Experimental study on the improvement of insomnia model mice's symptoms by Sanwei Anmian Decoction and its disassembled prescription

Yu-Kun Chen ,Fan Yun ,Xiao-Fan Wang ,Shi-Ya Feng ,Han Yang ,Shu-Guang Wei ,Nuo-Nan Zhang,Gui-Xiang Zhao,Yu Kong,Ke Dong,Jin-Chan Xia

1Longhua Hospital affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China;

2Hubei University of Chinese Medicine,Wuhan,430065,China;

3Dongfang Hospital Beijing University of Chinese Medicine,Beijing 100078,China;

4School of Life Sciences,Beijing University of Chinese Medicine,Beijing 100029,China;

5Dongzhimen Hospital Affiliated to Beijing University of Traditional Chinese Medicine,Beijing 100007,China;

6First Affiliated Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou 450000,China;

7The First Affiliated Hospital of Anhui University of Chinese Medicine,Hefei 230038,China;

8First Clinical Medical College of Henan University of Traditional Chinese Medicine,Zhengzhou 450046,China;

9College of Basic Medicine,Henan University of Traditional Chinese Medicine,Zhengzhou 450008,China.

Abstract Background:Insomnia is a sleep disorder,which seriously affects people's physical and mental health.Our experimental study will further explain the efficacy and mechanism of the traditional Chinese medicine prescription Sanwei Anmian Decoction as well as the single drug treatment for insomnia,and provide scientific basis for TCM decoction to cure more insomnia patients.Methods:42 mices,half male and half female,were randomly divided into 7 groups,6 in each group,Blank group,Model group,Positive group,Sanwei Anmian Decoction group (abbreviated as Sanwei group),Suanzaoren group,Maidong group,Yuanzhi group.Intraperitoneal injection (ip) of PCPA 350 mg·kg-1 in other groups except Blank group to establish a mice model of insomnia.On the next day after the successful modeling,10ml·kg-1 corresponding decoction was given to Sanwei group,Suanzaoren group,Yuanzhi group and Maidong group respectively,0.8mg · kg-1 estazolam for Positive group,Blank group and the Model group with the same amount of normal saline,three times a day for two days.The body weight and the number of autonomous activities of mice were observed.The sleep rate,sleep latency and sleep time were recorded after the last administration,and the contents of serum 5-hydroxytryptamine (5-HT) and dopamine (DA) were detected.Results:The changes of hair yellowing,alarm,food loss and weight loss in mice showed that the model was successful.After the last administration,compared with the Model group and the Blank group,the weight of mice in Sanwei Anmian Decoction and each disassembled prescription group increased,the autonomous activity decreased,the number of mice fell asleep increased,the concentration of 5-HT in serum increased and the concentration of DA decreased,P<0.01,which was statistically significant.Conclusions:Sanwei Anmian Decoction can effectively improve the symptoms of PCPA insomnia mice model.For single drug,Suanzaoren Decoction has the most obvious effect on the treatment of insomnia.

Keywords:Sanwei Anmian Decoction,Chlorophenylalanine,Insomnia,Mice,Experimental study

Introduction

Insomnia is a kind of sleep disorder disease,which is a common clinical manifestation of many kinds of physical,mental and behavioral diseases,which seriously affects people's physical and mental health,quality of life and work efficiency [1,2].Western medicine believes that the pathogenesis of insomnia mainly involves a large number of central nervous system neural network and series of neural media,neuroendocrine and neuroregulatory substances and other disorders [3,4].In the treatment of insomnia,drug-induced sleep is often used,which is more prominent in improving sleep quality and daytime function [5].However,long-term mass use can lead to drug resistance and drug dependence [6,7].Chinese medicine believes that insomnia is a kind of disease syndrome characterized by the loss of mind or restlessness,which often leads to the failure to obtain normal sleep,which is due to the lack of spirit preservation or being agitated [8].According to TCM,insomnia can be divided into five types:liver-fire disturbing heart type,phlegm-heat disturbing heart type,heart spleen deficiency type,heart-kidney disjunction type and heart courage Qi deficiency type[9,10].This experiment is to explore the experimental effect and molecular mechanism of Sanwei Anmian Decoction and its disassembled prescriptions in the treatment of insomnia mice caused by PCPA,so as to provide reference for the treatment of insomnia and other sleep disorders by traditional Chinese medicine in the future [11].

Materials and Methods

Animal

Forty-two healthy Kunming mice,male and female,each half,weighing (20 ± 2) g.

Drugs

Semen Ziziphi spinosae,Radix Maidongis and Radix Polygalae 60g each; para-chlorophenylalanine (PCPA,produced by sigma company of the United States);5-hydroxytryptamine (5-HT,Nanjing Jiancheng Institute of biology Co.,Ltd.); mice dopamine ELISA Kit (Nanjing Jiancheng Institute of biology Co.,Ltd.);estazolam (Linfen Baozhu Pharmaceutical Co.,Ltd.)and pentobarbital sodium 5g/ bottle (1 bottle); normal saline;distilled water; NaOH solution; 75% ethanol.

Instrument

Bas224s electronic balance,produced by saiduolis Scientific Instrument Co,Ltd(Beijing); yls-1b multi-functional rat independent activity recorder,equipment station of Shandong Academy of Medical Sciences; full automatic multi-functional enzyme labeling instrument,multi-skanmk3,thermo,USA;electric thermostatic incubator,dh4000a,Tianjin taist.

Trial preparation

The adult dosage of Sanwei Anmian Decoction was 15 g of Semen Ziziphi Spinosae,3 g of Ophiopogon japonicus and 3 g of Polygala Radix,which was converted into 2.25 mg/ g of Semen Ziziphi Spinosae,0.45 mg/ g of Ophiopogon japonicus and 0.45 mg/ g of Polygala Radix in mice.That is to say,in mice three flavor group,Semen Ziziphi Spinosae (13.5g),Ophiopogon japonicus (2.7g),Polygala Radix (2.7g)(when the weight of mice is 20g).Decoct Sanwei Anmian Decoction at the concentration of 0.1g/ml crude drug.First add cold water (95ml) equal to 5 times of the amount of medicinal materials to soak for 1h,decoct for 30min,then add 5 times of water (95ml)to decoct for 30min,then filtrate,and filter the liquid with double gauze.Other traditional Chinese medicine groups were the same as above,the final Decoction of Sanwei group was 9.45ml,that of Suanzaoren group was 6.75ml,that of Maidong and Yuanzhi groups was 1.35ml,stored at 4 ℃,and pre heated at 20 ℃[4,12].

Grouping and drug administration

42 mice were randomly divided into Blank control group,Model group,Positive control group,Sanwei group,Suanzaoren group,Maidong group and Yuanzhi group.Each group was fed with 10 mice for 2 days.The drug was administered 2 days later,and the body weight was weighed every day.①The Blank group normal feeding,no modeling,and infused with the same volume of normal saline as the traditional Chinese medicine groups.② The Model group,mice were injected with PCPA 15ml · kg-1intraperitoneally,with the concentration of 2%,and then infused with the same volume of normal saline as the traditional Chinese medicine group.③The Positive group,successfully established the insomnia model,and then infused with 2ml · kg-1,the concentration of the drug was 0.4% estazolam for treatment.④ In the three taste group,10 ml · kg-1was infused into the stomach after the insomnia model was established successfully,and the concentration of the drug was 200%,among which the ratio of Semen Ziziphi Spinosae,Ophiopogon japonicus and Polygala Radix was 5:1:1.⑤In the Suanzaoren group,after successfully establishing a mice model of insomnia,gavage 10 mL·kg-1with 200% jujube seed for treatment.⑥ In the Maidong group,after successfully establishing a mice model of insomnia,gavage 10 mL·kg-1with 200%Ophiopogon japonicus for treatment.⑦ In the Yuanzhi group,after successfully establishing a mice model of insomnia,gavage 10 mL·kg-1with a concentration of 200% Polygala for treatment [8,13].

Model preparation

Intraperitoneal injection of PCPA 350mg · kg-1in other groups except blank to establish insomnia mice model.The specific methods are as follows:firstly,20mg · mL-1PCPA suspension was prepared [14].According to the accurate pH test paper,NaOH is diluted to a weak alkaline solution with pH value of about 7.8,and PCPA and weak alkaline solution are mixed with a mixer to prepare a PCPA suspension with concentration of 20mg · ml-1.Then,PCPA is injected intraperitoneally in the proportion of 15ml · kg-1for each mice,for two consecutive days,after the model was established,the mice developed yellow hair,alarm,food loss,weight loss,and so on,which indicated that the model was successful [8].

Detection index

Test the body weight,autonomous activity,sleep rate,sleep latency,sleep time,5-HT,DA of mice.Before and after the establishment of the model and the treatment,the autonomic activities of the mice were recorded [15,16].

Detection methods

The sleep rate,sleep latency and sleep time of the mice were detect by the subthreshold hypnotic dose test of pentobarbital sodium and the threshold dose hypnotic dose test of pentobarbital sodium [17,18].The changes of body weight of mice in each group were observed before and after the first administration every day.The mice in each group were put into the spontaneous activity instrument before administration and adapted to it for 5 minutes.Then the spontaneous activity of 5min mice was observed and the values were recorded.After the first administration of the drug on the third day,the spontaneous activity of mice was observed by the above-mentioned method,the values (decimal spontaneous activity count and standing times) were recorded,and the defecation of mice (defecation granules) was observed.Subthreshold hypnotic dose test of pentobarbital sodium.Selection of pentobarbital sodium dose:the subthreshold hypnotic dose of pentobarbital sodium is 34.5mg ·kg-1,that is,the maximum threshold dose of pentobarbital sodium that does not disappear from righting reflex in mice.The solution was made into aqueous solution according to 3mg ·mL-1,and the injection dose was 7.5ml ·kg-1.50 minutes after the first administration on the second day,the mice of each group were intraperitoneally injected with ip pentobarbital sodium solution at the dose of 7.5ml ·kg-1,and the disappearance of 30min inverted reflex was used as an index.When the mice is placed in the dorsal recumbent position,it can turn the body immediately.For example,if it cannot be turned for more than 30 seconds,the righting reflex disappears,that is,it goes to sleep; the righting reflex recovers,that is,the animal awakens.The sleep phenomenon of mice in each group was observed after administration,and the sleep number of mice in each group was recorded [19,20].Suprathreshold hypnotic dose test of pentobarbital sodium.Selection of pentobarbital sodium dosage:when pentobarbital sodium was given to mice,the disappearance of righting reflex above 1min in 30min indicated that it entered the sleep state.Before the experiment,the dose of pentobarbital sodium,which made the mice fall asleep completely but did not sleep too long (< 10min),was determined as 50.6mg ·kg-1,as solution according to 3.5mg ·mL-1,and the administration capacity was 11ml·mL-1.Each group was given ip pentobarbital sodium solution at the dose of 35mg·kg-1.The injection time of pentobarbital sodium,the time of falling asleep and the end of sleep in mice were recorded.The difference between the time of falling asleep and the time of injection was regarded as the latency of sleep,and the difference between the end of sleep and the time of falling asleep was counted as the time of sleep.The experiment was conducted alternately between the treatment group and the control group to observe the effects of the combination of tested drugs and pentobarbital sodium on shortening the sleep latency and prolonging the sleep time in mice [21].At the end of the experiment,the rats were killed in the head,the brain was taken out,the brain stem was quickly separated on the ice plate,the body weight was weighed,the ice bath homogenate was taken,centrifuged at 4℃ (3000r · min-1,10min),the supernatant was taken,and the 5-HT and DA were detected by ELISA [22,23].

Statistical methods

The experimental data were expressed as mean ±standard deviation (x ± s).All data were statistically processed by SPSS 21.0 software,P< 0.05,which was statistically significant.

Results

Establishment of insomnia mice model induced by PCPA

We refer to Jalili et al's "establishing insomnia mice model by intraperitoneal injection of PCPA",and establish a mice insomnia model by intraperitoneal injection of PCPA [24].After 24 hours of modeling,the characterization and autonomous activity of the mice were observed,and it was found that compared with the blank group,the mice in the Model group showed symptoms such as lethargy,slow action,huddling,fur standing and dull,shortness of breath,dull and haggard coat color,etc.,which indicated that the model was successful.

Effect of S anwei Anmian Decoction and i ts disassembled prescription on bo dy weight of insomnia mice

Body weight is an important index to reflect the sleep state of mice,mice insomnia will appear food loss,weight loss and other changes,mice sleep state improvement,eating increase,weight gain [25,26].From Table 1 and Figure 1 that the overall weight loss of mice after modeling,for example,the weight of mice in the Model group decreased to 94.65% of the original.After giving the corresponding traditionalChinese medicine,the body weight of mice in Sanwei group,Suanzaoren group,Maidong group and Yuanzhi group increased in varying degrees,for example,the body weight of mice in Sanwei group increased from 94.69% before administration to 99.59%

Table 1 Changes of body weight of mice before and after modeling and administration (unit:g)

Figure 1 Changes of body weight of mice before and after modeling and administration

Effects of Sanwei Anmian Decoction and its disassembled prescriptions on autonomous activity and standing number in insomniac mice

There is an important relationship between the number of autonomous activities and insomnia in mice [27].Insomnia mice are easy to startle and increase their autonomous activities [28].From Table 2 and Figure 2,we found that the number of autonomous activities of mice increased after modeling,such as the Model group,and the autonomous activity increased from 100% to 122.91% after modeling.After the administration of the corresponding Chinese medicine,the independent activity of mice in Sanwei group,Suanzaoren group,Maidong group and Yuanzhi group decreased in varying degrees,for example,the autonomous activity of mice in Sanwei group decreased from 126% to 95% before administration.

Table 2 Changes of autonomic activity in mice before and after modeling and administration (unit:time/5min)

Figure 2 Changes of autonomic activity in mice before and after modeling and administration

Effects of Sanwei Anmian Decoction and its disassembled prescriptions on Sleep latency and Sleep duration induced by threshold dose of pentobarbital Sodium in mice

Sleep latency and sleep time are important factors to reflect the sleep quality of mice [21].The shorter the sleep latency,the faster the mice fall asleep,the longer the sleep time,the better the sleep quality of mice[16,27,29].As can be seen from Table 3 and Figure 3,Sanwei Anmian Decoction and its disassembled prescription group can shorten the sleep latency and prolong the sleep time.For example,compared with the Model group,the sleep latency of Sanwei Anmian Decoction (4 ±1.4min) was lower than that of the Model group (8 ±5.7 8.4min),while the sleep time (23±8.4min) of the Model group was longer than that of the Model group (18 ±min).

Table 3 Changes of sleep time and sleep latency in each group after administration

Figure 3 Changes of sleep time and sleep latency in each group after administration

Effects of Sanwei Anmian Decoction and its disassembled prescriptions on the contents of DA and 5-HT in Serum of Insomnia mice

The mice in Sanwei group,Suanzaoren group,Maidong group and Yuanzhi group were all increased in weight and the times of autonomous activity,the sleeping rate was increased,the sleep latency was shortened,and the sleeping time was prolonged.Among them,Sanwei group has the most obvious effect on improving mice's autonomous activities,followed by Suanzaoren group,Positive group,Yuanzhi group and Maidong group.It can be seen from Table 4 that the concentration of 5-HT in Sanwei group is higher than that in the Model group,and the concentration of DA is lower than that in the Model group,P < 0.01.DA concentration of Suanzaoren group was lower than that of Model group (P < 0.01),5-HT concentration of Maidong group was higher than that of Model group (P < 0.01),DA concentration was lower than that of Model group (P < 0.05).DA concentration in Yuanzhi group was lower than that in Model group (P < 0.05).By comparing the content of DA and 5-HT between the Model group and the treatment groups,we can see the therapeutic effect of Sanwei Anmian Decoction and its disassembled prescriptions on insomnia in mice.

Table 4 The concentration of 5-HT and DA in each group after administration

Discussion

Significance of changes in body weight and voluntary activities of insomnia mice

After intragastric administration in each group,the body weight of the mice increased at the end of the experiment and was similar to the body weight before modeling,and the final weight between the groups was also similar,indicating that the insomnia condition of the mice was improved.It can also show that Sanwei Anmian Decoction and its separate components have no adverse effects on the weight gain of mice.

Through the analysis of the influence of Sanwei Anmian Decoction and its decomposed formulas on the number of autonomous activities and standing times of insomnia mice,it is concluded that after modeling,the number of autonomous activities and standing times of the drug-administered group showed an overall increasing trend,and the sleep time was significantly reduced.On the first day of gavage and the second day of gavage,after the respective groups were given intragastric administration,the number of autonomous activities and standing times of the mice had a significant decrease.Among them,Sanwei Anmian Decoction had the greatest impact on the number of autonomous activities of mice.Followed by Suanzaoren Decoction,Positive group,Yuanzhi Decoction and Maidong Decoction.Experiments have shown that these substances can reduce the number of autonomous activities and standing times of mice,and prolong sleep time.Modern pharmacological studies have shown that steroidal saponins are one of the main active ingredients of Ophiopogon japonicus [30,31].The high isoflavones of Ophiopogon japonicus can improve the sleep status of mice to varying degrees,and have little effect on autonomous activities.The Sanwei Anmian Decoction is a combination of Chinese traditional Chinese medicines such as Semen Ziziphi Spinosae,Ophiopogon japonicus and Polygala Radix.Its effects can inhibit the central nervous system,have a relatively constant sedative effect,and have obvious effects.Therefore,the number of autonomous activities of the mice is significantly reduced.

Significance of falling asleep rate,sleep latency and sleep time in insomnia mice

It can be seen from the results that the Sanwei Anmian Decoction and its decomposed formulas increased the falling asleep rate of the mice,prolonged the sleep time of the mice to various degrees,and shortened the sleep latency of the mice.The comparison of experimental data shows that the efficacy of Sanwei group and Yuanzhi group is more obvious,followed by the Suanzaoren group,Positive group,and Maidong group.In recent years,scholars at home and abroad have conducted a large number of studies on the chemical composition of Semen Ziziphi Spinosae,which shows that in addition to general nutrients,such as carbohydrates,protein and oil,Semen Ziziphi Spinosae also contain a variety of biologically active ingredients,such as saponins and flavonoids [32].Compounds,alkaloids,triterpenes,etc.Jujube seed contains up to 32% fatty oil.The saponins contained in Semen Ziziphi Spinosae are unanimously considered as the effective part of the sedative and hypnotic effect of jujube seed [33].The results showed that compared with the blank control group,mice in the wild jujube seed oil intragastric administration group had a shorter sleep latency,and sleep longer.It shows that Suanzaoren Decoction can increase the falling asleep rate and prolong the falling asleep time of mice.Modern pharmacological studies have shown that steroidal saponins are one of the main medicinal active ingredients of Ophiopogon japonicus Ophiopogon japonicus high isoflavones,which can improve the sleep status of mice to varying degrees,but experimental data show that Ophiopogon japonicus is effective in insomnia mice.The improvement effect of the condition is weak and not statistically significant.Polygala Radix increased the falling asleep rate of mice,significantly prolonged the sleep time of mice,and shortened the sleep latency of mice.

Significance of changes in serum DA and 5-HT levels in insomnia mice

By comparing the contents of DA and 5-HT in the Model group and the treatment groups,we can clearly see the therapeutic effect of Sanwei Anmian Decoction and its decomposed formulas on insomnia in mice.By comparing the Positive group and the traditional Chinese medicine group,we can see that the therapeutic effect of Sanwei Anmian Decoction is extremely significant.The curative effect of Suanzaoren group,Maidong group and Yuanzhi group was slightly weaker than that of the Positive group.In single herb groups,Suanzaoren Decoction was the most obvious,and Yuanzhi decoction was the weakest.Comprehensive comparison,Sanwei Anmian Decoction had the best effect [34,35].

Prospects and reflections

After continuous exploration by scholars all over the world in recent years,the methods of traditional Chinese medicine in the treatment of insomnia are gradually increasing,which further shows the superiority of traditional Chinese medicine.But the existing problems cannot be ignored.The compound prescription of traditional Chinese medicine has the complex characteristics of multi-components and multi-systematics.In order to clarify its specific mechanism,it is necessary to make an in-depth study from the aspects of biochemistry and pharmacology,so as to guide the clinic.There are many treatment methods which are simple,cheap and suitable for long-term recuperation,but the promotion work is not enough.Traditional Chinese medicine has its own unique advantages in the treatment of insomnia,compared with western medicine,the curative effect is stable,not easy to relapse,and there is no addiction and dependence,but there are less drugs to directly promote sleep,more efficacy and less explanation of drug mechanism.PCPA insomnia model is currently recognized as an insomnia model,which has been widely used in the classical 5-HT animal model and its relationship with other transmitters [36,37].In recent years,a large number of studies on the chemical constituents of Semen Ziziphi Spinosae by scholars at home and abroad have shown that Semen Ziziphi Spinosae not only contains general nutrients,such as carbohydrates,protein oils,but also contains a variety of bioactive components,such as saponins,flavonoids,alkaloids,triterpenoids and so on.Semen Ziziphi Spinosae contains up to 32% fatty oil,and the saponins contained in Semen Ziziphi Spinosae are considered to be the effective parts of the sedative and hypnotic effect of Semen Ziziphi Spinosae [32,38].Therefore,the insomnia mice model was established by intraperitoneal injection of PCPA.The overall aspect is to explore the experimental effect and molecular mechanism of Sanwei Anmian Decoction in the treatment of mice with insomnia caused by chlorophenylalanine,and the single medicine is to obtain the specific data in the mice experiment through the control experiment.More specifically analyze which of the three traditional Chinese medicine decoction has a better sedative effect,in order to provide reference for the treatment of insomnia and other sleep disorders in the future.The results of this study show that Sanwei Anmian Decoction can significantly improve the curative effect of insomnia,and the curative effect of Semen Ziziphi Spinosae is the most obvious in terms of single traditional Chinese medicine.The mechanism of its treatment of insomnia may be related to the regulation of the balance of neurotransmitters in the brain [39,40].Our experimental study will further explain the efficacy and mechanism of traditional Chinese medicine prescription and single drug in the treatment of insomnia,and provide a scientific basis for Sanwei Anmian Decoction,a simple,cheap and effective traditional Chinese medicine decoction to cure more patients with insomnia.Thus it can make a contribution to the treatment of insomnia and provide an economic and practical choice.However,the experimental period is short,the dose of traditional Chinese medicine is small,and the effect is too late.There are some errors in the experimental data,which will affect the accuracy of the experimental results to a certain extent.

Conclusion

Sanwei Anmian Decoction can effectively treat the symptoms of PCPA insomnia model mice,prolong sleep time and shorten sleep latency.Suanzaoren Decoction has the most obvious effect in treating insomnia in the single Chinese medicine groups.In order to further verify the efficacy of Sanwei Anmian Decoction and single medicine,further research is needed.