AGRP基因在山羊组织表达及其对黑色素生成的作用机制

doi:10.11843/j.issn.0366-6964.2023.04.009

摘要/Abstract

摘要： 为了阐明AGRP基因在山羊皮肤表达模式以及对黑色素生成的作用机制。本研究通过qRT-PCR法检测AGRP基因在3只酉州乌羊不同组织和3只板角山羊皮肤组织表达情况。同时，通过体外试验分析不同浓度外源性AGRP蛋白对黑色素细胞影响，利用EdU法检测细胞活力、环磷腺苷（cyclic adenosine monophosphate，cAMP）试剂盒检测cAMP活性、并检测其对真黑色素含量和黑素生成相关基因黑素皮质素1型受体（melanocortin 1 receptor，MC1R）、酪蛋白酶（tyrosinase，TYR）、酪蛋白酶相关蛋白酶1（tyrosinase related protein 1，TYRP1）、多巴色素互变异构酶（dopachrome tautomerase，DCT） mRNA表达的影响。结果表明，AGRP基因在酉州乌羊组织均有表达，其中在垂体中表达最高，与其他组织相比差异显著（P<0.05）；板角山羊与酉州乌羊皮肤中AGRP表达量差异极显著（P<0.05）。添加不同浓度AGRP蛋白对黑色素生成和黑色素细胞增殖影响不显著（P>0.05）；随着AGRP浓度的升高，黑色素生成通路中cAMP活性显著下降（P<0.05）；200 nmol·L^-1 AGRP显著抑制MC1R mRNA表达且促进TYR、TYRP1 mRNA表达（P<0.05）；添加不同浓度外源性α-MSH均显著（P<0.05）挽救AGRP对黑色素细胞功能的影响。AGRP基因与酉州乌羊皮表型密切相关，确定AGRP在黑色素生成过程中的生物学功能为深入揭示AGRP对酉州乌羊皮肤黑色素沉积的作用机制奠定基础。

关键词: 酉州乌羊, AGRP, 基因表达, 黑色素细胞

Abstract: In order to elucidate the expression pattern of AGRP gene in goat skin and its action mechanism on melanin production. The tissue expression level of AGRP gene in different tissues of Youzhou dark goats and skin tissue of 3 Banjiao goats were detected by qRT-PCR. And the effects of exogenous AGRP protein at different concentrations on melanocytes were analyzed by in vitro experiments. The cell viability was detected by EdU method, and cAMP activity was detected by cyclic adenosine monophosphate kit, as well as its effects on the content of eumelanin and the mRNA expression of melanin-related genes MC1R, TYR, TYRP1 and DCT. This results showed that AGRP gene was expressed in different tissues of Youzhou dark goats and its expression in the pituitary gland was the highest (P<0.05). Meanwhile, the expression level of AGRP in the skin of Banjiao goat and Youzhou dark goat were significantly different (P<0.05). The addition of different concentrations of AGRP protein had no significant effect on the melanin production and proliferation of melanocytes (P>0.05). cAMP activity in the melanin production pathway significantly decreased (P<0.05) with the increasing of AGRP concentration. The 200 nmol·L^-1 AGRP significantly inhibited the expression of MC1R mRNA and enhanced the mRNA expression of TYR and TYRP1 (P<0.05). The addition of exogenous α-MSH at different concentrations could significantly (P<0.05) salvage the effects of AGRP on function of melanocyte. AGRP gene is closely related to that phenotype of Youzhou dark goat skin. Determining the biological function of AGRP in melanin production can lay the foundation for further revealing the mechanism of AGRP on melanin deposition in skin of Youzhou dark goat.

Key words: Youzhou dark goat, AGRP, gene expression, melanocyte

中图分类号:

S827.2

陈灿灿, 蒋婧, 孙晓燕, 任航行, 李杰. AGRP基因在山羊组织表达及其对黑色素生成的作用机制[J]. 畜牧兽医学报, 2023, 54(4): 1441-1451.

CHEN Cancan, JIANG Jing, SUN Xiaoyan, REN Hangxing, LI Jie. Expression of AGRP Gene in Goat Tissue and Its Action Mechanism on Melanin Production[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1441-1451.

参考文献 32

[1]	CÉSARINI J P, NSERM I. Melanins and their possible roles through biological evolution[J].Adv Space Res, 1996, 18(12):35-40.
[2]	ITO S, WAKAMATSU K, OZEKI H.Chemical analysis of melanins and its application to the study of the regulation of melanogenesis[J].Pigment Cell Res, 2000, 13(s8):103-109.
[3]	RZEPKA Z, BUSZMAN E, BEBEROK A, et al.From tyrosine to melanin:signaling pathways and factors regulating melanogenesis[J].Postepy Hig Med Dosw, 2016, 70:695-708.
[4]	D'MELLO S A N, FINLAY G J, BAGULEY B C, et al.Signaling pathways in melanogenesis[J].Int J Mol Sci, 2016, 17(7):1144.
[5]	马建青, 李祥龙, 周荣艳, 等.ASIP蛋白与AgRP相关蛋白生物信息比较分析[J].家畜生态学报, 2014, 35(1):9-14, 20.MA J Q, LI X L, ZHOU R Y, et al.Bioinformatics comparative analysis of agouti signaling protein and agouti-related protein[J].Acta Ecologiae Animalis Domastici, 2014, 35(1):9-14, 20.(in Chinese)
[6]	梁正翠.鸭的色素相关基因ASIP和AGRP的研究[D].扬州:扬州大学, 2010.LIANG Z C.Study on the melanogenic ASIP and AGRP gene of duck[D].Yangzhou:Yangzhou University, 2010.(in Chinese)
[7]	OLLMANN M M, LAMOREUX M L, WILSON B D, et al.Interaction of Agouti protein with the melanocortin 1 receptor in vitro and in vivo[J].Genes Dev, 1998, 12(3):316-330.
[8]	FLEMING K A, ERICSON M D, FREEMAN K T, et al.Structure-activity relationship studies of a macrocyclic AGRP-mimetic scaffold c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro] yield potent and selective melanocortin-4 receptor antagonists and melanocortin-5 receptor inverse agonists that increase food intake in mice[J].ACS Chem Neurosci, 2018, 9(5):1141-1151.
[9]	ZHANG J N, LI X, ZHOU Y W, et al.The interaction of MC3R and MC4R with MRAP2, ACTH, α-MSH and AgRP in chickens[J].J Endocrinol, 2017, 234(2):155-174.
[10]	LIMA L B, PEDROSO J A B, METZGER M, et al.Relationship of α-MSH and AgRP axons to the perikarya of melanocortin-4 receptor neurons[J].Brain Res, 2019, 1717:136-146.
[11]	黄萌, 贤明, 史明艳, 等.MC4R基因沉默对牛成纤维细胞内基因表达的影响[J].畜牧兽医学报, 2017, 48(3):403-415.HUANG M, XIAN M, SHI M Y, et al.Effects of MC4R gene silencing on gene expression in bovine fibroblast cells[J].Acta Veterinaria et Zootechnica Sinica, 2017, 48(3):403-415.(in Chinese)
[12]	GUNN T M, MILLER K A, HE L, et al.The mouse mahogany locus encodes a transmembrane form of human attractin[J].Nature, 1999, 398(6723):152-156.
[13]	盛微, 陈菲, 朱弘焱, 等.不同饲粮蛋氨酸水平鸡血清对鸡下丘脑神经元NPY和AgRp mRNA表达的影响[J].饲料研究, 2020, 43(4):34-38.SHENG W, CHEN F, ZHU H Y, et al.Effect of different dietary methionine levels on the expression of NPY and AgRp mRNA in hypothalamic neurons in chickens[J].Feed Research, 2020, 43(4):34-38.(in Chinese)
[14]	王梦远, 王俊博, 唐致恒, 等.AgRP/NPY神经元代谢调控功能研究进展[J].生理科学进展, 2019, 50(1):67-75.WANG M Y, WANG J B, TANG Z H, et al.Metabolic regulatory function of AgRP/NPY neurons[J].Progress in Physiological Sciences, 2019, 50(1):67-75.(in Chinese)
[15]	VOISEY J, CARROLL L, VAN DAAL A.Melanocortins and their receptors and antagonists[J].Curr Drug Targets, 2003, 4(7):586-597.
[16]	GODINO-GIMENO A, SÁNCHEZ E, GUILLOT R, et al.Growth performance after agouti-signaling protein 1(Asip1) overexpression in transgenic zebrafish[J].Zebrafish, 2020, 17(6):373-381.
[17]	SAKUMA Y, MATSUNAMI M, TAKADA T, et al.Multiple conserved elements structuring inverted repeats in the mammalian coat color-related gene Asip[J].Zoolog Sci, 2019, 36(1):23-30.
[18]	SHANG S Y, YU Y, ZHAO Y X, et al.Synergy between MC1R and ASIP for coat color in horses (Equus caballus)[J].J Anim Sci, 2019, 97(4):1578-1585.
[19]	ROYO L J, ÁLVAREZ I, ARRANZ J J, et al.Differences in the expression of the ASIP gene are involved in the recessive black coat colour pattern in sheep:evidence from the rare Xalda sheep breed[J].Anim Genet, 2008, 39(3):290-293.
[20]	韩吉龙, 岳耀敬, 郭健, 等.Agouti与MITF在不同颜色被毛藏羊皮肤组织中mRNA表达量研究[J].中国草食动物科学, 2012(S1):165-169.HAN J L, YUE Y J, GUO J, et al.mRNA expression of Agouti and MITF in skin tissue of Tibetan sheep with different color coat[J].China Herbivore Science, 2012(S1):165-169.(in Chinese)
[21]	BONILLA C, BOXILL L A, DONALD S A M, et al.The 8818G allele of the agouti signaling protein (ASIP) gene is ancestral and is associated with darker skin color in African Americans[J].Hum Genet, 2005, 116(5):402-406.
[22]	AINGER S A, JAGIRDAR K, LEE K J, et al.Skin pigmentation genetics for the clinic[J]. Dermatology, 2017, 233(1):1-15.
[23]	KINGO K, AUNIN E, KARELSON M, et al.Gene expression analysis of melanocortin system in vitiligo[J].J Dermatol Sci, 2007, 48(2):113-122.
[24]	RODRÍGUEZ-MARTÍNEZ S, GALVÁN I.A source of exogenous oxidative stress improves oxidative status and favors pheomelanin synthesis in zebra finches[J].Comp Biochem Physiol C Toxicol Pharmacol, 2020, 228:108667.
[25]	陈志强.鸡的色素相关基因TYR5'调控区和Agrp的SSCP研究[D].北京:中国农业大学, 2004.CHEN Z Q.Study on the melanogenic TYR5' regulational region and Agrp gene of chicken through SSCP[D].Beijing:China Agricultural University, 2004.(in Chinese)
[26]	KUROKAWA T, MURASHITA K, UJI S.Characterization and tissue distribution of multiple agouti-family genes in pufferfish, Takifugu rubripes[J].Peptides, 2006, 27(12):3165-3175.
[27]	SÁNCHEZ E, RUBIO V C, CERDÁ-REVERTER J M.Molecular and pharmacological characterization of the melanocortin type 1 receptor in the sea bass[J].Gen Comp Endocrinol, 2010, 165(1):163-169.
[28]	李健.Agouti、TYRP1、DCT在酉州乌羊和渝东白山羊皮肤组织中表达特性研究[D].重庆:西南大学, 2016.LI J.Study on the expression characteristics of Agouti, TYRP1 and DCT in the skin tissues of Youzhou dark goat and Yudong white goat[D].Chongqing:Southwest University, 2016.(in Chinese)
[29]	CHEN X L, XIAO Z N, CAI Y L, et al.Hypothalamic mechanisms of obesity-associated disturbance of hypothalamic-pituitary-ovarian axis[J].Trends Endocrinol Metab, 2022, 33(3):206-217.
[30]	BAI X Y, FU L, JIN N Q, et al.Rescue of obesity-induced infertility in female mice by silencing AgRP neurons[J].Biochem Biophys Res Commun, 2022, 623:32-38.
[31]	陈灿灿, 李杰, 马友记, 等.表皮黑色素细胞研究进展[J].黑龙江畜牧兽医, 2018(23):27-31.CHEN C C, LI J, MA Y J, et al.Progress in the study of epidermal melanocytes[J].Heilongjiang Animal Science and Veterinary Medicine, 2018(23):27-31.(in Chinese)
[32]	THIRUMOORTHY R, HOLDER J R, BAUZO R M, et al.Novel agouti-related-protein-based melanocortin-1 receptor antagonist[J].J Med Chem, 2001, 44(24):4114-4124.