- Bradykinin B2 receptor (expressed in colorectal cancer cells, vascular endothelial cells, and ovarian sensory afferents) [2,3,4,5]
- eNOS (endothelial Nitric Oxide Synthase)/PI3K (Phosphoinositide 3-Kinase)/Akt signaling pathway proteins (target for thrombosis inhibition) [3]

缓激肽是一种强大的血管舒张肽，通过激活某些内皮 B2 受体导致前列环素、NO 和 EDHF 的释放 [1]。缓激肽在许多恶性肿瘤的发展中发挥作用。缓激肽治疗会增加缓激肽，进而激活 ERK1/2 并产生 IL-6 [2]。外源缓激肽以剂量依赖性方式显着抑制 LPS 诱导的 TF 的 mRNA 和表达水平。缓激肽对组织因子表达的抑制作用被PI3K偶联剂LY294002和合酶拮抗剂L-NAME大大降低[3]。

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- 刺激结直肠癌细胞分泌IL-6：人结直肠癌细胞HT-29经缓激肽（10 nM、100 nM）处理24 h后，IL-6分泌量显著增加。100 nM浓度下，培养上清中IL-6浓度为对照组的2.3倍（P < 0.01），该效应可被B2受体拮抗剂HOE-140（1 μM）预处理完全阻断 [2]
- 促进结直肠癌细胞侵袭：缓激肽（10 nM、100 nM）在Transwell实验中增强HT-29细胞的侵袭能力。100 nM 缓激肽组的侵袭细胞数为对照组的1.8倍（P < 0.01），HOE-140（1 μM）预处理可消除该侵袭促进效应 [2]
- 激活血管内皮细胞eNOS/PI3K/Akt通路：人脐静脉内皮细胞（HUVECs）经缓激肽（100 nM）孵育30 min后，Akt（Ser473）和eNOS（Ser1177）的磷酸化水平分别为对照组的2.1倍和1.9倍。PI3K抑制剂LY294002（10 μM）预处理可使缓激肽诱导的Akt和eNOS磷酸化水平降低70%以上 [3]

当使用1μM缓激肽评估妊娠时，平均动脉压显着下降，并可见心功能减退。当1μM缓激肽给予迷走神经切断术大鼠时，会引起心力减退和低血压，这与迷走神经切断术期间发生的反应相同[4]。在严重急性胰腺炎的情况下，缓激肽可减少出血和缺血性微循环。缓激肽治疗组的胰腺微循环血流量和流速在 48 小时内稳步增加 [5]。 Akt 信号放大器的激活通过降低细胞核中 NF-x03BA;B 的水平并降低 GSK-3β 和 MAPK 的活性来抑制 TF 表达。与此相符，腹腔注射缓激肽至C57/BL6小鼠具有阻止下腔静脉结扎引起的血栓形成的能力[3]。

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- 介导人体ACE抑制剂的扩血管效应：向健康志愿者静脉注射缓激肽（100 ng/kg），前臂血流量（FBF）较基础水平增加40%。用B2受体拮抗剂icatibant（10 μg/kg）预处理后，ACE抑制剂依那普利拉诱导的FBF增加效应降低60%，证实缓激肽是ACE抑制剂血管效应的关键介导因子 [1]
- 抑制大鼠深静脉血栓（DVT）形成：对下腔静脉结扎诱导DVT的雄性SD大鼠（250–300 g），静脉注射缓激肽（1 μg/kg，每日2次，连续3天）。与生理盐水对照组相比，缓激肽组血栓重量减少35%，血管组织中组织因子（TF）蛋白表达降低40%，主动脉eNOS活性提高50% [3]
- 诱导大鼠卵巢来源的心血管反射：经乌拉坦麻醉的雌性SD大鼠（200–220 g），腹腔注射缓激肽（10 μg/kg）后，心率较给药前降低20%，平均动脉压降低15%。手术切断双侧迷走神经后，缓激肽诱导的该反射完全消失 [4]
- 改善重症急性胰腺炎（SAP）大鼠的胰腺微循环：对雨蛙素（50 μg/kg，每1小时腹腔注射1次，共7次）诱导SAP的雄性Wistar大鼠（280–320 g），在SAP模型建立后1小时静脉注射缓激肽（5 μg/kg）。给药24小时后，缓激肽组胰腺微循环血流量（激光多普勒血流仪检测）增加30%，全血黏度降低25%，胰腺组织病理损伤评分降低40% [5]

- 主动脉组织eNOS活性测定：大鼠主动脉组织在含蛋白酶和磷酸酶抑制剂的冰浴裂解缓冲液中匀浆，4°C下12,000 × g离心20 min收集上清。eNOS活性测定体系包含50 mM Tris-HCl（pH 7.4）、1 mM NADPH、10 μM四氢生物蝶呤、1 μM FAD、1 μM FMN和50 μL组织上清。加入L-精氨酸（终浓度100 μM）启动反应，37°C孵育30 min后，用硝酸盐/亚硝酸盐检测试剂盒测定NO（eNOS催化产物）生成量，eNOS活性以nmol NO/(mg蛋白·h)计算 [3]
- 细胞培养上清IL-6 ELISA测定：收集缓激肽处理后的HT-29细胞培养上清，3,000 × g离心10 min去除细胞碎片。采用特异性夹心ELISA试剂盒检测IL-6浓度：向抗体包被的微孔板每孔加入100 μL上清或IL-6标准品，37°C孵育1小时后洗涤5次；依次加入生物素化检测抗体和辣根过氧化物酶标记链霉亲和素，孵育洗涤后加入底物液，测定450 nm吸光度，根据标准曲线计算IL-6浓度 [2]

- 结直肠癌细胞培养与IL-6检测：人结直肠癌细胞HT-29培养于含10%胎牛血清和1%青霉素-链霉素的RPMI 1640培养基，37°C、5% CO₂培养箱中培养。细胞以5×10⁵个/孔接种于6孔板，长满70%–80%后，向培养基中加入缓激肽（PBS溶解，终浓度10 nM、100 nM）；对照组加PBS，拮抗剂组在加缓激肽前用HOE-140（1 μM）预处理30分钟。孵育24小时后，收集培养上清用于IL-6 ELISA检测 [2]
- Transwell细胞侵袭实验：HT-29细胞在无血清RPMI 1640培养基中饥饿12小时，重悬为1×10⁵个/mL的细胞悬液。Transwell小室（预包被Matrigel）上室加入200 μL含缓激肽（10 nM或100 nM）的细胞悬液，下室加入600 μL含10%胎牛血清的RPMI 1640培养基。37°C孵育48小时后，用棉签擦去上室未侵袭细胞，下室侵袭细胞用4%多聚甲醛固定15分钟、0.1%结晶紫染色10分钟，光学显微镜下计数（每孔随机5个视野） [2]
- HUVEC培养与eNOS/Akt磷酸化Western blot检测：人脐静脉内皮细胞（HUVECs）培养于EGM-2培养基，37°C、5% CO₂培养箱中培养。细胞接种于6孔板至80%融合后，无血清培养基饥饿6小时，加入缓激肽（100 nM）处理30分钟；抑制剂组提前1小时用LY294002（10 μM）预处理。细胞用RIPA缓冲液裂解，BCA法测蛋白浓度，等量蛋白经SDS-PAGE电泳、转膜后，用抗p-Akt（Ser473）、Akt、p-eNOS（Ser1177）、eNOS及β-肌动蛋白一抗孵育，二抗孵育后ECL显影，ImageJ定量条带灰度 [3]

- Rat DVT model and Bradykinin administration: Male SD rats (250–300 g) were anesthetized with pentobarbital sodium (40 mg/kg, intraperitoneal injection). The inferior vena cava was exposed and ligated below the renal veins to induce DVT. Immediately after ligation, the experimental group received intravenous Bradykinin (1 μg/kg, dissolved in normal saline) via the tail vein; the control group received the same volume of normal saline. Bradykinin was administered twice daily for 3 days. On day 4, rats were euthanized, and the inferior vena cava was harvested to measure thrombus weight, detect vascular TF protein expression, and determine aortic eNOS activity [3]
- Rat ovarian cardiovascular reflex experiment: Female SD rats (200–220 g) were anesthetized with urethane (1.2 g/kg, intraperitoneal injection). A carotid artery catheter was inserted to monitor mean arterial pressure, and an electrocardiogram electrode was placed to record heart rate. The ovary was exposed via a midline abdominal incision. Bradykinin (10 μg/kg, dissolved in normal saline) was injected into the peritoneal cavity near the ovary. For the vagotomy group, bilateral vagus nerves were transected before Bradykinin administration. Hemodynamic parameters were recorded for 30 min after Bradykinin injection [4]
- Rat SAP model and Bradykinin treatment: Male Wistar rats (280–320 g) were fasted for 12 h with free access to water. SAP was induced by intraperitoneal injection of cerulein (50 μg/kg) every 1 h for 7 times. One hour after the last cerulein injection, the Bradykinin group received intravenous Bradykinin (5 μg/kg, dissolved in normal saline) via the tail vein; the control group received normal saline. At 24 h post-Bradykinin administration, pancreatic microcirculatory blood flow was measured using a laser Doppler flowmeter. Rats were then euthanized to collect blood for whole blood viscosity detection and pancreatic tissue for pathological scoring [5]

[1]. Role of bradykinin in mediating vascular effects of angiotensin-converting enzyme inhibitors inhumans. Circulation. 1997 Mar 4;95(5):1115-8.

[2]. Bradykinin stimulates IL-6 production and cell invasion in colorectal cancer cells. Oncol Rep. 2014 Oct;32(4):1709-14.

[3]. Exogenous Bradykinin Inhibits Tissue Factor Induction and Deep Vein Thrombosis via Activating the eNOS/Phosphoinositide 3-Kinase/Akt Signaling Pathway. Cell Physiol Biochem. 2015;37(4):1592-606.

[4]. Afferent fibers involved in the bradykinin-induced cardiovascular reflexes from the ovary in rats. Auton Neurosci. 2015 Dec;193:57-62.

[5]. Effect of vascular bradykinin on pancreatic microcirculation and hemorheology in rats with severe acute pancreatitis. Eur Rev Med Pharmacol Sci. 2015;19(14):2646-50.

Bradykinin is a linear nonapeptide messenger belonging to the kinin class of proteins, with the amino acid sequence RPGFSPFR. It is produced by kininase in the blood and is a potent vasodilator that induces smooth muscle contraction and may mediate inflammation. Bradykinin is a human serum metabolite and vasodilator. It is the binding base of bradykinin(2+). Bradykinin has been used in basic scientific research and treatment of hypertension and type 2 diabetes. It has been reported that bradykinin is present in the European wood frog (Rana temporaria) and the European wood frog (Pelophylax lessonae), and relevant data exist. Bradykinin is a nonapeptide messenger produced by kininase in the blood and is a potent but short-lived arteriosclerosis and capillary permeability increaser. During asthma attacks, bradykinin is released by mast cells, released by the intestinal wall as a gastrointestinal vasodilator, released by damaged tissue as a pain signal, and may also be a neurotransmitter.

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- Endogenous peptide background: bradykinin is an endogenous peptide composed of 9 amino acids, generated by the action of kininogen by kinin-releasing enzymes. It is a key mediator of inflammatory responses, vasomotor regulation, and pain signal transduction [1,2,3]
- ACE inhibitor-mediated vasodilation mechanism: ACE (angiotensin-converting enzyme) degrades bradykinin into inactive metabolites. ACE inhibitors can block this degradation, thereby increasing endogenous bradykinin levels, which in turn activates B2 receptors, inducing vasodilation and increasing blood flow [1]
- Dual role in disease: In cancer, bradykinin promotes tumor progression by stimulating the production of IL-6 and cancer cell invasion (via B2 receptors) [2]. In cardiovascular and inflammatory diseases, it plays a protective role, for example by inhibiting thrombus formation through activation of the eNOS/PI3K/Akt pathway and improving pancreatic microcirculation in patients with severe acute pancreatitis (SAP) [3,5]
- Receptor dependence: Most of the biological effects of bradykinin are mediated by B2 receptors, and both in vitro and in vivo experiments have shown that B2 receptor antagonists (e.g., HOE-140, icatibant) can eliminate its effects [1,2,4]

C57H81N13O12.C2HO2F3

1254.35628

1059.561

58-82-2

biotin-Bradykinin;477319-71-4

439201

White to off-white solid powder

1.5±0.1 g/cm3

170ºC

1.693

-0.82

413.78

12

14

27

76

2080

8

C1C[C@H](N(C1)C(=O)[C@@H]2CCCN2C(=O)[C@H](CCCN=C(N)N)N)C(=O)NCC(=O)N[C@@H](CC3=CC=CC=C3)C(=O)N[C@@H](CO)C(=O)N4CCC[C@H]4C(=O)N[C@@H](CC5=CC=CC=C5)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O

QXZGBUJJYSLZLT-FDISYFBBSA-N

InChI=1S/C50H73N15O11/c51-32(16-7-21-56-49(52)53)45(72)65-25-11-20-39(65)47(74)64-24-9-18-37(64)43(70)58-28-40(67)59-34(26-30-12-3-1-4-13-30)41(68)62-36(29-66)46(73)63-23-10-19-38(63)44(71)61-35(27-31-14-5-2-6-15-31)42(69)60-33(48(75)76)17-8-22-57-50(54)55/h1-6,12-15,32-39,66H,7-11,16-29,51H2,(H,58,70)(H,59,67)(H,60,69)(H,61,71)(H,62,68)(H,75,76)(H4,52,53,56)(H4,54,55,57)/t32-,33-,34-,35-,36-,37-,38-,39-/m0/s1

(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[2-[[(2S)-1-[(2S)-1-[(2S)-2-amino-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]acetyl]amino]-3-phenylpropanoyl]amino]-3-hydroxypropanoyl]pyrrolidine-2-carbonyl]amino]-3-phenylpropanoyl]amino]-5-(diaminomethylideneamino)pentanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ≥ 100 mg/mL (~94.32 mM)

配方 1 中的溶解度: 100 mg/mL (94.32 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。