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Volume 38 Issue 3
May  2020
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Article Navigation >  Journal of Pharmaceutical Practice and Service  > 2020  >  38(3): 232-236

WU Qiuling, CHENG Yanqiong, LIU Aijun, ZHANG Weidong. The effect of four compounds of astragalus isoflavones on the differentiation of PC 12 cells[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(3): 232-236. doi: 10.12206/j.issn.1006-0111.202001045
Citation: WU Qiuling, CHENG Yanqiong, LIU Aijun, ZHANG Weidong. The effect of four compounds of astragalus isoflavones on the differentiation of PC 12 cells[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(3): 232-236. doi: 10.12206/j.issn.1006-0111.202001045
shu

The effect of four compounds of astragalus isoflavones on the differentiation of PC 12 cells

doi: 10.12206/j.issn.1006-0111.202001045
  • 1.

    School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China

  • 2.

    Department of Pharmacology, School of Pharmacy, Naval Medical University, Shanghai 200433, China

  • 3.

    Department of Natural Medicinal Chemistry, School of Pharmacy, Naval Medical University, Shanghai 200433, China

  • Received Date: 2020-01-10
  • Rev Recd Date: 2020-03-06
    • Available Online: 2020-05-20
  • Publish Date: 2020-05-01
  •   Objective  To investigate the effects of calycosin, formononetin, calycosin-7-glucoside and ononin on PC 12 cells differentiation.  Methods  PC 12 cells were cultured and treated with different concentrations of nerve growth factor (NGF), calycosin, formononetin, calycosin-7-glucoside and ononin for 5 days, once a day, 3 times in a row. The neurite outgrowth of PC 12 cells was observed and the expression of β III-tubulin were measured by immunofluorescence.  Results  Compared with the vehicle group, neurite outgrowth and the expression of β III tubulin in PC 12 cells had not promoted by calycosin, formononetin, calycosin-7-glucoside and ononin (0.01-10.00 μmol/L).  Conclusion  PC 12 cells differentiation could not be induced by calycosin, formononetin, calycosin-7-glucoside and ononin.
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    [2] 彭敏, 操瑶. 中西医结合治疗气虚血瘀型脑中风后遗症的临床效果观察[J]. 临床合理用药杂志, 2019, 12(23):44-45.
    [3] 劳祎林, 林吴, 媛伍, 等. 中医药疗法应用于脑梗死恢复期的研究进展[J]. 广西医学, 2019, 41(16):2102-2104.
    [4] 江泽珍. 生生不息的神经元[J]. 世界科学, 2019(8):22-23. doi:  10.3969/j.issn.1000-0968.2019.08.008
    [5] 陈国辉, 黄文凤. 黄芪的化学成分及药理作用研究进展[J]. 中国新药杂志, 2008, 17(17):1482-1485. doi:  10.3321/j.issn:1003-3734.2008.17.006
    [6] 郭超, 卞涛, 卜伟, 等. 毛蕊异黄酮通过抑制calpain-1的表达发挥抗脑缺血再灌注损伤的研究[J]. 现代生物医学进展, 2019, 19(4):631-635.
    [7] LI Z Z, DONG X H, ZHANG J F, et al. Formononetin protects TBI rats against neurological lesions and the underlying mechanism[J]. J Neurol Sci,2014,338(1-2):112-117. doi:  10.1016/j.jns.2013.12.027
    [8] REN M, WANG X D, DU G Q, et al. Calycosin-7-O-β-D-glucoside attenuates ischemia-reperfusion injury in vivo via activation of the PI3K/Akt pathway[J]. Mol Med Rep,2016,13(1):633-640. doi:  10.3892/mmr.2015.4611
    [9] 于玲, 王知斌, 王秋红, 等. 黄芪中黄酮类化合物药理作用研究进展[J]. 中医药信息, 2018, 35(2):104-108.
    [10] ALOE L, ROCCO M L, BALZAMINO B O, et al. Nerve growth factor: a focus on neuroscience and therapy[J]. Curr Neuropharmacol,2015,13(3):294-303. doi:  10.2174/1570159X13666150403231920
    [11] CUI W, CUI G Z, LI W M, et al. Bis(12)-hupyridone, a novel multifunctional dimer, promotes neuronal differentiation more potently than its monomeric natural analog huperzine A possibly through α7 nAChR[J]. Brain Res,2011,1401:10-17. doi:  10.1016/j.brainres.2011.05.042
    [12] WANG Y, REN Q Y, ZHANG X, et al. Neuroprotective mechanisms of calycosin against focal cerebral ischemia and reperfusion injury in rats[J]. Cell Physiol Biochem,2018,45(2):537-546. doi:  10.1159/000487031
    [13] WANG Y, DONG X H, LI Z Z, et al. Downregulated RASD1 and upregulated miR-375 are involved in protective effects of calycosin on cerebral ischemia/reperfusion rats[J]. J Neurol Sci,2014,339(1-2):144-148. doi:  10.1016/j.jns.2014.02.002
    [14] SONG L, LI X P, BAI X X, et al. Calycosin improves cognitive function in a transgenic mouse model of Alzheimer's disease by activating the protein kinase C pathway[J]. Neural Regen Res,2017,12(11):1870-1876. doi:  10.4103/1673-5374.219049
    [15] LIANG K, YE Y, WANG Y, et al. Formononetin mediates neuroprotection against cerebral ischemia/reperfusion in rats via downregulation of the Bax/Bcl-2 ratio and upregulation PI3K/Akt signaling pathway[J]. J Neurol Sci,2014,344(1-2):100-104. doi:  10.1016/j.jns.2014.06.033
    [16] RAO S N, PEARSE D D. Regulating axonal responses to injury: the intersection between signaling pathways involved in axon myelination and the inhibition of axon regeneration[J]. Front Mol Neurosci,2016,9:33.
    [17] MOSKOWITZ P F, OBLINGER M M. Sensory neurons selectively upregulate synthesis and transport of the beta III-tubulin protein during axonal regeneration[J]. J Neurosci,1995,15(2):1545-1555. doi:  10.1523/JNEUROSCI.15-02-01545.1995
    [18] SQUIZZATO A, BELLESINI M, TAKEDA A, et al. Clopidogrel plus aspirin versus aspirin alone for preventing cardiovascular events[J]. Cochrane Database Syst Rev,2017,12:CD005158.
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The effect of four compounds of astragalus isoflavones on the differentiation of PC 12 cells

doi: 10.12206/j.issn.1006-0111.202001045
  • 1. School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China
  • 2. Department of Pharmacology, School of Pharmacy, Naval Medical University, Shanghai 200433, China
  • 3. Department of Natural Medicinal Chemistry, School of Pharmacy, Naval Medical University, Shanghai 200433, China
  • Received Date: 2020-01-10
  • Rev Recd Date: 2020-03-06
  • Available Online: 2020-05-20
  • Publish Date: 2020-05-01

Abstract:   Objective  To investigate the effects of calycosin, formononetin, calycosin-7-glucoside and ononin on PC 12 cells differentiation.  Methods  PC 12 cells were cultured and treated with different concentrations of nerve growth factor (NGF), calycosin, formononetin, calycosin-7-glucoside and ononin for 5 days, once a day, 3 times in a row. The neurite outgrowth of PC 12 cells was observed and the expression of β III-tubulin were measured by immunofluorescence.  Results  Compared with the vehicle group, neurite outgrowth and the expression of β III tubulin in PC 12 cells had not promoted by calycosin, formononetin, calycosin-7-glucoside and ononin (0.01-10.00 μmol/L).  Conclusion  PC 12 cells differentiation could not be induced by calycosin, formononetin, calycosin-7-glucoside and ononin.

WU Qiuling, CHENG Yanqiong, LIU Aijun, ZHANG Weidong. The effect of four compounds of astragalus isoflavones on the differentiation of PC 12 cells[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(3): 232-236. doi: 10.12206/j.issn.1006-0111.202001045
Citation: WU Qiuling, CHENG Yanqiong, LIU Aijun, ZHANG Weidong. The effect of four compounds of astragalus isoflavones on the differentiation of PC 12 cells[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(3): 232-236. doi: 10.12206/j.issn.1006-0111.202001045
shu

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  • 脑中风是由于脑部血管突然破裂或因血管阻塞导致血液不能流入大脑而引起脑组织损伤的一类疾病[1]。大多数患者在治疗后会遗留不同程度的神经功能缺损，比如偏瘫、嘴歪眼斜、智力障碍、语言认知功能丧失等[2]。因而开发神经恢复剂，修复损伤神经功能尤为重要。修复受损神经功能的途径之一是促进被破坏或受损害的神经重塑[3]。神经重塑通过诱导神经元分化，促进突起向外生长，与其他神经元建立连接，重构皮层，从而修复损伤的神经功能[4]。

    黄芪异黄酮类化合物主要包括毛蕊异黄酮、刺芒柄花素、毛蕊异黄酮苷、芒柄花苷，结构如图1所示[5]。毛蕊异黄酮、刺芒柄花素、毛蕊异黄酮苷、芒柄花苷对急性脑缺血具有神经保护作用，对缺血后脑损伤神经功能的恢复作用未见报道[6-8]。

    PC 12细胞是来源于成年大鼠肾上腺髓质嗜铬细胞瘤的细胞系，培养PC 12细胞可动态观察其神经分化的过程[9]，因此可将PC 12细胞作为筛选促神经分化、恢复损伤神经功能药物的理想模型。本项研究工作使用PC 12细胞，观察毛蕊异黄酮、刺芒柄花素、毛蕊异黄酮苷、芒柄花苷是否具有诱导细胞神经分化的能力。神经生长因子(nerve growth factor, NGF)具有明确的促进神经元分化作用[10]，在本研究中用作阳性对照。

1.   材料与方法

    1.1.   试剂与仪器

  • 毛蕊异黄酮、毛蕊异黄酮苷、芒柄花苷（纯度≥98%，上海源叶生物科技有限公司）；刺芒柄花素（纯度≥98%，成都普瑞法科技开发有限公司）；二甲基亚砜(dimethyl sulfoxide，DMSO，Sigma公司）；多聚-L-赖氨酸（Biosharp公司）；胎牛血清（ScienCell公司）；特级马血清（索莱宝公司）；丙酮酸钠和RPMI 1640培养基（Gibco公司）；磷酸盐缓冲液（博光公司）；4',6-二脒基-2-苯基吲哚（DAPI）和Triton X-100（碧云天）；4%多聚甲醛（武汉赛维尔生物科技有限公司）；重组大鼠β-神经生长因子（NGF，R & D公司）；β微管蛋白（β III-tubulin，Abcam公司）；Alexa Fluor TM 568兔抗小鼠IgG(H+L)（Invitrogen公司）。

    6孔板培养皿和10 ml培养皿（美国Corning公司）；倒置荧光显微镜 (DMI3000 B，德国Leica公司）；精密电子天平（美国Ohaus公司）。

    • 1.2.   PC 12细胞培养

  • PC 12细胞购自上海中国科学院细胞库，经复苏后，加入含10%马血清、5%胎牛血清、1%丙酮酸钠、100 U/ml青霉素和100 μg/ml链霉素的RPMI 1640培养基，放置于培养箱（37 ℃，95%空气，5%CO2）中传代培养，每2 d传代1次。

    • 1.3.   NGF及黄芪异黄酮类化合物对PC 12细胞突起向外生长的影响

  • 将PC 12细胞以5×104 /ml的密度涂覆在多聚-L-赖氨酸包被过的6孔板中，放置于培养箱中孵育过夜。24 h后，换分化培养基（含1%马血清、1%胎牛血清、1%丙酮酸钠、100 U/ml青霉素和100 μg/ml链霉素），加不同浓度NGF（0.3~100.0 ng/ml）、毛蕊异黄酮、刺芒柄花素、毛蕊异黄酮苷、芒柄花苷（0.01~10.00 μmol/L）继续培养。每天更换一次上述化合物和分化培养基，连续3 d。培养5 d后在显微镜下观察各组PC 12细胞突起向外生长的情况。将长度超过一个细胞体的神经突记为阳性细胞[11]。在每孔中随机选择5个视野，分别计算神经突细胞的百分比，然后计算平均值。

    • 1.4.   NGF及黄芪异黄酮类化合物对PC 12细胞β III-tubulin蛋白表达的影响

  • PC 12细胞处理方式同“1.3”项，培养5 d后用免疫荧光染色方法检测β III-tubulin蛋白的表达。各组PC 12细胞经4%多聚甲醛固定20 min、Triton X-100透化15 min、5%脱脂牛奶封闭1 h、一抗β III-tubulin（1:100）4 ℃孵育过夜、二抗（1:500）室温孵育1 h、DAPI室温染色20 min后在荧光显微镜下观察β III-tubulin和DAPI的表达情况。在每孔中随机选择5个视野，分别统计β III-tubulin表达的阳性细胞数目，然后计算平均值。

    • 1.5.   数据分析

  • 采用Graphpad软件分析，数据采用（均值±标准误）表示，组间数据采用方差分析，以P＜0.05表示有显著性差异，P＜0.01表示有极显著性差异。

2.   结果

    2.1.   不同浓度NGF对PC 12细胞突起向外生长的影响

  • 如图2所示，与溶媒组相比，0.3 ng/ml NGF对PC 12细胞突起向外生长无促进作用。与溶媒组相比，NGF（1~100 ng/ml）显著促进PC 12细胞突起向外生长，且具有浓度依赖性。

    • 2.2.   不同浓度及黄芪异黄酮类化合物对PC 12细胞突起向外生长的影响

  • 如图3所示，与溶媒组相比，10 ng/ml NGF能显著促进PC 12细胞突起向外生长。与溶媒组相比，毛蕊异黄酮、刺芒柄花素、毛蕊异黄酮苷、芒柄花苷（0.01~10 μmol/L）对PC 12细胞突起向外生长无促进作用。

    • 2.3.   不同浓度NGF对PC 12细胞中β III-tubulin蛋白表达的影响

  • 如图4所示，与溶媒组相比，0.3 ng/ml NGF对PC 12细胞中β III-tubulin蛋白表达无影响。与溶媒组相比，NGF（1~100 ng/ml）显著促进PC 12细胞中β III-tubulin蛋白的表达，且具有浓度依赖性。

    • 2.4.   不同浓度黄芪异黄酮类化合物对PC 12细胞β III-tubulin蛋白表达的影响

  • 如图5所示，与溶媒组相比，10 ng/ml NGF能显著增加PC 12细胞中β III-tubulin蛋白的表达。与溶媒组相比，毛蕊异黄酮、刺芒柄花素、毛蕊异黄酮苷、芒柄花苷（0.01~10.00 μmol/L）对PC 12细胞中β III-tubulin蛋白表达无影响。

3.   讨论

  • 已有文献报道毛蕊异黄酮、刺芒柄花素、毛蕊异黄酮苷和芒柄花苷对急性脑缺血具有神经保护作用，通过抗自噬、抗凋亡、抗炎等发挥作用[7-8, 12-15]。毛蕊异黄酮、刺芒柄花素、毛蕊异黄酮苷和芒柄花苷对神经元分化、缺血后脑损伤神经功能的作用很少提及。本研究初步探讨了毛蕊异黄酮、刺芒柄花素、毛蕊异黄酮苷和芒柄花苷对PC 12细胞神经分化的影响，发现其未能诱导PC 12细胞分化。

    在特定因素刺激下，神经元的突起向外生长，促进受损神经元的分化，重新建立神经网络，恢复受损的神经功能[16]。β III-tubulin在微管元件中只表示神经元，是神经细胞分化的表型标志物，增加其表达可改变神经元骨架，促进突起向外生长[17-18]。在本研究中，我们主要以PC 12细胞突起向外生长和β III-tubulin蛋白表达作为主要的分化指标。

    在本研究中，用NGF（阳性对照）、毛蕊异黄酮、刺芒柄花素、毛蕊异黄酮苷、芒柄花苷分别刺激PC 12细胞，观察药物对细胞突向外生长的影响。利用免疫荧光染色来观察药物对PC 12细胞β III-tubulin蛋白表达的影响。通过以上两方面的初步研究，我们发现毛蕊异黄酮、刺芒柄花素、毛蕊异黄酮苷、芒柄花苷均没有促进PC 12细胞突起向外生长和β III-tubulin蛋白表达。

    综上得出结论：毛蕊异黄酮、刺芒柄花素、毛蕊异黄酮苷、芒柄花苷对PC 12细胞分化无促进作用。

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