|
|
|
| Baicalin Affects Glial Cell Line-derived Neurotrophic Factor(GDNF) and Stem Cell Factor (SCF) Expression in Heat-stressed Calf (Bos taurus) Sertoli Cells |
| , , , , , , , , |
|
|
|
|
Abstract Baicalin has many beneficial effects clinically such as heat-clearing bacteriostasis and anti-inflammatory. Its effects on improving the heat resistance of bovine(Bos taurus) sertoli cells(SCs) were studied by measuring expressions of glial cell line-derived neurotrophic factor(GDNF) and stem cell factor(SCF) of SCs treated with different concentrations of baicalin. Collagenase and typsin were sequentially used to separate SCs from calf testis, and then the differential adhesion method was used to purify the SCs(4 h). The obtained SCs were cultured in vitro and their GDNF and SCF mRNA expressions and satellite karyosomes were identified by RT-PCR and Feulgen staining, respectively. The SCs of logarithmic phase growth were selected and divided into different groups including control group(37 ℃), 42 ℃ heat stress group and 42 ℃ heat stress plus baicalin(0.1, 1, 10 and 20 μg/mL) treated group. The cell survival rate was observed with methyl thiazoletetrazolium(MTT) assay. The Real-time quantitative(qRT-PCR) and enzyme linked immunosorbent assay(ELISA) results showed that both marker genes of GDNF and SCF and special satellite karyosomes in SCs were all detected. The cell survival rate(P＜0.01) and the expressions of GDNF and SCF mRNA(P＜0.01) and protein(P＜0.05) reduced in the SCs in 42 ℃ heat stress group compared with the control group(37 ℃). However, compared with 42 ℃ heat stress group, the cell survival rates in the heat stress group treated with 1 μg/mL(P＜0.05) and 10 μg/mL(P＜0.01) of baicalin increased; the expressions of GDNF and SCF mRNA in the 42 ℃ heat group treated with 0.1~20 μg/mL baicalin very significantly(P＜0.01) increased, and so did the protein expressions of SCF(P＜0.01) and GDNF(P＜0.05) with 1 μg/mL baicalin, respectively. The results indicated that baicalin increased SCs survival rate under heat stress and improved mRNA and protein expressions of both SCF and GDNF. The present research revealed some heat-resistance mechanism of baicalin on SCs under heat stress, which provides theoretical basis for improving the cell resistance to heat shock in practice.
|
|
Received: 04 November 2014
Published: 01 March 2015
|
|
|
|
| 参考文献杜亚伟．黄芩提取物解热作用及机制研究[J]. 医学动物防制, 2010, (5): 442-442．(Efffect of extracts from scutellaria baicalensis georg on antipyresis and influence on mechanism[J]. Chinese Archives of Traditional Chinese Medicine, 2010, (5): 442-442.) (doi: 10.3969 / j.issn. 1003 - 6245 .2010. 05 .022)何红梅, 王新华, 金东岭等．黄芩苷对肝细胞损伤保护作用的形态学观察[J]．中国现代医学杂志, 2012, 22(7): 30-33．(Protection of baicalin on pathomorphology of hepatic tissues in hepatocyte injury[J]. China Journal of Modern Medicine, 2012, 22(7): 30-33.) (doi 见图13)刘闯, 赵晓娥, 马保华等．新生犊牛睾丸支持细胞的高效分离培养与鉴定[J]．西北农林科技大学学报(自然科学版), 2010, 01:6-10+16．(Efficient separation, culture and identification of Seroli cells in new-born calf [J]. Journal of North west A & F University (Nat. Sci. Ed.) 2010, 01:6-10+16.) (doi 见图9）刘慧莲．人参皂甙单体Rb1和Rg1对小鼠睾丸支持细胞影响的比较[D]．山东师范大学, 2006．(Comparison of the Effect of Ginsenoside Rb1 and Ginsenoside Rg1 on the Cultured Immature Sertoli Cells[D]. Shandong Normal University, 2006) (doi 见图3)李倩楠, 葛晓群．黄芩苷的解热作用及对细胞因子的影响[J]．中国中药杂志, 2010, 35( 4) : 1068-1072．(Efffect of baicalin on antipyresis and influence on cytokine[J]. China Journal of Chinese Materia Medica, 2010, 35( 4): 1068-1072.) (doi: 10.4268/cjcmm20100829)毛定安, 杨于嘉, 俞燕, 等．核因子-kB活性在大鼠脑水肿的变化及热休克反应对其影响的研究[J]．中国当代儿科杂志, 1999, l(5): 277-280．(NF-κB activity and effects of heat shock response on brain tissue in rats with infection brain edema[J].CHINESE JOURNAL OF CONTEMPORARY PEDIATRICS,1999, l(5): 277-280.) (doi 见图8）马文智, 王燕蓉, 田见晖．热应激处理对精原细胞增殖的影响[J]．宁夏医科大学学报, 2011, 12: 1131-1134．(Effect of Heat Shock Treatment on Proliferation of Spermatogonia[J]. Journal of Ningxia Medical University, 2011, 12: 1131-1134.) (doi 见图5)孙洁, 田林红, 王收宝等．黄芩苷对糖尿病大鼠肾脏细胞凋亡及其Bcl-2和Bax表达的影响[J]．实用医学杂志, 2011, 27(5): 757-760．(Effects of baicalin on apoptosis and expressions of Bax and Bcl-2 in renal cell in diabetic rats[J]. The Journal of Practical Medicine, 2011, 27(5): 757-760.) (doi: 10.3969/j.issn.1006-5725.105.1)史琨, 张晓丽, 张海玉, 等．脂多糖所致小鼠睾丸支持细胞GDNF表达减少对精原干细胞的影响[J]．山东大学学报(医学版), 2013, 03: 32-41+47．(Expression of GDNF in LPS-treated mouse Sertoli cells and its effects on spermatogonial stem cells[J]. (Journal of Shandong University (Health Sciences), 2013, 03: 32-41+47. ) (doi:10.6040/j.issn.1671-7554.2013.03.007)徐珊, 孟庆刚．黄芩提取物解热作用及机制研究进展[J]．中华中医药学刊, 2008, 06: 1179-1181．(Advance of Research on Antipyretic Effect and Mechanism of Scutellaria[J]. CHINESE ARCHIVES OF TRADITIONAL CHINESE MEDICINE, 2008, 06: 1179-1181.) (doi: 10.13193/j.archtcm.2008.06.60.xush.057)于磊, 郑鹏, 荣恩光等．新生牛睾丸支持细胞的体外培养及鉴定分析[J]．吉林农业大学学报, 2013,35(1): 63-66．(Culture and Identification of Test is Sertoli Cells in Calf in vitro[J].Journal of Jilin Agricul tural University,2013,35(1): 63-66.)(doi 见图10）杨明, 秦应和．热应激小鼠体内睾酮浓度的变化及其与支持细胞HSPs表达的关系[J]．中国畜牧杂志, 2013, 21: 22-25．(Androgen Concentration Changes in Heat-stressed Mice and Its Effects on Expression of HSPs mRNA in Sertoli Cell[J]. Chinese Journal of Animal Science, 2013, 21: 22-25.) (doi 见图11）俞燕, 杨于嘉, 陶永光, 等．黄芩苷、地塞米松对大鼠感染性脑水肿细胞因子的影响[J]．湖南医科大学学报, 2000, 25(6): 519-521．(Effect of baicalin and dexamethasone on cytokines in brain tissue of infectious brain edema[J]. BULL HUNAN MED UNIV, 2000, 25(6): 519-521.)(doi 见图7)赵红艳．黄芩甙对发热大鼠下丘脑 PGE2 和 cAMP 含量的影响[J]．中国应用生理学杂志, 2002, 18(2) : 139-141．(Effects of Baicalin on Contents of PGE2 and cAMP in Hypothalamus Fever Rats[J]. Chinese Journalof Applied Physiology,2002, 18(2) : 139-141.) (doi 见图6)Calamari L, Abeni F, Calegari F, et al. Metabolic conditions of lactating Friesian cows dairy during the hot season in the Povalley.2.Blood Minerals and Acid- base Chemistry[J]. International Journal of Biometeorology, 52(2): 97-107. (doi:10.1007/s00484-007-0097-4)Eddy E M. Male germ cell gene expression. Recent Prog Horm Res, 2002, 57(1): 103-128.(doi 见图1)Lee NP, Cheng CY. Adaptors, junction dynamics, and spermatogenesis[J].Biol Reprod, 2004, 71(2): 392-404．(doi:10.1095/biolreprod.104.027268)Mi Y, Zhang C, Xie M, Zeng W. Effects of follicle-stimulating hormone and rogen on proliferation of cultured testicular germ cells of embryonic chickens[J].General and comparative endocrinology 2004; 138(3): 237-46. (DOI: 10.1016/j.ygcen.2004.06.012)Jannes P, Spiessens C, Vander AuweraI, et al. Male subfertility induced by acute scrotal heating affects embryo quality in normal female mice[J]. HumReprod, 1998, 13(2) : 372-375．(doi: 10.1093/humrep/13.2.372)Kangasniemi M, Huhtaniemi I, Meistrich ML. Failure of spermatogenesis to recover despite the presence of a spermatogonia in the irradiated LBNF1 rat[J]. Biology of reproduction 1996; 54(6): 1200-8. (doi: 10.1095/biolreprod54.6.1200)Ohta H, Yomogida K, Dohmae K, et al．Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF[J]. Development, 2000, 127(10): 2125-2131. (doi 见图12)Schmidt JA, Avarbock MR, Tobias JW, et al. Identification of glial cell line-derived neurotrophic factor-regulated genes important for spermatogonial stem cell self-renewal in the rat[J]. Biol Reprod, 2009, 81:56-66. (doi: 10.1095/biolreprod.108.075358. )Setchell BP, D’OcchioMJ, Hall MJ, et al. Is embryonic mortality increased in normal female rats mated to subfertilemales[J]. J Reprod Fertil, 1988, 82(2):567-574.(doi: 10.1530/jrf.0.0820567)Tadokoro Y, Yomogida K, Ohta H, et al. Homestatic regulation of germinal stem cell proliferation by GDNF/FSH pathway[J]. Mech Dev, 2002, 113(1): 29-39.(doi 见图4)T.Ogawa, M.Ohmura, K.Ohbo. The niche for spermatogonial stem cells in the mammalian testis[J]. Int.J.Hematol. 2005, 82: 381–388.(doi 见图2)Unni SK, Modi DN, PathakSG, et al. Stage-specific localization and expression of c-kit in the adult human testis[J]. J Histochem Cytochem, 2009, 57: 861-869.(doi: 10.1369/jhc.2009.953737.) |
|
|
|
