Anshen Babu Ointment Improves Sleep Function of Insomnia Mice and Its Mechanism

Qin GONG, Xiaoxue GONG, Sen LIU, Yundan LI, Xi WANG

1. Department of Pharmacy, Shiyan Hospital of Traditional Chinese, Shiyan 442012, China; 2. Shiyan Taihe Hospital/Sleep Disorder Research Center, Affiliated Hospital of Hubei University of Medicine, Shiyan 442000, China

Abstract [Objectives] This study aimed to study the sleep-improving effect of Anshen Babu ointment on insomnia mice and its mechanism of action. [Methods] Mouse models of insomnia were established by p-chlorophenylalanine (PCPA) injection. They were given high, middle and low doses of Anshen Babu ointment, for 7 consecutive days, and then, the spontaneous activity of insomnia mice and the effect on hypnotic time and subthreshold hypnotic dose of pentobarbital sodium were observed, and the levels of 5-HT, IL-6 and TNF-α in the hippocampus of mice were determined. [Results] High and middle-dose Anshen Babu ointment significantly reduced the spontaneous activity of insomnia mice and shorten the hypnotic time of pentobarbital sodium; high and middle-dose Anshen Babu ointment increased the 5-HT content and inhibited the IL-6 and TNF-α expression in the hippocampus of mice. [Conclusions] Anshen Babu ointment has hypnotic and sedative effect on insomnia mice. The mechanism may be achieved by increasing 5-HT and reducing IL-6 and TNF-α levels.

Key words Anshen Babu ointment, Insomnia improvement, Pentobarbital sodium, 5-HT, Cytokines

1 Introduction

Sleep is a normal physiological activity to maintain the homeostasis of the organism[1]. In China, nearly 40% of the adult population has varying degrees of insomnia. Symptoms of insomnia include "being unable to take a nap", "being unable to fall asleep", "unwilling to sleep",etc., bringing great harm to human physical and mental health and social development[1-2]. The cause and pathogenesis of insomnia are not clear. At present, the treatment is mainly based on western medicine. Due to the resistance, dependence and obvious adverse reactions of western medicine, patient’s medication compliance and treatment effects are not ideal. Traditional Chinese medicine has sedative and hypnotic functions. In recent years, newly developed clinical reports for the treatment of insomnia are more common, and are widely favored by patients[3-4]. Anshen Babu ointment is a topical applicator-type soothing preparation independently developed by this research group. It is formulated with wild jujube seed, notoginseng flower, acanthopanax root,etc. according to the optimal ratio concluded by orthogonal experiment. In this study, the effect of Anshen Babu ointment on the spontaneous activity of insomnia mice, that is, its nerve-calming effect was investigated, so as to provide experimental basis for the treatment of insomnia.

2 Materials and methods

2.1Materials

2.1.1Drugs and reagents. P-Chlorophenylalanine (PCPA) (Sigma, batch No.1002094969); sodium pentobarbital (Sigma, batch No. P3761-25g); mouse TNF-α ELISA kit (Mitaka, code KE10002); mouse IL-6 kit (Mitaka, code KE10007); mouse 5-HT ELISA kit (Shanghai Kemin Biotechnology Co., Ltd., code KB2982).

Jujube seed, notoginseng flower, acanthopanax root, acorus rhizome, polygala root, anemarrhena rhizome, coptidis rhizome were concentrated in ethanol according to the proportion into an extract. After dried, it was mixed with a matrix and coated on Anmian paster (the formula was from Professor Liu Jishan, a famous Chinese medicine teacher in Hubei) to prepare Anshen Babu ointment.

2.1.2Animals. ICR male mice, SPF grade, 6 weeks old, weighing 17.3-21.6 g, were purchased from the Experimental Animal Center of Hubei University of Medicine[license SYXK (Hubei) 2016-0031]. There were 5 mice in each cage. The room temperature was controlled at 20-25 ℃. The light cycle was set as 12 h light/12 h dark. After 4 d of adaptation, the mice were used for experiment.

2.1.3Instruments and equipment. The main instruments and equipment used were YLS-1A multifunctional mouse spontaneous activity recorder (Beijing Zhongshi Dichuang Co., Ltd.), Epoch full-wavelength microplate reader (BioTek, USA) and electronic balance (METTLER TOLEDO, Switzerland).

2.2Methods

2.2.1Establishment of insomnia mouse models. Mouse models of insomnia were established by injecting p-chlorophenylalanine (PCPA) into mice. PCPA powder was dissolved in physiological saline (pH 7.4) according to 1 mL/100 g. At 9:00 am every day, 300 mg/kg of PCPA was injected intraperitoneally to the mice, for 2 consecutive days. After 30 h of the first injection, if it was observed that the overall behavior of the mice was significantly different from that of untreated mice, such as they were sensitive to sound and light, their day and night rhythm disappeared, their activities were active during the day, and the intake and defecation movements became disorderly, it indicated that the modeling was successful.

2.2.2Effect on spontaneous activity of mice. The ICR insomnia mice were randomly and evenly divided into control group and high, middle and low-dose Anshen Babu ointment groups, 10 mice in each group. According toPharmacologicalExperimentMethodology[5], the administration dose was calculated according to the body surface ratio of a 60 kg adult to a 6-week-old 20 g mouse. The normal dose (middle dose) was 5.8 mg/kg, and the high and low doses were set as 11.6 and 2.9 mg/kg. The mice in the blank group were given only matrix. Each of the mice was depilated from the Shenque acupoint[6], treated with Anshen Babu ointment, 1 h every day, for 7 consecutive days. After 1 h of the last treatment, the mice were placed in the multifunctional spontaneous activity recorder. After 5 minutes of acclimatization, the number of spontaneous activities of the mice within 3 min was recorded.

2.2.3Extending the hypnotic time of pentobarbital sodium. Forty ICR mice were treated in the same way as Section2.2.2. After 1 h of the last administration, sodium pentobarbital was injected intraperitoneally to the mice according to a dose of 47.5 mg/kg (conclusion of pre-experiment). The interval from giving pentobarbital sodium to the disappearance of the righting reflex (i.e., the latency to fall asleep) and the interval from the disappearance and the recovery of the righting reflex (i.e., the duration of sleep) were recorded.

2.2.4Effect on subthreshold hypnotic dose of pentobarbital sodium. Forty ICR mice were treated in the same way as Section2.2.2. After 1 h of the last administration, sodium pentobarbital was injected intraperitoneally to the mice at a dose of 32 mg/kg (conclusion of pre-experiment). The number of mice whose righting reflex disappeared for more than 1 min within 30 min was counted.

2.2.5Determination of serotonin and cytokine contents in mouse hippocampus. Forty ICR mice were treated in the same way as Section2.2.2. After the last administration, the mice were sacrificed by cervical dislocation, and the hippocampus tissue of the mice was quickly removed from the ice box and weighed. The hippocampus tissue was added with 1 mL of pre-cooled physiological saline, homogenized and centrifuged (4 ℃, 12 000 g, 30 min) to collect the supernatant. The contents of 5-HT and cytokines IL-6 and TNF-α were determined by ELISA.

3 Results and analysis

3.1EffectonthefrequencyofspontaneousactivityininsomniamiceCompared with the control group, the number of spontaneous activities in the low-dose Anshen Babu ointment group did not change significantly within 2 min (P>0.05), while the middle and high doses of Anshen Babu ointment significantly reduced the number of spontaneous activities of the mice (P<0.01) (Table 1).

Table 1 Effect of Anshen Babu ointment on spontaneous activity of insomnia mice and hypnotic time of pentobarbital sodium (n=10)

3.2EffectonprolongingthehypnotictimeofpentobarbitalsodiumAs shown in Table 1, low dose of Anshen Babu ointment showed no significant influence on the hypnotic time and sleep latency of pentobarbital sodium in the insomnia mice (P>0.05), while high and middle-dose Anshen Babu ointment increased the sleep duration and shortened the sleep latency of the mice (P<0.05,P<0.01).

3.3EffectonsubthresholdhypnoticdoseofpentobarbitalsodiuminmiceThe number of mice that had been fallen asleep in the control group and low, middle and high-dose Anshen Babu ointment groups was 2, 3, 7 and 6, with incidence of 20%; 30%, 70% and 60%, respectively (Table 2). There were significant differences between the middle and high-dose Anshen Babu ointment groups and the control group (P<0.01).

Table 2 Effect of Anshen Babu ointment on subthreshold hypnotic dose of pentobarbital sodium in mice (n=10)

3.4Effectonhippocampus5-HT,IL-6andTNF-αlevelsofinsomniamiceCompared with the control group, the 5-HT, IL-6 and TNF-α levels in the hippocampus tissue of the mice in the low-dose Anshen Babu ointment group did not change significantly (P>0.05), while in the middle and high-dose Anshen Babu ointment groups, the 5-HT content increased, and the IL-6 and TNF-α expression was inhibited (P<0.05,P<0.01) (Table 3).

Table 3 Effect of Anshen Babu ointment on 5-HT, IL-6 and TNF-α levels in hippocampus tissue of the mice (n=10, pg/mL)

4 Discussion

In this study, the paster preparation was adopted. Compared with traditional oral Chinese medicine, it has the following advantages: (i) large drug load and suitability for Chinese medicinal extract; (ii) good biocompatibility with the skin, breathability, sweat resistance, no allergization and no irritation; (iii) good drug release performance, conducive to the transdermal absorption of the drug; (iv) convenience. The drug is absorbed through the skin through the paster carrier and has a direct effect. Drug absorption is not affected by complex factors such as pH value in the digestive tract and food and drug movement time in the intestine, avoiding the first pass effect the liver and gastrointestinal side effects[6-7]. In modern clinical treatment, umbilical therapy is widely used in various clinical departments. Applying on Shenque acupoint can stimulate the qi of the meridian, so as to dredge the meridian, reconcile the qi and blood, and adjust the balance of yin and yang of the viscera[8].Therefore, the study used acupoint application to study the function of Anshen Babu ointment to improve sleep in mice with insomnia.

In recent years, wild jujube seeds have been widely reported to have the effect of improving sleep. The main active ingredients of jujube seeds are saponins and total flavonoids, which improve sleep status by increasing neurotransmitters such as DA, 5-HT, NE and GABA[9-10]. Studies have shown that Suanzaoren Decoction can improve the learning memory of sleep deprived rats[11]; Suanzaoren Decoction combined with acupoint application can effectively improve anxiety and insomnia[12]. Suanzaoren Decoction combined with acupuncture is used to treat menopausal insomnia[13]. Ciwujia Suanzaoren Tianma Capsules can effectively improve sleep in mice[14]. The Chinese medicinal preparation adopted in this study derived from the experience of external treatment of insomnia by Professor Liu Jishan, a famous teacher of traditional Chinese medicine in Hubei, and has solid and reliable clinical effect. Anshen Babu ointment is composed of wild jujube seed, notoginseng flower, acanthopanax root, acorus rhizome, polygala root, anemarrhena rhizome, coptidis rhizome, Chinese angelica, dens draconis, nacre mother of pearl, amber powder and cinnabar, which are extracted by ethanol, mixed with matrix according to the proportion and coated on paster, with functions of calming the liver, clearing away liver heat, clearing away heart heat, nourishing blood and calming the nerves. In this study, it was found that normal and high-dose Anshen Babu ointment effectively reduced the number of spontaneous activities in insomnia mice, increased the sleep duration and shortened the sleep latency of mice, and significantly increased the percentage of mice which had fallen asleep under the hypnotic dose of pentobarbital sodium, indicating that Anshen Babu ointment has a synergistic effect with pentobarbital sodium. According to theTechnicalSpecificationforHealthFoodInspectionandEvaluation(2003 Edition)[15], it is suggested that Anshen Babu ointment can effectively improve sleep.

In addition to participating in inflammation, cytokines are also closely related to brain development, sleep and endocrine regulation[16]. Studies have shown that the expression of IL-6 and TNF-α is negatively correlated with the body’s sleep quality. On the one hand, the shorter the sleep time and the shallower the sleep degree, the higher the serum IL-6 content. Artificial injection of IL-6 to normal people will cause a significant reduction in REMS, accompanied by fatigue and inattention[16-17]. After hypoxic hypothermia sleep deprivation in rats, the levels of TNF-α and IL-6 in serum were significantly increased[18-19]. On the other hand, while treating or improving the symptoms of insomnia through medication, the TNF-α and IL-6 levels in the body will also decrease accordingly[20-21]. The results of this experiment also show the levels of IL-6 and TNF-α in the hippocampus of mice in the middle-dose and high-dose groups of Anshen Babu ointment were significantly lower than those in the control group (P<0.05), indicating that the mechanism of Anshen Babu ointment to improve sleep may be related to the regulation of the expression of immune cytokines IL-6 and TNF-α.