Development Of The Specificity Antagonist For Miniature Pigs' Combined Anaesthetic And Study On Its Mechanisms Of Antagonism

Combined anaesthetic for miniature pigs (XFM) was developed on the basis of balanced anaesthesia theory and physiological characteristics of miniature pigs, and a series of scientific recipe testings. The results showed that XFM had rapid induction of general anesthesia, maintain anesthesia for long time and a much calmer recovery. XFM had stable anaesthetic effect, sure analgesia effect,better sedation and muscle relaxation, little impact on respiration and circulation system and no significant side effects were observed. XFM could provide 60⁷5min surgery time with no damage to heart and brain function. XFM can be used in correlated area scientific research, clinical diagnose and treatment. In order to popularize XFM in clinical application, ensure the physiological functions of miniature pigs rapid recovery after research, and accomplish anaesthesia time of XFM controllability. We planed to develop a efficient, security, no side effects specificity antagonist, and evaluate its antagonistic effect objectively, and the antagonism mechanism of specificity antagonist for miniature pigs' combined anaesthetic were systematically and extensively studied.Specificity antagonist for miniature pigs' combined anaesthetic was first developed by quadrature experimental and screening the best combinations in mice; acute toxicity, cumulative toxicity, tolerance, subchronic toxicity, local stimulation and drug stability were studied; and rats model were anaesthetized by XFM, they administrated specificity antagonist for miniature pigs' combined anaesthetic after being anaesthetized by XFM 10, 30 and 60 min individually, and the tissue samples of different brain regions in rats were collected at different recovery periods. Synaptosomes in cerebral cortex, hippocampus, thalamus, cerebellum and brainstem were separated through differential centrifugation. Effects of specificity antagonist for miniature pigs' combined anaesthetic on ATPases activity in synaptosome in different brain regions was measured by chromatometry. Molecular mechanism of transmembrane signal transduction in central nervous system cell was systematically explained, which was produced by specificity antagonist for miniature pigs' combined anaesthetic. Effects of specificity antagonist for miniature pigs' combined anaesthetic on NO/cGMP and cAMP signal transduction system in different brain regions was dynamically monitored by ELISA and chromatometry. Molecular mechanism of intracellular signal transduction in central nervous system produced by specificity antagonist for miniature pigs' combined anaesthetic was first systematically explained. Experimental results were such as follows:1.Development of specificity antagonist for miniature pigs' combined anaesthetic(1)Components of antagonists were determined by preliminary testing, scientific recipe testing, quadrature experimental verification and screening the best combinations testing, and the components were respectively atipamezole, flumazenil, naloxone with the best ration of 0.90: 0.79: 0.26, and named as specificity antagonist for miniature pigs' combined anaesthetic.(2)Chinese experimental miniature pigs were administrated 0.08 mL·kg-1 specificity antagonist for miniature pigs' combined anaesthetic after being anaesthetized by XFM 30 min,and then integrated monitoring of specificity antagonist for miniature pigs' combined anaesthetic were made. The results showed that it had short recovery time and calm recovery periods; it had ensure antagonistic effect, animals'posture, sedation, analgesia, muscle relaxation and auditory response had ideal and balanced recovery; it had little effect on respiratory and circulatory system; it had no obvious side effects; it had no obvious heart and brain functions damage. XFM and specificity antagonist for miniature pigs' combined anaesthetic can be uesed in related field science research and clinical diagnosis.2.Drug safety, drug combination effects and drug stability testings of specificity antagonist for miniature pigs' combined anaestheticThe effects of specificity antagonist for miniature pigs' combined anaesthetic on mice acute toxicity, accumulative toxicity, tolerance, subchronic toxicity were studied, and its effects on rabbits skin irritative reaction, intramuscular irritation reaction and eyes irritative reaction were also studied; the combination effects of components in specificity antagonist for miniature pigs' combined anaesthetic were studied; and antagonism effect was compared between specificity antagonist for miniature pigs' combined anaesthetic with and without drug accelerated stability testing, in order to judge its stability and effectively for quality guarantee period. Acute toxicity testing was carryed out by sequential method, ED₅₀ and LD₅₀ of specificity antagonist for miniature pigs' combined anaesthetic were measured, and specificity antagonist for miniature pigs' combined anaesthetic showed higher security, because its AI=4.04; specificity antagonist for miniature pigs' combined anaesthetic had mild accumulation effect, and its accumulation coefficient K=5.26, and tolerance was not produced in mice; mice in high doses group(l/5 LD₅₀)produced transient hyperventilate and excitement In the first four days, and side effects disappeared after four days; weigh gain were measured after being administrated specificity antagonist for miniature pigs' combined anaesthetic, and there had insignificant difference bewteen different groups in mice; there was no significant difference between experimental groups and control group in organ coefficient; there was no significiant difference between different groups in mice blood biochemical indicators; and there were no obvious pathological changes in lung, liver and kidney; according to the median effect principle, specificity antagonist for miniature pigs' combined anaesthetic had scientific recipe because of its CI=0.463; the results of drug stability testings showed that specificity antagonist for miniature pigs' combined anaesthetic was very stable, and its effective quality guarantee period was two years.3.Study on Antagonism mechanism of specificity antagonist for miniature pigs' combined anaesthetic(1)Na⁺,K⁺-ATPases activity was markedly activated by specificity antagonist for miniature pigs' combined anaesthetic in cerebral cortex and hippocampus, and the changes of Na⁺,K⁺-ATPases activity in rats brain regions were coincident with rats behavior changes. The results showed that antagonism mechanism of specificity antagonist for miniature pigs' combined anaesthetic maybe correlate with special brain regions Na⁺,K⁺-ATPases activity. And Na⁺,K⁺-ATPases activity in cerebral cortex and hippocampus might be one of target sites of specificity antagonist for miniature pigs' combined anaesthetic.(2)Ca²⁺,Mg²⁺-ATPase activity was markedly activated by specificity antagonist for miniature pigs' combined anaesthetic in brainstem and hippocampus, and the changes of Ca²⁺,Mg²⁺-ATPase activity in rats brain regions were coincident with rats behavior changes. The results showed that antagonism mechanism of specificity antagonist for miniature pigs' combined anaesthetic might be correlate with special brain regions Ca²⁺,Mg²⁺-ATPase activity. And Ca²⁺,Mg²⁺-ATPase activity in brainstem and hippocampus might be one of target sites of specificity antagonist for miniature pigs' combined anaesthetic.(3)cAMP content was markedly decreased by specificity antagonist for miniature pigs' combined anaesthetic in hippocampus and thalamus, it indicated that cAMP signal transduction system possibly participated in regulation of antagonism mechanism of specificity antagonist for miniature pigs' combined anaesthetic. And antagonistic effect of specificity antagonist for miniature pigs' combined anaesthetic might be correlate with inhibition cAMP signal transduction system in hippocampus and thalamus.(4)NOS activity, NO production and cGMP content were markedly activated by specificity antagonist for miniature pigs' combined anaesthetic in related brain regions. Observation of rats behavior changes showed that NOS activity in brainstem, NO production in thalamus and brainstem, cGMP content in cerebral cortex and brainstem played a main role in righting reflex recovery; NOS activity in cerebral cortex, NO production in cerebellum and brainstem, cGMP content in cerebral cortex, hippocampus, cerebellum and brainstem played a main role in rectilinear creeping recovery. It indicated that NO/cGMP signal transduction system possibly participated in regulation of antagonism mechanism of specificity antagonist for miniature pigs' combined anaesthetic, and its antagonistic effect might be correlate with activation NO/cGMP signal transduction system in related brain regions.