Effects of moxibustion on dynorphin and endomorphin in rats with chronic visceral hyperalgesia

AIM:To observe the analgesic effects of moxibustion in rats with chronic visceral hyperalgesia and its influence on the concentration of dynorphin(Dyn) and endomorphin(EM) in spinal cord.METHODS:The rat model of chronic visceral hyperalgesia was established by colorectal distention(CRD).In moxibustion(MX) group,moxibustion was applied once daily for 7 d;in sham moxibustion(SM) group,moxibustion was given to the same acupoints but with the nonsmoldered end of the moxa stick.Model control(MC) group and normal control group were also studied.The scoring system of abdominal withdrawal reflex was used to evaluate visceral pain for behavioral assessment.Enzyme linked immunosorbent assay was performed to determine the concentrations of Dyn and EM in spinal cord.RESULTS:Moxibustion significantly decreased visceral pain to CRD in this rat model,and no significant difference was detected between the SM group and the MC group.In MX group,moxibustion also increased the concentrations of Dyn and EM in spinal cord,and no significant difference was found between the SM group and the MC group.CONCLUSION:Moxibustion therapy can significantly enhance the pain threshold of rats with chronic visceral hyperalgesia,and the effect may be closely related to the increased concentration of Dyn and EM in spinal cord.

Inducible expression of endomorphins in murine dendritic cells

Bone marrow precursor cells were extracted from C57BL/6J mice aged 7-8 weeks, and dendritic cells were purified using anti-CD1 lc （a specific marker for dendritic cells） antibody-coated magnetic beads. Immunofluorescence staining revealed that the expression levels of endomorphin-1 and endomorphin-2 were upregulated in dendritic cells activated by lipopolysaccharide. An enzyme Jmmunoassay showed that lipopolysaccharide and other Toll-like receptor ligands promoted the secretion of endomorphin-1 and endomorphin-2 from activated dendritic cells. [3H]-thymidine incorporation demonstrated that endomorphin-1 and endomorphin-2 both inhibited the proliferation of T lymphocyte induced by activated dendritic cells. Furthermore, this immunosuppressive effect was blocked by CTOP, a specific antagonist of IJ-opioid receptors. Our experimental findings indicate that activated dendritic cells can induce the expression and secretion of endomorphins, and that endomorphins suppress T lymphocyte proliferation through activation of iJ-opioid receptors.

Neurochemical features of endomorphin-2-containing neurons in the submucosal plexus of the rat colon

AIM:To investigate the distribution and neurochemical phenotype of endomorphin-2(EM-2)-containing neurons in the submucosal plexus of the rat colon.METHODS:The mid-colons between the right and left flexures were removed from rats,and transferred into Kreb's solution. For whole-mount preparations,the mucosal,outer longitudinal muscle and inner circularmuscle layers of the tissues were separated from the submucosal layer attached to the submucosal plexus. The whole-mount preparations from each rat mid-colon were mounted onto seven gelatin-coated glass slides,and processed for immunofluorescence histochemical double-staining of EM-2 with calcitonin gene-related peptide(CGRP),choline acetyltransferase(Ch AT),nitric oxide synthetase(NOS),neuron-specific enolase(NSE),substance P(SP) and vasoactive intestinal peptide(VIP). After staining,all the fluorescence-labeled sections were observed with a confocal laser scanning microscope. To estimate the extent of the co-localization of EM-2 with CGRP,Ch AT,NOS,NSE,SP and VIP,ganglia,which have a clear boundary and neuronal cell outline,were randomly selected from each specimen for this analysis. RESULTS:In the submucosal plexus of the mid-colon,many EM-2-immunoreactive(IR) and NSE-IR neuronal cell bodies were found in the submucosal plexus of the rat mid-colon. Approximately 6 ± 4.2 EM-2-IR neurons aggregated within each ganglion and a few EM-2-IR neurons were also found outside the ganglia. The EM-2-IR neurons were also immunopositive for Ch AT,SP,VIP or NOS. EM-2-IR nerve fibers coursed near Ch AT-IR neurons,and some of these fibers were even distributed around Ch AT-IR neuronal cell bodies. Some EM-2-IR neuronal cell bodies were surrounded by SP-IR nerve fibers,but many long processes connecting adjacent ganglia were negative for EM-2 immunostaining. Long VIP-IR processes with many branches coursed through the ganglia and surrounded the EM-2-IR neurons. The percentages of the EM-2-IR neurons that were also positive for Ch AT,SP,VIP or NOS were approximately 91% ± 2.6%,36% ± 2.4%,44% ± 2.5% and 44% ± 4.7%,respectively,but EM-2 did not co-localize with CGRP. CONCLUSION:EM-2-IR neurons are present in the submucosal plexus of the rat colon and express distinct neurochemical markers.

Characteristic fragmentation behavior of the analogs of endomorphin-2 with phenylglycine in position 3 or 4 by ESI-FT tandem mass spectrometry

A series of analogs of endomorphin-2 （EM-2） with phenylglycine （Phg） in position 3 or 4 were synthesized. In electrospray ionization Fourier transform ion cyclotron resonance （ESI-Fr-ICR） MS/MS spectra of these compounds, some b, y, a, and internal ions were observed and slight mass differences between the calculated and observed results are obtained. Their sequences were derived successfully. However, the MS/MS patterns of these analogs with Dphg and Lphg were very similar. It is hard to distinguish them by MS/MS spectra. Moreover, if the third position was substituted by phenylglycine （L or D）, a rearrangement could occur in MS/MS experiment to lose proline residue.

Synthesis and Plasma Stability of Disulfide-Bridged Cyclic Endomorphin-1 Derivatives

Endomorphin-1 is an endogenous opioid peptide that mediates pain relief through interaction with the μ-opioid receptor in the central nervous system. To enhance the metabolic stability of this tetrapeptide, cyclisation through the formation of a disulfide bridge between the side chains of cysteine residues added to the sequence was explored. A further increase in stability was achieved through N-terminal modification with lipoamino acid and lactose succinamic acid, and the inclusion of D-amino acids. The latter also provided an alternative spatial arrangement of the aromatic side chains. The lipidated cyclic derivatives were insoluble in aqueous buffer, however, the cyclic peptides and glycopeptides showed greatly improved stability towards enzymatic degradation in human plasma.

Endomorphin-1对佐剂性关节炎作用的研究

目的 观察内吗啡肽 1 (endomorphin 1 ,EM 1 )对佐剂性关节炎动物的镇痛作用。 方法 制备大鼠佐剂性关节炎动物模型 ,采用钾离子透入法和辐射热 缩腿法两种测痛方法 ,观察腹腔注射EM 1 (50 μg/kg)的作用。 结果 EM 1对佐剂性关节炎急性期和慢性期的痛阈均有提高作用 ,并且EM 1的作用可被纳洛酮翻转。结论 EM

Endomorphin analogues with balanced affinity for bothμ- andδ-opioid receptors

Analogues of endomorphin and tripeptidcs modified at positions 4 and 3,respectively,with various phenylalanine analogues were synthesized and their affinities for opioid receptors were evaluated.Most of the peptides exhibited potentμ-receptor affinity and selectivity,among them,compound 7（Dmt-Pro-Tmp-Tmp-NH_2） exhibited potent affinity for bothμ-andδ-receptors （K_iμ= 0.47 nmol/L,K_iδ= 1.63 nmol/L）.

Short reaction of C-peptide, glucagon-like peptide-1, ghrelin and endomorphin-1 for different style diet in type 2 diabetic patients

Background Food composition and style is changing dramatically now, which causes inappropriate secretion of hormones from brain, gastrointestinal and endo-pancreas, may be related to unbalance of glucose in blood. The aim of this study was to explore the fast response of C-peptide, glucagon-like peptide-1 （GLP-1）, ghrelin and endomorphin-1 （EM-1） to the eastern and western style meals in patients with type 2 diabetes mellitus. Methods The study enrolled 57 patients with type 2 diabetes （20 men and 37 women, mean age （67.05±8.26） years）. Eastern style meal （meal A） and western style meal （meal B） were designed to produce the fullness effect. C-peptide, GLP-1, ghrelin and EM-1 were assessed before （0 hour） and after （2 hours） each diet. Results The delta （2h-0h） of C- peptide in meal A was significantly lower than that in meal B （P=0.0004）. C-peptide, GLP-1, ghrelin and EM-1 were obviously higher before meal B than those before meal A （P 〈0.0001, 〈0.0001, =0.001, =0.0004 respectively）. Blood glucose 2 hours and 3 hours after meal B were higher than those after meal A （P=0.0005, 0.0079 respectively）. Correlations between GLP-1 and ghrelin were strongly positive before both meals and 2 hours after both meals and also in relation to the delta of meal A and meal B （rA0h=0.7838, rB05=0.9368, rA25=0.7615, rB2h=0.9409, r A（2h-0h）=0.7531, rB（2h 05）=0.9980, respectively, P 〈0.0001）. Conclusion Western style meal （high fat and protein food） could make more response of C-peptide than eastern style meal, and could stimulate more gut hormones （GLP-1, ghrelin） and brain peptide （EM-1） at the first phase of digestion.

Structure-activity relationship of endomorphins and their analogs

To study the structure-activity relationship of endomorphins (EMs), the action of opioid receptor binding (AORB), analgesic activity and vasodilator effects of EMs and their eight analogs were investigated, which were prepared by rationally replacing the 2-/3-amino acid (Aa) of EMs. The results showed: (i) The 2-Aa was comparatively more related to the selectivity of EMs while the 3-Aa to their affinity; (ii) the analgesia and vasodilatation of EMs and their analogs were not completely dictated by their AORB (in vitro), the action of [D-Pro2]EM-2 was unusual; (iii) EMs lost their analgesia in the central nervous system and their vasodilatation in the circulatory system with different mechanisms; the former was due to the degradation of some peptidase, and the latter possibly due to the feedback inhibition.

Sequence and modified group analysis on C-terminal modified analogs of endomorphin-2 using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometer

In this paper, a series of C-terminal modified analogs of endomorphin-2 is investigated using ESI-FT-ICR-MS. Some b, y″, a, and internal ions are found in the CID spectra and slight mass differ- ences between the calculated and observed results are obtained. Moreover, if the C-terminal modified group is t-butyloxy, it can lose butene through McLafferty rearrangement. FT-ICR MS shows its power in peptide sequencing successfully helping us obtain the structure of peptide analogs.

内吗啡肽2对神经病理性痛大鼠背根神经节内μ-阿片受体表达的影响

目的探讨在内吗啡肽2(EM2)的镇痛过程中,μ-阿片受体(MOR)的表达变化及胞内囊泡转运对其变化的影响。方法成年雄性SD大鼠,随机分为3组:正常对照组、病理性痛组和药物组,病理性痛为坐骨神经分支选择性损伤(SNI)诱导的神经病理性痛(NP)。药物组为鞘内注射EM2的NP大鼠。采用免疫荧光单标染色和免疫印迹方法检测MOR总蛋白和磷酸化蛋白以及Rab7蛋白表达量的变化,采用免疫荧光双标染色方法检测MOR/Rab7和MOR/LAMP1免疫阳性标志物的表达变化。结果与正常对照组比较,病理性痛组大鼠背根神经节(DRG)内MOR总蛋白和磷酸化蛋白的表达量降低(P<0.05),Rab7蛋白的表达量明显增高(P<0.05);MOR/Rab7免疫阳性标志物占Rab7或MOR、MOR/LAMP1免疫阳性标志物占LAMP1或MOR阳性标志物的比例均增高(P<0.05)。鞘内多次注射EM2后,病理性痛大鼠的后足缩足反应阈值明显增高(P<0.01),DRG内MOR总蛋白和磷酸化蛋白的表达量增高(P<0.05),Rab7的表达量降低(P<0.05)。MOR/Rab7阳性双标产物占Rab7或MOR阳性标志物、MOR/LAMP1阳性双标产物占LAMP1或MOR阳性标志物比例均降低(P<0.05)。结论在镇痛过程中,EM2抑制SNI的NP大鼠DRG内Rab7的表达,减少MOR向溶酶体运输,促进MOR复敏。

μ阿片受体在大鼠结肠平滑肌细胞中的表达

目的通过培养大鼠结肠原代平滑肌细胞,探索μ阿片受体(MOR)在结肠平滑肌细胞中的表达特性。方法分离、培养和鉴定结肠平滑肌细胞;运用免疫荧光组织化学双重标记法观察MOR、内吗啡肽2(EM2)、钙调蛋白(CaM)在结肠平滑肌细胞的分布特性;施加乙酰胆碱(ACh)(1×10^(-3)mol/L)或EM2(2μmol/L)后,Western blot技术检测CaM蛋白表达变化;单独或依次施加ACh(1×10^(-3)mol/L)和EM2(2μmol/L)后,钙离子成像法观察平滑肌细胞内钙离子浓度的变化。结果鉴定结肠平滑肌细胞:α-肌动蛋白(α-SMA)标记的阳性细胞占细胞总数的95%以上;免疫荧光组织化学双重标记显示,在结肠平滑肌细胞有MOR和EM2的分布,且所有MOR和EM2阳性细胞均与α-SMA有共存;Western blot检测发现,施加ACh 10 min后,CaM表达升高(P<0.05);施加EM210 min后,CaM表达降低(P<0.05);钙离子成像结果显示,单独施加ACh,平滑肌细胞内钙离子浓度明显升高(P<0.05);单独施加EM2,结肠平滑肌细胞内钙离子浓度下调(P<0.05),依次施加ACh和EM2后,EM2显著抑制了ACh诱发的钙离子浓度升高(P<0.05)。结论MOR和EM2在结肠平滑肌细胞有表达,且EM2可通过MOR抑制CaM的表达与降低钙离子浓度。

单发佐剂性关节炎大鼠脊髓3种神经肽的变化规律

目的 :观察慢性佐剂性关节炎大鼠脊髓 3种神经肽含量及释放量的动态变化 ,以阐明其与炎症痛的关系。方法 :采用完全弗氏佐剂在大鼠右后肢造成单发性关节炎模型 ,用放射免疫分析法检测致炎前及致炎后 1～ 9周大鼠脊髓背半侧和脊髓灌流液中与疼痛有关的神经肽 :八肽胆囊收缩素 (cholecystokininoctapeptid ,CCK 8)、孤啡肽(orphaninFQ ,OFQ)、内吗啡肽 2 (endomorphin 2 ,EM 2 )的含量及释放量的动态变化。 结果 :(1)炎症期间 ,脊髓中CCK 8 ir释放量显著降低 ,脊髓背半侧含量于炎症第 2周呈一过性增高 ;(2 )OFQ ir释放量于炎症后的第 3、7周明显升高 ,而含量则在 3周后降至正常水平以下 ,并以患侧变化更为明显 ;(3)EM 2 ir释放量于第 1～ 2周明显降低 ,此后恢复正常 ,患侧脊髓含量 1～ 9周均显著降低。结论 :单发佐剂性关节炎大鼠脊髓背角和脊髓灌流液中有关的神经肽CCK 8、OFQ、EM 2均发生了可塑性变化。

侧脑室注射内吗啡肽-1对麻醉大鼠血压的影响

目的 观察侧脑室注射内吗啡肽 1(EM 1)对麻醉大鼠血压的影响 ,并初步探讨其作用机理。方法 侧脑室埋植导管给药 ,颈动脉插管测血压。结果 icvEM 1剂量依赖、纳洛酮敏感地降低麻醉大鼠的血压。icv或iv酚妥拉明、普萘洛尔和ivL NNA对EM 1引起的血压降低反应无影响 ;给予阿托品 (icv 2 5 μg·kg- 1 ;或iv 5 0 μg·kg- 1 )和切断双侧迷走神经减弱EM 1引起的血压降低反应。结论 icvEM 1可引起麻醉大鼠血压降低 ;此效应由阿片受体介导 ,有中枢M受体的参与 。

内吗啡肽研究进展

1997年发现的内吗啡肽 (Endomorphins、EMs) ,结构上属于四肽 ,被认为是 μ阿片受体 (MOR)的内源性配基 ,它通过与G蛋白偶联的MOR受体结合介导许多生理活动 ,本文结合本实验室对内吗啡肽的研究 ,对其神经系统分布、受体结合特性、生理作用。

内吗啡肽及其类似物对心血管系统的作用

目的 研究内吗啡肽 (EMs)及其类似物对心血管系统的影响 ,初步探讨其作用机理。方法 测定EMs及其类似物对大鼠平均动脉压和后肢血管阻力、蟾蜍肠系膜微动脉内径、兔离体胸主动脉条张力的影响。结果 EMs及其类似物剂量依赖 ( 10 -9- 10 -6mol·L-1,iv)且Nx敏感地降低麻醉大鼠平均动脉压、后肢血管灌流压和扩张蟾蜍肠系膜微动脉。EMs对去内皮兔离体胸主动脉条张力无影响 ;但剂量依赖地显著降低完整内皮胸主动脉条张力并被Nx和L NNA阻断。结论 EMs及其类似物通过降低外周阻力而显著降低动脉血压 。

内吗啡肽对小鼠树突状细胞免疫功能的影响

目的研究内吗啡肽-1和内吗啡肽-2对小鼠骨髓来源树突状细胞表型和免疫功能的影响。方法从7～8周龄的C57BL/6J小鼠提取骨髓前体细胞培养,经CD11c免疫磁珠分选,获得纯化的树突状细胞(dendritic cells,DC)。未成熟DC用脂多糖或脂多糖+不同浓度内吗啡肽共培养,流式细胞术检测DC的表型;检测内吗啡肽对DC趋化性的影响。在DC和T淋巴细胞共培养的条件下,用氚标记胸腺嘧啶核苷(3H-TdR)参入测定的方法检测T淋巴细胞的增殖能力。结果内吗啡肽抑制DC的趋化性;与T淋巴细胞体外共培养时,经内吗啡肽-1或内吗啡肽-2处理的DC能抑制T淋巴细胞的增殖反应。上述内吗啡肽的作用都能被Mu阿片受体特异性拮抗剂CTOP翻转或部分翻转,提示内吗啡肽的作用是由Mu受体介导的。结论内吗啡肽可通过作用于Mu阿片受体,改变树突状细胞的部分免疫功能,调节免疫反应。

海藻酸钠-壳聚糖固定化木瓜蛋白酶催化内吗啡肽的合成(英文)

反应体系以乙腈作为有机介质,在微水有机溶剂体系中以Boc-Trp-OH和Phe-NH2为底物,用海藻酸钠-壳聚糖固定化木瓜蛋白酶催化合成Trp-Phe-NH2时,产率为27.8%.在这一合成反应中,对pH值、离子强度、溶液含量、反应温度、酶用量和反应时间进行正交试验,证明pH是本合成过程的最重要影响因素.反应体系以乙腈为有机介质,在微水有机溶剂体系中以Boc-Tyr-Pro-OMe和Trp-Phe-NH2为底物,用IPSAC催化合成Tyr-Pro-Trp-Phe-NH2,产率为35.2%.

内吗啡肽-2的人工合成及其酶促降解

The recently discovered native endomorphin 2 plays an important role in opioid analgesia, but its metabolic fate in the organism remains relatively little known. The successful synthesis of endomorphin 2 was carried out by solution method. The C teminal dipeptide and the N terminal dipeptide have been synthesized, respectively, and then coupled successfully. High performance liquid chromatography and electrospray ionization mass spectrometry were used to identify the degradation products from the incubation of endomorphin 2 with brain synaptic membrane. The patterns of peptide metabolites indicated that brain synaptic membrane associated peptidases hydrolyzed first at the N terminal, and then split the peptide at the Phe Phe bond.

内吗啡肽在小鼠树突状细胞的诱导表达

目的研究内吗啡肽-1和内吗啡肽-2在小鼠树突状细胞的表达。方法从7～8周龄的C57BL/6J小鼠骨髓提取骨髓前体细胞培养,经CD11c(树突状细胞的特异性标记)免疫磁珠分选,获得纯化的树突状细胞。流式细胞仪分析表明,CD11c阳性细胞纯度达95%以上。免疫荧光染色研究内吗啡肽-1和内吗啡肽-2在树突状细胞的表达;酶免疫测定(enzyme immunoassay,EIA)的方法,检测培养树突状细胞上清液中的内吗啡肽-1和内吗啡肽-2的含量。结果免疫荧光染色表明,活化的树突状细胞内可分泌内吗啡肽-1和内吗啡肽-2;酶免疫测定检测结果表明,不同的Toll样受体(toll like receptor,TLR)配体活化树突状细胞促使分泌不同浓度的内吗啡肽-1和内吗啡肽-2。结论活化的树突状细胞可诱导内吗啡肽的表达。