白藜芦醇通过PROX1/SIRT1信号通路调控山羊肌纤维类型转化的机制研究

doi:10.11843/j.issn.0366-6964.2025.01.019

摘要/Abstract

摘要：

旨在解析白藜芦醇(resveratrol，RES)介导PROX1基因调控山羊骨骼肌肌纤维类型转化的分子机制。体内试验部分，试验选取12只180日龄去势努比亚山羊((28.25±0.26)kg)随机分为2组，每组6个重复。以补饲0、150 mg·kg^-1 RES组山羊背最长肌组织为试验样品，采用实时荧光定量PCR(RT-qPCR)及Western blot技术检测PROX1基因的表达量；体外试验，基于课题组前期试验所筛选出的20 μmol·L^-1 RES是细胞培养最佳浓度，通过转染PROX1干扰片段及干扰片段与20 μmol·L^-1 RES共处理山羊骨骼肌成肌细胞，检测CaN/NFAT通路关键基因与肌纤维四型标志基因在mRNA水平及蛋白水平的表达情况；同时，利用蛋白-蛋白对接技术检测SIRT1与PROX1蛋白是否互作，并通过SIRT1抑制剂EX-527和RES共处理山羊成肌细胞，验证SIRT1与CaN/NFAT通路的调控关系。结果表明，与对照组相比，补饲150 mg·kg^-1 RES处理山羊成肌细胞可使SIRT1基因及CaN/NFAT通路关键基因的mRNA表达量极显著上调(P < 0.001)，使MYH7蛋白的表达水平显著上调(P < 0.01)，MYH4蛋白的表达水平显著下降(P < 0.01)。而干扰PROX1基因后，CaN/NFAT通路关键基因mRNA表达水平及MYH7、MYH4基因mRNA表达水平与蛋白表达水平的表达情况都与上述相反，该情况则在干扰片段与RES共处理成肌细胞后发生改变。蛋白-蛋白对接结果表明，SIRT1蛋白与PROX1蛋白可以互作。SIRT1抑制剂处理成肌细胞后，CaN/NFAT通路关键基因(NFATc1、TNNC1、TNNI1、MYOZ2、SLN)的mRNA表达量较CON组显著下调(P < 0.01)；再经过RES干预后，TNNC1、TNNI1的mRNA表达量极显著上调(P < 0.01)。本研究初步揭示了RES通过SIRT1/PROX1/CaN/NFAT途径调控肌纤维类型转化的机制，为后续畜禽肉品质的改良提供理论依据。

关键词: 白藜芦醇(RES), CaN/NFAT信号通路, 山羊成肌细胞, 肌纤维转化

Abstract:

The aim of this study was to analyze the molecular mechanism of resveratrol (RES) regulating myofiber type transformation by mediating PROX1 gene in goat skeletal muscle. In vivo component, twelve 180-day-old depopulated Nubian goats ((28.25±0.26) kg) were randomly divided into two groups, with 6 replicates each, the PROX1 gene expression levels of in dorsal longest muscle tissues supplemented with 0 and 150 mg·kg^-1 RES were detected by real-time fluorescence quantitative PCR (RT-qPCR) and Western blot. In vitro test, based on the previous research results, 20 μmol·L^-1 RES was identified as the optimal concentration for cell culture. PROX1 interference fragments and a combination of interference fragments with 20 μmol·L^-1 RES were transfected into goat skeletal muscle myoblasts. The expression levels of key genes in the CaN/NFAT pathway and the marker genes for the four muscle fiber types were measured at both mRNA and protein levels. Meanwhile, protein-protein docking technology was used to detect whether SIRT1 interacts with the PROX1 protein interact, and the regulatory relationship between SIRT1 and the CaN/NFAT pathway was verified by co-treatment of goat myoblasts with the SIRT1 inhibitor EX-527 and RES. The results showed that, compared with the control group, the expression of the SIRT1 gene and key genes in the CaN/NFAT pathway at the mRNA level in goat myoblasts treated with 150 mg·kg^-1 RES was significantly upregulated (P < 0.001), the expression of MYH7 protein was significantly upregulated (P < 0.01), and the expression of MYH4 protein was significantly downregulated (P < 0.01). After PROX1 gene interference, the mRNA and protein expression levels of key genes in the CaN/NFAT pathway, MYH7 and MYH4 showed opposite trends, which were altered when the interference fragments and RES co-treated the myoblasts. The protein-protein docking results indicated that SIRT1 and PROX1 proteins could interact. After myoblasts treated with the SIRT1 inhibitor, the mRNA expression levels of key genes (NFATc1, TNNC1, TNNI1, MYOZ2, SLN) in the CaN/NFAT pathway were significantly downregulated compared to the control group (P < 0.01). Following RES intervention, the mRNA expression levels of TNNC1 and TNNI1 were significantly upregulated (P < 0.01). This study initially revealed the mechanism of RES regulating the transformation of muscle fiber types through SIRT1/PROX1/CaN/NFAT pathway, which provides a theoretical basis for the subsequent improvement of livestock and poultry meat quality.

Key words: resveratrol(RES), CaN/NFAT signaling pathway, goat myoblasts, myofiber transformation

中图分类号:

S827.1

吴少强, 刘雨帆, 韦义荣, 黄艳娜, 蒋钦杨. 白藜芦醇通过PROX1/SIRT1信号通路调控山羊肌纤维类型转化的机制研究[J]. 畜牧兽医学报, 2025, 56(1): 201-212.

WU Shaoqiang, LIU Yufan, WEI Yirong, HUANG Yanna, JIANG Qinyang. Mechanism of Resveratrol Regulating Myofiber Type Transformation through PROX1/SIRT1 Signaling Pathway in Goats[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 201-212.

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基因 Gene	登录号 Accession number	引物序列(5′→3′) Primer sequence	产物长度/bp Amplification length
MYH7	XM_018054604	F: AGTTCGTCAAGGCCACGATT R: CCTCCTTCACGGTCACTGTC	87
MYH2	XM_018064659	F: GATGGTGGAGAGGAGGGAGT R: TCAGCAGGGGCTTGATCTTG	113
MYH4	XM_018064656	F: GCACCAGCGCCTAATCAATG R: TCGGGATAGCTGAGACACCA	115
MYH1	XM_018064657	F: AATCCTGGGCTTCACTTCGG R: CTGGTTCTGCTTGCTCCTCA	116
SIRT1	NM_001314319	F: ATTTTCCATGGCGCTGAGGT R: GGACTCTGGCATGTTCCACT	98
NFATc1	XM_018039660	F: GTCTGCAACGGGAAGAGGAA R: TCGTAATCATCGCTGGGCTC	98
TNNC1	NM_001285572	F: TGACATCTACAAGGCTGCGG R: CTGCCATCCTCATCCACCTC	201
TNNI1	XM_018060884	F: GCCAACCTCAAGTCCGTGAA R: CCTCCACGTTCTTCCTCCAG	82
MYOZ2	NM_001287568	F: GTCATGCACCTGGGCAAAAA R: GGCACCACGGTTACTGAGAT	81
SLN	XM_005689416	F: GATCCACCCGGGAGATCTGT R: ATAGGACCTCACAAGGAGCCA	80
PROX1	XM_013970386	F: CTCTCTAGTACAGGCTCCGAAG R: GAATGGCTCATACCCCGGAT	191
GAPDH	XM_005680968	F: CCCGTTCGACAGATAGCCGTA R: AATCCGTTCACTCCGACCT	83