| BackgroundIn recent years, exogenous opioids (such as morphine, heroin) abuse hasbecame an increasingly serious social problem. Substance dependence includesphysical dependence and psychological dependence. There are various effectivemethods to reduce physical dependent symptoms, but there are not effective drug ormeasure to cure psychological dependent symptoms, such as the strong drug-seekingbehavior. Clinical research showed that majority patients of morphine dependencehave anxiety symptoms as their withdrawal symptoms disappeared. Anxiety is oneof the main withdrawal symptoms and protracted symptoms, drug-seeking behavioris positively correlated with anxiety. Aspiration and anxiety are the main reasonswhich cause patients relapse. So, it is very necessary to explore the changes ofanxiety of the morphine dependent patients and find a feasible treatment.Monoamine neurotransmitters, such as DA and 5-HT, NE, are closely relatedwith the occurrence of anxiety. The disorders of DA, 5-HT and NE system may bethe most fundamental neurobiology mechanism of drug-seeking behavior andanxiety after withdrawal. Therefore, if the disorders in monoamine neurotransmitter are regulated, there will have a good anti-anxiety effect.OBJECTIVETo establish the model of morphine dependent rats. The level of anxiety of ratsis measured with the elevated plus-maze (EPM), so effects of Qingfeng capsule onanxious behavior of morphine dependent rats are evaluated. Determine thecontents of NE, DA, 5-HT in the brainstems of rats with spectrofluorometry. order to explore the neurobiology mechanisms 01 antianxiety enects 01 Qinglengcapsule.METHODS1. Model and administration: 48 SD rats were divided into six groups atrandom: normal control group, model control group, diazepam (DZP) group (1mg-kg-1and QFJN high (2g-kg-1), medium (lg-kg-1), low (0.5g-kg-1) dosegroup(n=8). From dl to d5, rats of five modeling group(includmg model controlgroup, DZP group and QFJN high, medium, low dose group) were injected withmorphine according to a gradually increasing dose scheme. Rats of normal controlgroup were injected with normal saline. At d6, all rats were injected with naloxone(2.0 mg-kg-1) at 8:00. l0h after the administration of naloxane, all rats weretested in EPM. From d7 to d21, all rats were not treated, and ate and drank waterfreely. From d22 to d26, rats of five modeling group were injected with morphineaccording the same scheme as before. Rats of normal control group were injectedwith normal saline. At d27, all rats were injected with naloxone (2.0 mg-kg-1) at8:00. Then all rats were tested in EPM lOh after the injection of naloxone. Fromd27 to d31, rats of DZP group and QFJN high, medium, low dose group wereintragastric(ig) administered with DZP and QFJN once a day. Rats of normal andmodel control group were ig administered with normal saline.2. EPM test Test time: Before injection of morphine, lOh after the first injection of naloxone, 10h after the second injection of naloxone, 12h after the lastadministration. Test environment: Dim light, quiet, 24°C. Test method: At first,each rat was placed in a large box and free for 5 minutes. Then it was placed on theEPM central platform with it's head to one open arm, the following indexs of the ratwere recorded:①open arm entry (OE);②open arm time(OT);③close armentry(CE);④Close arm time(CT). Each rat was tested for 5 minutes. Calculatedthe following value according to①～④: (Dthe total of open arm entry and closearm entry(OE+CE): represent the locomotor activity of rats. ?the total of openarm time and close arm time(OT+CT);③The percent of open arm time(OE%):OE/(OE+CE)x100%;④The percent of open arm entry(OT%): OT/(OT+CT)x1OO%.3. Score the withdrawal symptoms of rats: after the first or the secondinjection of naloxone, the withdrawal symptoms of each rat were recorded for 15minutes and scored.4. Determination of the contents of NE, DA, 5-HT: After the last maze test,each rat was put to death with its head cut off. Then immediately take the rat brain.The rat brains were kept at -50°C. The rat brainstems which were taked and madeto sample solution by weighing, homogenate, centrifugalization. Determine thecontents of NE, DA and 5-HT by spectrofluorometry.5. Statistical method: Statistical analysis was performed by using the programSPSS11.5. In all statistical tests, significance level was set to p<0.05. The EPMdatas of all groups and each group were analysed by repeated measures ANOVA,and the data at each time were analysed by one-way ANOVA respectively, allANOVA followed by multiple post-hoc tests (Fisher's LSD). The datas of thecontents of NE, DA, 5-HT and scores of withdrawal were analysed by one-wayANOVA, then followed by multiple post-hoc tests (Fisher's LSD). RESULTS1. Before injiection of morphine, the value of OE + CE, OT + CT, OE%, OT%have no significant difference among all groups (P> 0.05). After the first or thesecond injection of naloxone, OE% and OT% of rats of five modelinggroups(including model control group, DZP group and QFJN high, medium, lowdose group) were lower than those of normal control group (P=0.002,0.000, 0.016,0.000, 0.001, 0.001, 0.000, 0.001, 0.000, 0.001) . Rats of all groups had nosignificant difference in the value of OE+CE, OT+CT (P> 0.05). That indicated themorphine dependent rats had obviously anxious symptoms. And the scores of thewithdrawal symptoms of five modeling groups rats were higher than those of thenormal control group (P=0.000).2. Effects of QFJN on anxious behavior of rats: After treatment with QFJNand DZP, rats of all groups had no significant difference in OE + CE, OT + CT(P> 0.05). OE% and OT% of all groups rats had significant difference (F=3.974,2.837; P=0.005,0.027) . OE% and OT% of model control group were lower thanthose of normal control group (P=0.01). OE% of QFJN high, medium dose groupand DZP group were higher than those of model control group (P=0.000, 0.025,0.026, OT% of QFJN high, low dose group and DZP group were higher than thoseof model control group (P=0.006, 0.02, 0.029) . OE% and OT% of QFJN high,medium dose group, OT% of the QFJN low dose group were not significantlydifferent with those of the DZP group (P > 0.05).3. The contents of NE, DA, 5-HT: There were significantly discrepancysamong all group (F=3.684, 2.889, 4.538; P=0.007, 0.025, 0.002) . For thecontents of NE, DA, 5-HT, model control group was higher than normal controlgroup (P=0.001, 0.005, 0.003) . For the content of NE and DA, QFJN high,medium dose group and DZP group were lower than model control group (P=0.005, 0.015,0.001, 0.007,0.008,0.011) . For the content of 5-HT, QFJN high, medium,low dose group and DZP group were lower than model control group (P=0.001,0.004,0.002,0.000) .CONCLUSION1. Before injiection of morphine, the locomotor activity and anxiety levels of allrats had no significant difference. After injiection of morphine, rats of fivemodeling groups had obviously withdrawal symptoms and anxiety. That mean themodeling method is effective and successful.2. QFJN and DZP can enhance OE% and OT% of the model rats and have noeffect on the locomotor activity of rats. That mean QFJN and DZP can obviouslyalleviate anxiety of morphine dependent rats and they will not suppress the centralnervous system. The strength of antianxiety effects of QFJN is similar to that ofDZP.3. After withdrawal the content of DA, NE and 5-HT in brainstem of themorphine dependent rats increase abnormally. It suggest that these monoaminetransmitters are closely related with anxiety. Qinfeng capsule can significantlyreduce the contents of DA, NE, 5-HT in brainstem of the morphine dependent rats.This effect may be one of the main neurobiological mechanism of antianxiety effectsof Qinfeng capsule.
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