- 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]

Bradykinin is a powerful vasodilator peptide that causes the release of prostacyclin, NO, and EDHF by activating certain endothelium B2 receptors [1]. Bradykinin plays a role in the development of many malignancies. Treatment with bradykinin increases bradykinin, which in turn activates ERK1/2 and produces IL-6 [2]. In a dose-dependent manner, exogenous bradykinin markedly suppressed the mRNA and expression levels of LPS-induced TF. Bradykinin's inhibitory effect on tissue factor expression was greatly reduced by the PI3K coupling agent LY294002 and the synthase antagonist L-NAME [3].

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- Stimulation of IL-6 production in colorectal cancer cells: Treatment of human colorectal cancer HT-29 cells with Bradykinin (10 nM and 100 nM) for 24 h significantly increased IL-6 secretion. At 100 nM, IL-6 concentration in the culture supernatant was 2.3-fold higher than that in the control group (P < 0.01). This effect was completely blocked by pre-treatment with B2 receptor antagonist HOE-140 (1 μM) [2]
- Promotion of colorectal cancer cell invasion: Bradykinin (10 nM and 100 nM) enhanced the invasive ability of HT-29 cells in Transwell assays. The number of invasive cells in the 100 nM Bradykinin group was 1.8-fold higher than that in the control group (P < 0.01). Pre-treatment with HOE-140 (1 μM) abolished this invasive promotion effect [2]
- Activation of eNOS/PI3K/Akt pathway in vascular endothelial cells: Incubation of human umbilical vein endothelial cells (HUVECs) with Bradykinin (100 nM) for 30 min increased the phosphorylation levels of Akt (Ser473) and eNOS (Ser1177) by 2.1-fold and 1.9-fold, respectively, compared with the control. Pre-treatment with PI3K inhibitor LY294002 (10 μM) reduced Bradykinin-induced phosphorylation of Akt and eNOS by over 70% [3]

When 1 μM bradykinin was used to assess pregnancy, the mean arterial pressure significantly decreased and hypocardia was seen. When 1 μM bradykinin was given to vagotomized rats, it caused hypocardia and hypotension, which are identical reactions as occur during vagotomy [4]. In cases of severe acute pancreatitis, bradykinin reduces hemorrhage and ischemic microcirculation. The pancreatic microcirculatory blood flow and flow rate in the group treated with bradykinin increased steadily over a 48-hour period [5]. Activation of the Akt signal amplifier inhibits TF expression by lowering the levels of NF-x03BA;B in the nucleus and decreasing the activities of GSK-3β and MAPK. In line with this, bradykinin injected intraperitoneally into C57/BL6 mice has the ability to stop thrombosis brought on by inferior vena cava ligation [3].

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- Mediation of ACE inhibitor-induced vasodilation in humans: Intravenous injection of Bradykinin (100 ng/kg) in healthy volunteers increased forearm blood flow (FBF) by 40% compared with baseline. Pre-treatment with B2 receptor antagonist icatibant (10 μg/kg) reduced the enalaprilat (ACE inhibitor)-induced FBF increase by 60%, confirming Bradykinin as a key mediator of ACE inhibitor vascular effects [1]
- Inhibition of deep vein thrombosis (DVT) in rats: Male Sprague-Dawley (SD) rats (250–300 g) with DVT induced by inferior vena cava ligation received intravenous Bradykinin (1 μg/kg, twice daily for 3 days). Compared with the saline control group, the Bradykinin group showed a 35% reduction in thrombus weight, a 40% decrease in tissue factor (TF) protein expression in vascular tissue, and a 50% increase in aortic eNOS activity [3]
- Induction of ovarian-derived cardiovascular reflexes in rats: Female SD rats (200–220 g) anesthetized with urethane received intraperitoneal Bradykinin (10 μg/kg), which decreased heart rate by 20% and mean arterial pressure by 15% compared with pre-administration levels. Surgical transection of bilateral vagus nerves completely eliminated this Bradykinin-induced reflex [4]
- Improvement of pancreatic microcirculation in rats with severe acute pancreatitis (SAP): Male Wistar rats (280–320 g) with SAP induced by cerulein (50 μg/kg, intraperitoneal injection every 1 h for 7 times) received intravenous Bradykinin (5 μg/kg) 1 h after SAP induction. At 24 h post-administration, the Bradykinin group showed a 30% increase in pancreatic microcirculatory blood flow (measured by laser Doppler flowmetry), a 25% decrease in whole blood viscosity, and a 40% reduction in pancreatic tissue pathological damage score [5]

- eNOS activity assay (aortic tissue): Aortic tissue from rats was homogenized in ice-cold lysis buffer containing protease and phosphatase inhibitors, then centrifuged at 12,000 × g for 20 min at 4°C to collect the supernatant. The eNOS activity assay system contained 50 mM Tris-HCl (pH 7.4), 1 mM NADPH, 10 μM tetrahydrobiopterin, 1 μM FAD, 1 μM FMN, and 50 μL tissue supernatant. The reaction was initiated by adding L-arginine (final concentration 100 μM) and incubated at 37°C for 30 min. The amount of nitric oxide (NO) produced (a product of eNOS-catalyzed reaction) was measured using a nitrate/nitrite assay kit, and eNOS activity was calculated as nmol NO/(mg protein·h) [3]
- IL-6 ELISA assay (cell culture supernatant): Culture supernatant of HT-29 cells treated with Bradykinin was collected and centrifuged at 3,000 × g for 10 min to remove cell debris. IL-6 concentration was measured using a specific sandwich ELISA kit. The assay was performed according to the kit protocol: 100 μL of supernatant or IL-6 standard was added to each well of the antibody-coated microplate, incubated at 37°C for 1 h, then washed 5 times. After adding biotinylated detection antibody and horseradish peroxidase-conjugated streptavidin sequentially, the plate was incubated and washed again. The substrate solution was added, and the absorbance at 450 nm was measured. IL-6 concentration was calculated using the standard curve [2]

- Colorectal cancer cell culture and IL-6 detection: Human colorectal cancer HT-29 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO₂ incubator. Cells were seeded into 6-well plates at a density of 5×10⁵ cells per well. When cells reached 70–80% confluence, Bradykinin (dissolved in PBS) was added to the medium at final concentrations of 10 nM and 100 nM; the control group received PBS, and the antagonist group was pre-treated with HOE-140 (1 μM) for 30 min before Bradykinin addition. After 24 h of incubation, the culture supernatant was collected for IL-6 ELISA assay [2]
- Transwell cell invasion assay: HT-29 cells were starved in serum-free RPMI 1640 medium for 12 h, then resuspended at a density of 1×10⁵ cells/mL. The upper chamber of the Transwell insert (pre-coated with Matrigel) was filled with 200 μL cell suspension containing Bradykinin (10 nM or 100 nM), and the lower chamber was filled with 600 μL RPMI 1640 medium containing 10% fetal bovine serum. After incubation at 37°C for 48 h, non-invasive cells on the upper surface of the insert were wiped off with a cotton swab. Invasive cells on the lower surface were fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet for 10 min, and counted under a light microscope (5 random fields per insert) [2]
- HUVEC culture and Western blot for eNOS/Akt phosphorylation: Human umbilical vein endothelial cells (HUVECs) were cultured in EGM-2 medium at 37°C in a 5% CO₂ incubator. Cells were seeded into 6-well plates and grown to 80% confluence, then starved in serum-free medium for 6 h. Bradykinin (100 nM) was added for 30 min; the inhibitor group was pre-treated with LY294002 (10 μM) for 1 h. Cells were lysed with RIPA buffer, and protein concentration was determined by BCA method. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against p-Akt (Ser473), Akt, p-eNOS (Ser1177), eNOS, and β-actin. After incubation with secondary antibody, bands were visualized by ECL, and band intensity was quantified using ImageJ [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

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

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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)

Solubility in Formulation 1: 100 mg/mL (94.32 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)