Mechanism of Shu-Mu Brain-Kidney Acupoint in Treating Oligoasthenospermia in Rats via the Hypothalamic-Pituitary-Testicular Axis

Summary

This study aims to verify the therapeutic effect of the Shu-Mu Brain-Kidney acupoint on oligoasthenospermia in rats and to explore its underlying mechanism through the hypothalamic-pituitary-testicular (HPT) axis.

Abstract

Oligoasthenospermia is a common cause of male infertility. The hypothalamic-pituitary-testicular (HPT) axis regulates gonadal differentiation and maturation through reproductive hormone synthesis and release, playing a vital role in male fertility. Disrupting HPT axis stability impairs sperm production, reducing semen quality. Investigating electroacupuncture's effect on HPT axis regulation may provide insights into treating oligoasthenospermia. Fifty 8-week-old male SD rats were randomly divided into blank, model, Shu-Mu Brain-Kidney, non-acupoint, and L-carnitine groups (n = 10 per group). Except for the blank group, rats received adenine intragastrically for 28 days to establish the model. Post-modeling, the Shu-Mu Brain-Kidney group underwent electroacupuncture at designated acupoints, while the non-acupoint group received sham treatment for 30 min daily. The L-carnitine group received L-carnitine (10 mL/kg) intragastrically once daily. Treatments continued for 28 days. General conditions, organ coefficients, and semen quality were assessed. HE staining analyzed tissue morphology, and ELISA detected serum hormone changes. Compared to the model group, the Shu-Mu Brain-Kidney, and L-carnitine groups exhibited significant improvements in spirit, diet, and bowel movements, with increased body weight, while the non-acupoint group showed no significant change. Renal organ coefficients decreased significantly in the Shu-Mu Brain-Kidney and L-carnitine groups but remained unchanged in the non-acupoint group. Testicular organ coefficients showed no significant differences among treatment groups. Sperm count, density, survival, and motility rates improved significantly in the Shu-Mu Brain-Kidney and L-carnitine groups, but not in the non-acupoint group. H&E staining showed ameliorated kidney and testicular tissue damage in the Shu-Mu Brain-Kidney and L-carnitine groups. ELISA revealed increased T, GnRH, and INHB and decreased LH, FSH, E2, and PRL levels in these groups (p < 0.001), with no significant changes in the non-acupoint group. These findings indicate that Shu-Mu Brain-Kidney acupoint therapy improves sperm quality by regulating the HPT axis, offering a potential treatment for oligoasthenospermia.

Introduction

Oligoasthenospermia, divided into "oligospermia" and "asthenospermia," is one of the common types of male infertility, with an incidence rate accounting for about 75% of male infertility cases¹. Oligoasthenospermia is mainly characterized by decreased sperm density and motility in the testes and is mostly clinically manifested as spermatogenesis disorder, reduced sperm count, and low sperm motility^2,3. In recent years, the incidence of oligoasthenospermia has increased year by year⁴. Without effective treatment, it will have long-term adverse effects on patients' physical and mental health and even family harmony⁵. Studies have shown that genetic problems⁶, reproductive endocrine disorders⁷, urogenital tract infections⁸, and chemical drugs⁹ can affect sperm production and maturation, leading to a decline in semen quality. Currently, improving the quality of damaged sperm has become a challenging problem in the medical field.

The hypothalamic-pituitary-testicular (HPT) axis is the main pathway of reproductive regulation in the body, which can regulate the differentiation and maturation of gonads by stimulating the synthesis and release of reproductive hormones related to the hypothalamus, pituitary gland, and testes, and plays an important role in maintaining normal male reproductive function¹⁰. If the stability of the HPT axis function is disrupted, it will cause the testicular secretion to be insufficient to reach the physiological concentration of testosterone, resulting in a decrease in the number and quality of sperm in the testis¹¹. Therefore, the treatment of oligoasthenospermia mainly aims to regulate the function of the HPT axis and achieve the purpose of maintaining the balance of related endocrine hormones, thereby increasing the number of sperm in semen, enhancing sperm activity, and improving semen quality.

In the clinical treatment of oligoasthenospermia, modern medicine mainly adopts methods such as surgery and drugs of anti-oxidation and enhance sperm motility^12,13, while electroacupuncture therapy combines traditional Chinese acupuncture and modern medical bioelectricity. It has the advantages of being fast and accurate, having an obvious curative effect, having few adverse reactions, a wide range of suitable diseases, and low treatment cost, and having a good treatment effect on oligoasthenospermia^14,15. "The back is yang, and the abdomen is yin." The Shu-Mu acupoint combination combines the Shu points with the Mu points, one in the front and one in the back, one yin and one yang, working together, also known as the front-back acupoint combination method. Traditional Chinese medicine (TCM) believes that the kidney is the root of prenatal essence, and the disease is located in the kidney. The Front-mu point of the kidney, Jingmen (GB25), and the Back-shu point, Shenshu (BL23), are selected, which has the effect of warming the kidney, assisting yang, and seeking yang within yin^16,17; the kidney stores essence and governs the production of marrow, the brain is the sea of marrow and the meeting place of all yang, and Taixi (KI3) is the source point of the foot Shaoyin kidney meridian, which can regulate and tonify the liver and kidney, nourish yin, and generate marrow¹⁸. Combined with Epangsanxian (MS4), it connects the kidney and brain, reflecting the idea of "Shu-Mu Brain-Kidney acupoint combination" in TCM¹⁹, and the therapeutic effect is more obvious than that of ordinary acupuncture.

Current treatment studies on semen quality primarily focus on improving testicular structure and function, while limited research has been conducted on the regulation of serum hormone levels across different tissues of the hypothalamic-pituitary-testicular (HPT) axis²⁰. In preliminary research, RNA sequencing (RNA-Seq) was used to identify candidate genes and regulatory pathways involved in semen quality regulation within the hypothalamic, pituitary, and testicular tissues of healthy rats and adenine-induced oligozoospermia model rats²¹. Additionally, semen quality testing and histological analysis of testicular tissues were performed in both groups. These findings provided insights into the molecular mechanisms underlying semen quality in adenine-induced oligoasthenospermia rats. Electroacupuncture therapy with distant-approximal acupoint could improve sperm quality in oligoasthenospermia rats by regulating the HPT axis, and the effect is related to the process of oxidative stress²². However, it is unknown whether the Shu-Mu Brain-Kidney acupoint can improve rats' semen quality by regulating the HPT axis. To further explore the regulatory mechanism of improving semen quality in oligoasthenospermia rats and effective treatment methods, this study used an adenine-induced rat model of oligoasthenospermia and selected four acupoints, Epangsanxian, Shenshu, Jingmen, and Taixi, to explore the related mechanism of the therapeutic effect of Shu-Mu Brain-Kidney acupoint on oligoasthenospermia in rats from the aspect of the HPT axis, providing a more scientific basis for clinical treatment.

Protocol

All animal experiments were reviewed and approved by the Animal Ethics Committee of Ningxia Medical University (approval number: IACUC-NYLAC-2021-130). The study was performed in strict accordance with the ARRIVE Guidelines 2.0 for animal experiments and adhered to the animal welfare principles described in the NIH Guide for the Care and Use of Laboratory Animals. This randomized controlled animal study employed one-way analysis of variance and was conducted at the Animal Experiment Center of Ningxia Medical University from March to May 2023. Eight-week-old SPF-grade male SD rats weighing 180-220 g were used for this study. The details of the reagents and equipment used are listed in the Table of Materials.

1. Animal model establishment and treatment procedures

1. Experimental animals and groups
   1. Obtain fifty 8-week-old SPF-grade male SD rats weighing 180-220 g.
   2. House all rats under standard conditions, maintaining a room temperature of 22-25 °C, humidity of 50%-60%, and providing free access to standard rodent feed and water.
   3. After one week of adaptation, randomly divide the rats into a blank (B) group (n = 10) and a modeling group (n = 40) using a random number table method. Administer normal saline to the blank group and adenine to the modeling group by oral gavage.
   4. After 28 days, randomly divide the modeling group into a model (M) group, Shu-Mu Brain-Kidney (S) group, non-acupoint (N) group, and L-carnitine (L) group (n = 10 per group). Leave the model group untreated.
      1. Apply electroacupuncture intervention using the Shu-Mu Brain-Kidney acupoint-matching method to the Shu-Mu Brain-Kidney group. Administer non-acupoint treatment to the non-acupoint group and L-carnitine oral liquid by gavage to the L-carnitine group. Treat all groups continuously for 28 days.
2. Model construction
   NOTE: Studies have shown that high doses of adenine can affect testicular function in rats, leading to abnormal secretion of sex hormones, disruption of the functional stability of the HPT axis, and a decline in sperm quality²³. Therefore, adenine is commonly used to establish male animal infertility models.
   1. Administer normal saline (1 mL/100 g body weight) to the blank group and adenine (20 mg/100 g body weight) to the model group by oral gavage (Figure 1A).
   2. Ensure continuous agitation during gavage to prevent adenine precipitation and blockage of the gavage needle. Administer the treatment once daily for 28 days.
3. 1.3 Treatment procedure
   1. Shu-Mu Brain-Kidney group
      1. Begin intervention on the first day after modeling. Perform acupuncture with disposable sterile acupuncture needles on bilateral points. After fixing the rat, expose and disinfect the acupuncture site.
      2. Fix the skin of the acupuncture site with the left hand and quickly pierce the acupuncture point with the disposable sterile acupuncture needle in the right hand. Connect an electrical stimulator. Apply dilatational waves with an electroacupuncture frequency of 2 Hz, a stimulation intensity of 1.5 mA, and a slight tremor of the local muscle.
      3. Perform electroacupuncture treatment for 30 min each time, once daily, for 28 consecutive days (Figure 1B).
         NOTE: Select acupuncture points in rats with reference to Yong Tang's Experimental Acupuncture and Moxibustion Science, Appendix II, "Acupuncture Points Commonly Used in Experimental Animals," combined with a comparative anatomical point selection method¹⁹. The following points were selected: (1) Epangsanxian (MS4): Located 3 mm below and 4 mm outside the Touwei point. Operation: Prick 1-2 mm straight; (2) Shenshu (BL23): Located in the spine area, on both sides of the lower spinous process of the second lumbar vertebra, 6 mm beside the median dorsal line. Operation: Prick 6 mm straight; (3) Jingmen (GB25): Located on the side waist, at the free end of the 13th rib on the lower side. Operation: Prick 1-2 mm straight; (4) Taixi (KI3): Located on the inner side of the foot, in the depression between the high point of the medial ankle and the posterior edge of the Achilles tendon. Operation: Prick 3 mm straight.
   2. Non-acupoint group
      1. Select and operate non-acupoints 1, 2, and 3 in the same manner as the Shu-Mu Brain-Kidney group (Figure 1B).
         NOTE: Because the non-acupoints do not follow the principle of point selection along the meridians, and according to Traditional Chinese Medicine (TCM) theory and long-term clinical observation, these three acupoints have little therapeutic effect on patients with oligoasthenozoospermia²⁴. (1) Non-acupoint 1: Located at the midpoint of the line between the elbow tip and the armpit on the inner side of the elbow. Operation: Prick 1-2 mm straight; (2) Non-acupoint 2: Located at the midpoint between the medial epicondyle of the humerus and the wrist of the ulna, on the ulnar margin. Operation: Prick 1-2 mm straight; (3) Non-acupoint 3: Located at the intersection of the anterior deltoid muscle and the biceps muscle on the inner arm. Operation: Prick 1-2 mm straight.
   3. L-carnitine group
      1. Administer levocarnitine oral liquid by oral gavage at a dose of 10 mL/kg body weight, once daily, for 28 consecutive days (Figure 1A).
4. Euthanasia
   1. After treatment, allow all rats to fast for 24 h, anesthetize them by intraperitoneal injection of 2% pentobarbital sodium (40 mg/kg body weight, following institutionally approved protocols), and collect blood from the apex of the heart using a disposable intravenous blood collection needle^21,24.
   2. After death by decapitation, remove one side of the epididymis to measure semen quality. Harvest the kidney and testicular tissues, weigh and record them, and fix them in paraformaldehyde fixative at room temperature for 48 h before embedding them in paraffin²⁴.

2. Observation indices

1. General observation: Observe the mental state, diet, and bowel movements of the rats, and monitor their body weight daily.
2. Organ coefficients: After death, carefully remove the surface fat and connective tissue from both the kidney and testicular tissues, rinse with normal saline, dry with filter paper, weigh on a balance, and record the measurements for the calculation of organ coefficients. Organ coefficient (%) = (organ weight [g] / body weight [g]) x 100%.
3. Semen quality
   1. After removing excess fat, remove and place one side of the epididymis from each rat in a constant temperature water bath at 37 °C for 5 min. Then, cut, filter, gently blow, and mix the sample well.
   2. Take a 10 µL aliquot of the diluted sperm suspension and drop it onto the edge of the cover glass of a pre-heated counting plate, allowing the sperm suspension to automatically permeate into the counting chamber.
   3. Measure sperm count, density, survival rate, and motility rate using a CASA (computer-assisted semen analysis) system²¹.
4. Hematoxylin and Eosin (H&E) staining
   NOTE: For details on the procedure, refer to Yang, N. et al.²¹.
   1. Rinse the fixed tissues with running water, dehydrate, and embed them.
   2. Cut paraffin sections (5 µm thick) using a microtome and bake at 65 °C for 4 h.
   3. Dewax the sections and rehydrate them with water.
   4. Stain the sections with hematoxylin staining solution for 3 min, then rinse with running water.
   5. Dehydrate the sections with 95% ethanol for 1 min, then stain with eosin solution for 2 min.
   6. After sealing the slides, perform scanning using a slide scanner, and collect and analyze the images.
5. Enzyme-Linked Immunosorbent Assay (ELISA)
   ​NOTE: For details on the procedure, refer to Yang, N. et al.²¹.
   1. To prepare the serum, add standard substances and serum of the corresponding concentrations to the standard substance wells and sample wells to be measured, followed by the addition of the enzyme-labeled reagent.
   2. Carry out incubation, washing, color development, and termination steps.
   3. Determine the absorbance at a wavelength of 450 nm using an enzyme-labeled instrument, and calculate the expression levels of testosterone (T), gonadotropin-releasing hormone (GnRH), inhibin B (INHB), luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), and prolactin (PRL).

3. Statistical analysis

1. Analyze the experimental data using statistical software, and present the results of each group as mean ± standard deviation (mean ± SD).
2. Use one-way ANOVA to compare sample means between multiple groups that meet the assumptions of normality and homogeneity of variance.
3. Use the t-test for comparisons between two groups, and non-parametric tests for data that do not meet the above assumptions.
4. Consider differences statistically significant when p < 0.05.

Results

Model evaluation

After modeling, observe the rats for lethargy, crouched posture, cold limbs, reluctance to eat, weight loss, increased urine volume, loose stools, and other symptoms, which generally align with the syndrome of kidney-yang deficiency.

After dissection, observe that the kidneys are tough, white, and heavier. H&E staining reveals that...

Discussion

In TCM, the "kidney" consists of the "inner kidney," referring to the functions of the urinary system in modern medicine, and the "outer kidney," referring to the hypothalamic-pituitary-gonadal axis and the anatomical external reproductive organs²⁵. The male seminal vesicles belong to the "outer kidney," and their components include the testes, epididymides, seminal vesicles, etc²⁶. TCM considers essence to be the material basis of the human ...

Materials

Name	Company	Catalog Number	Comments
Adenine	Beijing Solarbio Science & Technology Co. Ltd.	A8330
CASA computer-assisted semen analysis system	Nanning Songjing Tianlun Biotechnology Co., Ltd.	VICOS-SPERM
Centrifuge	Sartorius	A-14C
Differentiation fluid	Shanghai Biyuntian Biotechnology Co., Ltd	C0163M
Disposable sterile acupuncture needles	Hanyi brand	0.25mm*13mm
Drying machine	Leica	HI1220
Electrical stimulator	Hua Tuo Brand	SDZ-II
Electronic balance	Hangzhou Youheng Weighing Equipment Co., Ltd	HLD-6002
ELISA kit	Shanghai Jianglai Biotechnology Co. LTD	JL13251?JL11473?JL11525?JL12505?JL13256?JL12201
Enzyme-labeled instrument	Rayto	RT-6100
Eosin staining solution	Zhuhai Beso Biotechnology Co. LTD	BA-4024
Hematoxylin staining solution	Zhuhai Beso Biotechnology Co. LTD	BA-4041
Levocarnitine oral liquid	Northeast Pharmaceutical Group Shenyang First Pharmaceutical Co. LTD.	H19990372
Microtome	Leica	RM2255
Neutral balsam	Beijing Zhongshan Jinqiao Biotechnology Co., Ltd	Zli-9555
Paraffin embedding machine	Leica	HistoCore Arcadia H
SD rat, 8-week-old, Male, 180-220 g	144 Animal Center of Ningxia Medical University
Slide scanner	Leica	Aperio LV1
Vortex oscillator	Haimen Qilin Bell Instrument Manufacturing Co., Ltd	QL-902