内源性大麻肽类激动剂Hemopressin相关肽的生物活性研究
发布时间：2018-08-28 13:32
【摘要】：大麻系统在机体的痛觉调节中扮演着重要角色,越来越多研究表明大麻CB_1和CB_2受体是潜在的镇痛药物作用靶标。而传统的大麻配体主要有植物大麻、内源性脂类大麻和人工合成大麻三大类。近年来发现来源于血红蛋白降解片段的一类生物活性肽——Hemopressin及其相关肽能特异性地作用于大麻CB_1和CB_2受体,表现为大麻受体的新型肽类配体。然而,目前的研究主要集中于CB_1受体拮抗剂/反向激动剂Hemopressin的生物学功能方面,而对大麻受体激动剂Hemopressin相关肽的研究相对较少。本实验室已报道的研究发现CB_1受体激动剂(m)VD-hemopressin(α)在痛觉、体温、运动、胃肠道运动、摄食、奖赏、镇痛耐受和心血管活性方面具有一定的调节作用。本论文在前期研究基础上,开展了以下三方面的研究工作:(1)基于血红蛋白氨基酸序列同源性分析,预测出大鼠源大麻肽类激动剂(r)VD-hemopressin(α),并系统研究了该大麻肽在痛觉、体温、运动等方面的调节作用,及其中枢大麻样副作用;(2)在已报道的(m)VD-hemopressin(α)在光热甩尾急性痛中镇痛作用的研究基础上,探讨了中枢注射CB_1受体肽类激动剂(m)VD-hemopressin(α)对手术后痛、福尔马林痛和内脏痛等病理性痛模型中的调节活性及其作用机制;(3)利用光热甩尾急性痛、福尔马林痛和内脏痛等实验评价大麻受体非选择性肽类激动剂(m)VD-hemopressin(β)的痛觉调节作用及其作用机制。经分析预测,(r)VD-hemopressin(α)是一种来源于大鼠源血红蛋白α链的大麻肽类激动剂,本论文利用多肽固相合成法合成了十一肽(r)VD-hemopressin(α),并通过一系列的体内外生物活性实验全面鉴定了其药理学特性。体外研究结果发现(r)VD-hemopressin(α)可通过CB_1受体来促进Neuro2A细胞神经突生长。在小鼠光热甩尾实验中,(r)VD-hemopressin(α)能选择性地通过CB_1受体产生剂量依赖性中枢镇痛活性。并且,小鼠脊髓以上水平注射(r)VD-hemopressin(α)引起剂量依赖的降体温作用,该作用可被CB_1受体拮抗剂AM251部分拮抗。此外,侧脑室注射(r)VD-hemopressin(α)能抑制小鼠的运动活性,且该效应不受大麻和阿片受体拮抗剂的影响。最后,进一步评估了(r)VD-hemopressin(α)的中枢副作用,结果表明(r)VD-hemopressin(α)在有效镇痛剂量下可轻度抑制胃肠道运动,对奖赏作用无明显的调节,但是连续侧脑室注射会出现镇痛耐受现象。综上所述,本研究表明预测的大鼠源大麻肽类激动剂(r)VD-hemopressin(α)能通过不同的药理学机制而介导镇痛、体温下降和运动抑制作用。并且,(r)VD-hemopressin(α)在高效、低副作用大麻镇痛治疗中具有潜在的应用前景。此外,为了评价中枢注射(m)VD-hemopressin(α)对不同病理性痛的调节作用,本实验以经典大麻激动剂WIN55,212-2作为阳性对照,研究了侧脑室注射(m)VD-hemopressin(α)对手术后痛、福尔马林痛和内脏痛的影响。在小鼠切口手术后痛模型上,脊髓以上水平注射(m)VD-hemopressin(α)能剂量依赖性地降低切口所引起的机械性痛觉过敏,但该镇痛作用不能被大麻受体拮抗剂抑制。同样地,在福尔马林痛的第I相和醋酸扭体内脏痛模型中,小鼠侧脑室注射(m)VD-hemopressin(α)均能引起剂量依赖性镇痛作用,并且都不能被大麻受体拮抗剂所拮抗。而在福尔马林痛实验的第II相中,侧脑室注射(m)VD-hemopressin(α)无明显痛觉调节作用。然而,在这些病理性痛模型上,WIN55,212-2均能通过激活CB_1受体而介导中枢镇痛作用。总之,本实验研究表明在不同病理性痛模型中,CB_1受体的肽类激动剂(m)VD-hemopressin(α)能通过非大麻受体的机制而产生有效镇痛作用。因此,(m)VD-hemopressin(α)在正常小鼠和病理性痛中的镇痛作用可能是由不同的药理学机制来进行调节的。最后,本实验还检测了大麻CB_1和CB_2受体的非选择性激动剂(m)VD-hemopressin(β)的镇痛活性及其作用机制。在光热甩尾实验中,脊髓以上水平注射、鞘内注射、皮下注射和腹腔注射(m)VD-hemopressin(β)可以产生剂量依赖性镇痛作用,并且该镇痛作用能被同一水平注射的CB_1受体拮抗剂AM251选择性地阻断,但不受CB_2受体拮抗剂AM630的影响。并且,侧脑室注射时其镇痛作用效价最高。为了鉴定(m)VD-hemopressin(β)系统镇痛活性的作用位点,进一步检测发现其系统镇痛作用能被侧脑室、鞘内或皮下注射的AM251所拮抗,从而表明中枢和外周的CB_1受体均参与了(m)VD-hemopressin(β)的系统镇痛作用。此外,在醋酸扭体实验中,侧脑室注射(m)VD-hemopressin(β)能明显减少扭体次数,且该镇痛作用能被AM251部分拮抗。而在福尔马林痛实验中,侧脑室注射(m)VD-hemopressin(β)只能在第?相产生镇痛作用,且不能被大麻受体拮抗剂拮抗。本实验数据表明在上述痛觉检测模型中,(m)VD-hemopressin(β)具有显著的镇痛作用,并且该镇痛作用至少部分是由非大麻受体机制来介导的。综上所述,本论文揭示了大麻肽类激动剂(r)VD-hemopressin(α)、(m)VD-hemopressin(α)和(m)VD-hemopressin(β)的镇痛作用,但这些镇痛作用机制比较复杂,部分镇痛作用是通过非大麻受体机制来介导的。因此,仍需进一步的药理学实验来阐明其具体的镇痛作用机制。
[Abstract]:Cannabis system plays an important role in the regulation of pain. More and more studies have shown that CB1 and CB 2 receptors are potential targets for analgesic drugs. Traditional cannabis ligands mainly include plant cannabis, endogenous lipid cannabis and synthetic cannabis. Hemopressin-like bioactive peptides and its related peptides can specifically act on cannabis CB 1 and CB 2 receptors and represent novel peptide ligands for cannabis receptors. However, current studies have focused on the biological functions of CB 1 receptor antagonist/reverse agonist Hemopressin, while on hemopressin-related peptides, a cannabis receptor agonist. Our laboratory has reported that CB_1 receptor agonist (m) VD-hemopressin (a) plays a role in regulating pain, body temperature, exercise, gastrointestinal motility, feeding, reward, analgesic tolerance and cardiovascular activity. Homology analysis of the amino acid sequence of hemoglobin predicted that the cannabinoid peptides agonist (r) VD-hemopressin (a) from rats could regulate pain, body temperature and exercise, and its central hemp-like side effects; (2) the analgesic effect of (m) VD-hemopressin (a) on acute tail flick pain induced by photothermal therapy On this basis, the regulatory activity and mechanism of central injection of CB_1 receptor peptide agonist (m) VD-hemopressin (a) on pathological pain models such as postoperative pain, formalin pain and visceral pain were investigated; (3) The non-selective peptide agonist (m) VD-hem was evaluated by tail flick, formalin pain and visceral pain. It is predicted that, (r) VD-hemopressin (a) is a kind of cannabinoid peptide agonist derived from rat Hemoglobin alpha chain. In this paper, eleven peptides (r) VD-hemopressin (a) were synthesized by solid-phase synthesis of polypeptides and identified by a series of in vitro and in vivo bioactivity experiments. In vitro studies have shown that (r) VD-hemopressin (a) can promote the neurite growth of Neuro2A cells by CB_1 receptor. In the tail flick experiment in mice, VD-hemopressin (a) selectively produces dose-dependent central analgesic activity via CB_1 receptor. In addition, mice injected above spinal cord level (r) VD-hemopressin (a) can induce dose-dependent central analgesic activity. In addition, intraventricular injection of (r) VD-hemopressin (a) inhibited the motor activity of mice, and the effect was not affected by cannabis and opioid receptor antagonists. Finally, the central side effects of (r) VD-hemopressin (a) were further evaluated. It is concluded that (r) VD-hemopressin (a) can mildly inhibit gastrointestinal motility at an effective analgesic dose and has no significant effect on reward. However, analgesic tolerance may occur after continuous intraventricular injection. Moreover, (r) VD-hemopressin (a) has potential applications in the treatment of hemp with high efficacy and low side effects. In addition, in order to evaluate the effect of central injection of (m) VD-hemopressin (a) on the regulation of different pathological pain, the classical cannabis agonist WIN55, 212-2 was used as a positive control. The effects of intracerebroventricular injection (m) of VD-hemopressin (a) on postoperative pain, formalin pain and visceral pain were studied. In a mouse model of pain after incision surgery, injection above the spinal cord (m) of VD-hemopressin (a) could reduce mechanical hyperalgesia induced by incision in a dose-dependent manner, but the analgesic effect could not be inhibited by cannabis receptor antagonists. Similarly, in phase I of formalin pain and in the model of visceral pain induced by acetic acid writhing, the injection of (m) VD-hemopressin (a) into the lateral ventricle could induce dose-dependent analgesia in mice and could not be antagonized by cannabis receptor antagonists. In phase II of formalin pain experiment, the injection of (m) VD-hemopressin (a) into the lateral ventricle did not significantly modulate pain. However, WIN55, 212-2 can mediate central analgesia by activating CB_1 receptors in these pathological pain models. In conclusion, this study suggests that the peptide agonist (m) VD-hemopressin (a) of CB_1 receptors can produce effective analgesia through the mechanism of non-cannabis receptors in different pathological pain models. The analgesic effect of ressin (a) in normal mice and pathological pain may be regulated by different pharmacological mechanisms. Finally, the analgesic activity and mechanism of non-selective agonist (m) VD-hemopressin (beta) of CB_1 and CB_2 receptors in cannabis were tested. In the tail flick experiment, the analgesic effect of VD-hemopressin (beta) was injected above the spinal cord and in the sheath. Injection, subcutaneous injection and intraperitoneal injection of (m) VD-hemopressin (beta) can produce a dose-dependent analgesic effect, which can be selectively blocked by CB_1 receptor antagonist AM251 injected at the same level, but not by CB_2 receptor antagonist AM630. Moreover, the analgesic effect is most potent when injected into the lateral ventricle. Further studies showed that the analgesic effect of AM251 was antagonized by intrathecal, intraventricular or subcutaneous injection of AM251, indicating that both central and peripheral CB1 receptors were involved in the systemic analgesic effect of (m) VD-hemopressin (beta). Pressin (beta) can significantly reduce the number of writhing, and the analgesic effect can be partially antagonized by AM251. In formalin pain test, intraventricular injection of (m) VD-hemopressin (beta) can only produce analgesic effect in the first phase, and can not be antagonized by cannabis receptor antagonists. In summary, this paper reveals the analgesic effects of cannabinoid peptide agonists (r) VD-hemopressin (a), m VD-hemopressin (a) and (m) VD-hemopressin (b), but the mechanisms of these analgesic effects are complex and some of them are analgesic. Therefore, further pharmacological experiments are needed to elucidate the specific analgesic mechanism.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R96
，
本文编号：2209535
[Abstract]:Cannabis system plays an important role in the regulation of pain. More and more studies have shown that CB1 and CB 2 receptors are potential targets for analgesic drugs. Traditional cannabis ligands mainly include plant cannabis, endogenous lipid cannabis and synthetic cannabis. Hemopressin-like bioactive peptides and its related peptides can specifically act on cannabis CB 1 and CB 2 receptors and represent novel peptide ligands for cannabis receptors. However, current studies have focused on the biological functions of CB 1 receptor antagonist/reverse agonist Hemopressin, while on hemopressin-related peptides, a cannabis receptor agonist. Our laboratory has reported that CB_1 receptor agonist (m) VD-hemopressin (a) plays a role in regulating pain, body temperature, exercise, gastrointestinal motility, feeding, reward, analgesic tolerance and cardiovascular activity. Homology analysis of the amino acid sequence of hemoglobin predicted that the cannabinoid peptides agonist (r) VD-hemopressin (a) from rats could regulate pain, body temperature and exercise, and its central hemp-like side effects; (2) the analgesic effect of (m) VD-hemopressin (a) on acute tail flick pain induced by photothermal therapy On this basis, the regulatory activity and mechanism of central injection of CB_1 receptor peptide agonist (m) VD-hemopressin (a) on pathological pain models such as postoperative pain, formalin pain and visceral pain were investigated; (3) The non-selective peptide agonist (m) VD-hem was evaluated by tail flick, formalin pain and visceral pain. It is predicted that, (r) VD-hemopressin (a) is a kind of cannabinoid peptide agonist derived from rat Hemoglobin alpha chain. In this paper, eleven peptides (r) VD-hemopressin (a) were synthesized by solid-phase synthesis of polypeptides and identified by a series of in vitro and in vivo bioactivity experiments. In vitro studies have shown that (r) VD-hemopressin (a) can promote the neurite growth of Neuro2A cells by CB_1 receptor. In the tail flick experiment in mice, VD-hemopressin (a) selectively produces dose-dependent central analgesic activity via CB_1 receptor. In addition, mice injected above spinal cord level (r) VD-hemopressin (a) can induce dose-dependent central analgesic activity. In addition, intraventricular injection of (r) VD-hemopressin (a) inhibited the motor activity of mice, and the effect was not affected by cannabis and opioid receptor antagonists. Finally, the central side effects of (r) VD-hemopressin (a) were further evaluated. It is concluded that (r) VD-hemopressin (a) can mildly inhibit gastrointestinal motility at an effective analgesic dose and has no significant effect on reward. However, analgesic tolerance may occur after continuous intraventricular injection. Moreover, (r) VD-hemopressin (a) has potential applications in the treatment of hemp with high efficacy and low side effects. In addition, in order to evaluate the effect of central injection of (m) VD-hemopressin (a) on the regulation of different pathological pain, the classical cannabis agonist WIN55, 212-2 was used as a positive control. The effects of intracerebroventricular injection (m) of VD-hemopressin (a) on postoperative pain, formalin pain and visceral pain were studied. In a mouse model of pain after incision surgery, injection above the spinal cord (m) of VD-hemopressin (a) could reduce mechanical hyperalgesia induced by incision in a dose-dependent manner, but the analgesic effect could not be inhibited by cannabis receptor antagonists. Similarly, in phase I of formalin pain and in the model of visceral pain induced by acetic acid writhing, the injection of (m) VD-hemopressin (a) into the lateral ventricle could induce dose-dependent analgesia in mice and could not be antagonized by cannabis receptor antagonists. In phase II of formalin pain experiment, the injection of (m) VD-hemopressin (a) into the lateral ventricle did not significantly modulate pain. However, WIN55, 212-2 can mediate central analgesia by activating CB_1 receptors in these pathological pain models. In conclusion, this study suggests that the peptide agonist (m) VD-hemopressin (a) of CB_1 receptors can produce effective analgesia through the mechanism of non-cannabis receptors in different pathological pain models. The analgesic effect of ressin (a) in normal mice and pathological pain may be regulated by different pharmacological mechanisms. Finally, the analgesic activity and mechanism of non-selective agonist (m) VD-hemopressin (beta) of CB_1 and CB_2 receptors in cannabis were tested. In the tail flick experiment, the analgesic effect of VD-hemopressin (beta) was injected above the spinal cord and in the sheath. Injection, subcutaneous injection and intraperitoneal injection of (m) VD-hemopressin (beta) can produce a dose-dependent analgesic effect, which can be selectively blocked by CB_1 receptor antagonist AM251 injected at the same level, but not by CB_2 receptor antagonist AM630. Moreover, the analgesic effect is most potent when injected into the lateral ventricle. Further studies showed that the analgesic effect of AM251 was antagonized by intrathecal, intraventricular or subcutaneous injection of AM251, indicating that both central and peripheral CB1 receptors were involved in the systemic analgesic effect of (m) VD-hemopressin (beta). Pressin (beta) can significantly reduce the number of writhing, and the analgesic effect can be partially antagonized by AM251. In formalin pain test, intraventricular injection of (m) VD-hemopressin (beta) can only produce analgesic effect in the first phase, and can not be antagonized by cannabis receptor antagonists. In summary, this paper reveals the analgesic effects of cannabinoid peptide agonists (r) VD-hemopressin (a), m VD-hemopressin (a) and (m) VD-hemopressin (b), but the mechanisms of these analgesic effects are complex and some of them are analgesic. Therefore, further pharmacological experiments are needed to elucidate the specific analgesic mechanism.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R96
，
本文编号：2209535
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