目的 采用轉(zhuǎn)基因血管熒光斑馬魚研究蘆丁、甘草酸、漢防己甲素、葛根素、柚皮素、鞣花酸、黃芩苷、大黃素、穿心蓮內(nèi)酯、苦參堿促進血管新生的作用。方法 選取48 hpf轉(zhuǎn)基因血管熒光Fli-1品系斑馬魚3 480尾于六孔板中,每孔均處理30尾(實驗組),分別水溶給予蘆丁81.90~1 637.95 μmol·L^-1、甘草酸60.76~1 215.17 μmol·L^-1、漢防己甲素5.02~80.29 μmol·L^-1、葛根素120.08~2 161.49 μmol·L^-1、柚皮素3.67~734.59 μmol·L^-1、鞣花酸0.21~3.31 μmol·L^-1、黃芩苷28.00~448.07 μmol·L^-1、大黃素0.19~740.08 μmol·L^-1、穿心蓮內(nèi)酯35.67~570.69 μmol·L^-1、苦參堿50.33~805.28 μmol·L^-1,同時設(shè)置對照組(養(yǎng)魚用水處理斑馬魚),處理24 h后觀察記錄斑馬魚的毒性表型和死亡情況,確定供試品對斑馬魚的最大耐受濃度(MTC)。用10個化合物的MTC與斑馬魚共培養(yǎng)24 h,檢測腸下血管面積和腸下血管出芽數(shù)。結(jié)果 與對照組比較,蘆丁組、甘草酸組、漢防己甲素組、葛根素組、柚皮素組、黃芩苷組、穿心蓮內(nèi)酯組、苦參堿組斑馬魚腸下血管面積像素升高,其中甘草酸組、漢防己甲素組、黃芩苷組、穿心蓮內(nèi)酯組差異顯著(P<0.05、0.01、0.001);鞣花酸組、大黃素組斑馬魚腸下血管面積像素降低,差異不顯著;蘆丁組、漢防己甲素組、葛根素組、柚皮素組、鞣花酸組、苦參堿組斑馬魚腸下血管出芽數(shù)比對照組多,蘆丁組差異顯著(P<0.05);甘草酸組、黃芩苷組、穿心蓮內(nèi)酯組斑馬魚腸下血管出芽數(shù)比對照組少,大黃素組斑馬魚腸下血管出芽數(shù)與對照組相當。結(jié)論 蘆丁、甘草酸、漢防己甲素、黃芩苷和穿心蓮內(nèi)酯對斑馬魚具有促進血管新生作用;葛根素、柚皮素、鞣花酸、大黃素和苦參堿對血管新生無明顯影響。

Objective The effects of rutin, glycyrrhizic acid, tetrandrine, puerarin, naringin, ellagic acid, baicalin, emodin, androgynolide and matrine on angiogenesis were studied by transgenic fluorescein vascular zebrafish. Methods A total of 3 480 zebrafish of 48 hpf transgenic fluorescein FlI-1 strain were selected and placed in six well plates, with 30 fish in each well (experimental group). Rutin of 81.90—1 637.95 μmol·L^-1, glycyrrhizic acid of 60.76—1 215.17 μmol·L^-1, tetrandrine of 5.02— 80.29 μmol·L^-1, puerarin of 120.08—2 161.49 μmol·L^-1, naringin of 3.67—734.59 μmol·L^-1, ellagic acid of 0.21—3.31 μmol·L^-1, baicalin of 28.00—448.07 μmol·L^-1, emodin of 0.19—740.08 μmol·L^-1, androgynolide of 35.67—570.69 μmol·L^-1, matrine 50.33— 805.28 μmol·L^-1 were given respectively in aqueous solution, and the control group (zebrafish treated with fish culture water) were set up at the same time. After 24 h of treatment, the toxic phenotype and death of zebrafish were observed and recorded. The maximum tolerance concentration (MTC) of zebrafish was determined. Zebrafish were co-cultured with MTC of 10 compounds for 24 h, and the area and sprouted number of subintestinal vessels were detected. Results Compared with control group, the rutin group, the glycyrrhetinic acid group (P<0.05), the hanfangenin group (P<0.001), the gerberoside group, the naringenin group, the baicalin group (P<0.01), the andrographolide group (P<0.001), and the bitter ginseng base group had higher subintestinal area pixels in zebrafish, ellagic acid group, rhodopsin group zebrafish subintestinal vascular area pixel was lower. The number of subintestinal vascular outgrowths in zebrafish was more in the rutin group (P<0.05), hanfanganin, geranium group, naringenin group, ellagic acid group, and bitter ginseng base group than that in control group. The number of subintestinal vascular sprouts in zebrafish of glycyrrhizic acid group, baicalin group, and andrographolide group was less than that in the normal control group, and the number of subintestinal vascular sprouts in the rutin group was comparable to that in the rhodopsin group. Conclusion Rutin, glycyrrhizic acid, hanfenacin, baicalin, and andrographolide had pro-angiogenic effects on zebrafish. Puerarin, naringenin, ellagic acid, rhodopsin and bitter ginseng alkaloids had no significant effect on angiogenesis.

R285.5

重大新藥創(chuàng)制科技重大專項(2017ZX09304003)