【摘要】：目的：擬在前期RhoA蛋白小分子抑制劑HL07號化合物的基礎(chǔ)上，通過結(jié)構(gòu)優(yōu)化，在細胞、組織、整體水平篩選出在蛛網(wǎng)膜下腔出血后腦血管痙攣模型上有舒張作用的化合物，為發(fā)現(xiàn)新的有利于心血管疾病治療的先導(dǎo)化合物提供基礎(chǔ)，并為推動RhoA相關(guān)通路在疾病病理生理中作用機制的研究提供理論依據(jù)。 方法：1）人腦血管平滑肌細胞（thehuman cerebrovascular smooth muscle cells,HBVSMCs）的培養(yǎng)，采用G-LISA方法；定量評價結(jié)構(gòu)優(yōu)化的33個化合物對溶血磷脂酸（LPA）引起RhoA激活作用的抑制，觀察化合物對RhoA蛋白GTP結(jié)合域競爭性抑制活性；2）采用SD大鼠離體血管條實驗方法觀察化合物HL47/47K、HL79/79K、HL82/82K對預(yù)收縮血管條的張力的影響；3）大鼠SAH-CVS動物模型采用視交叉池二次注血法，實驗隨機分為7組：正常對照組、SAH模型組、SAH模型組+DMSO、SAH模型組+Fasudil、SAH模型組+HL47（L）、SAH模型組+HL47（M），，SAH模型組+HL47（H），每組9只，比較各組大鼠基底動脈(basilar artery, BA)細胞形態(tài)學(xué)及內(nèi)徑周長的差異。 結(jié)果：1）細胞篩選實驗中，33個結(jié)構(gòu)改造后的化合物中，篩選出3個小分子抑制劑活性強于HL07（IC50=1.79±0.17μM），分別是HL47（IC50=1.66±0.28μM）、HL79（IC50=1.26±0.13μM）和HL82（IC50=1.17±0.19μM）；2）血管條組織水平：HL47（IC50=70.90±1.33μM）、HL82（IC50=73.43±2.61μM）對100nM苯腎上腺素（phenylephrine, PE）預(yù)收縮的離體去內(nèi)皮胸主動脈（thoracic aorta, TA）血管條呈現(xiàn)濃度依賴的明顯舒張作用（P0.05），且活性均強于HL0（7IC50=123.22±8.93μM）;HL79（0~180μM）舒張作用微弱；HL47K（IC50=59.92±5.40μM）、HL82K（IC50=66.64±2.32μM）、HL79K（IC50=111.88±15.71μM）較未成鹽時，活性均有不同程度的增強，HL79K活性增加最為顯著（P0.05）；3）大鼠SAH-CVS動物模型實驗中，各組血管內(nèi)徑周長比較：SAH模型組（386.25±11.25μm）較正常對照組（472.50±53.47μm）、SAH模型組+Fasudil（468.83±43.53μm）減小，其差異有統(tǒng)計學(xué)意義（P0.05）；SAH模型組與SAH模型組+DMSO（390.93±20.60μm）之間差異無統(tǒng)計學(xué)意義（P0.05）；、SAH模型組+HL47(L、M、H)（464.00±50.46μm、475.60±63.79μm、513.42±40.15μm）均較SAH模型組增大，其差異有統(tǒng)計學(xué)意義(P0.05)，與SAH模型組+Fasudil（468.83±43.53μm）差異無統(tǒng)計學(xué)意義（P0.05）。SAH模型組+HL47(L)、SAH模型組+HL47(M)、SAH模型組+HL47(H)三組之間相互差異均無統(tǒng)計學(xué)意義（P0.05）。 結(jié)論：1）部分經(jīng)過HL07結(jié)構(gòu)改造后的化合物（HL47、HL79、HL82），其抑制LPA對RhoA激活作用的活性有所增強；2）HL47/47K、HL82/82K對PE預(yù)收縮的血管條有顯著的舒張作用，且活性均強于HL07，結(jié)果與細胞實驗一致；小分子抑制劑的水溶性對其活性有較大的影響；3）HL47對大鼠腦血管痙攣有緩解作用。 本實驗結(jié)果提示：小分子化合物HL47可更好地通過抑制RhoA蛋白活性，舒張大鼠蛛網(wǎng)膜下腔出血后腦血管痙攣。
[Abstract]:Objective: on the basis of HL07, a small molecular inhibitor of RhoA protein, we selected the vasodilator on the model of cerebral vasospasm after subarachnoid hemorrhage by structural optimization, cell, tissue and whole level. It provides the basis for the discovery of new leading compounds which are beneficial to the treatment of cardiovascular disease and provides the theoretical basis for the study of the mechanism of RhoA related pathway in pathophysiology of the disease. Methods (thehuman cerebrovascular smooth muscle cells of human vascular smooth muscle cells (thehuman cerebrovascular smooth muscle cells) were cultured by G-LISA method. The inhibitory effects of 33 compounds on RhoA activation induced by lysophosphatidic acid (LPA) were evaluated quantitatively. To observe the effect of compounds on the competitive inhibitory activity of RhoA protein GTP binding domain in SD rat vascular strips in vitro, we observed the effect of HL47 / 47K / 79KN HL82 / 82k on the tension of preconstricted vascular strips. The rat model of SAH-CVS was injected with the second injection of blood into the optic chiasma cistern. The experiment was randomly divided into 7 groups: normal control group: SAH model group: DMSO SAH model group, Fasudiline SAH model group, HL47 (L) group, SAH model group, HL47 (M) group, SAH model group, HL47 (H), group, 9 rats in each group. The differences of (basilar artery, BA) cell morphology and inner diameter perimeter of basilar artery in each group were compared. Results in the cell screening experiment, three small molecular inhibitors (IC50=1.79 鹵0.17 渭 M),) were found to be HL47 (IC50=1.66 鹵0.28 渭 M), HL79 (IC50=1.26 鹵0.13 渭 M) and HL82 (IC50=1.17 鹵0.19 渭 M);) in 33 structurally modified compounds. 2)琛

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