| Objective:To investigate the effects of cytotoxin ingredients CTXn and CTX1 in cobra venom on drug-abuse and the possible initial mechanism.Methods:(1) Different doses of crude venomⅣof snake venom(0.027-0.08 mg/kg, ip) were given to test the effect of crude venomⅣof snake venom on morphine-induced CPP after the establishment of morphine-dependent CPP model, and including the intervention and therpeutic action for the withdrawal syndroms induced by naloxone.(2) Five ingredients were separated from Crude venomⅣ,then each component was isolated and selected. Male SD rats were administered with morphine(10 mg/kg, sc)for eight days to test CPP; Rats were randomly divided into seven group:morphine control groups, saline control groups and rats treated with differernt ingredients of crude venom groups. Rats treated by intraperitoneal injection (ip) different ingredients in crudeⅣof snake venom were to study the effects of different ingredients on morphine induced CPP; Determination of the amino acid sequence of the effective components and make a identification.(3) To study the effects of CTXn on the acquisition and maintenance of morphine-induced conditioned place preference in rats.①Estaplished a CPP model induced by morphine (10 mg/kg,sc) for successive 8 days in rats, and then different doses of CTXn(0.5,1.0,2.0 mg / kg,IP)was administrated to the rats for 5 successive days, CPP and pain threshold were tested, the brain slices, immunohistochemical staining, Using LSCM to study the expression of TNF-αin the hippocampus dentate gyrus.②Rats were administered with CTXn (0.5,1.0,2.0 mg/kg, IP), or vehicle once a day 30 min before morphine (10 mg/kg, SC) treatment for 8 days,Test the effect of CTXn on CPP and locmotor activity induced by morphine. TNF-αexpression in hippocampus were carried by immunohistochemical staining.③Rats were administered with CTXn (0.5,1.0,2.0 mg/kg, IP) or vehicle once a day, for 18 days, observe the CPP induced by itself. SD rats were given intraperitoneal CTXn (1.0mg/kg) group or to give intrathecal CTXn (25μg/kg), continuous medication 24 days in the d0, d2, d4, d6, d8, d10, d12, d14, d16, d18, d20, d22, d24 pain threshold testing in rats to observe the changes in the pain threshold, while serum antibody titer were performed by using ELISA assay in the d0, d4, d8, d12, d16, d20, d24.(4) To study the effects of CTX1 on the acquisition and maintenance of morphine-induced conditioned place preference in rats.①Different doses of CTX1(0.1,0.2,0.4 mg/kg, IP)was administrated to the rats for 5 successive days after estaplishment a CPP model induced by morphine, CPP and pain threshold were tested, the brain slices, immunohistochemical staining,using LSCM to study the expression of TNF-αin hippocampus.②Rats were administered CTX1 (0.1,0.2,0.4 mg/kg, IP), or vehicle once a day 30 min before morphine (10 mg/kg, SC) treatment for 8 days, test the effect of CTX1 on CPP and locmotor activity induced by morphine. TNF-αlevels were performed and TNF-αexpression in hippocampus were carried by immunohistochemical staining, and LTP were performed in morphine, saline control group and rats treatment with CTX1 group.③Rats were administered CTX1 (0.1, 0.2, 0.4 mg/kg, IP) or vehicle once a day, for 8 days,observe the CPP induced by itself.④SD rats were given intraperitoneal CTX1 (0.2 mg/kg) group or intrathecal CTX1 (10μg/kg) group, continuous medication 24 days in the d0, d2, d4, d6, d8, d10, d12, d14, d16, d18, d20, d22, d24 pain threshold testing in rats to observe the changes in the pain threshold, while serum antibody titer were performed by using ELISA assay in the d0, d4, d8, d12, d16, d20, d24. Results:(1) 10 mg/kg morphine could induce rats acquiring a significant place preference, and both doses of shake venom could not induced CPP; Administered (ip) of crude venomⅣafter morphine-induced CPP blocked the development of morphine-induced CPP and the withdrawal symptoms induced by Naloxone could be inhibited .(2) Five components are separateed and purified from crude venomⅣ:Ⅳ1,Ⅳ3,Ⅳ4,Ⅳ5,Ⅳ7 peak respectively, the ingredients ofⅣ4 andⅣ5 could obviously inhibit CPP induced by morphine, and suppressed the withdrawal syndromes induced by naloxone, The ingredients ofⅣ4 andⅣ5 show signifficant different compared with the rats treated with the ingredients ofⅣ1,Ⅳ3 andⅣ7; an average weight increase of rats treated with the ingredients ofⅣ4 andⅣ5 were 15.5% and 17.1%. However, rats treated with the ingredients ofⅣ1,Ⅳ3 andⅣ7 were 6.2%, 4.9% and 8.8% respectively,amino acid sequence analysis showedⅣ4 is CTXn,Ⅳ5 peak for CTX1.CTXn array:LKCNQ LIPPF YKTCA AGKNL CYKMF MVAAP KVPVK RGCID VCPKS SLLVK YVCCN TDRCNCTX1 array:LKCNK LIPIA SKTCP AGKNL CYKMF MMSDL TIPVK RGCID VCPKS NLLVK YVCCN TDRCN(3) Effects of CTXn on the acquisition and maintenance of morphine-induced conditioned place preference in rats.①CTXn post-treatment on rats'addiction can be inhibited morphine-induced CPP and hyperalgesia in a dose-dependent post-processing. CPP values of administration different dose of CTXn groups (0.5, 1.0, 2.0 mg / kg) were from the addiction to the time of 451.8±49.2,450.8±72.3 and 450.7±91.2s fell to 164±49.5,77.4±41.1 and 68.2±37.2s respectively; Rat pain sensitivity threshold from the addiction when they were 15.5±1.6,15.2±3.2 and 15.6±2.3s, increased to 22.4±1.7,27.5±2.8 and 28.6±3.6s respectively;②Post-treatment with differernt doses of CTXn can inhibited the morphine-induced CPP, locomotor activity and analgesic effect in a dose dependent manner.CTXn post-processing can be significantly increased TNF-αexpression in the dentate gyrus and restore functions of neurons in rat dentate gyrus;③CTXn (0.5-2.0 mg/kg) before morphine administration for 8 consecutive days could mitigate morphine-induced CPP and locomotor activity;④The continued use of cytotoxin CTXn showed no reaward effect, while continuous administration of CTXn for 24 days could induce antibody production following analgesic tolerance, intrathecal injection of 0.025 mg/kg CTXn had no such effect.(4) Effects of CTX1 on the acquisition and maintenance of morphine-induced conditioned place preference in rats.①CTX1 post-treatment on rats'addiction can inhibite morphine-induced CPP in a dose-dependent and hyperalgesia;②after morphine addiction, CTX1 post-treatment significantly increased the TNF-αexpression in hippocampal CA region;③CPP values were not significantly increased in rats pre-treatment with CTX1,CTX1 could dose-dependently inhibit morphine-induced CPP acquisition,and shows an inhibitory role in morphine addiction; CTX1 significantly increased the levels of TNF-αand the expression of hippocampal CA area, and significantly chang the inhibition of morphine on LTP;④CTX1 itself does not produce CPP, CTX1 abdominal medication 16 days later, serum antibody titers gradually increased, a corresponding decrease analgesic effects; but antibody production and analgesic tolerance.were not seen in rats treatment with CTX1 (intrathecal).Conclusion:(1) Purification of cytotoxicⅣin the monomer material CTXn and CTX1 dose-dependent inhibition of morphine-induced CPP maintenance in rats, which can supress morphine addiction.(2) CTXn and CTX1 not addictive itself does not produce CPP, CTXn and CTX1inhibited morphine-induced CPP acquisition, the prevention of morphine addiction, morphine hyperalgesia has a good analgesic effect, but long-term use will produce antibodies that induce analgesic tolerance to analgesic effect,The phenomenon can be avoided by intrathecal drug delivery.(3) CTXn could promote TNF-αexpression in the hippocampus dentate gyrus, and promote neuronal function recovery.CTX1 can increase the levels of TNF-αto promote TNF-αexpression in the hippocampus in the hub can be lifted of morphine on the inhibition of LTP. (4) CTXn and CTX1 have better suppression and prevention morphine addiction,CTXn and CTX1 have good effect of drug addiction. |
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