Efficacy of stimulating Mingmen (GV4) and Guanyuan (CV4) on kidney Yang deficiency in rat model: laser irradiation vs traditional moxibustion

Abstract

OBJECTIVE:

To investigate the effect of 10.6-μm laser moxibustion (LM) and traditional moxibustion (TM) on kidney Yang deficiency (KYD) model rats.

METHODS:

Forty rats were randomly divided into normal, model, 10.6-μm LM, and TM groups. KYD was induced through intramuscular hydrocortisone injections.

RESULTS:

Hydrocortisone injections decreased the body weight, activity, and temperature and induced changes in the organ coefficients. Compared with the model group, TM and LM treatment prevented body weight loss and improved organ coefficients after treatment (P < 0.05), while the body weight remained lower compared to the normal group (P < 0.01). Additionally, LM and TM significantly increased the levels of urine 17-hydroxycorticosteroid (17-OHCS) compared with the model group (P < 0.01). The LM and TM groups showed a greater distance travelled across the central area, time spent in the central area, and 5-min total distance than the model group in the open field test (P < 0.01). The model group had the lowest anal or surface temperature (P < 0.05); there were no differences among the other three groups (P > 0.05). TM treatment increased the temperature of Mingmen (GV4) and Guanyuan (CV4) compared to the model group (P < 0.05, P < 0.01). LM treatment increased Mingmen (GV4) temperature (P < 0.05), while there were no obvious differences in Guanyuan (CV4) temperature compared with the normal group before and after treatment (P > 0.05). There was a positive correlation between Mingmen (GV4)/Guanyuan (CV4) temperature and 17-OHCS levels.

CONCLUSIONS:

LM and TM treatment can improve KYD symptoms, and acupoint temperature changes may be an objective indicator of KYD.

1. INTRODUCTION

Kidney Yang deficiency (KYD) is a common Traditional Chinese Medicine (TCM) symptom pattern caused by hereditary weakness, chronic illness, excessive sexual activity, overwork, and old age.¹ Its clinical characteristics include weakness of the waist and knee, chills, tinnitus, impotence, and premature ejaculation.^2,3 Unlike other organs, the kidney Yang is the root of yang. KYD can affect one or more of the aforementioned organs and is found in chronic diseases, including rheumatoid arthritis, hypertension, and diabetes, posing a considerable challenge to the medical system.⁴ An epidemiological survey revealed that KYD incidence was higher among people aged > 60 years,⁵ posing a potential threat to the quality of life of older people.

Moxibustion has been used to treat many diseases in China for thousands of years.⁶ It involves burning the moxa herb and applying thermal stimulation to specific acupoints on the human body to warm and soothe Qi and blood, restore Yang, rescue from Yang collapse, and strengthen the body. It is specifically used for chronic diseases, diseases caused by Yang deficiency, and deficiency cold patterns.⁷ However, it may cause air pollution, unpleasant smell, and skin burning. Some hazards have been identified in moxa smoke that could affect human health, especially in individuals highly responsive to moxa smoke.^8,9

Infrared laser moxibustion is an emerging, non-invasive, and painless treatment technology.¹⁰ Specific infrared radiation wavelengths produced by moxibustion are as potent as those of thermal radiation.¹¹ A 10.6-μm laser can yield similar thermal effects as moxibustion.^12,13 Moreover, it is a good substitute for traditional moxibustion since it exerts similar therapeutic effects without the smoke and smell.

Thus far, extensive research on the pharmacological activity of reinforcing kidney Yang herbs is ongoing. This study aimed to newly determine whether traditional and laser moxibustion can improve KYD symptoms and compared their effects.

2. MATERIALS AND METHODS

2.1. Animals

Male Sprague-Dawley rats [(220 ± 20) g] were purchased from the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine. The animals were housed in humidity-controlled environments maintained at (20 ± 2) ºC under a 12/12 h light-dark cycle (lights on 08:00 am to 08:00 pm). Food and water were provided ad libitum. All study procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institution of Animal Care and Use Committees of the Laboratory Animal Welfare and Ethics Committee of Shanghai University of Traditional Chinese Medicine (approval number: PZSHUTCM190322001).

2.2. KYD model

Forty rats were randomly divided into the normal group (n = 10) and KYD group (n = 30). The KYD group received intramuscular hydrocortisone injections (25 mg/kg) in the hind limb once a day (08.00-10.00 a.m.) on alternate sides for 15 d (following a classical method of establishing the KYD model.² ) The normal group received intramuscular injections of NaCl (25 mg/kg); all other procedures were similar for both groups. Subsequently, the KYD group was randomly divided into the model, laser moxibustion (LM), and traditional moxibustion TMgroups (n = 10 per group).

2.3. Animals treatment

The normal and model groups received no treatment. The LM-group rats were immobilized and received laser irradiation on Mingmen (GV4) and Guanyuan (CV4) for 10 min once every other day for 10 sessions. The carbon dioxide laser device (wavelength: 10.6 𝜇m; radiant power: 80 mW spot area: 2 mm in diameter; and irradiation provided at 2 cm away from the skin surface) was obtained from SX10-C1, Shanghai Wonderful Opto-Electrics Tech Co., Ltd., Shanghai, China.

The traditional moxibustiongroup received moxibustion on Mingmen (GV4) and Guanyuan (CV4) for 10 min once every other day for 10 sessions.

2.4. Measurement index and methods

To validate the establishment of the KYD model, we measured the following indicators.

2.4.1. Body weight, 24-h water intake, and urine volume

Body weight was examined during model establishment and treatment. Subsequently, the rats were kept in separated cages with free access to water and food deprivation for 24 h, during which, water intake and urine volume were recorded.

2.4.2. Determination of 24-h urine 17-hydroxycorti-costeroid (17-OHCS) levels

The urine 17-OHCS level is a criterion for assessing KYD in TCM;^14,15 therefore, we examined urine 17-OHCS levels. Collected urine was centrifuged at 2000 r/min at 4 ℃ for 10 min; subsequently, the supernatant was divided into 1.5-mL EP tubes. Urine 17-OHCS levels were quantified using enzyme-linked immune-osorbent assay following the manufacturer’s instructions.

2.4.3. Behavioral tests

Yang also refers to excitement and promotion in TCM theory.¹⁶ According to a study, KYD rats present with a dull reaction, decreased activity, and listlessness. Therefore, we performed an open field test to confirm these observations. After the last treatment, the LM and TM groups were placed in an open field apparatus (100 × 100 × 40 cm) and allowed to move freely for 5 min. The total distance traveled, distance travelled across the central area, and time spent in the central area were recorded using ANY-maze.

2.4.4. Anal, surface, and acupoint temperature

KYD is characterized by decreased body temperature.¹⁷ Anal temperature was measured using an electronic thermometer for animals (Linhai Medical Instrument Factory, Linhai, China 200930194084.7). Body temperature was measured using an infrared thermal imager. Specifically, an infrared thermogram of the body surface was scanned using FLIR T420 infrared thermography (Shanghai Jianling Electronic Technology Co., Ltd., Shanghai, China). To determine the relationship between acupoint temperature change and therapeutic effect, we measured the pre- and post-treatment Mingmen (GV4) and Guanyuan (CV4) temperatures using a digital thermocouple thermometer (UNI-T, UT325, Dongguan, China).

2.4.5. Organ coefficients

All rats were anesthetized using sodium pentobarbital (40 mg/kg) and sacrificed 24 h after the last treatment. The kidneys, adrenal glands, testicles, and epididymis were immediately dissected on an ice plate, washed in normal saline at 4 ℃, and weighed on the precision balance. Subsequently, we calculated the organ coefficients (organ coefficient = organ weight/body weight × 100%).

2.5. Statistical analysis

Statistical analyses were performed using SPSS Statistics for Windows (Version 24, IBM Corp., Armonk, NY, USA). Data are presented as mean ± standard deviation ($\bar{x} \pm s$). An independent samples t-test was used to analyze body weight, water intake, urine volume, and urine 17-OHCS levels. Data collected at multiple time points were analyzed using repeated-measures analysis of variance. Temperature data were analyzed using one-way analysis of variance with the post-hoc least significant difference test. Significant within-group changes were analyzed using the paired-samples test before and after treatment. Pearson’s correlation was used to determine the relationship between urine 17-OHCS levels and acupoint temperature. Significance and high statistical significance were set at P < 0.05 and P < 0.01, respectively. All figures were created using Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA).

3. RESULTS

Intramuscular hydrocortisone injection for 15 d induced similar KYD symptoms in rats, including decreased body mass and body temperature, reduced activity, slow reactions, hunched back, sparse and loss of luster of body hair, and reduced water intake,¹⁸ indicating that the KYDS model was successfully established for subsequent experiments.

3.1. Body weight, 24-h water intake, and 24-h urine volume

After the 5th day of modeling with hydrocortisone, the KYD group showed significantly lower body-weight gain than the normal group (Figure 1A, P < 0.01). Regarding body-weight measurements during treatment (Figure 1B), there was no significant difference in body weight among the model, LM, and TM groups (P > 0.05). However, after the last treatment, the body weight in the LM and TM groups was higher than that in the model group (P < 0.05). There were significant differences between the normal and treatment groups, with the body weight being higher in the normal group (P < 0.01).

Figure 1. Changes in body weight, 24-h water intake, urine volume, and 17-OHCS levels in the different groups.

A: the body weights of rats after hydrocortisone injection; B: the body weights of rats after treatment; C: 24-h water intake and 24-h urine after hydrocortisone injection; D: post-treatment 24-h water intake and 24-h urine levels; E: urine 17-OHCS levels after hydrocortisone injection; F: urine 17-OHCS levels after treatment. The normal group: normal rats without modeling; the KYD group received intramuscular hydrocortisone injections (25 mg/kg) (n = 30); the model group: KYD rats without treatment (n = 10); the LM-group: KYD rats were immobilized and received laser irradiation on Mingmen (GV4) and Guanyuan (CV4) for 10 min once every other day for 10 sessions (n = 10); the TM group: KYD rats received moxibustion on Mingmen (GV4) and Guanyuan (CV4) for 10 min once every other day for 10 sessions (n = 10). KYD: kidney Yang deficiency; LM: laser moxibustion; TM: traditional moxibustion. Significant differences compared with the normal group were designated as ^aP < 0.05 and the model group as ^bP < 0.05.

The 24-h water intake and urine volume in the KYD group were lower than those in the normal group after model establishment (Figure 1C, 1D, P < 0.01, < 0.01). After LM and TM, there was increase in urine and water levels, but with no significant difference compared with the normal group. Furthermore, there was no significant change in the model group in urine volume and 24-h water intake.

3.2. 24-h urine 17-OHCS

After hydrocortisone injection, there was a significant decrease in 17-OHCS levels in the KYD group (Figure 1E, P < 0.01). However, LM and TM treatment significantly increased the 17-OHCS levels (Figure 1F, P < 0.01). There was no significant difference among the normal, LM, and TM groups (P > 0.05).

3.3. Behavioral tests

As shown in Figure 2, the model group showed significantly less distance traveled across the central area and time spent in the central area than the normal group (both, P < 0.01). There were no significant differences among the normal, LM, and TM groups (P > 0.05). The total 5-min distance, distance across the central area, and time spent in the central area showed the same trend of significant differences (P < 0.01).

Figure 2. Open field test in the different groups.

A: distance across the central area after treatment; B: time spent in the central area after treatment; C: the total 5-min distance after treatment; D: the course map of each group within the open field area. D1: Normal group; D2: Model group; D3: LM group; D4: TM group. The red dot indicates that the mouse begins to move, and the blue point indicates that the mouse stops. The normal group: normal rats without modeling; the KYD group received intramuscular hydrocortisone injections (25 mg/kg) (n = 30); the model group: KYD rats without treatment (n = 10); the LM-group: KYD rats were immobilized and received laser irradiation on Mingmen (GV4) and Guanyuan (CV4) for 10 min once every other day for 10 sessions (n = 10); the TM group: KYD rats received moxibustion on Mingmen (GV4) and Guanyuan (CV4) for 10 min once every other day for 10 sessions (n = 10). KYD: kidney Yang deficiency; LM: laser moxibustion; TM: traditional moxibustion. Significant differences compared with the normal group were designated as ^aP < 0.05and the model group as ^bP < 0.05.

3.4. Temperature

3.4.1. Anal and surface temperature

On the last day of modeling, rectal temperature was significantly lower in the KYD model group than in the normal group (Table 1, P < 0.05). After three treatment sessions, there was increase in the rectal temperature of the LM and TM groups; however, there was no significant difference compared with the normal group (Table 2, P > 0.05). However, there were significant differences between the model and normal groups (Table 2, P < 0.05).

Table 1.

Anal temperature during modeling time (℃, $\bar{x} \pm s$)

Group	n	Day 1	Day 5	Day 10	Day15
Normal	10	37.5±0.4	37.1±0.4	37.1±0.4	37.0±0.3
KYD	30	37.5±0.5	36.8±0.6	36.5±0.7a	36.3±0.6a

Notes: The KYD group received intramuscular hydrocortisone injections (25 mg/kg). KYD: kidney Yang deficiency. ^aP < 0.05, compared with the normal group.

Table 2.

Anal temperature during treatment time (℃, $\bar{x} \pm s$)

Group	n	1st	3rd	5th	8th	10th
Normal	10	37.01±0.21	37.00±0.18	37.11±0.47	37.21±0.50	37.28±0.32
Model	10	36.33±0.49a	36.41±0.87	36.40±0.78	36.30±0.53a	36.15±0.34a
LM	10	36.35±0.30a	36.49±0.63	36.80±0.64	36.82±0.32	37.02±0.36b
TM	10	36.32±0.23a	36.90±0.34	37.34±0.43b	37.00±0.23b	37.13±0.09b

Notes: the normal group: normal rats without modeling; the KYD group received intramuscular hydrocortisone injections (25 mg/kg) (n = 30); the model group: KYD rats without treatment (n = 10); the LM-group: KYD rats were immobilized and received laser irradiation on Mingmen (GV4) and Guanyuan (CV4) for 10 min once every other day for 10 sessions (n = 10); the TM group: KYD rats received moxibustion on Mingmen (GV4) and Guanyuan (CV4) for 10 min once every other day for 10 sessions (n = 10). LM: 10.6-μm laser moxibustion; TM: traditional moxibustion; KYD: kidney Yang deficiency. ^aP < 0.05, compared with the normal group; ^bP < 0.05, compared with the model group.

As shown in Table 3, there were significant differences between the model and the other three groups in head-neck temperature and back and tail root temperature (P < 0.05). Additionally, the temperature was significantly higher in the LM and TM groups than in the model group (P < 0.05). Compared with the normal group, the LM and TM groups showed significantly higher temperatures in the back and tail root (P < 0.05). The temperature of the tail root was the lowest in all groups.

Table 3.

Surface temperature after treatment (℃, $\bar{x} \pm s$)

Group	n	Head and neck	Back	Tail root
Normal	10	32.73±0.56	33.67±0.80	28.44±0.05
Model	10	27.72±0.69a	27.93±0.53a	23.02±0.65a
LM	10	31.01±0.91b	31.33±1.71a,b	27.84±0.92a,b
TM	10	31.08±0.53b	32.37±1.00a,b	28.99±0.46a,b

Notes: the normal group: normal rats without modeling; the KYD group received intramuscular hydrocortisone injections (25 mg/kg) (n = 30); the model group: KYD rats without treatment (n = 10); the LM-group: KYD rats were immobilized and received laser irradiation on Mingmen (GV4) and Guanyuan (CV4) for 10 min once every other day for 10 sessions (n = 10); the TM group: KYD rats received moxibustion on Mingmen (GV4) and Guanyuan (CV4) for 10 min once every other day for 10 sessions (n = 10). LM: 10.6-μm laser moxibustion; TM: traditional moxibustion; KYD: kidney Yang deficiency. ^aP < 0.05, compared with the normal group; ^bP < 0.05, compared with the model group.

3.4.2. Temperature of Mingmen (GV4) and Guanyuan (CV4) and Pearson’s correlation analysis

Before treatment, the Mingmen (GV4) temperature of the normal group was higher than those of the other three groups. After treatment, the Mingmen (GV4) temperature of the LM and TM groups showed significant increase compared with the model group (Figure 3A, P < 0.01). However, there were no significant differences in Guanyuan (CV4) between the normal and LM groups before and after treatment (Figure 3B, P > 0.05). The Guanyuan (CV4) temperature of the TM group showed significant increase compared with that of the model group (P < 0.01).

Figure 3. Mingmen (GV4) and Guanyuan (CV4) temperatures in the different groups and Pearson’s correlation analysis results.

A: Mingmen (GV4) temperature in the different groups; B: Guanyuan (CV4) temperature in the different groups. ^aP < 0.05, compared with the normal group. ^bP < 0.05, compared with the model group. C: correlation of 17-OHCS levels with the Mingmen (GV4) and Guanyuan (CV4) temperatures after modeling; D: correlation of 17-OHCS levels with Mingmen (GV4) and Guanyuan (CV4) temperatures after treatment. The normal group: normal rats without modeling; the KYD group received intramuscular hydrocortisone injections (25 mg/kg) (n = 30); the model group: KYD rats without treatment (n = 10); the LM-group: KYD rats were immobilized and received laser irradiation on Mingmen (GV4) and Guanyuan (CV4) for 10 min once every other day for 10 sessions (n = 10); the TM group: KYD rats received moxibustion on Mingmen (GV4) and Guanyuan (CV4) for 10 min once every other day for 10 sessions (n = 10). KYD: kidney Yang deficiency; LM: laser moxibustion; TM: traditional moxibustion. Significant differences compared with the normal group were designated as ^aP < 0.05 and the model group as ^bP < 0.05.

As shown in Figure 4C and 4D, Pearson’s correlation analysis revealed a significant positive correlation between urine 17-OHCS levels and Mingmen (GV4) temperature (after modeling: R = 0.42, P < 0.01; after treatment: R = 0.40, P < 0.05). There was a positive linear correlation between 17-OHCS levels and Guanyuan (CV4) temperature (after modeling: R = 0.32, P < 0.05; after treatment: R = 0.35, P < 0.05).

Figure 4. Organ coefficients in the different groups.

A: kidneys in the different groups; B: Adrenal glands in the different groups; C: testicles in the different groups; D: epididymis in the different groups. ^aP < 0.05, compared with the normal group; ^bP < 0.05, compared with the model group. The normal group: normal rats without modeling; the KYD group received intramuscular hydrocortisone injections (25 mg/kg) (n = 30); the model group: KYD rats without treatment (n = 10); the LM-group: KYD rats were immobilized and received laser irradiation on Mingmen (GV4) and Guanyuan (CV4) for 10 min once every other day for 10 sessions (n = 10); the TM group: KYD rats received moxibustion on Mingmen (GV4) and Guanyuan (CV4) for 10 min once every other day for 10 sessions (n = 10). KYD: kidney Yang deficiency; LM: laser moxibustion; TM: traditional moxibustion. Significant differences compared with the normal group were designated as ^aP < 0.05 and the model group as ^bP < 0.05.

3.5. Organ coefficients

We calculated the rat organ coefficients as the ratio of organ weight to rat body weight, which can reflect the extent of organ damage, such as edema or atrophy. As shown in Figure 4A, the kidneys showed higher organ coefficients in the model group than in the normal and LM groups (P < 0.01). There were significant differences in the coefficients of the adrenal gland, testicles, and epididymis between the normal and model groups. LM and TM suppressed organ-coefficient reduction (Figure 4B-4D). The decline of the adrenal gland coefficient may be related to adrenalcortical insufficiency.

4. DISCUSSION

Previous research has suggested that KYD is associated with damage and functional dysfunction of the hypothalamic pituitary target gland axes (adrenal, thyroid, and gonad).^19,20 High-dose hydrocortisone injection inhibits the release of the adrenocorticotropic hormone in the anterior pituitary and disturbs the hypothalamic-pituitary-adrenal axis, thereby inducing a state of hyperfunction in energy metabolism, with the animals exhibiting signs of exhaustion similar to those seen in KYD patterns, such as weight loss and decreased activity.^21,22 Hence, hydrocortisone injection is a commonly used method for preparing a KYD model. Our findings indicate that LM and TM improved KYD symptoms; thus, LM and TM could be effective for KYD treatment. Furthermore, acupoint temperature was positively correlated with urine 17-OHCS levels.

TCM considers the kidneys’ store to be the congenital essence that determines our fundamental constitution; therefore, it is considered as “The Root of Life.” Kidney Yang can maintain a normal metabolic rate to satisfy the normal physiological activities of the human body, and serum metabolic profiling analysis of KYD rats revealed significant downregulation in metabolic activities.²³ KYD pattern is similar to glucocorticoid withdrawal symptom pattern regarding its clinical characteristics.²⁴

Moxibustion is among the oldest therapies for KYD, with its origin traceable back to primitive society.⁶ Based on ancient Chinese literature, the therapeutic effects of moxibustion are associated with treating chronic symptoms related to “yang deficiencies” and preventing human disorders.²⁵ Increasingly, studies have shown that moxibustion affects both humans and animals and almost all major physiological systems, especially in the fields of analgesics, immunity, and anti-aging.^26,⇓-28 Furthermore, the warm effect and light radiation produced by moxibustion are among the most important factors.²⁹ The radiation spectra of moxa burning mainly include near-infrared wavelengths.³⁰ Near-infrared radiation can penetrate deep tissue, increase microcirculation, promote metabolism, and improve immune function.^31,⇓-33

The radiation wavelength of indirect moxibustion and human acupoints is approximate to 10 μm.³⁴ The 10.6-μm LM mimics this effect without producing smoke and smell. Irradiation of the forearms with 10.6-μm LM can improve microcirculation, with skin blood perfusion measured using a laser speckle image.³⁵ Microcirculation is crucially involved in providing nutrients and removing metabolic byproducts from virtually all living cells in the body.³⁶ Numerous diseases are intimately associated with microvascular dysfunction. Currently, regulating microcirculation is considered crucial to the acupuncture effect.^37,38 We observed a positive correlation of 17-OHCS levels with the Mingmen (GV4) and Guanyuan (CV4) temperatures. Additionally, the Mingmen (GV4) temperature in the TM and LM groups increased by 0.55℃ and 0.62℃, respectively, compared with those before treatment. There was no significant difference between the TM, LM, and normal groups. The acupoint temperature has been reported to vary greatly across different physiological and pathological conditions; furthermore, it is positively correlated with disease severity.^39,40 We found that Mingmen (GV4) temperature increased with KYD improvement, indicating that temperature can reflect the state of health and Yang Qi. Conversely, Guanyuan (CV4) temperature did not display an obvious change. Thus, Mingmen (GV4) appears to have a better specificity for the detection of temperature changes associated with Yang deficiency pattern compared to Guanyuan (CV4). This is consistent with TCM theory. According to TCM theory, the GV—located at the dorsal midline—increases the body’s resistance and elimination of the cold-damp sensation, which is regarded as “the sea of yang meridian”.⁴¹ Mingmen (GV4) can activate and inspire Yang Qi, which is the root of Yuan Qi and the gateway of life.⁴² Modern studies have confirmed that blood-flow perfusion volumes in the skin and deeper tissues along GV are significantly higher than those of their bilateral corresponding control points; additionally, the Mingmen (GV4) temperature rise was the most significant.^43,44 Prior research in this area primarily focused on blood perfusion and microcirculation.³⁸

As the symptoms of KYD improve, Mingmen (GV4) temperature rises. Temperature changes at Mingmen (GV4) may be used as an objective indicator for KYD assessment. Further studies are needed to examine the clinical efficacy of LM and TM. Additionally, the un-derlying mechanism of action regarding KYD imp-rovement and regulation of microcirculation at acupoints remains unclear. Therefore, further inves-tigation is warranted.

In conclusion, our findings suggest that laser moxi-bustion may have thermal effects and could effectively improve KYD symptoms. There may be a specific relationship between temperature increase in the acupoints and the therapeutic effect.