Zhu Shi-peng, Luo Li, Sun Zhi-fang, Ding Na, Zhang Qing-qing, He Yu-wei, Zhang Lu-fen, Li Xiao-hong

1 Beijing University of Traditional Chinese Medicine, Beijing 100029, China

2 Huguosi Hospital Affiliated to Beijing University of Traditional Chinese Medicine, Beijing 100035, China

Effect of Preventive Acupuncture and Moxibustion on Hypothalamus and Ovarian Leptin and Blood Lipid Levels in Menopausal Rats

Zhu Shi-peng1, Luo Li1, Sun Zhi-fang1, Ding Na1, Zhang Qing-qing1, He Yu-wei2, Zhang Lu-fen1, Li Xiao-hong1

1 Beijing University of Traditional Chinese Medicine, Beijing 100029, China

2 Huguosi Hospital Affiliated to Beijing University of Traditional Chinese Medicine, Beijing 100035, China

Objective: To observe the changes of hypothalamus, ovarian leptin levels and blood lipid in 12-month-old rats with natural menopause, and the effect of preventive acupuncture and moxibustion at Guanyuan (CV 4). To explore the impact of preventive acupuncture and moxibustion on the internal environment of early menopause disorder.

Methods: Menopausal female rats were screened by vaginal smear. Rat model of menopause was obtained by natural aging method. Preventive acupuncture and moxibustion at Guanyuan (CV 4) was used as intervention methods. Hypothalamus and ovarian leptin levels were detected by radioimmunoassay. Plasma total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL) and high density lipoprotein (HDL) levels were determined by biochemical methods.

Results: Compared with the young control group, leptin levels in the hypothalamus and ovarian increased (P＜0.01), plasma TC increased (P＜0.05), LDL increased (P＜0.01), and HDL reduced in 12-month-old rats. Hypothalamus and ovary leptin levels in 12-month-old rats with preventive acupuncture and moxibustion were significantly decreased (P＜0.01); plasma TC and LDL levels decreased and HDL levels were increased in rats with preventive acupuncture (P＜0.05).

Conclusion: Preventive acupuncture and moxibustion can improve leptin resistance state and restore the abnormal lipid levels in menopausal body, and therefore can slow down the disorder of inner environment in early menopause.

Acupuncture-moxibustion Therapy; Moxa Cone Moxibustion; Climacteric; Rats

Menopause is a phase of life in women that signifies the end of their reproductive period. In this period the kidney qi gradually declines, and the internal environment of body changes, which result in multi-system dysfunction. Menopause obesity caused by leptin resistance and lipid metabolism disorders often occurs in this period. ‘Preventive acupuncture and moxibustion’ is one important therapy to preventively treat diseases, that through certain stimulus it produces ‘benign pre-stress’ to the body, in order to decrease the injury degree caused by adverse stimuli. Our previous animal studies have confirmed that ‘preventive acupuncture and moxibustion’ can moderately stimulate stress proteins and hormones. Through the nerveendocrine-immune network, it initiates the endogenous protective mechanism and plays a preventive role in the subsequent environmental disturbancecaused by menopause[1-3]. This study aims to explore the influence and mechanism of ‘preventive acupuncture and moxibustion’ on menopausal obesity by observing hypothalamus and ovarian leptin and blood lipid levels changed by preventive acupuncture and moxibustion at Guanyuan (CV 4) in natural menopausal rats.

1 Experimental Materials

1.1 Animals

Clean female SD rats were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. [License No: SCXK (Beijing) 200223]. Experimental animals were raised in clean cabinets, with laboratory temperature (20±1)℃, humidity 50 %, normal diet, and free drinking water.

1.2 Main instruments

Electronic scales; Hitachi 7600 automatic biochemical analyzer (Hitachi, Japan); DY98-1 electric glass homogenizer (Ningbo Xinzhi Biotechnology Co., Ltd., China); ZK380 low temperature centrifuge (Hermle, Germany); ultra-low temperature freezer (Sanyo, Japan).

1.3 Main reagents and materials

Leptin kit (Sunbio Biomedical Technology Co., Ltd., China); total cholesterol (TC) kit, triglycerides (TG) kit, high density lipoprotein (HDL) kit, and low density lipoprotein (LDL) kit (Beijing Sino-uk Institute of Biological Technology, China).

Moxa cone was 0.7 cm in diameter, 1 cm in height and 2 g in weight (Fig.1). The needle was 0.35 mm in diameter and 25 mm in length.

Fig.1 Moxa cone

2 Experimental Methods

2.1 Animal grouping

The menopause was determined through vaginal smear method[4]. Histological changes of vaginal smears in 9.5-month-old female SD rats, stained with alkaline methylene blue solution, were observed daily under the microscope for three estrous cycles (15 d). According to cell morphology, type and quantity, twenty eight 10-month-old female SD rats were screened, which had begun to emerge estrous cycle disorders (indicating imminent or has begun to enter menopause), and were divided into four groups according to the random number table, namely 10-month-old group, 12-monthold group, preventive moxibustion 12 month-old group and preventive acupuncture 12 month-old group, with seven in each group. Seven 3.5-month-old female SD rats were as young control group.

2.2 Acupuncture and moxibustion methods

Guanyuan (CV 4) was located according to rat standard acupoint mapping, i.e, the midpoint of the two hind legs roots.

Moxibustion: Rat hair in the region of 2 cm in diameter around Guanyuan (CV 4) was cut and the skin was exposed. The rats were supine on the palm of operator’s hand and were quiet awake. A lit moxa cone was directly placed on the acupoint until it was burned out.

Acupuncture: The rats were supine on the palm of the operator’s hand and were quiet awake. The needle was directly pierced into Guanyuan (CV 4) about 0.2 cun and punctured upward. The needle was retained for 20 min, during which the rat was free to move.

2.3 Grouping and treatment

Young control group and 10-month-old group received no treatment.

Preventive moxibustion 12-month-old group and preventive acupuncture 12-month-old group received moxibustion and acupuncture respectively, from 10 months of age, twice a week for 8 weeks. The 12-month-old rats were only grabbed as those in the preventive acupuncture and moxibustion groups from 10 months of age without other treatment for 8 weeks.

2.4 Samples obtaining

At the end of the last treatment, blood of rats in each group was immediately collected with rapid decapitation, placed in 2 mL anticoagulated tubes [with 50 μL Ethylene Diamine Tetraacetic Acid (EDTA), 40 μL Aprotinin], and centrifuged (3 500 r/min, 15 min). The plasma was stored at -20 ℃.

The above decapitated rats were placed on sterile ice. The hypothalamus and ovaries were rapidly removed, boiled in saline for 5 min, weighed and placed in a glass homogenizer tube. One millilite hydrogen chloride (1 mol/mL) was added. The tissues were homogenized on ice sufficiently, placed at room temperature for 100 min, and 0.8 mL sodium hydroxide (NaOH, 1 mol/mL) was added to neutralize to a certain pH. Then it was centrifugated (3 500 r/min, 15 min) and the supernatant was stored at -70 .℃

2.5 Detection indicators and methods

2.5.1 Hypothalamus and ovarian leptin levels

Radioimmunoassay was operated in strict accordance with the kit instructions.

2.5.2 Blood lipids

These tests include plasma TC, TG, LDL and HDL levels measurements. Biochemical methods were used in accordance with kit instructions.

2.6 Statistical methods

Conventional statistical software was used for statistical analysis. Measurement data were expressed as mean ± standard deviation (), nonparametric test was used for inter-group comparison, andP＜0.05 was statistically significant.

3 Results

3.1 Hypothalamus and ovarian leptin changes in natural menopausal rats and the effects of preventive acupuncture and moxibustion

Compared with the young control group, hypothalamus and ovarian leptin levels of rats in the 10-month-old group and the 12-month-old group showed an increasing trend, and those in the 12-month-old group were significantly higher (P＜0.01). Compared with the 12-month-old group, the hypothalamus and ovarian leptin levels of rats in the preventive moxibustion 12-month-old group and the preventive acupuncture 12-month-old group were significantly decreased (P＜0.01), (table 1).

3.2 Blood lipids levels in natural menopausal and the impact of preventive acupuncture

Compared with the young control group, the plasma TC and LDL levels in the 10-month-old group and the 12-month-old group showed an increasing trend, and these in the 12-month-old group were significantly increased (P＜0.05); whereas plasma HDL levels decreased. Compared with the 12-month-old, plasma levels of TC and LDL decreased, and HDL levels increased in the preventive moxibustion 12-month-old group and the preventive acupuncture 12-month-old group, the trend in the latter was more significant (bothP＜0.05 ), (table 2).

Table 1. Comparison of hypothalamus and ovarian leptin levels (, ng/mL)

Table 1. Comparison of hypothalamus and ovarian leptin levels (, ng/mL)

Table 2. Comparison of blood lipids in each group (, mmol/L）

Table 2. Comparison of blood lipids in each group (, mmol/L）

Note: Compared with the young control group, 1)P＜0.05, 2)P＜0.01; compared with the 12-month-old group, 3)P＜0.05

4 Discussion

Menopausal lipid metabolism disorders and abnormal body fat distribution are not only closely associated with common syndromes such as menopausal hot flashes, depression, muscle and joint pain, sweating[5], but also the risk of multiple diseases such as hypertension, hyperlipidemia, diabetes, osteoporosis, cardiovascular disease[6-7]. Blood lipid is a most basic indicator of the body metabolic state. TC is the total content of LDL, HDL and very low density lipoprotein (VLDL). LDL particles are small and easyto enter the arterial intima and oxidized. They were swallowed by macrophage evolved from blood mononuclear cells and become foam cells, which accumulates to fat lines. The cell rupture releases large amount of cholesterol and becomes the main component of the lipid plaque core. By combining with cholesterol in arterial wall, serum HDL promotes cholesterol outward transporting to liver catabolized to bile acid and excreted out of the body[8]. Elevated serum TG levels can lead to small dense LDL particles and increased HDL levels, which form high TG, low HDL and LDL particles triad, resulting in excessive lipids accumulation. Menopause is a period during which women switch from vigorously sexual maturity to old age. In this period dramatic internal environment changes lead to multiple body system dysfunction. Studies have confirmed that lipid metabolism disorder is one characteristics of menopause[9]. Estrogen levels decline in menopausal women, which is an important factor in lipid metabolism disorder[10-11]. Studies have shown that TC, TG and weight of postmenopausal women increased significantly compared with those in premenopausal women[12-14].

Leptin is secreted primarily by white adipose tissue. Its amount is proportionally to the body fat. The physiological effects are mediated by leptin receptor B, which is mainly expressed in the hypothalamus. Leptin joins cerebrospinal liquid through choroid plexus of the brain and enters the third ventricle. Leptin then diffuses into the hypothalamus through the ependymal layer, and binds to receptors on arcuate nucleus and paraventricular nucleus to regulate the hypothalamus satiety center, and reduce fat amount by inhibiting neuropeptide Y (NPY) transcription[15]. In addition, leptin can act directly on adipose tissue to consume fat.

Leptin levels in obese people were significantly higher than non-obese, indicating that there may be an endogenous leptin resistance in human obesity[16], and leptin resistance is an important reason of abdominal fat accumulation in menopausal women[17]. In situ hybridization techniques have shown OB-Rb expressions in ovary cells, indicating that leptin affects ovarian function directly through OB-Rb[18]. Studies have shown that leptin levels and weight increase during menopause[19]. Leptin also exerts important influence on lipid metabolism. Leptin levels were positively correlated with LDL, TG and apolipoprotein B (ApoB) in menopausal women[20-22]. Hormone replacement therapy can improve the leptin levels in postmenopausal women, thus reduce LDL, TG and increase HDL levels[23].

In this study, TC and LDL levels in 12-month-old rats were significantly higher than those in the young control group and the 10-month-old group, while HDL levels decreased, suggesting the abnormal lipid levels in menopause as a trend of fat accumulation. Meanwhile, hypothalamus and ovarian leptin in the 12-month-old rats were significantly higher than those in the young control group and the 10-month-old group, which may be due to endogenous leptin resistance caused by menopause disorders and fat accumulation, i.e., less leptin receptor binding resulting in its inadequate physiological function. Compared with the nature 12-month-old rats, plasma TC and LDL levels decreased, and HDL levels increased in those in the preventive acupuncture 12-month-old and the preventive moxibustion 12-month-old group, indicating that acupuncture and moxibustion can adjust the dyslipidemia in menopausal rats, prevent fat accumulation, and the effects of preventive acupuncture was more obvious. After preventive acupuncture and moxibustion therapy, hypothalamus and ovarian leptin levels maintain at the level of the young control group, suggesting preventive acupuncture and moxibustion reduces leptin resistance. Combined with previous findings[1-3], we believe that preventive acupuncture and moxibustion have a broad regulatory role from centre to the peripheral. Through peripheral stimulation, its benign pre-stress signals was transmitted to the centre and integrated, causing signal activation of nervous, endocrine and immune systems, and thus play an active role in the prevention and treatment of subsequent inner environment disorder.

In summary, preventive acupuncture and moxibustion can adjust the hypothalamus and ovarian leptin levels and restore its physiological function to prevent fat accumulation trend, and thus play a preventive and therapeutic role in menopausal metabolic disorder and slow the early menopausal inner environment disorder. Multi-system and multitarget regulation from the central to the peripheral is a notable feature of preventive acupuncture and moxibustion.

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Translator: Feng Xiao-ming

R2-03

A

Date: May 6, 2013

Author: Zhu Shi-peng, doctoral degree candidate

Li Xiao-hong, M.D., professor, doctoral supervisor.

E-mail: xiaohong_li@sina.com