PI3K

PDGFR 740Y-P 肽刺激肌肉细胞的有丝分裂反应。 740Y-P 肽刺激有丝分裂的能力具有高度特异性，而不是细胞渗透性 SH2 结构域结合肽的一般特征[2]。通过众所周知的 PI 3-激酶-Akt 存活级联，740Y-P 在促进神经元细胞存活方面与生长因子 (FGF2) 一样有效。 PDGFR740Y-P 肽不需要胰岛素，可以刺激神经元细胞存活[1]。

740 YP 增加阿尔茨海默病大鼠模型中 AKT 和 PI3K 磷酸化的程度，并降低用 A(25-32) 治疗的海马组织中 ROS 水平的程度。研究人员发现，GABAB受体激活可以恢复AD大鼠的空间记忆和学习能力，并通过激活PI3K/Akt信号通路抑制神经元凋亡和海马萎缩。此外，GABAB受体激活通过激活PI3K/Akt信号通路，降低MDA水平，增加SOD、GSH-Px和CAT水平，从而减轻氧化应激损伤。 结论：综上所述， 研究结果表明，GABAB受体激活通过激活PI3K/Akt信号通路抑制AD大鼠神经元氧化应激损伤，这可能是AD潜在的新治疗靶点。

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此外，成功建模的大鼠分别给予巴氯芬（AD +巴氯芬组）、740-Y-P（PI3K/Akt信号通路激动剂）（AD + 740-Y-P组）和LY294002 （AD + LY294002组）治疗，每组10只。对大鼠海马提取物进行不同处理，通过western blot分析检测PI3k和Akt的磷酸化程度（图3B， C）。与正常组相比，AD +巴氯芬组、AD +强bbb740 - y - p 组、AD + LY294002组和AD +巴氯芬+ LY294002组大鼠PI3k和Akt的磷酸化程度下降。此外，与AD组相比，AD +巴氯芬组和AD + 740-Y-P组PI3k和Akt磷酸化程度增强，而AD + LY294002组PI3k和Akt磷酸化程度降低。[3]
此外，我们采用western blot分析各组海马提取物中Bax、Bcl-2、cleaved caspase 3和caspase-3的表达情况（图3B、D）。结果显示，AD +巴氯芬组和AD + 740-Y-P组中Bcl-2和caspase-3的表达升高，Bax和cleaved caspase-3的表达降低；而在AD + LY294002组中，Bcl-2和Caspase-3的表达显著降低，Bax和cleaved - Caspase-3的表达显著升高。AD +巴氯芬+ LY294002组大鼠Bcl-2和Caspase-3表达下调，Bax和cleaved - Caspase-3表达上调。综上所述，GABAB受体可激活PI3K/Akt信号通路，抑制海马细胞凋亡。[3]

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与a β处理海马组织相比，巴氯芬或740-Y-P处理a β处理海马组织的ROS水平降低。ELISA结果显示，与AD组、AD +巴氯芬组、AD + 740-Y-P组、AD + LY294002组、AD +巴氯芬+ LY294002组相比，正常组MDA水平明显降低，SOD、GSH-Px、CAT水平明显升高（p < 0.05）。AD +巴氯芬或AD +740-Y-P组MDA水平显著降低，SOD、GSH-Px、CAT水平显著升高，AD + LY294002组MDA水平显著升高，SOD、GSH-Px、CAT水平显著降低（均p < 0.05）。[3]

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采用流式细胞术检测巴氯芬、740-Y-P、LY294002、Aβ联合用药对培养海马神经元凋亡的影响。结果显示（图6B），与对照组相比，其他各组凋亡细胞数量均显著增加，与Aβ组相比，Aβ+巴氯芬组和Aβ+740-Y-P组的凋亡率降低，而Aβ+ LY294002组的凋亡率高于Aβ+ LY294002组。［3］

小磷酸肽与PI3-激酶p85调节亚基的SH2结构域的结合可以在体外激活该酶。在本研究中，已经评估了特异性结合p85的SH2结构域的细胞可渗透肽在C2肌肉细胞系中刺激促有丝分裂反应的能力。与其他四种SH2结合肽相比，该肽在刺激进入S期方面与血清、EGF和FGF一样有效。wortmannin和雷帕霉素抑制了对p85结合肽（而不是FGF）的反应，表明该肽激活了PI3-激酶/S6激酶信号通路。肽反应不受MEK抑制剂（PD098059）的抑制，也不刺激Erk磷酸化。因此，在p85结合肽激活的途径和p42/p44 MAPK级联之间似乎没有直接的串扰[2]。

740-Y-P增加阿尔茨海默病大鼠模型中 AKT 和 PI3K 磷酸化的程度，并降低用 A(25-32) 处理的海马组织中 ROS 水平的程度。NIH HBSS（含 10% FCS、Ca2+ 和 Mn2+ 游离），以及将 50 μg/ml 740-Y-P肽或等体积的 PBS 作为对照与 2 106 个 3T3 细胞悬浮液在 37°C 下孵育。 2小时后将细胞离心、清洗并用胰蛋白酶消化以分解非内化的肽。然后，将细胞重悬于裂解缓冲液（50 mM Tris pH 7.4、150 mM NaCl、10% 甘油、2% NP40、0.25% 脱氧胆酸盐、1 mM EDTA、1 mM 钒酸盐、Boehringer-Mannheim 的蛋白酶抑制剂“完整”混合物中）并在 4°C 下孵育 1 小时。通过以 1.4×104g 离心 5 分钟澄清裂解物，然后将上清液与链霉亲和素-琼脂糖珠一起孵育 1 小时。之后，将珠子在裂解缓冲液中洗涤 3 次，在 SDS 样品缓冲液中煮沸，在 12% 凝胶上通过 SDS-PAGE 解析，转移到硝酸纤维素上，并用 p85 单克隆抗体进行免疫印迹。

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海马神经元细胞的分离培养[3]
直率地分离海马体，同时切除血管和脑膜。海马切成直径0.4 mm的小块。切片用0.25%胰蛋白酶和0.04 DNA酶反应12 min，加入马血清终止反应。用移液管滋养10次分散细胞。细胞悬液在含10%胎牛血清、5%马血清、25 mmol/L KCI、10 mmol/L HEPES、105 U/L青霉素和0.1 g/L链霉素的MEM中培养。经直径为75μm的尼龙筛网过滤器过滤后，将样品置于涂有聚l -赖氨酸氢溴化物载玻片的35 mm培养皿中，在5% CO2和95% O2中37℃孵育，密度为0.6×10~9 L-1。第3天，在培养液中添加5μmol/L阿糖胞苷，每24 h更换一次。培养后第7天，采用SP法进行神经元特异性烯醇化酶免疫细胞化学染色，鉴定海马神经元细胞。分别用Aβ（终浓度为25μmol/L）、巴氯芬（终浓度为25μmol/L）、740-Y-P（终浓度为20μmol/L）、LY294002（终浓度为10μmol/L）单独或联合作用培养细胞。每次处理持续24 h。以未处理的细胞为对照。

To evaluate this hypothesis, a rat AD model was established by intraperitoneal injection of the GABAB receptor agonist (baclofen), PI3K/Akt signaling pathway agonist (740-Y-P), and antagonist (LY294002), respectively. The effects of GABAB activation on spatial memory and learning ability in the AD rats were measured by Morris water maze. Whereas the effects of GABAB and PI3K/Akt signaling pathway on apoptosis and oxidative stress injury were determined in vivo and in vitro using primary neuronal cultures [3].

Seventy healthy male adult SD rats [specific pathogen-free (SPF); age, 2– 3 months, weighed 32.10±24.70 g were housed with free access to water and food in a 12/12 h day/night cycle at 25±2°C. After seven days of acclimatization, a total of 60 rats were randomly grouped with 10 rats each group. AD modeled rats were intraperitoneally injected with GABAB receptor agonist baclofen (2.0 mg/kg), PI3K/Akt signaling pathway agonist 740-Y-P (10 mg/kg), or PI3K/Akt signaling pathway inhibitor LY294002 (20 mg/kg), or both baclofen (2.0 mg/kg) +LY294002 (20 mg/kg). Rats were treated differently for consecutive 6 weeks. The experimental protocol for treatments and behavioral tests can be seen in Fig. 1. [3]

[1]. Mol Cell Neurosci. 1999 Apr;13(4):272-80.

[2]. Biochem Biophys Res Commun. 1998 Oct 9;251(1):148-52.

[3]. J Alzheimers Dis. 2020;76(4):1513-1526.

PI 3-kinase has emerged as a key enzyme for regulating neuronal cell survival. However, it has not as yet been demonstrated whether activation of the endogenous pool of the enzyme, that is regulated by the p85 subunit, is sufficient to promote a survival response. It is also not known whether the FGF family of growth factors promote survival via a PI 3-kinase-dependent pathway. We have previously developed a cell permeable p85 binding peptide and shown that it can stimulate a mitogenic response in muscle cells that is dependent on a PI 3-kinase/p70 S6 kinase pathway. In the present study we show that this peptide can rescue cerebellar granule cells from death induced by serum deprivation and that this response is comparable to a growth factor response (FGF2). Experiments with wortmannin, LY294002, and rapamycin suggest that the peptide survival response is dependent on PI 3-kinase activity, but not p70 S6 kinase activity. The peptide response was correlated with a PI 3-kinase-dependent phosphorylation of Akt, an established downstream effector in the PI 3-kinase survival cascade. In contrast to the survival response stimulated by the p85 binding peptide, the response stimulated by FGF2 was not inhibited by wortmannin or LY294002, nor was it associated with phosphorylation of Akt. Thus we can conclude that activation of the endogenous pool of PI 3-kinase that is regulated by p85 is sufficient for cell survival; however, growth factors such as FGF2 can clearly support survival in a PI 3-kinase-independent manner.[1]

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The binding of small phosphopeptides to the SH2 domains of the p85 regulatory subunit of PI 3-kinase can activate the enzyme in vitro. In the present study a cell-permeable peptide that binds specifically to the SH2 domains of p85 has been evaluated for its ability to stimulate a mitogenic response in the C2 muscle cell line. This peptide, in contrast to four other SH2-binding peptides, was as effective as serum, EGF, and FGF at stimulating entry into S-phase. The response to the p85 binding peptide, but not FGF, was inhibited by wortmannin and rapamycin, indicating that the peptide activates the PI 3-kinase/S6 kinase signalling pathway. The peptide response was not inhibited by the MEK inhibitor (PD098059) and did not stimulate Erk phosphorylation. Thus, there would appear to be no direct cross-talk between the pathway activated by the p85 binding peptide and the p42/p44 MAPK cascade.[2]

C141H222N43O39PS3.XC2HF3O2

3270.70 (free acid)

1236188-16-1

Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Ser-Asp-Gly-Gly-{Tyr(PO2)}-Met-Asp-Met-Ser

RQIKIWFQNRRMKWKKSDGG-{PO2Y}-MDMS

Typically exists as solid at room temperature

(3S)-3-[[(2S)-2-[[(2S)-2-[[2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3S)-2-[[(2S)-6-amino-2-[[(2S,3S)-2-[[(2S)-5-amino-2-[[(2S)-2-amino-5-carbamimidamidopentanoyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]amino]hexanoyl]amino]-3-methylpentanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-3-phenylpropanoyl]amino]-5-oxopentanoyl]amino]-4-oxobutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-carbamimidamidopentanoyl]amino]-4-methylsulfanylbutanoyl]amino]hexanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]hexanoyl]amino]hexanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]acetyl]amino]acetyl]amino]-3-(4-phosphonooxyphenyl)propanoyl]amino]-4-methylsulfanylbutanoyl]amino]-4-[[(2S)-1-[[(1S)-1-carboxy-2-hydroxyethyl]amino]-4-methylsulfanyl-1-oxobutan-2-yl]amino]-4-oxobutanoic acid TFA salt

740 Y-P TFA; 740 YP; 740YPDGFR TFA; PDGFR740Y-P TFA; 740 Y P; 740-YPDGFR; PDGFR 740 Y-P; 740 YPDGFR; PDGFR 740Y-P; H-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Ser-Asp-Gly-Gly-Tyr(PO3H2)-Met-Asp-Met-Ser-OH; Alternative Name: PDGFR740Y-P; 740 Y-P?; PDGFR 740Y-P

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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));
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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；
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