Abstract：Baicalin is a kind of flavonoid, can promotes posttransplant fetal growth and development, and hasn't toxicity to the fetus development. The objectives of the present study were to investigate the effects of baicalin on the development of mouse (Mus musculus) embryos in vitro and it's possible molecular mechanism. Mouse embryos cultured in vitro were randomly divided into control group(CZB cultural medium) and experimental group (supplemented with 4 μg/mL of baicalin). The development rates of embryos in their different stages were recorded, respectively, using a stereoscopic microscope, and also the nuclei of blastocysts were stained with orcein for cell number counting. The expressions of gap junction protein alpha-1(GJA1), heat Shock 70 (Hsp70), B-cellymphoma/leukemia-2(Bcl-2) and Bcl-2 associated X protein (Bax) mRNA were detected by using qRT-PCR. The results showed that baicalin treatment, as compared to control group, significantly improved (P＜0.05) 2-cell and 4-cell embryonic cleavage rates, morula, blastocyst and expanded blastocyst development rates and blastocyst hatching rates, and also significantly (P＜0.05) improved the cell number of expanded and hatched blastocysts. In addition, the expression of GJA1 mRNA was significantly increased (P＜0.05), and the expressions of Hsp70 and Bax mRNA were significantly decreased (P＜0.05), and also the ratio between Bax/Bcl-2 was significantly reduced (P＜0.05). We concluded that baicalin improves the blastocyst development rates and the quality of blastocysts of mouse embryo in vitro, probably, by upregulating GJA1 mRNA expression and downregulating Hsp70 and Bax mRNA expressions. The study reveals the possible mechanism of action that the baicalin promotes embryonic development, and provides basic data for the study of baicalin tocolysis role.
陈健伟, 国晓瞳, 田文儒. 灯盏花乙素(Scu)对猪肾小管上皮细胞(LLC-PK1)细胞热休克蛋白72(HSP72)及凋亡相关基因表达的影响[J]. 农业生物技术学报, 2014, 22(10): 1261-1268.（CHEN Jian-Wei, GUO Xiao-Tong, TIAN Wen-Ru. Expression of Heat Shock Protein 72 (HSP72) and Apoptosis Genes in Pig Kidney Proximal Tubular (LLC-PK1) Cells Treated with Scutellarin[J]. Journal of Agricultural Biotechnology, 2014, 22(10): 1261-1268.）（doi: 10.3969/j.issn.1674-7968.2014.10.009）傅文栋. 小檗碱、黄芩苷、黄芪多糖对小鼠2-细胞胚胎体外培养及移植效果的研究[D]. 新疆农业大学,2005.高建明, 孙玉成, 苏辉等. 黄芩苷、川芎嗪对小鼠体外培养胚胎冷冻-解冻及移植效果的研究[J]. 畜牧兽医学报, 2007, 10:1120-1125.（GAO Jian-ming, S UN Yu-cheng,MU Xiang et al.Study on Effects of Baicalin and Ligustrazine on Early Embryos in vitro Culture and the Froze Embryos Transfer in Mice[J]. Acta Veterinaria et Zootechnica Sinica.2007, 10:1120-1125）（doi:0366-6964(2007)10-1120-06）Hribal R, K Jewgenow, BC Braun and P Comizzoli. Influence of Culture Medium Composition on Relative mRNA Abundances in Domestic Cat Embryos[J]. Reprod Dom Anim, 2013, 48, 245–251. （doi:10.1111/j.1439-0531.2012.02139.x）Kampinga HH, Craig EA. The HSP70 chaperone machinery: J proteins as drivers of functional specificity[J]. Nat Rev Mol Cell Biol, 2010, 11: 579–592. Lonergan P, T. Fair,D. Corcoran, A.C.O. Evants. Effect of culture environment on gene expression and developmental characteristics in IVF-derived embryos[J]. Theriogenology, 2006, Jan, 7;65(1):137-52.(doi:10.1016/j.theriogenology.2005.09.028)Niemann H, Wrenzycki. Alterations of expression of development entally important genes in preimplantation bovine embryos by in vitro culture conditions:implications for subsequent development. Theriogenology, 2000, Jan 1; 53(1):21-34.Pomar FJ, Teerds KJ, Kidson A, Colenbrander B, Tharasanit T, Aguilar B, Roelen BA. Differences in the incidence of apoptosis between in vivo and in vitro produced blastocysts of farm animal species: a comparative study. Theriogenology, 2005, 63: 2254–2268.（DOI:?http://dx.doi.org/10.1016/j）Romy Hribal, Alexandra Hachen et al.The influence of recombinant feline oviductin on different aspects of domestic cat (Felis catus) IVF and embryo quality[J].Theriogenology, 2014 Sep 15; 82(5):742-9. (doi: 10.1016/j.theriogenology)宋殿荣, 郭洁, 王跃飞等. 双黄连冻干粉透过妊娠大鼠胎盘屏障的药物成分研究[J]. 中国中药杂志. 2010, 35( 12) : 1626-1629.（Song DR, Guo J, Wang YF et al. Ingredients of Shuanghuanglian injection powder permeation through placental barrier of rat in pregnancy[J]. China J Chin Mater Med, 2010, 35( 12) : 1626-1629.(doi:10.4268 /cjcmm20101227)孙春玲, 国晓瞳, 赵园, 陈健伟等. 黄芩苷对热应激条件下猪近端肾小管(LLC-PK1)细胞凋亡率及B细胞淋巴瘤-2基因(Bcl-2)和Bcl-2相关X蛋白基因(Bax)表达的影响[J]. 农业生物技术学报. 2014, 22(12): 1553~1560.（SUN Chun-Ling, GUO Xiao-Tong, ZHAO Yuan, CHEN Jian-Wei et al.Effects of Baicalin on Expression of B-cellymphoma-2 (Bcl-2) and Bcl-2 Assaciated X Protein Gene (Bax) and Apoptosis Rates of Pig Kidney Proximal Tubular (LLC-PK1) Cells Subjected to Heat Stress[J]. Journal of Agricultural Biotechnology 2014, 22(12): 1553~1560.(doi: 10.3969/j.issn.1674-7968.2014.12.010)Tesfaye, D, P Lonergan, M. Hoelker et al. Suppression of connexin 43 and E-cadherin transcripts in in vitro derived bovine embryos following culture in vitro or in vivo in the homologous bovine oviduct[J]. Mol. Reprod. 2007, Dev. 74:978–988.(DOI:?10.1002/mrd.20678）Thanida Sananmuang, Nawapen Phutikanit et al. In Vitro Culture of Feline Embryos Increases Stress-induced Heat Shock Protein 70 and Apoptotic Related Genes[J]. Journal of Reproduction and Development, 2013; 59(2):180-8.（doi:org/10.1262/jrd.2012-116）WANG Ming-zhi, TIAN Wen-ru, JIANG Tong-quan, REN Deng-liang, TIAN Zhong-jie, QU Ping-ping. Effect of RNA Interference Hsp72 Gene Expression on Development of Mouse preimplantation Embryos[J]. Agricultural Sciences in China. 2009, 8(2):223-230.Waurich R, J Ringleb, B C Braun and K Jewgenow. Embryonic gene activation in in vitro produced embryos of the domestic cat (Felis catus)[J]. Reproduction, 2010,140, 531–540. (doi: 10.1530/REP-10-0298)Wrenzycki C et al. Analyse der Genexpressionsmuster zur Beurteilung der Embryonenqualitat[J]. Reproduktionsmed Endokrinol, 2007, 4, 234–239.Zhong Jian Bao, Shuan Zhao, Ihtesham UI Haq and Shen-Ming Zeng. Recombinant bovine interferon-τ enhances in vitro development of bovine embryos by upregulating expression of connexin 43 and E-cadherin[J]. J Dairy Sci, 2014 Nov; 97(11):6917-25.(doi: 10.3168/jds.2014-8106)
