Effects of Sini San used alone and in combination with fluoxetine on central and peripheral 5-HT levels in a rat model of depression
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- Edgar Fitzgerald
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1 Online Sumissions: J Trdit Chin Med 2013 Octoer 15; 33(5): info@journltcm.com ISSN JTCM. All rights reserved. EXPERIMENTAL STUDY TOPIC Effects of Sini Sn used lone nd in comintion with fluoxetine on centrl nd peripherl 5-HT levels in rt model of depression Yuo Li, Yuxiu Sun, Xueling M, Xioxing Xue, Wenting Zhng, Zhiqin Wu, Yulin Ouyng, Jinxin Chen, Weiming Wng, Shuzhen Guo, Wei Wng Yuo Li, Xueling M, Xioxing Xue, Wenting Zhng, Zhiqin Wu, Yulin Ouyng, Jinxin Chen, Weiming Wng, Shuzhen Guo, Wei Wng, School of Bsic Medicl Sciences, Beijing University of Chinese Medicine, Beijing , Chin Yuxiu Sun, School of Acupuncture & Moxiustion nd Tuin, Beijing University of Chinese Medicine, Beijing , Chin Supported y Grnt from the Ntionl Bsic Reserch Progrm of Chin (973 Progrm No. 2011CB505106) Correspondence to: Prof. Wei Wng, School of Bsic Medicl Sciences, Beijing University of Chinese Medicine, Beijing , Chin. wngwei@ucm.edu.cn Telephone: ; Accepted: April 1, 2013 Astrct OBJECTIVE: To investigte the effects of Sini Sn nd fluoxetine on the levels of centrl nd peripherl 5-HT in rt model of depression, nd provide new insight into the tretment of depression with integrted Chinese-Western Medicine. METHODS: A rt model of depression ws estlished y chronic mild stress (CMS). Model rts received either Sini Sn, fluoxetine, comintion of the two drugs, or no drug tretment. Helthy nive rts were used s controls. Open field nd sucrose preference tests were used to ssess depression-like ehvior. ELISA nd immunohistochemistry were used to determine centrl nd peripherl levels of 5-HT. RESULTS: In the group with no drug tretment, centrl 5-HT expression decresed while peripherl 5-HT concentrtions incresed s CMS continued. Four weeks fter CMS, Sini Sn lone ws less effective in reducing depression-like ehvior thn fluoxetine lone or in comintion with Sini Sn, ut comined use ws more effective thn fluoxetine lone. Eight weeks fter CMS, Sini Sn lone or in comintion with fluoxetine ws more effective in reducing depression-like ehvior thn fluoxetine lone. Furthermore Sini Sn nd fluoxetine used lone or in comintion notly incresed centrl 5-HT expression nd decresed peripherl 5-HT levels in the rt model. CONCLUSION: The results of the present study indicte tht there is synergistic ction etween the two medicines in the tretment of depression. Sini Sn exhiited reltively long lg efore its effects were oserved; however, y eight weeks the Trditionl Chinese Medicine ppered t lest s effective s fluoxetine. We suggest tht Sini Sn cn replce fluoxetine in the lter stges of depression tretment to minimize side effects oserved with long-term fluoxetine dministrtion JTCM. All rights reserved. Key words: Fluoxetine; Stress; Depression; Serotonin; Sini Sn INTRODUCTION Depression is mood disorder chrcterized y persistent low mood, decresed motivtion, nd pthy. The suicide rte in depressed ptients is 20 times higher thn tht in the norml popultion. 1 Modern medicl investigtion hs demonstrted tht the onset of depres- JTCM www. journltcm. com 674
2 sion is relted to lck of serotonin (5-HT), 2 neurotrnsmitter involved in the regultion of emotion, memory, ppetite nd sexul function. Chronic mild stress (CMS) is widely ccepted method of estlishing rt model of depression. In this prdigm, nimls re exposed to series of unpredictle mild stressors which reflect the vried, low-intensity environmentl triggers tht cn led to the development of depression. As exposure to CMS continues, the nimls' consumption of food nd wter decreses, reflecting core symptom of depression, nhedoni, 3,4 nd mimicking the onset nd development of depression in mn. Fluoxetine is currently first-line tretment for depression in Western Medicine; however, serious side effects my occur fter long-term use. Therefore, Trditionl Chinese Medicine or integrted trditionl Chinese nd Western Medicine re widely dopted in the clinicl tretment of depression in Chin. Sini Sn is commonly prescried for the tretment of depression in Trditionl Chinese Medicine. In the present study, we oserved the effects of Sini Sn nd fluoxetine, dministered lone or together, on the ehvior nd centrl nd peripherl 5-HT levels in rts exposed to CMS, to provide new insight into the ppliction of integrted trditionl Chinese nd Western Medicine for the tretment of depression. MATERIALS AND METHODS Experimentl nimls nd grouping A totl of 200 mle, specificlly pthogen-free Sprgue Dwley rts, ged 6 weeks nd weighing (200 ± 10) g, were provided y Beijing Weitong Lihu Experimentl Animl Technology Co., Ltd. (experimentl niml production license: SCXK Beijing ). The rts were fed nd housed in the niml room of the Scientific Reserch Center of Beijing University of Chinese Medicine, t n mient temperture of nd under 12 h light/drk cycle. Rts were hituted to the housing room for two weeks, then rndomly divided into five groups y rndom numer tle method (n=40 per group). Four groups were exposed to CMS: stress-only group (exposed to CMS with no susequent drug tretment); Sini Sn group (exposed to CMS then received Sini Sn); the fluoxetine group (CMS then fluoxetine); nd comined Sini Sn nd fluoxetine group (CMS then comined dministrtion of Sini Sn nd fluoxetine, hereinfter referred to s the comintion group). Nive rts were used s controls. All rts in the test groups were housed in isoltion. Control rts were housed in groups of three, except during the sucrose preference test when they were lso housed singly. Regents Rt 5-HT enzyme-linked immunosorent ssy (ELI- SA) kits were purchsed from Americn R&D Co. (Minnepolis, MN, USA); the primry ntiody (mouse nti-rt 5-HT monoclonl IgG) ws purchsed y Snt Cruz Biotechnology, Inc (Dlls, TX, USA); the secondry ntiody (iotin-leled got nti-mouse IgG) nd the immunohistochemistry detection kits were purchsed y Gene Technology Co., Ltd. (Shnghi, Chin). Estlishment of rt model of depression y exposure to CMS Rts were exposed dily to rndom selection of three stressors from the following: no food or wter for 24 h; cge tilted t 30 overnight; wet housing overnight; 5 min swim in wter t 4 ; heting of home cge to 45 for 5 min; restrint for 2 h; til clmping, in which hemosttic clmp ws plced on the til ner the se for 1 min, with enough pressure to cuse the rt to voclize; nd 36 sessions of electricl stimultion tht ws dministered s inescple foot shock in chmer t 1.5 ma intensity consisting of 30 shocks in 1 min, with n intervl of 30 s etween sessions. CMS ws crried out for four weeks in the first experiment, nd eight weeks in the second experiment. The CMS strted nd ended t the sme time for the ll groups. Sini Sn nd fluoxetine dministrtion Sini Sn, composed of Chihu (Rdix Bupleuri Chinensis), Zhishi (Fructus Aurntii Immturus), Chisho (Rdix Peonie Rur) nd Gnco (Rdix Glycyrrhize), ws purchsed from Chin Beijing Tongrentng Group Co., Ltd. (Beijing, Chin), nd identified ccording to the Phrmcopoei of the People's Repulic of Chin (2010 edition) y the Chin-Jpn Friendship Hospitl phrmcy in Beijing. Ech ingredient (200 g) ws roken into soyen-like prticles, soked for 24 h in 95% lcohol, nd refluxed nd extrcted three times per hour in 95% lcohol. The extrcts were then concentrted nd resuspended in 1000 ml 0.5% sodium croxymethyl cellulose to mke Sini Sn suspension contining ech ingredient t concentrtion of 0.24 g/ ml. The humn to rt dose conversion coefficient is 0.16, 5 ssuming ody weights of 60 kg for n dult humn nd 0.2 kg for n dult rt. Humn doses re 6 g/ dy for ech ingredient, so n equivlent dose in the rt is 0.12 g for ech ingredient. Fluoxetine ws purchsed s 20 mg cpsules from Lili Suzhou Phrmceuticl Co., Ltd. (Jingsu, Chin) nd 10 mg/ml solution in norml sline ws prepred. Immeditely efore the dily exposure to CMS, rts in the Sini Sn group were fed with Sini Sn suspension (2 ml/kg), those in the fluoxetine group were given fluoxetine (2.5 ml/kg i. p.), nd those in the comintion group received oth Sini Sn (2 ml/kg p.o.) nd fluoxetine (2.5 ml/kg i.p.). Sucrose preference test The sucrose preference test ws crried out every two weeks, strting two weeks prior to CMS exposure. Animls were deprived of food nd wter for 12 h efore the test. Two 100 ml ottles of sucrose solution (2%) JTCM www. journltcm. com 675
3 were plced in ech cge for 12 h; 12 h lter the ottles were chnged to one 100 ml ottle of sucrose (2% ) nd one 100 ml ottle of wter. The ottles were then weighed to determine the mount of sucrose, wter nd totl liquid consumed, 6 nd the preference percentge of sucrose ws clculted ccording to the formul: preference percentge=(sucrose consumption/totl liquid consumption) 100%. Ethologicl oservtion A squre open field ws used, mesuring cm with lck wlls 35 cm high nd 4 4 cm grid drwn on the floor. A digitl cmer ws plced 2 m ove the open field to cpture the whole field. During the test, the rts were plced in the center of the field nd recorded using smll niml ehvior recording nd nlysis system (Pnl Smrt version 1.0) for 3 min. Locomotor ctivity ws determined from the frequency of gridline crosses nd the totl distnce moved. In order to prevent nimls hituting to the open field, the test ws crried out every four weeks from the strt of CMS exposure. Blood nd tissue smpling A 2 ml lood smple ws collected from nimls in ech group using retro-oritl leeding, efore CMS nd 2, 4 nd 6 weeks fter the strt of CMS. Twenty rts were rndomly selected from ech group to otin rin tissue smples fter 4 weeks of CMS. For the remining rts, rin tissue ws otined fter 8 weeks of CMS; rts were nesthetized with 10% chlorl hydrte, 5 ml of dominl lood ws tken, nd the rin nd smll intestine were immeditely removed on ice. Tissues were fixed in 4% prformldehyde for 48 h, nd then emedded in prffin. Hippocmpl CA2 ws exmined t the following coordintes reltive to regm: 3.8 mm posterior, 5.20 mm lterl. Determintion of 5-HT serum levels The ELISA kit (Americn R&D Co., Minnepolis, MN, USA) ws rought to room temperture, 10 μl of serum from ech niml ws dded to the wells. A stndrd curve ws prepred y dding 10 μl of protein stndrds of known concentrtions to seprte wells. Mouse nti-rt 5-HT IgG (40 μl, Snt Cruz Biotechnology Inc, Dlls, TX, USA) t dilution ws dded to ech well nd iotinylted nti-rt IgG (40 μl) were then dded to ech well. The plte ws seled nd shken gently for 30 s efore incution t room temperture for 45 min. The liquid ws removed, nd the plte ws wshed y dding 350 μl wshing liquid to ech well nd removing it, totl of five times. Sustrte solution (100 μl) ws dded to ech well for color development nd the plte ws gently mixed for 10 seconds efore incution t room temperture for 20 min. The rection ws terminted using 100 μl stop solution nd shking the plte gently for 30 s. The opticl density (OD) of ech well ws red t 450 nm within 30 min of terminting the rection. Serum smple concentrtions were determined y compring the OD of ech smple ginst the stndrd curve. Determintion of 5-HT expression in the rin nd smll intestine Expression of 5-HT in the rin nd smll intestine ws detected y immunohistochemistry. The prffin-emedded sections were preheted nd dewxed, then dehydrted in grded series of lcohols, nd microwved for 6 min, After cooling to room temperture, the sections were plced into 0.3% H 2O 2 for 15 min, rinsed with phosphte-uffered sline (PBS) for 5 min three times, nd incuted overnight in mouse nti-rt 5-HT IgG t 4. After rinsing gin with PBS, the sections were then incuted with horserdish peroxidse-leled secondry ntiody for 1 h t room temperture. DAB ws used to visulize stining, nd the sections were counterstined with hemtoxylin, dehydrted, clered nd seled. For the negtive control, sections were incuted without primry ntiody with ll remining steps crried out s descried. Imge-Pro Plus version 6.0 (Medi Cyernetics, Inc., Bethesd, MD, USA) imge nlysis softwre ws used to nlyze the dt. Three rin sections nd three smll intestine sections were selected for ech rt, nd four non-overlpping views in the hippocmpl CA2 region were selected for ech section under light microscope t 200 mgnifiction. Men OD for ech niml ws the vlue included in the nlysis. Sttistics All dt re expressed s men ± stndrd dervition (SD). SPSS 17.0 softwre (SPSS v.17.0 for Windows; SPSS Inc., Chicgo, IL, USA) ws used to nlyze the dt. Groups were compred using two-wy nlysis of vrince (ANOVA). The lest significnt difference (LSD) post-hoc test ws used for dt with homogeneity of vrince, nd Tmhne's T2 test ws used for those with heterogeneity of vrince. P<0.05 ws considered sttisticlly significnt. RESULTS Sucrose preference Before CMS exposure there were no differences in sucrose preference mong the five groups (P>0.05). After 2 weeks of CMS, sucrose preference y rts tht hd received Sini Sn, fluoxetine, or oth comined, ws lower thn tht in the nive control group (P=0.040). After 4 weeks of CMS, sucrose preference in the Sini Sn group nd the stress-only group ws lower thn tht in the nive control group (P=0.035); sucrose preference in the fluoxetine nd comintion groups ws not significntly different from the nive control group (P> 0.05), ut higher thn tht in the stress-only group (P= 0.022); rts tht hd received Sini Sn consumed less JTCM www. journltcm. com 676
4 sucrose thn those in the fluoxetine group (P=0.025). After 6 weeks of CMS, the consumption in the Sini Sn group remined lower thn tht in the nive control group nd the fluoxetine group (P=0.012), ut ws higher thn tht in the stress-only group (P= 0.023); the fluoxetine nd comintion groups showed no significnt difference in sucrose preference compred with the norml control group (P>0.05), ut consumed more thn the stress-only group (P=0.048). After 8 weeks of CMS, the Sini Sn, fluoxetine, nd comintion groups showed no significnt differences compred with the nive control group (P>0.05), nd hd higher level of consumption thn the stress-only group (P=0.003). The chnges oserved within ech group s CMS continued were s follows: in the stress-only group, sucrose preference decresed grdully throughout the experiment (P=0.025); in the Sini Sn group, consumption reduced until 4 weeks (P= 0.046) ut rose gin etween 4 nd 8 weeks of CMS (P=0.019); consumption in the fluoxetine group reduced fter 2 weeks of CMS (P=0.020) rose gin fter 4 weeks (P=0.031) nd reched plteu t 6-8 weeks of CMS (P>0.05); the pttern of chnge in sucrose preference in the comintion group followed tht in the fluoxetine group, reducing fter 2 weeks of CMS (P=0.039), incresing fter 4 weeks (P=0.022) nd stilizing from 6 weeks until the end of the experiment (P>0.05) (Figure 1). Sucrose preference (%) c c Locomotor ctivity Before the stress, no significnt differences were found mong the five groups of rts in the frequency of gridline crosses or the totl distnce moved in the open field (P>0.05). After 4 weeks of CMS exposure, the gridline-crossing frequency nd the totl distnce moved in the stress-only group, Sini Sn group, fluox- 100 etine group nd comintion group were significntly lower thn those of the nive control group (P=0.034); the gridline-crossing frequency nd the totl distnce moved y rts in the Sini Sn group, fluoxetine group nd comintion group were significntly higher thn those in the stress-only group (P=0.021), ut oth prmeters were lower in the Sini Sn group thn in the fluoxetine group (P=0.013), nd no significnt difference ws found etween the comintion nd fluoxetine groups (P>0.05). After 8 weeks of CMS, the gridline-crossing frequency nd totl distnce moved in the stress-only group, Sini Sn group, fluoxetine group nd comintion group were significntly lower thn those in the nive control group (P=0.027); the gridline-crossing frequency nd totl distnce moved in the Sini Sn group, fluoxetine group nd the comintion group were significntly higher thn those in the stress-only group (P=0.032), with no significnt differences mong the three drug-treted groups (P>0.05). Progression over time in the two locomotor ctivity prmeters etween 4 nd 8 weeks of CMS within ech group ws s follows: gridline-crossing frequency nd totl distnce moved in stress-only rts decresed t oth time points (P=0.030); the Sini Sn group showed decrese in oth prmeters t 4 weeks fter CMS (P=0.022) nd rise gin t 8 weeks (P=0.029); the fluoxetine nd comintion groups showed decrese in locomotor ctivity 4 weeks fter the stress (P= 0.042) which remined stle until eight weeks (P> 0.05) (Tles 1 nd 2). Hippocmpl expression of 5-HT After 4 weeks of CMS, 5-HT expression in the hippocmpl CA2 region ws determined in 20 rts of ech group using immunohistochemistry. Levels of 5-HT in the four experimentl groups were lower thn in the Before stress (n=40) After 2 weeks of After 4 weeks of After 6 weeks of After 8 weeks of 0 Nive control group Stress-only group Sini Sn group Fluoxeting group Comintion group Figure 1 Sucrose preference (%) in rts undergoing chronic mild stress Nive rts were used s controls; the stress-only group were exposed to chronic mild stress (CMS) with no susequent drug tretment; the Sini Sn group were exposed to CMS then received Sini Sn; the fluoxetine group were exposed to CMS then fluoxetine; the comintion group were exposed to CMS then comined dministrtion of Sini Sn nd fluoxetine. JTCM www. journltcm. com 677
5 nive control group (P=0.024); those in the Sini Sn group were not significntly different from those in the stress-only group (P>0.05), while the remining groups hd higher 5-HT expression levels thn the stress-only group (P=0.036); Sini Sn-treted rts showed lower 5-HT levels thn the fluoxetine group (P=0.011), wheres the comintion group ws not different from the fluoxetine group (P>0.05). After 8 weeks of stress, 5-HT expression in CA2 ws determined in the remining 20 rts in ech group. Expression levels in the four experimentl groups were lower thn those in the nive control group (P=0.034); the Sini Sn, fluoxetine nd comintion groups showed significntly greter 5-HT expression thn the stress-only group (P=0.008); 5-HT expression in the Sini Sn nd comined groups ws significntly higher thn tht in the fluoxetine group (P=0.017). Chnges oserved in 5-HT expression within ech group etween 4 nd 8 weeks post-cms were s follows: decrese in the stress-only group (P=0.036), n increse in the Sini Sn group (P=0.025), nd no chnge in the fluoxetine, comintion nd control groups (P>0.05) (Tle 2 nd Figure 2). 5-HTexpression in smll intestine After 4 weeks of stress, the expression of 5-HT in the smll intestine ws mesured in 20 rts of ech group using immunohistochemistry. The four experimentl groups showed greter expression of 5-HT thn the Li YB et l. / Experimentl Study Tle 1 Gridline-crossing frequency (totl numer of crosses in 3 min control group (P=0.001); expression levels in the Sini Sn group were not significntly different from those in the stress-only group (P>0.05); the levels of 5-HT in the fluoxetine nd comintion groups were significntly lower thn those in the stress-only group (P= 0.029); in the Sini Sn group, the expression of 5-HT ws significntly higher thn tht in the fluoxetine group (P=0.017); nd 5-HT expression in the fluoxetine group ws not significntly different from tht in the comintion group (P>0.05). After 8 weeks of stress, 5-HT expression in the smll intestine ws mesured in the remining 20 rts of ech group. The levels of 5-HT in the four experimentl groups were higher thn those in the nive control group (P=0.027); the Sini Sn, fluoxetine nd comintion groups hd significntly lower 5-HT expression thn the stress-only group (P=0.036); the levels of 5-HT in the Sini Sn group nd the comintion group were lower thn those in the fluoxetine group (P=0.041). The chnges in 5-HT expression in the smll intestine within ech group from 4 to 8 weeks fter the stress were s follows: n increse in the stress-only group (P=0.022), decrese in the Sini Sn nd fluoxetine groups (P= 0.014); nd no significnt chnge in the comintion or nive control groups (P>0.05) (Tle 3 nd Figures 3). Serum 5-HT concentrtions Before CMS, no significnt differences were found in serum 5-HT content mong the five groups of rts (P> Group Before 4 weeks fter 8 weeks fter stress (n=20) Nive control group 71.8± ± ±6.3 Stress-only group 76.1± ± ±2.4 Sini Sn group 77.4± ±6.8 c 59.8±9.5 Fluoxetine group 79.6± ± ±11.2 Comintion group 73.6± ± ±13.9 Notes: nive rts were used s controls; the stress-only group were exposed to chronic mild stress (CMS) with no susequent drug tretment; the Sini Sn group were exposed to CMS then received Sini Sn; the fluoxetine group were exposed to CMS then fluoxetine; the comintion group were exposed to CMS then comined dministrtion of Sini Sn nd fluoxetine. P<0.05, compred with the stress-only group; P<0.05, compred with the norml control group; c P<0.05, compred with the fluoxetine group. Tle 2 Men opticl density of 5-HT in hippocmpl CA2 in rts undergoing chronic mild stress (n=20) Group Nive control group Stress-only group Sini Sn group Fluoxetine group After 4 weeks of stress 431±26 308±36 321±24 c 391±50 After 8 weeks of stress 497±50 123±32 402±26 c 372±35 Comintion group 398±46 410±70 c Notes: nive rts were used s controls; the stress-only group were exposed to chronic mild stress (CMS) with no susequent drug tretment; the Sini Sn group were exposed to CMS then received Sini Sn; the fluoxetine group were exposed to CMS then fluoxetine; the comintion group were exposed to CMS then comined dministrtion of Sini Sn nd fluoxetine. P<0.05, compred with the stress-only group; P<0.05, compred with the control group; c P<0.05, compred with fluoxetine group. JTCM www. journltcm. com 678
6 A B C D E F G H I Figure 2 Expression of 5-HT in rt hippocmpl CA2 (Immunohistochemicl stining, 200) A: stress-only group fter 4 weeks of stress; B: stress-only group fter 8 weeks of stress; C: nive control group; D: Sini Sn group fter 4 weeks of stress; E: fluoxetine group fter 4 weeks of stress; F: comintion group fter 4 weeks of stress; G: Sini Sn group fter 8 weeks of stress; H: fluoxetine group fter 8 weeks of stress; I: comintion group fter 8 weeks of stress. Tle 3 Men opticl density vlues of 5-HT in rt smll intestine Group Nive control group Stress-only group Sini Sn group Fluoxetine group After 4 weeks of stress (n=20) 68±20 234±42 251±60 c 101±61 After 8 weeks of stress (n=20) 87±37 495± ±132 c 126±95 Comintion group 93±39 104±61 c Notes: nive rts were used s controls; the stress-only group were exposed to chronic mild stress (CMS) with no susequent drug tretment; the Sini Sn group were exposed to CMS then received Sini Sn; the fluoxetine group were exposed to CMS then fluoxetine; the comintion group were exposed to CMS then comined dministrtion of Sini Sn nd fluoxetine. P<0.05, compred with the stress-only group; P<0.05, compred with the control group; c P<0.05, compred with the fluoxetine group. 0.05). After 2 weeks of stress, serum concentrtions of 5-HT in ll four experimentl groups were higher thn in the nive control group (P=0.002), with lower concentrtion oserved in the comintion group thn in the stress-only nd fluoxetine groups (P=0.036). After 4 weeks of stress, serum levels of 5-HT in ll experimentl groups were higher thn in the nive control group (P=0.041); rts treted with fluoxetine or oth drugs in comintion hd lower serum concentrtions of 5-HT thn those in the stress-only group (P= 0.020), nd 5-HT serum concentrtions in rts of the Sini Sn group were not significntly different from those of the stress-only group (P>0.05) ut higher thn those of the fluoxetine group (P=0.031). After 6 weeks of stress, serum 5-HT levels were still higher in the Sini Sn group thn in the control nd fluoxetine groups (P=0.014), with no significnt difference from the stress-only group (P>0.05); nd the fluoxetine nd comintion groups hd higher 5-HT levels thn the control group (P>0.05). After 8 weeks of stress, serum 5-HT levels in the Sini Sn nd comintion groups were significntly lower thn those in the stress-only JTCM www. journltcm. com 679
7 A B C D E F G H I Figure 3 Expression of 5-HT in rt smll intestine (Immunohistochemicl stining, 200) A: stress-only group fter 4 weeks of stress; B: stress-only group fter 8 weeks of stress; C: nive control group; D: Sini Sn group fter 4 weeks of stress; E: fluoxetine group fter 4 weeks of stress; F: comintion group fter 4 weeks of stress; G: Sini Sn group fter 8 weeks of stress; H: fluoxetine group fter 8 weeks of stress; I: comintion group fter 8 weeks of stress. Tle 4 Serum 5-HT concentrtions Before the stress Group (n=40) Nive control group 119±18 After 2 weeks of 120±26 group (P=0.026) nd not significntly different from the control group (P>0.05). Fluoxetine-treted rts hd higher serum 5-HT levels thn rts in the Sini Sn or comintion groups (P<0.05), ut lower levels thn the stress-only group (P=0.012). Within ech group, the chnges oserved in serum 5-HT levels cross the experiment were s follows: consistent increse in the stress-only group (P=0.026); n increse in the Sini Sn group until 4 weeks of stress (P=0.034) followed y reduction etween 6 nd 8 weeks of stress (P= 0.026); nd pek in oth the fluoxetine nd comintion groups fter 2 weeks of stress (P=0.020) followed y decrese fter 4 weeks (P=0.034), nd stilizing etween 6 to 8 weeks (P>0.05) (Tle 4). DISCUSSION Modern medicl reserch hs demonstrted tht the iologicl sis for depression is relted to lck of the monomine neurotrnsmitter 5-HT, which plys role in the regultion of emotion, memory, ppetite, sexul function, nd ehvior in mmmls. 7 The physiologi- After 4 weeks of 125±20 After 6 weeks of stress (n=20) 114±26 After 8 weeks of stress (n=20) 120±25 Stress-only group 126±27 178±36 188±30 209±23 239±13 Sini Sn group 120±21 161±30 179±42 c 160±49 c 118±53 c Fluoxetine group 121±20 168±24 151±34 130±40 129±14 Comintion group 123±23 160±34 c 145±36 120±39 117±10 c Notes: nive rts were used s controls; the stress-only group were exposed to chronic mild stress (CMS) with no susequent drug tretment; the Sini Sn group were exposed to CMS then received Sini Sn; the fluoxetine group were exposed to CMS then fluoxetine; the comintion group were exposed to CMS then comined dministrtion of Sini Sn nd fluoxetine. P<0.05, compred with the stress-only group; P<0.05, compred with the control group; c P<0.05, compred with the fluoxetine group. JTCM www. journltcm. com 680
8 cl roles of 5-HT lso include the stimultion of intestinl secretion nd direct ction on smooth muscle, thus regulting gstrointestinl motility nd secretion; conversely, physiologicl stimultion (eting) cn promote the relese of 5-HT. 8 In the present study, the persistent increse of 5-HT in the serum nd smll intestine in rts exposed to CMS suggests tht chronic, unpredictle low-intensity stress cn promote the relese of 5-HT, resulting in high sensitivity of the viscerl fferent nerves nd the enteric nervous system, which my ctivte vriety of neurl ctive sustnces. This my in turn disrupt the chemicl signls involved in the regultion of the rin-gut xis, resulting in norml gstrointestinl motility nd viscerl hypersensitivity, mnifesting s dirrhe in rts. Chnges in the levels of centrl nd peripherl 5-HT my e useful iomrkers for the clinicl dignosis nd tretment of depression. Modern medicine hs proved tht 5-HT minly comes from the centrl nervous system nd the intestinl trct. The centrl nd peripherl metolism of 5-HT occurs in two independent systems ecuse it cnnot cross the lood-rin rrier. 9 In the present study, the level of 5-HT in the hippocmpus of rts in the stress-only group decresed s CMS continued, while tht in serum nd the smll intestine stedily incresed, suggesting tht stress differentilly ffects centrl nd peripherl 5-HT. Depression is n ffective disorder chrcterized y persistent low mood. In Chinese medicine, depression flls into the ctegory of Yu Zheng. The Trditionl Chinese Medicine Sini Sn relieves the depressed liver nd hrmonizes the functions of liver nd spleen. The unique lw of comptiility, the therpeutic effects nd the sfety of Sini Sn hve een confirmed y long-term clinicl prctice. 10 The results of the present study indicte tht Sini Sn cn e used in comintion with fluoxetine to relieve symptoms of depression. In the lter stges of tretment, fluoxetine cn e decresed or stopped while Sini Sn is continued, to void the side-effects experienced fter long-term use of the fluoxetine in the clinicl tretment of depression. REFERENCES 1 Shen Y. Psychitry. Beijing: People's Medicl Pulishing House, 1995: Yng GL, Wng WP, Wng J, et l. Clinicl epidemiology investigtion on the TCM syndromes of depression. Lioning Zhong Yi Z Zhi 2008; 35(2): Fores NF, Stewrt CA, Mtthews K, et l. Chronic unpredictle mild stress nd sucrose consumption: vlidity s model of depression. Physiol Behv 1996; 60(6): Americn Psychitric Assocition. Dignostic nd sttisticl mnul of psychitric disorders, 4th ed (DSM-IV). Wshington D. C: Americn Psychitric Assocition, 1994: Shi XY. Medicl Lortory Animl Science. first edition. Shnxi: Shnxi Science nd Technology Press, 1989: Ycoui ME, Bouli S, Pop D, et l. Behviorl, neurochemicl, nd electrophysiologicl chrcteriztion of genetic mouse model of depression. Proc Ntl Acd Sci USA 2003; 100(10): Butterweck V, Wll A, Lieflnder Wulfu, et l. Effects of the totl extrct nd frctions of hypericum perforte in niml ssys for ntidepressnt ctivity. Phrmcopsychitry 1997; 30(S2): Muchimpur S, Fulford AJ, Mson R, et l. Isoltion rering in the rt disrupts the hippocmpl response to stress. Neuroscience 2002; 112(3): Linthorst AC, Penlv RG, Flchskmm C, et l. Forced swim stress ctivtes rt hippocmpl serotonergic neurotrnsmission involving corticotrophin-relesing hormone receptor-dependent mechnism. Eur J Neurosci 2002; 16 (12): Sun SC, Ji FY, Zhng JF. Modified Sini Sn: its long-term toxicity test nd sfety evlution. Shnxi Zhong Yi Xue Yun Xue Bo 2008; 31(3): JTCM www. journltcm. com 681
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