Drug Evaluation Research 40 3 2017 3 307 1 2 2 2 1 1 1 1. 310051 2. 250101 10.0 33.3 100.0 µg/ml NIDS-Element s 10.0 33.3 100.0 µg/ml 2% 24% 50% P 0.001 3% 29% 48% P 0.05 44% P 0.05 49% P 0.01 93% P 0.001 36% P 0.01 37% P 0.001 41% P 0.001 R965 A 1674-6376(2017)03-0307 - 07 DOI: 10.7501/j.issn.1674-6376.2017.03.004 Protective and regeneration-promoting effects of Ruyi Zhenbao Pill on nerve injury in zebrafish ZHU Xiao-yu 1, WANG Hong-yue 2, LI Huai-ping 2, WANG Hai-ping 2, LAN Xiao-hong 1, SONG Ru-shun 1, LI Chun-qi 1 1. Hangzhou Hunter Biotechnology Co., Ltd., Hangzhou 310051, China 2. Shandong ARURA Pharmaceutical R&D Co., Ltd, Jinan 250101, China Abstract: Objective To observe the protective and regeneration-promoting effects of Ruyi Zhenbao Pill (RZP) on nerve injury in zebrafish. Methods The zebrafish model of central nervous injury was induced by mycophenolate mofetil, the model of peripheral motor nerve and axonal injury was induced by ethanol, and the model of myelin damage was induced by ethidium bromide. The variations of central nerve, axon and myelin sheath fluorescence intensity, and peripheral motor nerve length in zebrafish, which exposed to the different concentration (10.0, 33.3, and 100.0 µg/ml) of RZP, were observed with fluorescence microscope. The effective protection rates of RZP on zebrafish central nerve and axone, and the regeneration-promoting effect on peripheral motor nerve and myelin sheath were analyzed and calculated with the image processing software NIDS-Element s. Results In 10.0, 33.3, and 100.0 µg/ml RZP groups, the zebrafish central nervous injury protective rates were 2%, 24%, and 50% (P < 0.001), respectively, the peripheral nerve regeneration promoting rates were 44% (P < 0.05), 49% (P < 0.01), and 93% (P < 0.001), the axonal injury recovery rates were 3%, 29% and 48% (P < 0.05), and the myelin sheath regeneration promoting rates were 36% (P < 0.01), 37% (P < 0.001), and 41% (P < 0.001), compared with model group. Conclusions RZP could not only protect the central nervous and axonal injury, but also promote the regeneration of peripheral nerve and myelin sheath in zebrafish. Key words: Ruyi Zhenbao Pill; nerve injury protection; zebrafish; regeneration; NOAEL; central nerve; axone; myelin sheath 2016-10-08 2014C03009 1985 E-mail: jackli@ zhunter.com
308 Drug Evaluation Research 40 3 2017 3 [1] [2] [3] [1] [4-5] [6-7] [8] [9] 2 3 d [10] [2] 4 d 4 dpf [11] [12-14] 1 1.1 0.5 g/ 20150129 64-17-5 128794-94-5 70-18-8 T3/T4 55-03-8 G1220028 1.2 SZX7 OLYMPUS Japan AZ100 Nikon 6 Nest Biotech Aladdin Shanghai China 1.3 AB 1 2 dpf 1 dpf SYXK 2012-0171 28 1 L 200 mg 480 510 μs/cm ph 6.9 7.2 53.7 71.6 mg/l CaCO 3 AAALAC [14-15] 2 2.1 NOAEL 1 dpf AB 0.25 μmol/l 24 h 100 250 500 1 000 µg/ml 30 24 h 2.2 1 dpf AB 0.25 μmol/l 24 h 500 μmol/l 10.0 33.3 100.0 µg/ml 30 AO 15 NIDS-Element s S
Drug Evaluation Research 40 3 2017 3 309 S S / S S 2.3 1 dpf AB 1.5% 48 h T3/T4 T3/T4 0.04 μmol/l 10.0 33.3 100.0 µg/ml 30 48 h Axon 15 NIDS-Element s S S S / S S 2.4 1 dpf 1.5% 48 h T3/T4 T3/T4 0.04 μmol/l 10.0 33.3 100.0 µg/ml 24 h 30 15 NIDS-Element s L /L 2.5 2 dpf AB 75 μmol/l 2 dpf AB 96 h T3/T4 T3/T4 0.04 μmol/l 10.0 33.3 100.0 µg/ml 30 96 h 15 NIDS-Element s S S S / S S 2.6 x ± s GraphPad 5.0 Dunnett s-t 3 3.1 NOAEL 1 OriginPro 8.0-1 MNLC 444 μg/ml 444 μg/ml NOAEL 100 μg/ml 10.0 33.3 100.0 μg/ml 3 1 - Table 1 Concentration-mortality data of RZP /(μg ml 1 ) / 100 0 0 250 0 0 500 0 0 1 000 15 50.0 1 500 28 93.3 2 000 30 100.0 100 80 60 40 20 0 0 500 1 000 1 500 2 000 /(μg ml 1 ) 1 - Fig. 1 Concentration-mortality curve of RZP 3.2 10.0 33.3 100.0 μg/ml 2% 24% 50% P 0.001 100 μg/ml 97% P 0.001 2 2 3.3 10.0 33.3 100.0 µg/ml 3% 29% 48% P 0.05 100 µg/ml
310 Drug Evaluation Research 40 3 2017 3 /(μg ml 1 ) 2 Fig. 2 Protective effect of RZP on central nervous system injury 2 n 15 Table 2 Protective effect of RZP on central nervous system injury (n 15) / (μg ml 1 ) / 88 932±3 028 *** 21 0861±11 873 500 93 161±5 155 *** 97 10.0 213 564±14 359 2 33.3 181 481±12 054 24 100.0 150 375±5 839 *** 50 *** P 0.001 *** P < 0.001 vs model group 100µg/mL T3/T4 66% P 0.01 3 3 3.4 10.0 33.3 100.0 µg/ml 44% P 0.05 49% P 0.01 93% P 0.001 T3/T4 46% P 0.01 4 4 /(μg ml 1 ) 3 Fig. 3 Protective effect of RZP on axon injury 3.5 10.0 33.3 100.0 µg/ml 36% P 0.01 37% P 0.001 41% P 0.001 T3/T4
Drug Evaluation Research 40 3 2017 3 311 3 n 15 Table 3 Protective effect of RZP on axon injury (n 15) / 78 136±2 433 *** 61 509±1 919 T3/T4 0.04 μmol L 1 73 734±2 680 ** 66 10.0 μg ml 1 64 442±2 310 3 33.3 μg ml 1 67 791±1 652 29 100.0 μg ml 1 70 328±2 164 * 48 * P 0.05 ** P 0.01 * P < 0.05 ** P < 0.01 vs model group 47% P 0.001 5 5 4 NRF [15] [16] EGb50 [17] bfgf NGF AKT/PKB VEGF Flk-1 /(μg ml 1 ) 4 Fig. 4 Promotion effect of RZP on peripheral motor neuron regeneration 4 n 15 Table 4 Promotion effect of RZP on peripheral motor neuron regeneration (n 15) / 309±4.40 *** 263±4.78 T3/T4 0.04 μmol L 1 284±5.74 * 46 10.0 μg ml 1 283±5.01 * 44 33.3 μg ml 1 286±4.42 ** 49 100.0 μg ml 1 306±3.87 *** 93 * P 0.05 ** P 0.01 *** P 0.001 * P < 0.05 ** P < 0.01 *** P < 0.001 vs model group [18-21] MBP [22] [23-24] [25] [26] [27] [28]
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