Dipeptidyl peptidase 4 (DPP-4)

虽然 CXCR4（SDF-1α 受体）阻滞剂或 Src 抑制剂会削弱这种连接，但二蛋白 A 会增加人内皮细胞中 Src 和血管内皮钙粘蛋白 (VE-cadherin) 的磷酸化，从而破坏内皮细胞与细胞之间的连接。 [1]。 在 100 μM CXCR4 阻滞剂或 Src 氧化处理后 30 分钟，暴露于含氧量正常和 H/R 条件的人内皮细胞会发生 Src [Tyr 416] 和 VE-钙粘蛋白 [Tyr731] 的磷酸化。它会氧化并分解 CXCR4（SDF-1α 受体）发泡剂或 Src 偶联的内皮细胞与内皮细胞连接 [1]。

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DPP4抑制剂破坏内皮细胞的完整性并增加其通透性[1]
为了测试DPP4抑制剂是否会增加血管通透性，我们用Diprotin A（DipA；Ile-Pro-Ile）处理hECs，并评估Src和VE-cadherin的磷酸化。hECs上的H/R诱导了Src[Tyr 416]和VE-caadherin[Tyr731]的磷酸化（图3a-c）。DPP4抑制剂进一步增加Src[Tyr416]和VE-cadherin[Tyr731]的磷酸化。为了研究DPP4抑制剂对VE钙粘蛋白[Tyr731]的磷酸化是否由SDF-1α/CXCR4配体/受体相互作用和下游信号分子Src激酶介导，我们评估了CXCR4阻断剂（AMD3100）和Src抑制剂（PP2）对Src和VE钙粘素磷酸化的抑制作用。CXCR4阻断剂和Src抑制剂都降低了DPP4抑制剂诱导的人胚胎干细胞中Src[Tyr416]和VE-cadherin[Tyr731]的磷酸化（图3a-c）。然后，我们对人胚胎干细胞中的VE-cadherin进行了免疫荧光染色，以确定DPP4抑制剂诱导的VE-caadherin磷酸化是否真的导致内皮完整性的破坏。DPP4抑制剂破坏了用VE-cadherin标记的内皮细胞间连接。CXCR4阻断剂或Src抑制剂可以阻止这种连接的破坏（图3d，e）。我们使用体外经孔内皮通透性测定法，逐步评估了免疫荧光图像上内皮细胞间连接中断与内皮单层“实际泄漏”的相关性。通过测量FITC葡聚糖（异硫氰酸荧光素偶联葡聚糖；40 kDa）从上腔到下腔通过内皮单层的通道（图3f；详细实验方案见补充图S3）。在上腔中加入DPP4抑制剂后，下腔室中代表内皮通透性的FITC葡聚糖含量显著增加，这被CXCR4阻断剂或Src抑制剂治疗所阻止（图3g）。

在早产视网膜血管模型中，二蛋白 A 会增加视网膜血管分布和渗漏。此外，在糖尿病视网膜病变小鼠模型中，二蛋白 A 会加剧视网膜血管渗漏并增强 Src 和 VE-钙粘蛋白的磷酸化。总体而言，SDF-1α/CXCR4/Src/VE-钙粘蛋白信号通路被二蛋白 A 增强，从而导致血管渗漏 [1]。 使用二蛋白 A（腹腔注射；70 μg/kg；每天两次；7 天）会导致大量血管渗漏以及 Src 和 VE-钙粘蛋白的磷酸化增加。总之，SDF-1α/CXCR4/Src 被二蛋白 A 增强。

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多剂量Diprotin A（DipA；Ile-Pro-Ile）和西格列汀的内皮通透性 为了探索剂量反应关系，我们测试了多剂量的<强>Diprotin A（DipA；Ile-Pro-Ile）（1、10、100μM）。由于糖尿病患者的DPP4抑制是通过格列汀实现的，我们还研究了西格列汀对Src和VE-cadherin磷酸化以及内皮通透性的影响。使用0.1、1和10μM的西格列汀浓度来反映服用西格列汀25、100和600的人类志愿者的实际血浆浓度 mg，分别为32，DipA和西格列汀以剂量依赖的方式增加Src[Tyr 416]和VE钙粘蛋白[Tyr731]的磷酸化（图5a，b）。内皮通透性也以剂量依赖的方式增加（DipA和西格列汀的p<0.001；图5c）。两组6个可能的成对比较的p值均<0.01。

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DPP4抑制剂体内内皮渗漏[1]
为了评估DPP4抑制剂是否会增加体内血管通透性，我们使用小鼠耳朵进行了Miles通透性测定（补充图S4）。注射SDF-1α的耳朵因全身给药的伊文思蓝染料外渗而变蓝（图6a，b）。腹膜内注射DPP4抑制剂（Diprotin A（DipA；Ile-Pro-Ile））5天后，SDF-1α引起的渗漏加重。然而，CXCR4阻断剂（AMD3100）或Src抑制剂（PP2）显著减少了DPP4抑制剂和SDF-1α的血管渗漏（图6a，b）。

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DPP4抑制剂引起的视网膜毛细血管渗漏：早产儿视网膜病变模型[1]
我们使用早产小鼠视网膜病变模型测试了DPP4抑制剂对视网膜血管通透性的体内影响。如实验方案（图7a）所示，新生小鼠幼崽从第7天到第12天暴露于高氧（75%）（高氧情况），然后恢复到正常空气（20%）中5天（相对缺氧情况）。在采集眼睛之前，系统地施用了两种不同的染料；用于血管检查的TRITC偶联的简单斑叶菌凝集素（BS-1凝集素）和用于血管渗漏检查的FITC葡聚糖。与高氧下的新生儿或出生后第12天视网膜相比，出生后第17天小鼠在相对缺氧下的视网膜新生血管增加（补充图S5）。DPP4抑制剂（Diprotin A（DipA；Ile-Pro-Ile））的全身给药不仅增加了血管（补充图S6），还增加了血管渗漏（图7b-d），而CXCR4阻断剂（AMD3100）可以预防血管渗漏。

体外渗透性测定[1]
FITC-葡聚糖（40kDa）是一种易于检测的示踪剂。通过FITC-葡聚糖穿过hECs单层来评估内皮膜的渗透性。实验前两天，将hECs接种到包被纤维连接蛋白的0.4μm孔24孔大小的细胞培养插入物上。将细胞在添加了0.5%胎牛血清的EBM中在标准培养条件下（37°C、95%加湿空气和5%二氧化碳）饥饿培养18小时。在实验开始时，用CXCR4阻断剂（AMD3100；1μg/ml）或Src抑制剂（PP2；1μM）预处理培养基。DPP4抑制剂（Diprotin A（DipA；Ile-Pro-Ile），Ile-Pro-Ile；100μM）在CXCR4阻断剂或Src抑制剂治疗后30分钟施用。DPP4抑制剂处理后60分钟，将浓度为20μg/ml的FITC-葡聚糖加入上腔（Diprotin a（DipA；Ile-Pro-Ile）的药物处理时间：60分钟）。在37°C下孵育20分钟后，从下腔室抽取100μl培养基（渗透时间：20分钟）。为了探索剂量反应关系，在上腔中加入多剂量的<强>Diprotin A（DipA；Ile-Pro-Ile）（1，10，100μM）和西格列汀（0.1，1，10μM）。30分钟后，将FITC葡聚糖加入上腔（药物处理时间：30分钟）。在FITC-葡聚糖处理后5分钟抽取下腔室培养基（渗透时间：5分钟）。通过荧光分光荧光计测定下腔室的荧光。

蛋白质印迹分析[1]
细胞类型： 人内皮细胞[1]
测试浓度： 100 μM
孵育时间： 使用 CXCR4 阻滞剂或 Src 抑制剂治疗后 30 分钟
实验结果： hEC [Tyr731] 中 Src [Tyr 416] 和 VE-钙粘蛋白的磷酸化诱导。

Animal/Disease Models: Streptozotocin-induced wild-type C57/BL6 mouse diabetic retinopathy model [1]
Doses: 70 μg/kg
Route of Administration: intraperitoneal (ip) injection; /VE-cadherin signaling induces leaky blood vessels [1]. twice (two times) daily; 7 days
Experimental Results: Induces vascular leakage by enhancing SDF-1α/CXCR4/Src/VE-cadherin signaling pathway.

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In-vivo permeability assay [1]
Miles assay was performed in wild-type C57/BL6 mice. Three groups of mice received an intra-peritoneal injection of DPP4-inhibitor (Diprotin A (DipA; Ile-Pro-Ile); 70 μg/kg twice daily), and another group received vehicle for 5 days. The specific dose of DPP4-inhibitor used in this study has been shown to mediate therapeutic effects in murine models20,51. After 5 days, 2 groups of mice injected with DPP4-inhibitor received an intra-peritoneal injection of CXCR4-blocker (AMD3100; 7.5 mg/kg, once per day) or Src-inhibitor (PP2; 1 mg/kg, once per day). Thirty minutes later, each mouse was injected with PBS into its right ear and SDF-1α (250 ng) to its left ear, and this was followed by an injection of intra-cardiac 0.5% Evans blue dye. Photographs of the ears were obtained, and the mice were euthanized 30 minutes later. Mouse ear tissue was collected with an 8 mm skin punch and was incubated in 300 μl of formamide at 56 °C for 48 hours. The quantity of Evans blue dye in the tissues and standards was determined by assessing the optical density at 600 nm.

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Retinopathy of prematurity model [1]
The oxygen exposure protocol placed oxygen-exposed mouse pups with their nursing mothers in the same covered plastic box with 75% oxygen from postnatal day 7 through postnatal day 12 as previously described42. The oxygen was delivered at 75 ± 2%, and it was monitored at least three times daily during the oxygen exposure period. Oxygen concentrations were measured with an oxygen monitor. On postnatal day 12, the animals were returned to room air and were subsequently sacrificed by a lethal intra-peritoneal injection of chloral hydrate (360 mg/kg) on postnatal day 17. DPP4-inhibitor injection (Diprotin A (DipA; Ile-Pro-Ile); 70 μg/kg, twice daily) was administered from postnatal day 12 to 17. The control mice were injected with PBS in the same manner as DPP4-inhibitor. CXCR4-blocker (AMD3100; 7.5 mg/kg, once per day) was also injected in the same manner as that for the DPP4-inhibitor. BS-1 Lectin (Sigma-Aldrich, St. Louis, MO, USA) was infused systemically for vascularity examination and FITC-dextran or permeability examination. Both eyes of each animal were used for examination of the retinal vascular pattern after flat mounting of the retina.

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Streptozotocin-induced diabetic retinopathy model [1]
To induce diabetes mellitus, 180 mg/kg of intra-peritoneal STZ was injected into 7-week-old C57/BL6 mice. Blood sugar levels from tail vein blood samples and body weight were monitored weekly. Before the mice were sacrificed, 250 μl of whole blood were drained from the heart for hemoglobin A1c (HbA1c) examination. Two weeks after STZ injection, the mice were confirmed to be diabetic if the BST level was greater than 500 mg/dl. These mice were divided into 4 groups: STZ only; STZ + DPP4-inhibitor; STZ + DPP4-inhibitor + CXCR4-blocker; and STZ + DPP4-inhibitor + Src-inhibitor. Intra-peritoneal DPP4-inhibitor (Diprotin A (DipA; Ile-Pro-Ile); 70 μg/kg twice daily) was injected for 7 days beginning from 6 weeks after STZ injection. Single doses of intra-peritoneal CXCR4-blocker (AMD3100; 7.5 mg/kg, once per day) and Src-inhibitor (PP2; 1 mg/kg, once per day) were also injected in the same manner as that for the DPP4-inhibitor. TRITC-conjugated BS-1 Lectin was infused for vascularity examination and FITC-dextran for permeability examination. Both eyes of each mouse were used for examination of the retinal vascular pattern after flat mounting of the retina. Images were obtained with a Nikon DS-Qi2 CMOS camera head mounted on a Nikon Ti-E motorized inverted microscope. To quantify the fluorescence intensity of FITC-dextran, captured images from each experiment were analyzed using NIS-Elements and ROI statistics software. Using the black-green interface, we created region of interest on each captured image. By ROI statistics, we calculated the mean intensity of FITC-dextran in the region of interest of each captured image. After the retinal vascularity examination, western blot analysis for phosphorylated Src and VE-cadherin was performed using the retinal tissue.

mouse LD intravenous >250 mg/kg Journal of Antibiotics., 37(422), 1984 [PMID:6427168]

[1]. Dipeptidyl Peptidase-4 Inhibitor Increases Vascular Leakage in Retina through VE-cadherin Phosphorylation. Sci Rep. 2016 Jul 6;6:29393.

(2S,3S)-2-[[[(2S)-1-[(2S,3S)-2-amino-3-methyl-1-oxopentyl]-2-pyrrolidinyl]-oxomethyl]amino]-3-methylpentanoic acid is a peptide.
diprotin A has been reported in Bacillus cereus with data available.

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The inhibitors of CD26 (dipeptidyl peptidase-4; DPP4) have been widely prescribed to control glucose level in diabetic patients. DPP4-inhibitors, however, accumulate stromal cell-derived factor-1α (SDF-1α), a well-known inducer of vascular leakage and angiogenesis both of which are fundamental pathophysiology of diabetic retinopathy. The aim of this study was to investigate the effects of DPP4-inhibitors on vascular permeability and diabetic retinopathy. DPP4-inhibitor (Diprotin A (DipA; Ile-Pro-Ile)or sitagliptin) increased the phosphorylation of Src and vascular endothelial-cadherin (VE-cadherin) in human endothelial cells and disrupted endothelial cell-to-cell junctions, which were attenuated by CXCR4 (receptor of SDF-1α)-blocker or Src-inhibitor. Disruption of endothelial cell-to-cell junctions in the immuno-fluorescence images correlated with the actual leakage of the endothelial monolayer in the transwell endothelial permeability assay. In the Miles assay, vascular leakage was observed in the ears into which SDF-1α was injected, and this effect was aggravated by DPP4-inhibitor. In the model of retinopathy of prematurity, DPP4-inhibitor increased not only retinal vascularity but also leakage. Additionally, in the murine diabetic retinopathy model, DPP4-inhibitor increased the phosphorylation of Src and VE-cadherin and aggravated vascular leakage in the retinas. Collectively, DPP4-inhibitor induced vascular leakage by augmenting the SDF-1α/CXCR4/Src/VE-cadherin signaling pathway. These data highlight safety issues associated with the use of DPP4-inhibitors.

C19H32F3N3O6

455.469096183777

455.224

209248-71-5

Diprotin A;90614-48-5

137319704

White to off-white solid powder

150

4

10

8

31

552

5

C(F)(F)(F)C(=O)O.C(N1CCC[C@H]1C(=O)N[C@H](C(=O)O)[C@@H](C)CC)(=O)[C@@H](N)[C@@H](C)CC

KAUYVJOUHGUZNN-BABXMESMSA-N

InChI=1S/C17H31N3O4.C2HF3O2/c1-5-10(3)13(18)16(22)20-9-7-8-12(20)15(21)19-14(17(23)24)11(4)6-2;3-2(4,5)1(6)7/h10-14H,5-9,18H2,1-4H3,(H,19,21)(H,23,24);(H,6,7)/t10-,11-,12-,13-,14-;/m0./s1

(2S,3S)-2-[[(2S)-1-[(2S,3S)-2-amino-3-methylpentanoyl]pyrrolidine-2-carbonyl]amino]-3-methylpentanoic acid;2,2,2-trifluoroacetic acid

Diprotin A (TFA); Diprotin A TFA; 209248-71-5; Diprotin A TFA (Ile-Pro-Pro (TFA)); (2S,3S)-2-[[(2S)-1-[(2S,3S)-2-amino-3-methylpentanoyl]pyrrolidine-2-carbonyl]amino]-3-methylpentanoic acid;2,2,2-trifluoroacetic 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 : ≥ 77.5 mg/mL (~170.15 mM)

配方 1 中的溶解度: 100 mg/mL (219.55 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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