Effects of Chailong Jieyu Pill on Behavior, Monoamine ... · Monoamine Neurotransmitters.-Hydroxytryptamine, dopamine, and norepinephrine contents were reduced in each administration

Research ArticleEffects of Chailong Jieyu Pill on Behavior MonoamineNeurotransmitters and Corticosteroid Receptors in a RatModel of Anxiety Disorder

Guang-kui Feng 1 Xian-junMa1 Yin-yi Chen1 Guang-rong Bian1

Chao Yang2 and Bao-dong Gu1

1Department of Encephalopathy Lianyungang Affiliated Hospital Nanjing University of Chinese MedicineLianyungang 222004 China2Department of Rehabilitation Wuhan No 1 Hospital Wuhan 430000 China

Correspondence should be addressed to Guang-kui Feng lygfgk163com

Received 22 January 2018 Revised 21 April 2018 Accepted 7 May 2018 Published 31 May 2018

Academic Editor Youn C Kim

Copyright copy 2018 Guang-kui Feng et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Chailong Jieyu Pill (CJP) is composed of Radix Bupleuri Radix Scutellariae Rhizoma Pinelliae Preparata Radix Codonopsis RadixGlycyrrhizae preparata keel Concha Ostreae Concha Margaritifera Usta Rhizoma Zingiberis Recens and Fructus Jujubae CJPhas shown good clinical effects on improving anxiety disorders However as the mechanism underlying such benefits remainsunclear the aim of this study was to investigate the mechanism of action for CJP on anxiety-related behaviors in a rat model ofanxiety disorder After establishing a rat model of anxiety disorder using uncertain empty bottle stimulation rats were divided intocontrol model citalopram low-dose CJP and high-dose CJP groups After 1 month of administration effects of treatments on ratappearance body weight and open-field test scores were observed In addition hippocampal monoamine neurotransmitter (5-hydroxytryptamine dopamine and norepinephrine) contents were measured with an enzyme-linked immunosorbent assay andmRNAexpression ofmineralocorticoid receptor (MR) and glucocorticoid receptor (GR)weremeasuredwith reverse transcription-polymerase chain reaction CJP increased rat weight and this effect was increased in the high-dose CJP group compared with thecitalopram group (P lt 005) CJP also elevated open-field test scores compared with the citalopram group (P lt 005) While CJPdecreased monoamine neurotransmitter contents in rat hippocampus the regulatory effect of CJP on 5-hydroxytryptamine wasreduced compared with citalopram (P lt 001) CJP upregulated GR mRNA expression in both low-dose (P lt 005) and high-dose(P lt 001) CJP groups but only the latter significantly downregulatedMRmRNAexpression and showed enhanced effects comparedwith citalopram (P lt 005) Thus CJP likely exerted its significant antianxiety effect by diminishing monoamine neurotransmittersand regulating mRNA expression of MR and GR in the hippocampus of our rat model of anxiety disorder

1 Introduction

The incidence of anxiety disorder is increasing yearly witha lifetime prevalence of anxiety disorder of approximately41 in China [1] and up to 30 in Europe [2] Anxi-ety disorder is mainly induced by emotional discomfortdepression and stagnation of qi Indeed stagnation of liverqi and qi stagnation are key for pathogenesis Dispersingstagnated liver qi to relieve qi stagnation is the basic prin-ciple of treatment Our hospital preparation Chailong JieyuPill (CJP) was approved and licensed by Jiangsu Province

Hospital in 2004 CJP can disperse depressed liver energyregulate vital energy clear liver heat invigorate the spleen toremove phlegm and suppress tranquilize and calm adverse-rising energy Through 10 years of clinical practice we havediscovered that CJP elicits good clinical effects for improvinganxiety and insomnia This includes a study using a placebocontrol whereby 61 anxiety patients were observed usinga randomized double-blind method HAMA and self-madeTraditional Chinese Medicine (TCM) symptom scales wereutilized to assess symptoms before and after treatment whilethe Treatment Emergent Symptom Scale (TESS) was applied

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 5489215 9 pageshttpsdoiorg10115520185489215

2 Evidence-Based Complementary and Alternative Medicine

to evaluate adverse reactions Results of a 6-week observationperiod demonstrated that CJP could improve HAMA andself-made TCM symptom scale scoresMoreover effects weresignificant at 2 4 and 6 weeks indicating a progressive effectcompared with the placebo group Importantly the effectiverate was 931 and no adverse reactions were observed [3]

CJP is composed of Radix Bupleuri Radix ScutellariaeRhizoma Pinelliae Preparata Radix Codonopsis Radix Gly-cyrrhizae preparata keel Concha Ostreae Concha Margari-tifera Usta Rhizoma Zingiberis Recens and Fructus JujubaeModern research has confirmed that saikoside an importantcomponent of Radix Bupleuri plays a role in cholinergiceffects and regulates the digestive and nervous systems bysuppressing cholinesterase [4] Moreover Radix Bupleuridecoction elicited improvements in physical symptomsdepression and irritability [5 6] which are mainly associ-ated with regulation of hypothalamic-pituitary-adrenal axishyperfunction and brain monoamine neurotransmitters [5]Keel and Concha Ostreae have sedative and anticonvulsanteffects [7] whereas total glycosides extracted from highlycompatibleRadix Bupleuri keel andConchaOstreae can dra-matically reduce hippocampal neuronal cell death rates [8]inhibit anxiety and depression and improve rat behaviors [9]Radix Codonopsis can elicit resistance to fatigue and hypoxiaand enhance immunity [10 11] Ginger propanol extract canhave a cholagogic effect and inhibit 5-hydroxytryptamineIn addition to generating sedative antihypertensive andanticonvulsant effects Fructus Jujubae can increase ani-mal weight enhance muscle strength and evoke a certainantagonistic effect on 5-hydroxytryptamine and histamineHowever previous research has mainly focused on the roleand effect of CJP on depression indeed to our knowledgefew studies have examined the effect of CJP on anxiety In thisstudy we explored themechanism underlying the antianxietyeffects of CJP by observing effects in a rat model of anxietydisorder

2 Materials and Methods

21 Materials Clean male Sprague-Dawley rats were pro-vided by the Animal Experimental Center of NanjingUniver-sity of Chinese Medicine of China [License number SCXK(Su) 2013-0003] All rats were acclimated for 1 week Fortyrats weighing 200 plusmn 20 g with similar behaviors as screenedby open-field test were selected and randomly assignedto control (no treatment or anxiety model induction) andanxiety model groups model (untreated) citalopram low-dose CJP and high-dose CJP All procedures were approvedby the Animal Ethics Committee of Lianyungang AffiliatedHospital Nanjing University of Chinese Medicine (NanjingChina)

CJP was provided by the Manufacturing LaboratoryLianyungang Affiliated Hospital Nanjing University of Chi-nese Medicine Drug preparation (1) Radix Bupleuri RadixScutellariae Radix Glycyrrhizae preparata keel ConchaOstreae Concha Margaritifera Usta Rhizoma ZingiberisRecens and Fructus Jujubae were decocted twice with waterfirst for 2 h and then for 1 h The filtrate was mixed and

concentrated to a moderate amount (2) Rhizoma PinelliaePreparata and Radix Codonopsis were triturated into powderwhich was mixed with the above-described concentratedsolution A bolus was prepared dried and stored in bottles(No Z04000043 60 gbottle) Citalopram (No Z56666580320 mgpill 14 pillsbox) was purchased from Xian JanssenPharmaceutical (Xian China)

22 Methods

221 Model Establishment Rat models of anxiety disorderwere established using empty bottle stimulation in accor-dance with a previous study [12] Briefly rats in all groupsexcept the control groupwere given access to regular drinkingwater only twice a day at 900mdash910 and 2100mdash2110 for1 week This entrainment was followed by a stress testuncertain empty bottle stimulation was performed once forthe above-described time period (ie rats could not drinkwater once) for 2 consecutive weeks [13ndash15] (Table 1)

222 Drug Administration One day after model establish-ment rat appearance [16] weight [17] and open-field behav-iors [18] were measured and administration was carried outAdministration dosages for rats were 1620 mgkg for thelow-dose CJP group (equivalent to the dosage commonlyused in adults) 3240 mgkg for the high-dose CJP group(equivalent to a double dosage in adults) and 18 mgkgcitalopram hydrobromide Medicines were intragastricallyadministered according to weight (1 mL100 g) once dailyFor untreated control and model groups an equal volume ofphysiological saline was administered daily Administrationwas conducted from 800 to 1200 Thirty days later ratbehaviors were evaluated Subsequently rats were decapitatedand the hippocampus was obtained

23 Behavioral Indices

231 General Observation of Rats General appearanceincluded hair color body posture mental state activity colorof the auricle and resistance to restraint stress Rats wereweighed before and after administration

232 Open-Field Test The open-field test is a widely usedmodel to evaluate anxious behavior in animals [19 20] A self-made open box was used the inner wall and undersurface ofthe cardboard box (80 cm times 80 cm times 80 cm) were coveredwith black oil paper and fixed with adhesive tapeThe groundwas equally divided into 25 squares with chalk Rats wereplaced in the center of an open-field box to observe theirhorizontal and verticalmovementsThe frequency of crossingthe undersurface squares was used to measure horizontalmovement and the frequency of being upright on hindlimbs was used to measure vertical movement The detectiontime was 5 min and each rat was examined once Scoringcriterion for horizontal movement half of the body enteringthe other square was scored as 1 Scoring criterion of verticalmovement forelimbs were 1 cm above the ground standingup on the hind limbs once was scored as 1

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Empty bottle stimulus schedule

Time 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21900 ES ES ES ES ES ES N N N ES N N ES N ES ES N ES N N ES2100 ES ES ES ES ES ES ES ES ES N ES ES N ES N N ES N ES ES NNote ES water supply N empty bottle

Table 2 Comparison of weight changes of rats in each group (mean plusmn SD n = 40 g)

Time (day) Blank control group Model group Citalopram group Low-dose Chailong JieyuPill group

High-dose Chailong JieyuPill group

0 19638plusmn524 19538plusmn652 19963plusmn537 19513plusmn1045 19738plusmn52622 31738plusmn2802 28388plusmn1235lowastlowast 28463plusmn1166lowastlowast 28450plusmn938lowastlowast 28938plusmn2296lowast

52 44325plusmn2039 40275plusmn1203lowastlowast 41575plusmn971lowastlowast998771 41438plusmn915lowastlowast998771 43113plusmn1340998771998771IelowastP lt 005 lowastlowastP lt 001 versus blank control group 998771P lt 005 998771998771P lt 001 versus model groupIP lt 005 citalopram group eP lt 005 versus low-dose ChailongJieyu Pill group

233 Specimen Extraction and Index Determination Ratswere intraperitoneally injected with 10 chloral hydrate (4mL100 g) and decapitated Hippocampi (approximately 15mg) were obtained cut and triturated Approximately 500120583L of physiological saline was added and mixed followed bycentrifugation at 5000 rpm for 10 min After removing thesupernatant samples were stored at minus80∘C

234 Monoamine Neurotransmitter Detection Blank andstandard wells were set and 10 120583L of samples (final dilutionof the sample was 5-fold) was added to the bottom ofthe microplate After lightly shaking 50 120583L of standardpreparation and 100 120583L of detected sample were addedto the reaction wells Biotin-labeled antibody (50 120583L) wasimmediately added and a cover was placed on the plate Afterlightly shaking and mixing samples were incubated at 37∘Cfor 45 minThe liquid in the well was discarded and cleaningsolution was added to each well for a 30-second shakingThecleaning solution was discarded and the well was dried withabsorbent paper After four washes 100 120583L of streptavidin-horseradish peroxidase was added to each well and then theplate was lightly shaken and incubated at 37∘C for 30 minfollowed by four washes as above Substrates A and B (50 120583Lof each) were added to each well then the plate was lightlyshaken mixed and incubated at 37∘C for 5 min in the darkMicroplates were then taken out and the reaction was rapidlyterminated by adding 50 120583L of stop buffer

235 Detection ofMR andGRmRNAExpression Equipmentand kitswere as follows cDNAFirst-Strand Synthesis Kit TapDNA Polymerase Regular Agarose G10 polymerase chainreaction (PCR) cycler and a nucleic acid electrophoresisapparatus In accordance with the manufacturerrsquos instruc-tions one-step RT-PCR was used to measure mRNA expres-sion MR mRNA upstream 31015840mdashAAC AAA ATG CCC CACGGT TAmdash51015840 (20 bp) downstream 31015840mdashGGG ACG ATGCAA TGG ACT GTmdash51015840 (20 bp) GR mRNA upstream31015840mdashGGA TTT CCA GAG CCC ACC ATmdash51015840 (20 bp)downstream31015840mdashCATTCCTGATGGTCACCTCGmdash51015840 (20bp)

24 Statistical Analysis Data were analyzed using SPSS 190software Measurement data are expressed as mean plusmn SDMultivariate analysis of variance was used to test the signif-icance of group differences Hypotheses were tested using atwo-sided test Values of P lt 005 were considered statisticallysignificant Values of P lt 001 were considered remarkablystatistically significant

3 Results

31 General Conditions After model establishment rats inthe control group showed good spirit quick actions brightand clean hair and a normal diet Rats in the model citalo-pram low-dose CJP and high-dose CJP groups presentedlistlessness messy hair slow responses violent resistancescreaming and struggling In the citalopram low-dose CJPand high-dose CJP groups calls were soft resistance andconfrontationwereweak and the frequencies of breaking freeand biting were reduced In the model group the reaction torestraint was intense including struggling biting the cagebraying and trying to break free In addition rats in themodel group had loose stools Rats in control citalopramlow-dose CJP and high-dose CJP groups had moderate stoolshapes

32 Changes in Rat Weight The weight of rats was lower inmodel citalopram low-dose CJP and high-dose CJP groupscompared with the control group (P lt 005) indicatingsuccessful model establishment The weight of rats in citalo-pram low-dose CJP and high-dose CJP groups was increasedcompared with the model group (P lt 001 P lt 005) Therewas no difference in the weight change of rats in low-doseCJP and citalopram groups (P gt 005) The weight of rats inthe high-dose CJP group was increased compared with low-dose CJP and citalopram groups (P lt 005) and similar to thecontrol group (P gt 005 Table 2 Figure 1)

33 Open-Field Test Scores for horizontal and verticalmove-ments were significantly lower in model citalopram low-dose CJP and high-dose CJP groups compared with the


Figure 1 Rat body weight changes in each group lowastP lt 005 lowastlowastP lt 001 versus blank control group 998771P lt 005 998771998771P lt 001 versus modelgroup IP lt 005 citalopram group eP lt 005 versus low-dose Chailong Jieyu Pill group

Figure 2 Comparison of horizontal movements at different timepoints in rats from each group lowastP lt 005 lowastlowastP lt 001 versus blankcontrol group 998771P lt 005 998771998771P lt 001 versus model group IP lt 005versus citalopram group

control group (P lt 005) After administration horizontalmovement scores were higher in the citalopram group thanin control and model groups (P lt 005) while verticalmovement scores were more improved than in the controlgroup (P lt 005) Horizontal movement scores were signif-icantly higher in the low-dose CJP group than in model andcitalopram groups (Plt 001P lt 005) and verticalmovementscores were also increased compared with the model group(P lt 005) Scores for horizontal and vertical movements inthe high-dose CJP group were significantly higher than inthe model group (P lt 001 P lt 005) and were improvedcompared with the citalopram group (P lt 005) Scores ofhorizontal and vertical movements were not significantly

different between low-dose and high-dose CJP groups (P gt005 Table 3 Figures 2 and 3)

34 Monoamine Neurotransmitters 5-Hydroxytryptaminedopamine and norepinephrine contents were reduced ineach administration group with levels in the citalopramgroup being lower than in the model group (P lt 001)but still higher than in the control group Contents of allthree neurotransmitters were higher in the low-dose CJPgroup compared with the citalopram group Interestingly5-hydroxytryptamine contents were significantly different(P lt 001) but dopamine and norepinephrine contentswere not (P gt 005) between low-dose CJP and citalopramgroups or between high-dose CJP and citalopram groups5-Hydroxytryptamine dopamine and norepinephrine con-tents were not significantly different between low-dose andhigh-dose CJP groups (P gt 005 Table 4 Figure 4)

35 MR and GR mRNA Expression MR mRNA expressionwas upregulated but GR mRNA expression was significantlydownregulated in the hippocampi of rats in the modelgroup compared with control group rats (P lt 001) AfteradministrationMRmRNAexpressionwas downregulated (Plt 005) but GRmRNA expression was upregulated (P lt 001)in the citalopram group compared with the model groupMR mRNA expression was not significantly downregulatedin the low-dose CJP group Moreover MRmRNA expressionwas not significantly different between low-dose CJP andcitalopram groups (P gt 005) GR mRNA expression inthe low-dose CJP group was significantly upregulated (P lt005) however this level remained significantly lower thanobserved in the citalopram group (P lt 005) In the high-dose CJP group MR mRNA expression was significantlydownregulated while GRmRNA expression was upregulated(P lt 001) Downregulation of MR mRNA expression wasenhanced in the high-dose CJP group compared with citalo-pram (P lt 005) and low-dose CJP (P lt 005 Table 5 Figure 5)groups


Figure 3 Comparison of vertical movements at different time points in rats from each group lowastP lt 005 lowastlowastP lt 001 versus blank controlgroup 998771P lt 005 998771998771P lt 001 versus model group IP lt 005 versus citalopram group

Figure 4 Comparison of monoamine neurotransmitters in the brains of rats in each group lowastP lt 005 lowastlowastP lt 001 versus blank control group998771P lt 005 998771998771P lt 001 versus model group IIP lt 001 citalopram group

4 Discussion

In the present study we used empty bottle stimulation toestablish an anxiety model in rats Empty bottle stimulationis a mature easily operated and generally accepted methodfor the preparation of anxious animals The method ofempty bottle stimulation used to establish our model ofanxiety disorder was first proposed by Izquierdo et al [21]Briefly rats are trained to drink water at regular times toproduce a conditioned reflex and then empty water bottlesare irregularly placed in cages to simulate anxiety responseswithin rats General conditions weight changes and open-field test results demonstrated that ratweight slowly increased(P lt 001) and scores for horizontal and vertical movementwere reduced (P lt 005) in the model group indicating

that rats presented anxious behaviors and the model wassuccessfully established The weight of rats in the low-doseCJP group was increased and identical to animals in thecitalopram group (P gt 005) but still lower than observedin the control group (P lt 001) Rat weight was increasedin the high-dose CJP group compared with the citalopramgroup (P lt 001) and identical to that of the control group(P gt 005) This may be associated with the ability of CJPto disperse depressed liver energy calm nerves with heavymaterial invigorate the spleen and stomach relieve tensionimprove anxiety state and increase appetite In the open-fieldtest horizontal movement scores were significantly increased(P lt 001) and vertical movement scores were increased (Plt 005) in the low-dose CJP group Scores for horizontaland vertical movements were identical between low-dose


Figure 5 Comparison of MR and GR gene expression in brains of rats in each group lowastP lt 001 versus blank control group 998771P lt 005 998771998771P lt001 versus model group IP lt 005 versus citalopram group eP lt 005 versus low-dose Chailong Jieyu Pill group MR mineralocorticoidreceptor GR glucocorticoid receptor

Table 3 Comparison of open-field test results at different time points in each group (mean plusmn SD score n = 40)

Group Before modeling After modeling After administrationHorizontal movement Blank control 4675plusmn680 4313plusmn591 4400plusmn595

Model 4650plusmn655 2688plusmn591lowastlowast 2588plusmn770Citalopram 4763plusmn1151 2763plusmn723lowastlowast 3513plusmn860lowast998771

Low-dose Chailong Jieyu Pill 4638plusmn1220 3013plusmn801lowastlowast 4275plusmn396998771998771I

High-dose Chailong Jieyu Pill 4438plusmn912 2713plusmn726lowastlowast 4388plusmn726998771998771I

Vertical movement Blank control 1150plusmn321 1000plusmn346 1075plusmn427Model 1113plusmn270 500plusmn227lowastlowast 525plusmn238

Citalopram 1088plusmn436 575plusmn311lowast 688plusmn230lowast

Low-dose Chailong Jieyu Pill 1138plusmn410 588plusmn290lowast 925plusmn388998771

High-dose Chailong Jieyu Pill 088plusmn309 575plusmn392lowast 975plusmn276998771998771IlowastP lt 005 lowastlowastP lt 001 versus blank control group 998771P lt 005 998771998771P lt 001 versus model group IP lt 005 versus citalopram group

Table 4 Comparison of monoamine neurotransmitter contents in rats of each group (mean plusmn SD ngmL n = 40)

Group 5-Hydroxytryptamine Dopamine NorepinephrineBlank control 2285plusmn0445 5578plusmn0618 1244plusmn0638Model 4115plusmn0459lowastlowast 7568plusmn0438lowastlowast 15462plusmn1157lowastlowast

Citalopram 2754plusmn0225lowast998771998771 6084plusmn0528998771998771 13790plusmn0986lowastlowast998771998771

Low-dose Chailong Jieyu Pill 3456plusmn0284lowastlowast998771998771II 6615plusmn1171lowast998771 14077plusmn0829lowastlowast998771

High-dose Chailong Jieyu Pill 3494plusmn0106lowastlowast998771998771II 6781plusmn0863lowastlowast998771 14045plusmn1263lowastlowast998771lowastP lt 005 lowastlowastP lt 001 versus blank control group 998771P lt 005 998771998771P lt 001 versus model group IIP lt 001 citalopram group

Table 5 Comparison of MR and GR mRNA expression in the brain of rats from each group (mean plusmn SD n = 40)

Group MR GRBlank control 0160plusmn0052 0618plusmn0121Model 0470plusmn0186lowast 0303plusmn0080lowast

Citalopram 0272plusmn0089lowast998771 0557plusmn0103998771998771

Low-dose Chailong Jieyu Pill 0312plusmn0107lowast 0422plusmn0103lowast998771I

High-dose Chailong Jieyu Pill 0185plusmn0058998771998771Ie 0562plusmn0107998771998771elowastP lt 001 versus blank control group 998771P lt 005 998771998771P lt 001 versus model group IP lt 005 versus citalopram group eP lt 005 versus low-dose ChailongJieyu Pill group MR mineralocorticoid receptor GR glucocorticoid receptor


CJP and control groups (P gt 005) Scores for horizontaland vertical movements in the high-dose CJP group wereincreased (P lt 001) better than observed in the citalopramgroup (P lt 005) and identical to the control group (P gt005) Vertical movement indicates active behavior whichdecreases with anxiety disorder Vertical movement scoresof rats were lower than horizontal movement scores aftermodeling Therefore elevated vertical movement scores aremore meaningful than elevated horizontal movement scoresImprovement in the vertical movement was not significant inthe citalopram group (P gt 005) but was significant in bothlow-dose and high-dose CJP groups (P lt 005) Thus it isevident that CJP has significant antianxiety effects in anxiousrats

The current diagnosis of anxiety is still based on clinicalmanifestations and scales as the main diagnostic criteria asa result of inaccurate etiology and lack of relevant objec-tive laboratory indicators While the diagnosis of anxietyis mainly based on symptoms [22] the cause of anxietyremains unclear However imbalances in various neurotrans-mitters including gamma-aminobutyric acid (GABA) andthe monoamines dopamine norepinephrine and serotoninhave been implicated as important mechanisms leading toanxiety [23] The incidence of anxiety disorders is associatedwith excessive release of monoamine neurotransmitters inthe brain Indeed drugs that reduce 5-hydroxytryptaminedopamine and norepinephrine exert an anxiolytic effect [24]An increased 5-hydroxytryptamine concentration is directlyproportional to anxiety attacks [25] Neuroleptic agentscan alleviate clinical manifestations of anxiety disorder andreduce dopaminergic transmission suggesting a potentiallyimportant role for the dopaminergic system in pathogenesisof anxiety disorders [26]While the role of norepinephrine inanxiety disorders is not well understood research examiningcerebrospinal fluid blood and urine observed noradrenergicactivity during an anxiety attack [27] Low-dose and high-dose CJP remarkably reduced monoamine neurotransmittercontents in the rat hippocampus However the ability ofCJP to reduce 5-hydroxytryptamine content was less thanthat of citalopram (P lt 001) while the ability of thesetwo treatments to decrease dopamine and norepinephrinecontents were similar (P gt 005) It is hypothesized thatCJP exerted an antianxiety effect by reducing the release ofmonoamine neurotransmitters in the hippocampus How-ever this is not exactly the case because the degree of reduced5-hydroxytryptamine which is closely related to anxiety wassignificantly weaker than observed for citalopram althoughthe improvement of behavioral index was better than citalo-pram These results suggest that the beneficial effects of CJPon anxiety are not limited to monoamine transmitters theeffects of CJP on the metabolites of these neurotransmittersneed to be further investigated Moreover the effects ofhigh-dose and low-dose CJP on monoamine neurotrans-mitters were not significantly different suggesting that theeffects of CJP on anxiety disorder were not significantlydose-dependent While the reason for an absence of dosedependence is not yet clear it may be due to the fact thatthe difference between high and low dosages was too smallIndeed as a 1-fold higher dose of CJP was used in the present

study the effect of a 3- to 5-fold higher dose of CJP on anxietyneeds to be further investigated

Hippocampal neurons contain abundant MR and GRwith the balance between these two receptors playing animportant role in neuronal excitability and stress responses[28] MR content determines the level of cortisol whichis associated with fear whereas GR inhibits hypothalamic-pituitary-adrenal axis overreaction under stress [29] Patientswith chronic emotional stimuli exhibit sustained low cortisolresponses hippocampal imaging changes and enhancednegative feedback control of the hypothalamic-pituitary-adrenal axis [30] Few studies have examined the effect ofChinese medicine preparation on MR and GR expression Inthis study we observed the effects of CJP on MR and GRmRNA expression we found that MR downregulation wasnot significant in the low-dose CJP group (P gt 005) but MRmRNA expression was not significantly different between thelow-dose CJP and citalopram groups (P gt 005) GR mRNAexpression in the low-dose CJP group was significantlyupregulated (P lt 005) but remained significantly less thanobserved in the citalopram group (P lt 005) In the high-dose CJP group MR mRNA expression was significantlydownregulated but GR mRNA expression was upregulated(P lt 001) The downregulation of MR mRNA expressionwas enhanced in the high-dose CJP group compared withcitalopram (P lt 005) and low-dose CJP (P lt 005) groupsbut similar to the control group (P gt 005) Taken togetherCJP could obviously regulate MR and GR mRNA expressionin a dose-dependent manner This effect was enhanced inthe high-dose CJP group compared with the citalopramgroup and was close to normal This may be the reasonthe regulatory effect of CJP on 5-hydroxytryptamine is lessthan citalopram but the improvement on rat behaviors isbetter Our results suggest that CJP has a moderate effecton regulating neurotransmitters and a significant effect onregulating gene expression which are different from that ofcitalopram Moreover MR and GR mRNA expression aresensitive to CJP treatment although the exact mechanismremains to be elucidated

5 Conclusions

Our results verified that CJP could remarkably improveanxious behavior increase weight reduce hippocampalmonoamine neurotransmitters and regulate MR and GRmRNA expression Nevertheless we still poorly understoodwhy the regulatory effect of low-dose CJP on MR and GRmRNA expression was not significant but the effect ofhigh-dose CJP was very significant This requires furtherinvestigation

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare no conflicts of interest


Acknowledgments

The authors would like to thank all the research and medicalstaff of the Department of encephalopathy in LianyungangHospital of Traditional Chinese Medicine for their prepa-ration before the experiment The authors thank ProfessorLiu Tao for giving help and support during the experimentThanks are due to Wang Wei for the selfless help a biologyresearch company in Nanjing Thanks are due to ChenYexiang Zheng Tao Wu Dongling Xu Ronglong and JiangHuili for their great help during the course of the projectThestudy was supported by the subject of science and technologyproject of Jiangsu Provincial Bureau of Traditional ChineseMedicine ldquoEffects of Chailong Jieyu Pill on BehaviorsMonoamine Neurotransmitters and Corticosteroid Recep-tors in Rat Models of Anxiety Disorderrdquo (YB2015117) JiangsuProvincial Bureau of Traditional Chinese Medicine-KangChinese Medicine Science and Technology Innovation FundProject (no HZ1019KY)

References

[1] X J Li Y Guo and J P Hu ldquoCorrelation among copingstyle social support and mental health in generalized anxietydisorderrdquo Shenjing Jibing yu JingshengWeisheng vol 13 pp 187ndash189 2013

[2] B Bandelow and S Michaelis ldquoEpidemiology of anxiety disor-ders in the 21st centuryrdquoDialogues in Clinical Neuroscience vol17 no 3 pp 327ndash335 2015

[3] G K Feng Y Y Chen L J Li et al ldquoClinical Research ofChailong Jieyu Pill in Treating Generalized Anxiety DisorderrdquoNanjing Zhongyiyao Daxue Xuebao vol 3 pp 214ndash217 2015

[4] J Y Zhang and X J Zhang ldquoExperimental study on effectsof Bupleurum chinense on acetylcholine metabolism andhistomorphology in hippocampus of depression model ratsrdquoHeilongjiang Zhongyiyao Daxue Xuebao vol 4 pp 506ndash5082011

[5] YHu andMHong ldquoStudy of PrescriptionsContaining Bupleu-rum as Monarch for Treatment of Depressionrdquo ZhongguoShiyan Fangjixue Zazhi vol 16 pp 247ndash249 2010

[6] H X Yuan C LWei and Y Cheng ldquoStudy on Anti-depressionEffects of Xiaochaihu Tangrdquo Zhongguo Shiyan Fangjixue Zazhivol 18 pp 190-191 2012

[7] H Zhang L Zhang and Y Liu ldquoStudies on chemical compo-nents and pharmacological activities of Os Draconis (Longgu)andOstreae Conchardquo Zhongguo Zhongyao Zazhi vol 36 no 13pp 1839-1840 2011

[8] Y Liu S Ma and R Qu ldquoSCLM total saponins extracted fromChaihu-jia-longgu-muli-tang reduces chronic mild stress-induced apoptosis in the hippocampus inmicerdquoPharmaceuticalBiology vol 48 no 8 pp 840ndash848 2010

[9] WZhu SMa RQu andDKang ldquoAntidepressant-like effect ofsaponins extracted from Chaihu-jia-longgu-muli-tang and itspossible mechanismrdquo Life Sciences vol 79 no 8 pp 749ndash7562006

[10] J-H Liu Y-M Bao J-J Song and L-J An ldquoCodonopsispilosula (Franch) Nannf total alkaloids potentiate neurite out-growth induced by nerve growth factor in PC12 cellsrdquo ActaPharmacologica Sinica vol 24 no 9 pp 913ndash917 2003

[11] P Zhang L Hu R Bai et al ldquoStructural characterizationof a pectic polysaccharide from Codonopsis pilosula and its

immunomodulatory activities in vivo and in vitrordquo Interna-tional Journal of Biological Macromolecules vol 104 pp 1359ndash1369 2017

[12] W J Lin W W Wang and F Shao ldquoEfects of ChronicEmotional Stress on Behavior Neuroendocrine and ImmuneResponses in Rats A New Emotional Stress Modelrdquo KexueTongbAO vol 48 pp 926ndash929 2003

[13] X Zhang The clinical and experimental research on the treat-ment of anxiety with AnShenfang Guangdong Southern Medi-cal University 2012

[14] J Wang L P Chen F W Wang Z G Sun and C Y Xu ldquoTheEffect of Shuyuningxin Decoction on the Behavior and SerumLevels of Neuropeptide Y and Corticosterone of Anxiety RatsrdquoZhongguo Zhongyi Jizheng vol 22 pp 1459-1460 2013

[15] B Zhang andY L Li Improvement Role of AnemarrheanaWaterDecoction for the Rats with Chronic Emotional Stress Inducedby Empty Bottles Heilongjiang University of ChineseMedicineHeilongjiang China 2012

[16] N Dong Q S Tang R Z Zhao et al ldquoEffects of liver-soothingheat-clearing and spleen-fortifying method on the ex-pressionsof cerebral bax and bcl-2 in rats with generalized anxietydiseaserdquo Beijing Zhongyiyao Daxue Xue bao vol 38 no 6 pp383ndash387 2015

[17] H W Wei W Jiao Y Z Zhang et al ldquoEffects of exercising onthe ethological expressions and body weight of chronic stressinduced depression model ratsrdquo Tiyu Xuekan vol 17 pp 100ndash105 2010

[18] Y Huang Y-T Yang X-X Liu et al ldquoEffect of herbal-partitioned moxibustion at Tianshu (ST 25) and Qihai (CV6) on pain-related behavior and emotion in rats with chronicinflammatory visceral painrdquo Journal of Acupuncture and TuinaScience vol 13 no 1 pp 1ndash8 2015

[19] L Prut and C Belzung ldquoThe open field as a paradigm tomeasure the effects of drugs on anxiety-like behaviors a reviewrdquoEuropean Journal of Pharmacology vol 463 no 1ndash3 pp 3ndash332003

[20] MAngrini and J Leslie ldquoTheopenfield drinking test as amodelfor the assessment of anxiolytic actionrdquo Journal of MedicinalChemistry vol 52 no 24 pp 7958ndash7961 2003

[21] I Izquierdo and J L Mcgaugh ldquoEffect of novel experiences onretention of inhibitory avoidance behavior in mice The influ-ence of previous exposure to the same or another experiencerdquoBehavioral and Neural Biology vol 47 no 2 pp 109ndash115 1987

[22] A J Mitchell ldquoClinical utility of screening for clinical depres-sion and bipolar disorderrdquo Current Opinion in Psychiatry vol25 no 1 pp 24ndash31 2012

[23] M J Millan ldquoThe neurobiology and control of anxious statesrdquoProgress in Neurobiology vol 70 no 2 pp 83ndash244 2003

[24] J J Li J L Tang L L Hu et al ldquoEffect of rhynchophyllatotal alkaloids on behavior and contents of monoamine neuro-transmitters in brain tissues of anxiety model ratsrdquo Disan JunyiDaxue Xuebao vol 35 pp 237ndash240 2013

[25] Y Sun and K Z Bai ldquoA review of 5-HT receptors and theirfunction in anxiety disorderrdquo Shenjing Jibing yu JingshengWeisheng vol 10 pp 528-529 2010

[26] X H Liu Z Q Yang S X Xian et al ldquoEffect ofWendan Tabletson Behaviors andMonoamine Neurotransmitters in Rat Modelof Anxietyrdquo Guangzhou Zhongyiyao Daxue Xuebao vol 29 pp674ndash678 2012

[27] Y G Yuan A Q Wu X B Zhang and S N Zhang ldquoAcontrolled study of plasma monoamine neurotransmitters in


comorbid anxiety and depressionrdquo Linchuang Jingsheng YixueZazhi vol 11 article 129 2001

[28] M S Oitzl D L Champagne R van der Veen and ER de Kloet ldquoBrain development under stress hypotheses ofglucocorticoid actions revisitedrdquo Neuroscience amp BiobehavioralReviews vol 34 no 6 pp 853ndash866 2010

[29] A M Rozeboom H Akil and A F Seasholtz ldquoMineralocorti-coid receptor overexpression in forebrain decreases anxiety-likebehavior and alters the stress response in micerdquo Proceedings ofthe National Acadamy of Sciences of the United States of Americavol 104 no 11 pp 4688ndash4693 2007

[30] H Winter and E Irle ldquoHippocampal volume in adult burnpatients with and without posttraumatic stress disorderrdquo TheAmerican Journal of Psychiatry vol 161 no 12 pp 2194ndash22002004

Stem Cells International

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to evaluate adverse reactions Results of a 6-week observationperiod demonstrated that CJP could improve HAMA andself-made TCM symptom scale scoresMoreover effects weresignificant at 2 4 and 6 weeks indicating a progressive effectcompared with the placebo group Importantly the effectiverate was 931 and no adverse reactions were observed [3]

CJP is composed of Radix Bupleuri Radix ScutellariaeRhizoma Pinelliae Preparata Radix Codonopsis Radix Gly-cyrrhizae preparata keel Concha Ostreae Concha Margari-tifera Usta Rhizoma Zingiberis Recens and Fructus JujubaeModern research has confirmed that saikoside an importantcomponent of Radix Bupleuri plays a role in cholinergiceffects and regulates the digestive and nervous systems bysuppressing cholinesterase [4] Moreover Radix Bupleuridecoction elicited improvements in physical symptomsdepression and irritability [5 6] which are mainly associ-ated with regulation of hypothalamic-pituitary-adrenal axishyperfunction and brain monoamine neurotransmitters [5]Keel and Concha Ostreae have sedative and anticonvulsanteffects [7] whereas total glycosides extracted from highlycompatibleRadix Bupleuri keel andConchaOstreae can dra-matically reduce hippocampal neuronal cell death rates [8]inhibit anxiety and depression and improve rat behaviors [9]Radix Codonopsis can elicit resistance to fatigue and hypoxiaand enhance immunity [10 11] Ginger propanol extract canhave a cholagogic effect and inhibit 5-hydroxytryptamineIn addition to generating sedative antihypertensive andanticonvulsant effects Fructus Jujubae can increase ani-mal weight enhance muscle strength and evoke a certainantagonistic effect on 5-hydroxytryptamine and histamineHowever previous research has mainly focused on the roleand effect of CJP on depression indeed to our knowledgefew studies have examined the effect of CJP on anxiety In thisstudy we explored themechanism underlying the antianxietyeffects of CJP by observing effects in a rat model of anxietydisorder

2 Materials and Methods

21 Materials Clean male Sprague-Dawley rats were pro-vided by the Animal Experimental Center of NanjingUniver-sity of Chinese Medicine of China [License number SCXK(Su) 2013-0003] All rats were acclimated for 1 week Fortyrats weighing 200 plusmn 20 g with similar behaviors as screenedby open-field test were selected and randomly assignedto control (no treatment or anxiety model induction) andanxiety model groups model (untreated) citalopram low-dose CJP and high-dose CJP All procedures were approvedby the Animal Ethics Committee of Lianyungang AffiliatedHospital Nanjing University of Chinese Medicine (NanjingChina)

CJP was provided by the Manufacturing LaboratoryLianyungang Affiliated Hospital Nanjing University of Chi-nese Medicine Drug preparation (1) Radix Bupleuri RadixScutellariae Radix Glycyrrhizae preparata keel ConchaOstreae Concha Margaritifera Usta Rhizoma ZingiberisRecens and Fructus Jujubae were decocted twice with waterfirst for 2 h and then for 1 h The filtrate was mixed and

concentrated to a moderate amount (2) Rhizoma PinelliaePreparata and Radix Codonopsis were triturated into powderwhich was mixed with the above-described concentratedsolution A bolus was prepared dried and stored in bottles(No Z04000043 60 gbottle) Citalopram (No Z56666580320 mgpill 14 pillsbox) was purchased from Xian JanssenPharmaceutical (Xian China)

22 Methods

221 Model Establishment Rat models of anxiety disorderwere established using empty bottle stimulation in accor-dance with a previous study [12] Briefly rats in all groupsexcept the control groupwere given access to regular drinkingwater only twice a day at 900mdash910 and 2100mdash2110 for1 week This entrainment was followed by a stress testuncertain empty bottle stimulation was performed once forthe above-described time period (ie rats could not drinkwater once) for 2 consecutive weeks [13ndash15] (Table 1)

222 Drug Administration One day after model establish-ment rat appearance [16] weight [17] and open-field behav-iors [18] were measured and administration was carried outAdministration dosages for rats were 1620 mgkg for thelow-dose CJP group (equivalent to the dosage commonlyused in adults) 3240 mgkg for the high-dose CJP group(equivalent to a double dosage in adults) and 18 mgkgcitalopram hydrobromide Medicines were intragastricallyadministered according to weight (1 mL100 g) once dailyFor untreated control and model groups an equal volume ofphysiological saline was administered daily Administrationwas conducted from 800 to 1200 Thirty days later ratbehaviors were evaluated Subsequently rats were decapitatedand the hippocampus was obtained

23 Behavioral Indices

231 General Observation of Rats General appearanceincluded hair color body posture mental state activity colorof the auricle and resistance to restraint stress Rats wereweighed before and after administration

232 Open-Field Test The open-field test is a widely usedmodel to evaluate anxious behavior in animals [19 20] A self-made open box was used the inner wall and undersurface ofthe cardboard box (80 cm times 80 cm times 80 cm) were coveredwith black oil paper and fixed with adhesive tapeThe groundwas equally divided into 25 squares with chalk Rats wereplaced in the center of an open-field box to observe theirhorizontal and verticalmovementsThe frequency of crossingthe undersurface squares was used to measure horizontalmovement and the frequency of being upright on hindlimbs was used to measure vertical movement The detectiontime was 5 min and each rat was examined once Scoringcriterion for horizontal movement half of the body enteringthe other square was scored as 1 Scoring criterion of verticalmovement forelimbs were 1 cm above the ground standingup on the hind limbs once was scored as 1













3 Results














4 Discussion





























5 Conclusions


Data Availability





Acknowledgments


References






































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of































3 Results














4 Discussion





























5 Conclusions


Data Availability





Acknowledgments


References






































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





























4 Discussion





























5 Conclusions


Data Availability





Acknowledgments


References






































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















4 Discussion





























5 Conclusions


Data Availability





Acknowledgments


References






































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of













































5 Conclusions


Data Availability





Acknowledgments


References






































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























5 Conclusions


Data Availability





Acknowledgments


References






































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Acknowledgments


References






































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Documents

Effects of Chailong Jieyu Pill on Behavior, Monoamine ... · Monoamine Neurotransmitters.-Hydroxytryptamine, dopamine, and norepinephrine contents were reduced in each administration