Effects Of XFM And Its Antagonist On The LKB1-AMPK-mTOR Signaling Pathway And The Orexin System

XFM and its antagonist have the characteristics of indeed effect, safety, less side effect and easy to use. But the anesthesia and analeptic mechanism is not fully understood. Latest research shows that intracellular signal transduction system is closely related to animal anesthesia and analeptic mechanism. LKB1-AMPK-m TOR signaling pathway plays an important role in the regulation of local dendritic excitability and long-term regulation of anesthesia. It suggests m TOR might be involved in the regulation of analgesic. Orexin system can regulate sleep and arousal. Presumably orexin system may be involved in the regulation of anesthesia and analeptic. To fully understand the interaction of XFM and its antagonist in the central nervous system of rats, LKB1-AMPK-mTOR signaling pathway and orexin system were studied. First, Select key protein LKB1, AMPK and downstream transcription factors 4EBP1 of mTOR signal transduction system to study the analgesic effect of XFM and its antagonists by the model of incisional pain. The interaction mechanisms of XFM and its antagonist are discussed from the molecular level. Second, select Orexin A and Orexin B to study the awakening mechanism of XFM and its antagonist. Analysis the interaction of XFM and its antagonists inthe lateral hypothalamus region of rats.This experiment were divided into two part, about mTOR signaling system and the orexin system. 126 SD rats were divided into control, incision pain control group, incision pain saline group, anesthetic group, antagonist group and interactive group. Incisional pain model was established after rats were intraperitoneal injected with 0.5% pentobarbital sodium. The drug compounds were injected at the corresponding time point and the rats were euthanized by cervical dislocation. Five brain structures and spinal cord were dissected immediatedly. Quantitative real-time PCR and western blot were performed to measure the mRNA expressions and phorsphorylation of LKB1, AMPKα1, AMPKα2 and 4EBP1. 78 SD rats were divided into control, anesthetic group, antagonist group and interactive group. Lateral hypothalamus area was dissected immediatedly after rats were euthanized by cervical dislocation. Prepro-orexin m RNA expression was measured by PCR and Orexin A and Orexin B protein expression by western blot.The results are as follows:(1) Effects of XFM and its antagonist on the expression of LKB1 in incisional pain rats.XFM significantly activated LKB1 in cerebellum, thalamus, hippocampus and brainstem, the phosphorylation level is consistent with the m RNA expression. Injection of the antagonist inhibited mRNA and protein expression of LKB1 in cerebral cortex, cer ebellum, hippocampus and spinal cord. After antagonist waked XFM, LKB1 phosphorylation of incisional pain significantly reduced in cerebral cortex and cerebellar(P<0.01), indicating that XFM may induce specific brain regions LKB1 mRNA transcription and protein phosphorylation.(2) Effects of XFM and its antagonist on the expression of AMPK in incisional pain rats.XFM significantly activated AMPKα1 and AMPKα2 mRNA levels in cerebellum, thalamus, hippocampus and brainstem and spinal cord(P<0.01), the phosphorylation level is consistent with the mRNA expression. Injection of the antagonist inhibited mRNA and protein expression of AMPK in cerebellum, hippocampus and brainstem. After antagonist waked XFM, AMPK phosphorylation of incisional pain significantly reduced in cerebellar, brainstem and spinal cord(P<0.01), indicating that XFM may induce specific brain regions AMPK mRNA transcription and protein phosphorylation.(3) Effects of XFM and its antagonist on the expression of 4EBP1 in incisional pain rats.XFM markedly elevated the mRNA transcript and phosphorylation of 4EBP1 in five brain areas and spinal cord of incisional pain rats. the antagonists significantly reduced the mRNA and phosphorylation expression of 4EBP1 in cerebellum, thalamus, hippocampus, brainstem of rats. After antagonist waked XFM, the phosphorylation level was significantly inhibited in cerebra l cortex, cerebellum and thalamus(P<0.05), indicating that the anaesthesia effect of XFM may be related to mRNA transcription and phosphorylation in the central nervous system.(4) Effects of XFM and its antagonist on the expression of Prepro-orexin mRNA and Orexin A, Orexin B in different brain regions of rats.XFM can inhibit Orexin A, Orexin B protein expression and prepro-orexin m RNA transcription in LHA regions. Intraperitoneal injection of antagonists significantly activate the prepro-orexin mRNA transcription, promote Orexin A and Orexin B protein expression in LHA regions. After antagonist waked XFM, prepro-orexin mRNA and Orexin A expression were signigicantly inhibited, indicating that the inhibition was conducted through the reduction of orexin expression and this kind of effect was reversed by the antagonist.Conclusion:(1) XFM has a activating effect on LKB1-AMPK-mTOR signaling pathway and the downstream factor. The promoting role may be related to cerebellum, thalamus, brainstem, which may be the pain target of anesthesia and analgesia that LKB1-AMPK-m TOR signaling pathway involved in.(2) The antagonist of XFM is capable of inhibiting the LKB1-AMPK-mTOR signaling pathway and downstream factor in different brain regions and spinal cord, espe cially cerebral cortex, cerebellum and spinal cord. Antagonist efficacy is achieved through LKB1-AMPK-mTOR signaling pathway.(3) XFM and its antagonist have a contrary effect on LKB1-AMPK-mTOR signaling pathway, which may reflect the interaction of XFM and its antagonist at the molecular level. However, antagonists has not been fully reverse the effects XFM caused, indicating that LKB1-AMPK-mTOR signaling pathway is not the sole mechanism in regulation of anaesthesia and analgesia.(4) XFM produces suppressing effect in the LHA regions to arousal caused by the orexin system. The antagonists can reverse the inhibitory effect in LHA of rats generated by XFM, suggesting that the LHA may be one of the waking site orexin system involved in anaesthesia and arousal of XFM and its antagonists.