Combined Auricular Electrostimulation and Sunshi Dingtong Decoction Relieve Primary Dysmenorrhea With Qi Stagnation and Blood Stasis in Rats

Summary

Transcutaneous auricular electrostimulation combined with Sunshi Dingtong decoction alleviated uterine pathological injury and inflammation-related changes in a rat model of primary dysmenorrhea with qi stagnation and blood stasis, accompanied by altered mRNA expression of PPAR-γ2 and COX-2.

Abstract

Primary dysmenorrhea with a qi stagnation and blood stasis pattern is associated with uterine inflammation and pain, and current pharmacologic therapies may cause adverse effects. This study evaluated the combined therapeutic effects of transcutaneous auricular point electrostimulation (TEAP) and Sunshi Dingtong decoction (SDD) in a rat model of qi stagnation and blood stasis primary dysmenorrhea. Thirty-six female Sprague–Dawley rats were randomly allocated to the blank, model, SDD, TEAP, TEAP plus SDD, and ibuprofen groups. The model was induced using estradiol benzoate sensitization, epinephrine hydrochloride combined stress stimulation, and oxytocin administration. Rats received normal saline, SDD, ibuprofen, or TEAP at defined auricular points for seven days. Uterine morphology was examined by hematoxylin and eosin staining. Uterine levels of IL-1β, IL-6, TNF-α, PGF2α, and PGE2 were measured by ELISA, and uterine PPAR-γ2 and COX-2 mRNA expression was quantified by real-time PCR. Model rats showed uterine epithelial degeneration, neutrophil infiltration, increased IL-1β, IL-6, TNF-α, PGF2α, and COX-2 mRNA expression, and decreased PGE2 and PPAR-γ2 mRNA expression. TEAP or SDD alone partially reversed these pathological and molecular changes, whereas combined TEAP plus SDD produced more pronounced improvements, with effects comparable to those observed in the ibuprofen group. These findings suggest that TEAP combined with SDD alleviates uterine pathological injury and inflammation-related changes in qi stagnation and blood stasis primary dysmenorrhea (QSBSPD) rats, and that these effects are associated with the regulation of PPAR-γ2 and COX-2 mRNA expression.

Introduction

Primary dysmenorrhea (PD), alternatively termed functional menstrual pain, is characterized by the occurrence of dysmenorrhea without any identifiable organic pathology within reproductive organs¹. Female patients experiencing severe dysmenorrhea frequently find themselves unable to perform daily tasks, which significantly impacts their quality of life and necessitates pharmacological intervention for symptom relief². Extant literature indicates that it is associated with various factors, including prostaglandins (PGs), estrogen, progesterone, vasopressin, oxytocin (OT), the immune system, neuroendocrine effects, and alterations in microelement calcium levels³. Simultaneously, inflammatory factors, such as tumor necrosis factor (TNF) and interleukin-6 (IL-6), can induce the synthesis or secretion of PGs. This process leads to the hypercontraction of uterine muscles, subsequently resulting in ischemic pain and PD⁴. Currently, the management of PD in contemporary medicine is primarily categorized into general treatment, pharmacological treatment, and surgical intervention. Among the pharmacological options, non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen are frequently utilized. Nevertheless, the use of NSAIDs is correlated with significant gastrointestinal side effects, and prolonged or heavy use can result in complications such as peptic ulcers and upper gastrointestinal bleeding⁵. Thus, identifying and developing safer and more efficacious strategies for PD treatment are imperative and necessary.

Increasing evidence suggests that primary dysmenorrhea is frequently associated with an excess pattern in traditional Chinese medicine, among which qi stagnation and blood stasis are commonly observed clinically⁶. In this pattern, impaired qi movement and blood circulation are considered to contribute to uterine stasis, excessive uterine contraction, and pain. Therapeutic strategies that regulate qi, activate blood circulation, warm the meridians, and relieve pain are therefore commonly used for this condition. Auricular point therapy is an important non-pharmacological intervention in traditional Chinese medicine. It is simple to apply and has been used to relieve pain by modulating neuroendocrine and endogenous analgesic pathways⁷. Sunshi Dingtong decoction (SDD), a clinical experience-based formula developed by the Gynecology Department of Fuzhou Hospital of Traditional Chinese Medicine, has been used for dysmenorrhea characterized by qi stagnation and blood stasis. The formula is based on the therapeutic principle of regulating qi and blood, promoting blood circulation, warming the meridians, and relieving menstrual pain. SDD contains Shixiao San, which consists of Pollen typhae (Puhuang) and Faeces Trogopterori (Wulingzhi), together with Rhizoma Cyperi (Xiangfu), Myrrh (Moyao), Ramulus cinnamomi (Guizhi), Radix et Rhizoma Clematidis Chinensis (Weilingxian), Herba Salivae Chinensis (Shijianchuan), Rhizoma Corydalis Yanhusuo (Yanhusuo), Radix Angelicae Sinensis (Danggui), Herba Artemisiae Anomalae (Liujinu), and Fructus foeniculi (Xiaohuixiang). However, the experimental efficacy of SDD, particularly when combined with transcutaneous auricular point electrostimulation, has not been systematically evaluated in a controlled animal model of qi stagnation and blood stasis primary dysmenorrhea.

Peroxisome proliferator-activated receptors (PPARs), belonging to the nuclear hormone receptor family, are ligand-activated receptors integral to the regulation of numerous intracellular metabolic processes. There are three distinct subtypes of PPARs, known as PPAR-α, PPAR-β/δ, and PPAR-γ⁸. Recent findings indicate that PPAR-γ2 is expressed across a diverse range of tissues and exhibits a significant association with inflammatory processes⁹. According to the report, PPAR-γ suppresses the expression of IL-6, cyclooxygenase-2 (COX-2), endothelin-1, nitric oxide synthase, matrix metalloproteinase-9, gelatinase, and adhesion molecules through inactivating nuclear factor κB, activated protein-1, and signal transducer and activator of transcription signaling pathways, thereby inhibiting the inflammatory response¹⁰. In addition, Huang et al.¹¹ indicate that COX-2 and PPAR-γ2 may serve as critical targets implicated in the pathophysiology of qi stagnation and blood stasis (QSBS) disorders.

Therefore, this study investigated the therapeutic effects of transcutaneous auricular point electrostimulation (TEAP), SDD, and their combined application in a rat model of qi stagnation and blood stasis primary dysmenorrhea. By integrating uterine histopathology, inflammatory mediator profiling, prostaglandin measurement, and PPAR-γ2/COX-2-related mRNA analysis, this study aimed to evaluate whether combined internal and external traditional Chinese medicine-based therapy could provide enhanced protection against dysmenorrhea-associated uterine inflammation. The novelty of this work lies in the experimental assessment of TEAP combined with a clinical experience-based SDD formula in a controlled qi stagnation and blood stasis primary dysmenorrhea (QSBSPD) model and in the exploration of its potential association with the PPAR-γ2/COX-2 inflammatory regulatory axis.

Protocol

Ethical statement

All animal procedures were reviewed and approved by Fujian Ambry Biotechnology Laboratory Animal Ethics Committee approval number:IACUC  FJABR2024031202). Perform all animal experiments in accordance with the approved institutional animal care and use protocol.

SDD and ibuprofen preparation

SDD consists of Shixiao San (9 g), Xiangfu (9 g), Moyao (6 g), Guizhi (9 g), Weilingxian (12 g), Shijianchuan (20 g), Yanhusuo (12 g), Danggui (9 g), Liujinu (15 g), and Xiaohuixiang (5 g). Shixiao San consists of Puhuang (4.5 g) and Wulingzhi (4.5 g). All herbal components were used as concentrated granules and are listed in the Table of Materials. Prepare SDD by dissolving 106 g of concentrated granules in 200 mL of boiling water to obtain a final concentration of 0.53 g/mL crude drug equivalent. Mix thoroughly until the granules are completely dissolved, allow the solution to cool to room temperature, and administer it by intragastric gavage at 2.5 mL/kg per administration. Prepare the ibuprofen suspension according to the required concentration for intragastric gavage. Administer ibuprofen at 0.02 g/kg as the positive control treatment.

Animals and grouping

Use 36 healthy adult female Sprague-Dawley rats of specific pathogen-free grade, aged 2–3 months and weighing 200 ± 20 g, with no history of pregnancy or mating. Maintain the rats under a 12 h/12 h light/dark cycle at 22 ± 2 °C and 50%–70% relative humidity, with free access to standard chow and drinking water. After 1 week of acclimatization, randomly assign the rats into six groups (n = 6 per group): blank, model, SDD, TEAP, SDD + TEAP, and ibuprofen groups.

QSBSPD model preparation and administration

In this experiment, the widely accepted model of OT-induced dysmenorrhea was employed. In brief, rat estrous cycles were assessed through vaginal smears. Rats were deemed eligible for inclusion in the study if their estrous cycle duration was determined to be 4–5 days.

With the exception of the blank group, the rats in the experimental groups were administered subcutaneous injection of estradiol benzoate and epinephrine hydrochloride daily for 10 days. After injection of epinephrine hydrochloride at a dosage of 0.9 mg/kg daily, the rats were exposed to various combined stimulation (A: Acoustic stimulation: rats were exposed to acoustic stimuli (90 dB, 1 kHz) for 5 min/time, 3–4 times daily, with a 2-h interval between each time. B: Light stimulation: rats were exposed to flashing light stimuli for 5 min/time, 3–4 times daily, with a 2-h interval between each time. C: Electrical stimulation: rats were subjected to electrical stimulation (biphasic square-wave pulses) at 30–35 V, with alternating current applied for 0.3 s every 2 s. This stimulation was administered for 5 min every 2 h, 2–4 times daily. D: Restraint: rats were placed in a restraint cage constructed from barbed wire, measuring approximately 25 cm in length and 5 cm in diameter. Once inside the cage, the ends were clamped to immobilize the rats and restrict their movement for 2 h, once per day). In addition, on days 1 and 10, the rats were subcutaneously injected with 5.0 mg/kg/day of estradiol benzoate, and on days 2–9, they received 2.5 mg/kg/day.

Starting from the 5th day of estradiol benzoate injection, the rats received intragastric administration of different drugs for 7 days, twice daily at 10:00 a.m. and 4:00 p.m., except the ibuprofen group, which received once daily at 10:00 a.m. In the blank, model, and TEAP groups, the rats were administered 2.5 mL/kg of normal saline. In SDD and TEAP+SDD groups, the rats received 2.5 mL/kg of SDD (prepared as 106 g of crude drug dissolved in 200 mL of water, 0.53 g/mL); this dose was converted from the adult clinical daily dose of 106 g based on body surface area equivalence, resulting in a rat-equivalent dose. In the ibuprofen group, the rats received 0.02 g/kg of ibuprofen suspension. In TEAP and TEAP+SDD groups, the rats received TEAP stimulation starting from the 5th day following estradiol benzoate injection. Attach the electrodes to the bilateral Jiaogan, Shenmen, Neishengzhiqi, and Neifenmi auricular points, as defined according to a previously reported standard for rat acupoint localization¹². These points are situated in the bilateral ear conchal cavity, innervated by the vagus nerve. The electrodes were connected to an electronic needle therapy instrument, delivering a continuous wave for 20 min (frequency: 20 Hz; intensity: 1 mA), once per day. The TEAP+SDD group rats underwent TEAP after the first SDD gavage every day. The final TEAP stimulation was conducted prior to the intraperitoneal injection of OT.

At 24 h after the last estradiol benzoate injection, that is, on the 11th day, 1 h after the last intragastric administration, except for the blank and TEAP groups, the rats in other groups received 2 U of OT per rat with intraperitoneal injection. The rats in the TEAP group underwent intraperitoneal injection of OT at a dosage of 2 U per rat after the final TEAP stimulation. The rats in the blank group did not receive OT injection. This protocol successfully established a rat model of QSBSPD. The overall animal selection, model preparation, drug administration schedule, TEAP stimulation parameters, and auricular point locations are summarized in Figure 1.

Wright-Giemsa staining

The estrous cycle was determined by Wright-Giemsa staining of vaginal smears. A sterile cotton swab was moistened with 0.9% sodium chloride solution and gently inserted 0.5–1.0 cm into the vaginal canal. The swab was rotated 1–2 times to collect vaginal exfoliated cells, and the sample was evenly smeared onto a clean glass slide in one direction. The smear was air-dried at room temperature. Wright-Giemsa staining solution was added to cover the smear and incubated for 5 min. An equal volume of phosphate-buffered saline was then added to the staining solution, and staining was continued for another 5 min. The slide was gently rinsed three times with ultrapure water, air-dried, and examined under a light microscope. The stage of the estrous cycle was identified according to the dominant cell types, including nucleated epithelial cells, cornified epithelial cells, and leukocytes.

Sample collection

Rats were placed in an anesthesia induction box with isoflurane concentration set at 3% to 5%. After the rats lost consciousness, they were transferred to the operating table and maintained under anesthesia with a mask on. The concentration was reduced to 1.5% to 2.5%. The abdominal fur of the rat was trimmed using scissors and subsequently disinfected with a topical application of alcohol. An incision of 2–3 cm was made along the median abdominal line. One side of the uterus, along with its ligaments, was exteriorized, and the adipose tissue adhering to the uterine surface was excised. The uterine tissue was then carefully isolated. If the rat is still alive after sampling, euthanize it using CO₂ asphyxiation.

Hematoxylin and eosin (HE) staining

Uterine tissues were fixed, embedded in paraffin, and sectioned at a thickness of 5 µm. The sections were dewaxed in xylene twice for 10 min each. The sections were then rehydrated sequentially in absolute ethanol twice for 5 min each, followed by 95%, 90%, 80%, and 70% ethanol for 5 min each. The sections were rinsed in ultrapure water. Hematoxylin staining was performed for 5 min, and the sections were rinsed with water until no excess dye remained. The sections were immersed in phosphate-buffered saline for 5 min for bluing. The sections were then stained with eosin for 1 min and rinsed with water. Subsequently, the sections were dehydrated in 95% ethanol for 10 min and absolute ethanol for 10 min, cleared in xylene for 10 min, and mounted with neutral resin. Representative images were acquired using a light microscope or digital slide scanner.

Enzyme-linked immunosorbent assay (ELISA)

Fresh uterine tissues were rinsed with pre-cooled phosphate-buffered saline to remove residual blood. Each tissue sample was weighed, cut into small pieces, and homogenized in pre-cooled phosphate-buffered saline at a tissue-to-buffer ratio of 1:9. The homogenate was centrifuged at 5000 × g for 5–10 min at 4 °C, and the supernatant was collected for subsequent analysis. Levels of IL-1β, IL-6, TNF-α, PGF2α, and PGE2 were measured using rat ELISA kits according to the manufacturer’s instructions. All reagents and microplate strips were equilibrated to room temperature before use. Standards or samples (50 µL) were added to the designated wells. Horseradish peroxidase-conjugated detection antibody (100 µL) was added to each well except the blank wells, and the plate was sealed and incubated at 37 °C for 60 min. The liquid was discarded, and each well was washed five times with diluted washing buffer. Subsequently, 50 µL of substrate A and 50 µL of substrate B were added to each well, followed by incubation at 37 °C in the dark for 15 min. Stop solution (50 µL) was then added to each well, and the optical density was measured at 450 nm within 15 min. Sample concentrations were calculated from the standard curves.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from uterine tissues using a total RNA extraction reagent according to the manufacturer’s instructions. Briefly, the tissue was ground in liquid nitrogen, and 1 mL of RNA extraction reagent was added. The lysate was incubated at room temperature for 5 min. Chloroform (200 µL) was then added, and the mixture was shaken vigorously for 15 s, incubated at room temperature for 5 min, and centrifuged at 12,000 × g for 12 min at 4 °C. The aqueous phase was transferred to a new tube, mixed gently with an equal volume of isopropanol, and incubated at −20 °C for 15 min. The samples were centrifuged at 12,000 × g for 10 min at 4 °C, the supernatant was discarded, and the RNA pellet was washed with 75% ethanol. The pellet was air-dried and dissolved in RNase-free water. RNA concentration and purity were determined using a microvolume spectrophotometer. Genomic DNA was removed, and cDNA was synthesized according to the reverse transcription reagent instructions. For genomic DNA removal, total RNA was incubated with the genomic DNA removal reagent at 42 °C for 5 min and then placed on ice. Reverse transcription was performed at 50 °C for 15 min, followed by 75 °C for 5 min to terminate the reaction.

Real-time PCR was performed using a SYBR Green-based qPCR mix. Each 20 µL reaction contained 2 µL of cDNA, 0.4 µL of forward primer, 0.4 µL of reverse primer, 10 µL of 2× SYBR qPCR mix, and 7.2 µL of nuclease-free water. The qPCR was run using the following cycling program: 95 °C for 1 min; 40 cycles of 95 °C for 20 s, 56 °C for 20 s, and 72 °C for 38 s; followed by melting curve analysis. GAPDH was used as the internal control, and relative mRNA expression was calculated using the 2^−ΔΔCt method. Each biological sample was measured in technical triplicate. The mean Ct value of the technical triplicates was used to calculate ΔCt and relative mRNA expression for each biological replicate. The primer sequences are as follows: PPAR-γ2, forward 5′-CGGAAGCCCTTTGGTGACTT-3′ and reverse 5′-CTCGATGGGCTTCACGTTCA-3′; COX-2, forward 5′-AACCAGGTGAACTTCGTCTATTAG-3′ and reverse 5′-GGTCGGTTTGATGTCACTGTA-3′; GAPDH, forward 5′-GGTTGTCTCCTGCGACTTCA-3′ and reverse 5′-GGTGGTCCAGGGTTTCTTACTC-3′.

Statistical analysis

Data were analyzed using statistical software. All quantitative data were presented as the mean ± standard deviation. For qRT-PCR analysis, technical triplicates were first averaged for each biological replicate, and statistical analysis was performed using the biological replicate values. Comparisons among multiple groups were performed using one-way analysis of variance followed by Bonferroni post hoc tests when the data met the assumptions of normality and homogeneity of variance. Statistical significance was defined as P < 0.05.

Results

Screening of the estrous cycle in rats

The results of Wright-Giemsa staining showed that the vaginal smears in preoestrus predominantly exhibited nucleated epithelial cells, with a small amount of keratinocytes. The nuclei of these epithelial cells appeared lightly stained, enlarged, and showed signs of dissolution. In oestrus, the smears were characterized by a predominance of large, deciduous keratinized epithelial cells. In postestrus, there was a substantial presence of ke...

Discussion

PD is the lower abdominal pain that manifests before and during menstruation without any identifiable organic damage in the genital or pelvic regions¹. According to the survey, approximately 45%–90% of women globally experience dysmenorrhea, with 10% to 25% suffering from severe dysmenorrhea¹³. Therefore, it is imperative to identify safer and more effective interventions for PD and to further elucidate their underlying mechanisms.

Currentl...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% sodium chloride solution	Heilongjiang Qiqin Animal Health Products Co., Ltd.	240314A03	Tissue rinsing and sample preparation
1× phosphate-buffered saline (PBS)	Biosharp	BL601A	Tissue rinsing, ELISA homogenization, and staining procedures
Acoustic and light stimulation system	Custom-built in animal facility	Custom-built	Combined stress stimulation during QSBSPD model construction
Adhesive microscope slides	Jiangsu Citotest Labware Manufacturing Co., Ltd.	80312-3161	Preparation of paraffin sections and vaginal smears
Automated digital slide scanner	Guangzhou Guangying Cell Technology Co., Ltd.	GCell-60	Digital imaging of HE-stained sections
Automatic tissue dehydrator	Hubei Xiaogan Kuohai Medical Technology Co., Ltd.	KH-TS	Tissue dehydration before paraffin embedding
Biological microscope	Nikon	E200MV	Observation of vaginal smears and stained tissue sections
CO2 gas cylinder and euthanasia chamber	Fuzhou Hospital of Traditional Chinese Medicine Animal Center	Institutional CO2 system	Animal euthanasia
Cold plate for embedding station	Hubei Xiaogan Kuohai Medical Technology Co., Ltd.	KH-BL	Paraffin embedding
Cryogenic grinder	Shanghai Jingxin Industrial Development Co., Ltd.	TISS-24	Tissue homogenization
Danggui (Radix Angelicae Sinensis)	Guangdong Yifang Pharmaceutical Co., Ltd.	A1070343	SDD ingredient; concentrated granules
DEPC-treated water	Meilunbio	MA0018	qRT-PCR reagent
Electronic acupuncture treatment instrument	Suzhou Medical Appliance Factory Co., Ltd.	SDZ-II	TEAP stimulation and electrical stimulation
Electronic analytical balance	Sartorius Balance Beijing Co., Ltd.	BS 224S	Sample and reagent weighing
Eosin	Epredia	7111	HE staining
Epinephrine hydrochloride	TargetMol	119091	QSBSPD model construction
Estradiol benzoate	Sigma-Aldrich	E8515	QSBSPD model construction
Ethanol	Xilong Scientific Co., Ltd.	240403A1	Tissue processing and dehydration
Guizhi (Ramulus Cinnamomi)	Guangdong Yifang Pharmaceutical Co., Ltd.	A2060533	SDD ingredient; concentrated granules
Hematoxylin	Epredia	7211	HE staining
Ibuprofen suspension	Shanghai Johnson & Johnson Pharmaceutical Co., Ltd.	H20000359	Positive control drug; 20 mg/mL
Liujinu (Herba Artemisiae Anomalae)	Guangdong Yifang Pharmaceutical Co., Ltd.	A210A413	SDD ingredient; concentrated granules
Low-speed mini centrifuge	Hunan Xiangyi Laboratory Instrument Development Co., Ltd.	WTL-4K	Sample preparation
Micro refrigerated centrifuge	Beckman Coulter	Microfuge 22R	RNA extraction and tissue homogenate preparation
Microplate reader	Guangdong Danli Technology Co., Ltd.	K3	ELISA absorbance measurement at 450 nm
Microscope cover slips	Jiangsu Citotest Labware Manufacturing Co., Ltd.	80340-1630	Microscopy slide preparation
Mold incubator	Shanghai Lichen Bangxi Instrument Technology Co., Ltd.	W0704	Incubation during staining or assay procedures
Moyao (Myrrh)	Guangdong Yifang Pharmaceutical Co., Ltd.	1107853	SDD ingredient; concentrated granules
Neutral balsam	Meilunbio	MB9899	Mounting of stained tissue sections
NovoScript Plus All-in-one 1st Strand cDNA Synthesis SuperMix	Novoprotein	E047-01A	Reverse transcription
NovoStart SYBR qPCR SuperMix Plus	Novoprotein	E096-01B	qRT-PCR amplification
Oxytocin injection	Anhui Fengyuan Pharmaceutical Co., Ltd.	220906-4	QSBSPD model induction
Paraffin wax	Hualing	20220918	Paraffin embedding
PCR system	Bio-Rad	PTC-100	PCR-related procedures
Puhuang (Pollen Typhae)	Guangdong Yifang Pharmaceutical Co., Ltd.	A2092143	SDD ingredient; concentrated granules; component of Shixiao San
qRT-PCR primers for PPAR-γ2, COX-2, and GAPDH	Sangon Biotech (Shanghai) Co., Ltd.	Custom synthesis	qRT-PCR primers
Rat IL-1β ELISA kit	Meimian Biotechnology	MM-0047R2	ELISA kit
Rat IL-6 ELISA kit	Meimian Biotechnology	MM-0190R2	ELISA kit
Rat PGE2 ELISA kit	Meimian Biotechnology	MM-0068R2	ELISA kit
Rat PGF2α ELISA kit	Meimian Biotechnology	MM-0230R2	ELISA kit
Rat restraint cage	Custom-built in animal facility	Custom-built	Restraint stress during QSBSPD model construction
Rat TNF-α ELISA kit	Meimian Biotechnology	MM-0180R2	ELISA kit
Real-time PCR system	Applied Biosystems	7300	qRT-PCR analysis
RNAiso Plus	TaKaRa	9109	Total RNA extraction
Rotary microtome	Thermo Fisher Scientific	HM325	Paraffin sectioning
Shijianchuan (Herba Salviae Chinensis)	Guangdong Yifang Pharmaceutical Co., Ltd.	A3052113	SDD ingredient; concentrated granules
Sprague–Dawley rats	Beijing Vital River Laboratory Animal Technology Co., Ltd.	SCXK (Jing) 2019-0010	Animal model construction
Tissue embedding cassette	Jiangsu Citotest Labware Manufacturing Co., Ltd.	23033	Paraffin sectioning consumable
Tissue embedding machine	Hubei Xiaogan Kuohai Medical Technology Co., Ltd.	KH-BL	Paraffin embedding
Tissue flotation bath and slide warmer	Hubei Xiaogan Kuohai Medical Technology Co., Ltd.	KH-P2	Paraffin section mounting
Ultra-low temperature freezer	Zhongke Meiling Cryogenics Co., Ltd.	DW-HL340	Sample storage
Ultramicro UV-Vis spectrophotometer	Beijing BioTeke Corporation	ND5000	RNA concentration and purity measurement
Ultrapure water system	Sichuan Delishi Technology Co., Ltd.	SP-D2-V-10LS	Preparation of ultrapure water
Weilingxian (Radix et Rhizoma Clematidis Chinensis)	Guangdong Yifang Pharmaceutical Co., Ltd.	A2030543	SDD ingredient; concentrated granules
Wright-Giemsa stain	Solarbio	G3005	Estrous cycle screening
Wulingzhi (Faeces Trogopterori)	Guangdong Yifang Pharmaceutical Co., Ltd.	A2082223	SDD ingredient; concentrated granules; component of Shixiao San
Xiangfu (Rhizoma Cyperi)	Guangdong Yifang Pharmaceutical Co., Ltd.	A2069413	SDD ingredient; concentrated granules
Xiaohuixiang (Fructus Foeniculi)	Guangdong Yifang Pharmaceutical Co., Ltd.	A1125153	SDD ingredient; concentrated granules
Xylene	Xilong Scientific Co., Ltd.	240124A2	Dewaxing and tissue clearing
Yanhusuo (Rhizoma Corydalis Yanhusuo)	Guangdong Yifang Pharmaceutical Co., Ltd.	A2032983	SDD ingredient; concentrated granules