate cyclooxygenase to increase the level of platelet endogenous camp, it can inhibit platelet aggregation and release induced by ADP, collagen, and AA (Kido et al., 2007), expand blood vessels, prevent platelet aggregation and thrombosis in local blood vessels, relieve dysmenorrhea symptoms, reduce the level of TXB 2 in plasma, increase the content of 6-k-PGF1 α, and reduce the ratio of TXB 2/6-k-PGF1 α so as to inhibit platelet aggregation, expand blood vessels, improve microcirculation, and restore the blood supply of the uterus by activating blood circulation and removing blood stasis so as to relieve dysmenorrhea (Shi et al., 2011).
The expression of TNF-a mRNA, protein, and its receptor in human endometrial decidua and trophoblast cells is regulated by estrogen and progesterone (Fluhr et al., 2007). TNF-a can inhibit endometrial hyperplasia, induce apoptosis, cause the loss of cadherin catenin actin complex, and promote the injury of endothelial cells. TNF-α, IL-6, and IL-4 can stimulate the AA metabolism of chorionic villi, the latter producing prostaglandins, thromboxane, and leukotrienes. The results showed that the serum levels of IL-6 and IL-10 in dysmenorrhea women were significantly higher than those in non-dysmenorrhea women (Huang et al., 2016; Szebeni et al., 2019). NO is a vasoactive substance, which is the main component of the endothelium relaxing factor. It can dilate blood vessels, and it can reduce thrombosis by inhibiting platelet adhesion, aggregation, and TXB2 release. When the synthesis and release of endogenous NO decrease, the NO-mediated biological information transmission appears abnormal, and the regulation of vasoactive substances is maladjusted, which leads to dysfunction of endothelin function, thus causing pain. As a result, the blood supply of ischemic tissue is gradually restored, and the degree of pain is reduced (Jia et al., 2016).
According to modern medical research, the formation of primary dysmenorrhea may be related to the ischemia–reperfusion injury of the uterine muscle, which causes intracellular Ca2+ overload. On the one hand, it will aggravate the contraction of blood vessels and the myometrium and cause the blood supply of the endometrium to be insufficient; on the other hand, it will cause the imbalance of nuclear Ca2+ homeostasis, which will lead to depletion of cell energy and damage of the cell membrane, thus causing the production of dysmenorrhea. It can reduce the Ca2+ level in uterine smooth muscle cells and relax uterine smooth muscle (Hsia et al., 2008; Thaina et al., 2009; Xiong et al., 2019). β - EP is a class of neuropeptides with morphine-like activity, which is in the endometrial stroma, glandular epithelium, and muscle nerve fibers. It has an endogenous analgesic effect and participates in the regulation of reproductive endocrine. The content of β - EP in peripheral blood of patients with primary dysmenorrhea is significantly higher than that of the NC group (Jia et al., 2006).
VCAS can reduce the ratio of PGF2 a/PGE2, TXB2/6-k-PGF1 α, TNF-α, and IL-6 levels, promote the synthesis and release of endogenous NO, reduce the level of Ca2+ in uterine smooth muscle cells so as to inhibit platelet aggregation, dilate blood vessels, improve microcirculation, and restore the blood supply of the uterus by activating blood circulation and removing blood stasis so as to relieve dysmenorrhea. The effect of RCAS on PGF2 α, TNF-α, NO, Ca2+, and β - EP was not obvious, so the therapeutic effect of RCAS on dysmenorrhea was weaker than that of VCAS.
The results of plasma metabolomics showed that the improvement of dysmenorrhea caused by blood stasis was mainly related to the metabolism of glycerin phospholipid, phenylalanine, glutathione, and tryptophan. The results of urine metabolomics showed that the improvement of dysmenorrhea caused by blood stasis was mainly related to the metabolism of glycerophosphatide, phenylalanine, steroid hormone, cysteine and methionine, arginine, and proline. The results of fecal metabolomics showed that the improvement of dysmenorrhea in blood stasis syndrome was mainly related to the glycerin phospholipid metabolic pathway, phenylalanine metabolic pathway, tyrosine metabolic pathway, and arachidonic acid metabolic pathway.
LysoPCs are important metabolites of lipid metabolism and play an important role in dyslipidemia, diabetes, cancer, atherosclerosis, inflammation, and other diseases (Jackson et al., 2008; Jiang et al., 2013). LysoPC can inhibit transcription activity, tissue factor and NF-κB during coagulation and participate in thrombosis by regulating the expression of tissue factors (Engelmann et al., 1999). Lysophosphatidic acid lysoPA (16:0/0:0) can promote the expression of cyclooxygenase-2 (Guimarães and Povoa, 2020), and cyclooxygenase-2 is the key enzyme for the conversion of arachidonic acid to prostaglandins. Compared with the NC group, the contents of lysoPCs and lysoPA (16:0/0:0) in the M group were significantly increased. After the intervention, the RCAS and VCAS had a downregulation effect on the glycerophosphatidylcholine metabolites, especially the VCAS. The results showed that RCAS and VCAS could reduce the glycerol phospholipid metabolites and inhibit cyclooxygenase-2 by regulating the glycerophosphatidic metabolic pathway. The effect of VCAS on dysmenorrhea was more significant.
Arachidonic acid is a type of polyunsaturated fatty acid, which can mediate the production of inflammatory mediators such as TNF-a and IL-1, PGD2, TXA2, and leukotriene, which can induce platelet aggregation, promote blood coagulation, smooth muscle contraction, leukocyte chemotaxis, production of inflammatory cytokines, and immune function (Ma et al., 2013; Burns et al., 2018). The changes of PGG2 and PGD2 metabolites are closely related to the occurrence of primary dysmenorrhea. PGE2 produced and released by the endometrium can inhibit the spontaneous activity of uterine smooth muscle, mediate the cAMP signal molecular transduction pathway, and regulate some neurotransmitters, thus leading to downstream pathway reactions (including reproduction and inflammation) (Iacovides et al., 2015; Guimarães and Povoa, 2020). 12 (s)—HETE is one of the six monohydroperoxy fatty acids produced by non-enzymatic oxidation of arachidonic acid (leukotriene), which produces more stable hydroxyl fatty acid (+/−) 12-HETE through reduction reaction and then participates in host defense reaction and pathophysiological conditions, such as allergy and inflammation. Compared with the endogenous metabolite table, the contents of arachidonic acid, PGE2, PGD2 and 12 (s)—HETE in the M group were significantly higher than those in the NC group. After the intervention of VCAS, the contents of arachidonic acid, PGE2, PGD2 and 12 (s)—HETE in the M group were significantly higher than those in the NC group. Therefore, it is speculated that the pro-inflammatory effect of arachidonic acid may be a contributing factor to the formation of blood stasis syndrome. The arachidonic acid metabolic pathway is one of the important pathways for VCAS to remove blood stasis and relieve pain.
Through UHPLC-Q/TOF-MS and multivariate statistical pattern discrimination, the results showed that normal rats and model rats were discernibly divided into two groups. They exhibited clustering state, which indicated the successful replication of PDM rats from the perspective of metabolomics. In addition, uterine histopathology, the pain factors PGF2 a/PGE2 and TXB2 related to dysmenorrhea/6-keto-PGF1 α, IL-6, TNF - α, NO, Ca2+, β - EP, and other pharmacodynamic indexes changed before and after the establishment of the model. After RCAS and VCAS intervention, the uterine tissue morphology of dysmenorrhea model rats was improved, and gland hypertrophy and myometrial hyperplasia were reduced as well as neutrophil content. By regulating pain-related factors, the metabolism of glycerophospholipid, glutathione, steroid hormone biosynthesis, cysteine and arginine, and arachidonic acid was regulated, while the spasm of the uterine smooth muscles was relieved.