Protective and regeneration-promoting effects of Ruyi Zhenbao Pill on nerve injury in zebrafish

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1 Drug Evaluation Research µg/ml NIDS-Element s µg/ml 2% 24% 50% P % 29% 48% P % P % P % P % P % P % P R965 A (2017) DOI: /j.issn 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 , China 2. Shandong ARURA Pharmaceutical R&D Co., Ltd, Jinan , 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 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 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 C jackli@ zhunter.com

2 308 Drug Evaluation Research [1] [2] [3] [1] [4-5] [6-7] [8] [9] 2 3 d [10] [2] 4 d 4 dpf [11] [12-14] g/ T3/T G SZX7 OLYMPUS Japan AZ100 Nikon 6 Nest Biotech Aladdin Shanghai China 1.3 AB 1 2 dpf 1 dpf SYXK L 200 mg μs/cm ph mg/l CaCO 3 AAALAC [14-15] NOAEL 1 dpf AB 0.25 μmol/l 24 h µg/ml h dpf AB 0.25 μmol/l 24 h 500 μmol/l µg/ml 30 AO 15 NIDS-Element s S

3 Drug Evaluation Research S S / S S dpf AB 1.5% 48 h T3/T4 T3/T μmol/l µg/ml h Axon 15 NIDS-Element s S S S / S S dpf 1.5% 48 h T3/T4 T3/T μmol/l µg/ml 24 h NIDS-Element s L /L dpf AB 75 μmol/l 2 dpf AB 96 h T3/T4 T3/T μmol/l µg/ml h 15 NIDS-Element s S S S / S S 2.6 x ± s GraphPad 5.0 Dunnett s-t NOAEL 1 OriginPro MNLC 444 μg/ml 444 μg/ml NOAEL 100 μg/ml μg/ml Table 1 Concentration-mortality data of RZP /(μg ml 1 ) / /(μg ml 1 ) 1 - Fig. 1 Concentration-mortality curve of RZP μg/ml 2% 24% 50% P μg/ml 97% P µg/ml 3% 29% 48% P µg/ml

4 310 Drug Evaluation Research /(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 ) / ±3 028 *** ± ±5 155 *** ± ± ±5 839 *** 50 *** P *** P < vs model group 100µg/mL T3/T4 66% P µg/ml 44% P % P % P T3/T4 46% P /(μg ml 1 ) 3 Fig. 3 Protective effect of RZP on axon injury µg/ml 36% P % P % P T3/T4

5 Drug Evaluation Research n 15 Table 3 Protective effect of RZP on axon injury (n 15) / ±2 433 *** ±1 919 T3/T μmol L ±2 680 ** μg ml ± μg ml ± μg ml ±2 164 * 48 * P 0.05 ** P 0.01 * P < 0.05 ** P < 0.01 vs model group 47% P 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/T μmol L 1 284±5.74 * μg ml 1 283±5.01 * μg ml 1 286±4.42 ** μg ml 1 306±3.87 *** 93 * P 0.05 ** P 0.01 *** P * P < 0.05 ** P < 0.01 *** P < vs model group [18-21] MBP [22] [23-24] [25] [26] [27] [28]

6 312 Drug Evaluation Research /(μg ml 1 ) 5 Fig. 5 Promotion effect of RZP on remyelination 5 n 15 Table 5 Promotion effect of RZP on remyelination (n 15) / ± *** ±3 160 T3/T μmol L ±5 643 *** μg ml ±7 317 ** μg ml ±9 140 *** μg ml ±3 921 *** 41 ** P 0.01 ** P * P < 0.01 ** P < vs model control group [29] [30] 50% 48% 93% 41% NGFBDNF [13] [1]. [J]., 2014, 35(4): [2]. [J]., 2007, 6(2): [3],,. [J]., 2007, 20(12): [4],. [J]., 2014, 20(6): [5]. [J]., 2009, 36(2): [6],,,.

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