| Objective: To separate and purify analgesic component, cobra venom analgesic factor(CVAF), from venom of Wannan area cobra and investigate its possible mechanism ofanalgesic effect.Methods:1. The active component CVAF was purified from crude venom of cobra using cationexchange chromatography and size-exclusion chromatography. Its purity and molecularweight were determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) andcapillary zone electrophoresis (CZE), respectively. LD50of analgesic component weredetected; Morphological changes were also observed in major organs.2. The analgesic activity of CVAF was analyzed through hot-plate test and acetic acidwrithing test, respectively. The mice were seperated to5groups randomly, including salinegroup (negative control), morphine group (positive control) and experimental groups(0.03mg/kg,0.1mg/kg, and0.3mg/kg). Analgesic activity of CVAF was also evaluated onthe mice stimulated by hot plate test after administrating with atropine, naloxone andL-NAME.3. Inflammatory pain model of rat (CFA group) was established using CFA (150μl, s.c.). Thechanges of thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT),induced by CVAF, were determined by Spurs pain and Mechanical pressure methods,respectively. The levels of interleukin (IL)-1and tumor necrosis factor (TNF)-α in spinal cordwere detected by enzyme-linked immunosorbent assay (ELISA). Analgesic activity of CVAFwas also evaluated on the CFA group after administrating with atropine, naloxone andL-NAME. Results:1. Single active component, CVAF, was obtained from crude cobra venom followed by IECand SEC. The molecular weight was nearly6500Da; LD50of CVAF was0.6289mg/kg.2. After administrating CVAF, the analgesic effect lasted from0.5h to8h. The writhinginhibition rates were27%,50%and70%, respectively, with intraperitoneal injection using0.03mg/kg,0.1mg/kg and0.3mg/kg in mice. Compared with morphine group, there wasno statistical different in0.3mg/kg group (P>0.05). At a certain extent, atropine (5mg/kg)and naloxone (4mg/kg) could reverse analgesic activity of CVAF in hot plate test. Anonselective inhibitor of nutric oxide synthase, L-NAME, could increase the analgesicactivity of CVAF.3. Compared with NC group, TWL and MWT in CFA group were decreased from1stday to14thday; thermal hyperalgesia and mechanical allodynia were also presented simultaneously.Moreover, the phenomena mentioned above were relieved after injecting CVAF. The levels ofIL-1and TNF-α from spinal cord in CFA group were significantly higher than that of NCgroup (P <0.01). Compared with CFA group, the levels of IL-1and TNF-α in CFA groupinjected intraperitoneally with CVAF were significantly reduced (P <0.01). The resultshowed CVAF played a role of analgesic activity via inhibition of inflammatory factor. Afterinjecting intraperitoneally CVAF followed by injecting L-NAME in CFA group, TWL andMWT of rats were arised obviously, than that of CFA+CVAF group (P <0.01). CVAF didnot affect TWL and MWT in CFA group before and after injecting atropine or naloxone (P>0.05). However, compared with CFA+CVAF group, atropine or naloxone could returnincreased TWL and MWT by CVAF (P <0.01). The results indicated that both opioidpeptide receptor system and cholinergic receptor system could be related to analgesic effect ofCVAF for inflammatory pain.Conclusion:1. Single active component, CVAF, was obtained from crude cobra venom successfully.The molecular weight is6500Da. The LD50of CVAF is0.6289mg/kg. 2. CVAF plays a role of analgesic effect with dose-dependent. Both opioid peptide receptorsystem and cholinergic receptor system could be related to analgesic effect of CVAF.3. CVAF plays a role of analgesic activity via inhibition of inflammatory factor. Both opioidpeptide receptor system and cholinergic receptor system could be related to analgesic effect ofCVAF for inflammatory pain. However, the exactly mechanism should be further analyzed. |
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