內(nèi)源性大麻肽類激動劑Hemopressin相關(guān)肽的生物活性研究
發(fā)布時間：2018-08-28 13:32
【摘要】：大麻系統(tǒng)在機體的痛覺調(diào)節(jié)中扮演著重要角色,越來越多研究表明大麻CB_1和CB_2受體是潛在的鎮(zhèn)痛藥物作用靶標(biāo)。而傳統(tǒng)的大麻配體主要有植物大麻、內(nèi)源性脂類大麻和人工合成大麻三大類。近年來發(fā)現(xiàn)來源于血紅蛋白降解片段的一類生物活性肽——Hemopressin及其相關(guān)肽能特異性地作用于大麻CB_1和CB_2受體,表現(xiàn)為大麻受體的新型肽類配體。然而,目前的研究主要集中于CB_1受體拮抗劑/反向激動劑Hemopressin的生物學(xué)功能方面,而對大麻受體激動劑Hemopressin相關(guān)肽的研究相對較少。本實驗室已報道的研究發(fā)現(xiàn)CB_1受體激動劑(m)VD-hemopressin(α)在痛覺、體溫、運動、胃腸道運動、攝食、獎賞、鎮(zhèn)痛耐受和心血管活性方面具有一定的調(diào)節(jié)作用。本論文在前期研究基礎(chǔ)上,開展了以下三方面的研究工作:(1)基于血紅蛋白氨基酸序列同源性分析,預(yù)測出大鼠源大麻肽類激動劑(r)VD-hemopressin(α),并系統(tǒng)研究了該大麻肽在痛覺、體溫、運動等方面的調(diào)節(jié)作用,及其中樞大麻樣副作用;(2)在已報道的(m)VD-hemopressin(α)在光熱甩尾急性痛中鎮(zhèn)痛作用的研究基礎(chǔ)上,探討了中樞注射CB_1受體肽類激動劑(m)VD-hemopressin(α)對手術(shù)后痛、福爾馬林痛和內(nèi)臟痛等病理性痛模型中的調(diào)節(jié)活性及其作用機制;(3)利用光熱甩尾急性痛、福爾馬林痛和內(nèi)臟痛等實驗評價大麻受體非選擇性肽類激動劑(m)VD-hemopressin(β)的痛覺調(diào)節(jié)作用及其作用機制。經(jīng)分析預(yù)測,(r)VD-hemopressin(α)是一種來源于大鼠源血紅蛋白α鏈的大麻肽類激動劑,本論文利用多肽固相合成法合成了十一肽(r)VD-hemopressin(α),并通過一系列的體內(nèi)外生物活性實驗全面鑒定了其藥理學(xué)特性。體外研究結(jié)果發(fā)現(xiàn)(r)VD-hemopressin(α)可通過CB_1受體來促進Neuro2A細(xì)胞神經(jīng)突生長。在小鼠光熱甩尾實驗中,(r)VD-hemopressin(α)能選擇性地通過CB_1受體產(chǎn)生劑量依賴性中樞鎮(zhèn)痛活性。并且,小鼠脊髓以上水平注射(r)VD-hemopressin(α)引起劑量依賴的降體溫作用,該作用可被CB_1受體拮抗劑AM251部分拮抗。此外,側(cè)腦室注射(r)VD-hemopressin(α)能抑制小鼠的運動活性,且該效應(yīng)不受大麻和阿片受體拮抗劑的影響。最后,進一步評估了(r)VD-hemopressin(α)的中樞副作用,結(jié)果表明(r)VD-hemopressin(α)在有效鎮(zhèn)痛劑量下可輕度抑制胃腸道運動,對獎賞作用無明顯的調(diào)節(jié),但是連續(xù)側(cè)腦室注射會出現(xiàn)鎮(zhèn)痛耐受現(xiàn)象。綜上所述,本研究表明預(yù)測的大鼠源大麻肽類激動劑(r)VD-hemopressin(α)能通過不同的藥理學(xué)機制而介導(dǎo)鎮(zhèn)痛、體溫下降和運動抑制作用。并且,(r)VD-hemopressin(α)在高效、低副作用大麻鎮(zhèn)痛治療中具有潛在的應(yīng)用前景。此外,為了評價中樞注射(m)VD-hemopressin(α)對不同病理性痛的調(diào)節(jié)作用,本實驗以經(jīng)典大麻激動劑WIN55,212-2作為陽性對照,研究了側(cè)腦室注射(m)VD-hemopressin(α)對手術(shù)后痛、福爾馬林痛和內(nèi)臟痛的影響。在小鼠切口手術(shù)后痛模型上,脊髓以上水平注射(m)VD-hemopressin(α)能劑量依賴性地降低切口所引起的機械性痛覺過敏,但該鎮(zhèn)痛作用不能被大麻受體拮抗劑抑制。同樣地,在福爾馬林痛的第I相和醋酸扭體內(nèi)臟痛模型中,小鼠側(cè)腦室注射(m)VD-hemopressin(α)均能引起劑量依賴性鎮(zhèn)痛作用,并且都不能被大麻受體拮抗劑所拮抗。而在福爾馬林痛實驗的第II相中,側(cè)腦室注射(m)VD-hemopressin(α)無明顯痛覺調(diào)節(jié)作用。然而,在這些病理性痛模型上,WIN55,212-2均能通過激活CB_1受體而介導(dǎo)中樞鎮(zhèn)痛作用。總之,本實驗研究表明在不同病理性痛模型中,CB_1受體的肽類激動劑(m)VD-hemopressin(α)能通過非大麻受體的機制而產(chǎn)生有效鎮(zhèn)痛作用。因此,(m)VD-hemopressin(α)在正常小鼠和病理性痛中的鎮(zhèn)痛作用可能是由不同的藥理學(xué)機制來進行調(diào)節(jié)的。最后,本實驗還檢測了大麻CB_1和CB_2受體的非選擇性激動劑(m)VD-hemopressin(β)的鎮(zhèn)痛活性及其作用機制。在光熱甩尾實驗中,脊髓以上水平注射、鞘內(nèi)注射、皮下注射和腹腔注射(m)VD-hemopressin(β)可以產(chǎn)生劑量依賴性鎮(zhèn)痛作用,并且該鎮(zhèn)痛作用能被同一水平注射的CB_1受體拮抗劑AM251選擇性地阻斷,但不受CB_2受體拮抗劑AM630的影響。并且,側(cè)腦室注射時其鎮(zhèn)痛作用效價最高。為了鑒定(m)VD-hemopressin(β)系統(tǒng)鎮(zhèn)痛活性的作用位點,進一步檢測發(fā)現(xiàn)其系統(tǒng)鎮(zhèn)痛作用能被側(cè)腦室、鞘內(nèi)或皮下注射的AM251所拮抗,從而表明中樞和外周的CB_1受體均參與了(m)VD-hemopressin(β)的系統(tǒng)鎮(zhèn)痛作用。此外,在醋酸扭體實驗中,側(cè)腦室注射(m)VD-hemopressin(β)能明顯減少扭體次數(shù),且該鎮(zhèn)痛作用能被AM251部分拮抗。而在福爾馬林痛實驗中,側(cè)腦室注射(m)VD-hemopressin(β)只能在第?相產(chǎn)生鎮(zhèn)痛作用,且不能被大麻受體拮抗劑拮抗。本實驗數(shù)據(jù)表明在上述痛覺檢測模型中,(m)VD-hemopressin(β)具有顯著的鎮(zhèn)痛作用,并且該鎮(zhèn)痛作用至少部分是由非大麻受體機制來介導(dǎo)的。綜上所述,本論文揭示了大麻肽類激動劑(r)VD-hemopressin(α)、(m)VD-hemopressin(α)和(m)VD-hemopressin(β)的鎮(zhèn)痛作用,但這些鎮(zhèn)痛作用機制比較復(fù)雜,部分鎮(zhèn)痛作用是通過非大麻受體機制來介導(dǎo)的。因此,仍需進一步的藥理學(xué)實驗來闡明其具體的鎮(zhèn)痛作用機制。
[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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R96
，
本文編號：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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R96
，
本文編號：2209535
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