AKR1B1介导AMPK/mTOR/S6通路调控猪骨骼肌卫星细胞增殖和分化

doi:10.11843/j.issn.0366-6964.2025.08.015

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (8): 3722-3733.doi: 10.11843/j.issn.0366-6964.2025.08.015

AKR1B1介导AMPK/mTOR/S6通路调控猪骨骼肌卫星细胞增殖和分化

胡金玲¹(), 钟奇祺¹, 黄程¹, 雷明刚¹^,²^,³^,*()

1. 华中农业大学动物科技学院, 农业动物遗传育种教育部重点实验室, 农业部猪遗传育种重点实验室, 武汉 430070
2. 华中农业大学国家畜牧工程技术研究中心, 武汉 430070
3. 生猪可持续生产协同创新中心生猪生产部, 武汉 430070

收稿日期:2025-01-15 出版日期:2025-08-23 发布日期:2025-08-28
通讯作者: 雷明刚 E-mail:jinling_hu@163.com;leimg@mail.hzau.edu.cn
作者简介:胡金玲(2000-)，女，广西桂林人，硕士，主要从事猪遗传育种与繁殖研究，E-mail：jinling_hu@163.com
基金资助:
国家现代农业产业技术体系资助(CARS-35-026)；优质猪新品种设计与培育(2023ZD04047)

AKR1B1 Regulates Proliferation and Differentiation of Porcine Skeletal Muscle Satellite Cells via the AMPK/mTOR/S6 Signaling Pathway

HU Jinling¹(), ZHONG Qiqi¹, HUANG Cheng¹, LEI Minggang¹^,²^,³^,*()

1. Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Education, Key Laboratory of Pig Genetics and Breeding of Ministry of Agriculture, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
2. National Center for Livestock Engineering and Technology Research, Huazhong Agricultural University, Wuhan 430070, China
3. Pig Production Department, Collaborative Innovation Center for Sustainable Pig Production, Wuhan 430070, China

Received:2025-01-15 Online:2025-08-23 Published:2025-08-28
Contact: LEI Minggang E-mail:jinling_hu@163.com;leimg@mail.hzau.edu.cn

摘要/Abstract

摘要：

旨在探讨醛酮还原酶家族1成员B1基因(aldo-keto reductase family 1 member b1, AKR1B1)对猪骨骼肌卫星细胞(porcine skeletal muscle satellite cells, PSCs)增殖和分化的影响。本研究利用生物信息学方法预测分析AKR1B1基因所编码蛋白的结构与理化性质；采集3日龄仔猪十二指肠、背最长肌和皮下脂肪等8个组织，提取RNA，进行实时定量PCR(qRT-PCR)，确定各组织中AKR1B1表达水平；从腿部肌肉和背最长肌中分离PSCs体外培养，分别收集增殖分化特定时间节点的样品；利用小干扰RNA(small interfering RNA, siRNA)抑制AKR1B1基因表达水平，使用qRT-PCR、细胞免疫荧光(immunofluorescence)及蛋白质印迹法(Western blot)等方法进行探索，评估AKR1B1基因在PSCs增殖与分化过程中的功能。AKR1B1蛋白由316个氨基酸组成，主要在细胞质中表达，经预测AKR1B1为稳定的、亲水性较强的酸性蛋白质。qRT-PCR结果显示，AKR1B1基因在猪的背最长肌和十二指肠mRNA表达水平较高；PSCs增殖期，AKR1B1表达逐渐上升；分化期AKR1B1表达水平随着分化进程逐步增加，第5天达到峰值后开始下降。增殖期干扰AKR1B1基因显著降低增殖标志物Ki67、细胞周期蛋白D(cyclin D1, ccnd1)(P＜0.05)的mRNA和蛋白表达水平，EdU⁺细胞(P＜0.05)比例显著减少。分化期干扰AKR1B1基因后，分化标志物肌细胞生成素(myogenin, MyOG)、肌球蛋白重链(myosin heavy chains, MyH3)(P＜0.05)mRNA和蛋白表达显著上调，细胞免疫荧光结果显示MyOG⁺细胞比例极显著提高(P＜0.01)，分化指数显著升高(P＜0.05)。干扰AKR1B1基因显著降低AMPK Ser182位点的磷酸化水平(P＜0.05)，显著升高TSC2、RAPTOR、mTOR Ser2448磷酸化水平(P＜0.05)，促使S6K1以及S6(P＜0.05)蛋白表达显著提高。综上所述，干扰AKR1B1基因抑制PSCs增殖，对分化产生正调控作用，同时发现AKR1B1介导AMPK/mTOR/S6通路调控PSCs分化。本研究旨在探索AKR1B1基因在猪骨骼肌生长发育过程中产生的具体影响，以期为健康畜牧提供理论依据和科学线索。

关键词: AKR1B1, 猪骨骼肌卫星细胞, 骨骼肌细胞增殖分化, AMPK/mTOR/S6通路

Abstract:

This study aimed to investigate the effects of the aldo-keto reductase family 1 member b1 (AKR1B1) gene on the proliferation and differentiation of porcine skeletal muscle satellite cells (PSCs). Bioinformatics tools were used to predict and analyze the structure and physicochemical properties of the AKR1B1-encoded protein. RNA was extracted from 8 tissues, including the duodenum, longissimus dorsi, and subcutaneous fat of 3-day-old piglets, and quantitative real-time PCR (qRT-PCR) was performed to determine AKR1B1 expression levels in each tissue. PSCs were isolated from leg muscles and longissimus dorsi and cultured in vitro, with samples collected at specific time points during proliferation and differentiation. Small interfering RNA (siRNA) was used to suppress AKR1B1 expression, and qRT-PCR, immunofluorescence, and Western blot analyses were conducted to evaluate AKR1B1′s role in PSCs proliferation and differentiation. The AKR1B1 protein consisted of 316 amino acids, was predominantly expressed in the cytoplasm, and was predicted to be a stable, highly hydrophilic, acidic protein. qRT-PCR results showed that AKR1B1 expression was highest in the longissimus dorsi and duodenum. During PSCs proliferation, AKR1B1 expression gradually increased, peaking on day 5 of differentiation before declining. Knockdown of AKR1B1 during proliferation significantly reduced the mRNA and protein levels of proliferation markers Ki67 and cyclin D1 (ccnd1) (P < 0.05) and decreased the proportion of EdU⁺ cells (P < 0.05). During differentiation, AKR1B1 knockdown significantly upregulated the mRNA and protein levels of differentiation markers myogenin (MyOG) and myosin heavy chain (MyH3) (P < 0.05). Immunofluorescence results showed a highly significant increase in the proportion of MyOG⁺ cells (P < 0.01) and an elevated differentiation index (P < 0.05). AKR1B1 knockdown significantly reduced the phosphorylation of AMPK at Ser182 (P < 0.05) while significantly increasing the phosphorylation of TSC2, RAPTOR, and mTOR at Ser2448 (P < 0.05). This led to a significant increase in the expression of S6K1 and S6 proteins (P < 0.05). In summary, AKR1B1 knockdown inhibited PSCs proliferation and positively regulated differentiation. AKR1B1 was found to mediate the AMPK/mTOR/S6 signaling pathway to regulate PSCs differentiation. This study provides insights into the specific effects of AKR1B1 on porcine skeletal muscle growth and development, offering a theoretical basis and scientific clues for healthy livestock management.

Key words: AKR1B1, porcine skeletal muscle satellite cells, skeletal muscle cell proliferation and differentiation, AMPK/mTOR/S6 pathway

中图分类号:

S828.1

胡金玲, 钟奇祺, 黄程, 雷明刚. AKR1B1介导AMPK/mTOR/S6通路调控猪骨骼肌卫星细胞增殖和分化[J]. 畜牧兽医学报, 2025, 56(8): 3722-3733.

HU Jinling, ZHONG Qiqi, HUANG Cheng, LEI Minggang. AKR1B1 Regulates Proliferation and Differentiation of Porcine Skeletal Muscle Satellite Cells via the AMPK/mTOR/S6 Signaling Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 3722-3733.

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参考文献 38

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名称 Name	来源 Source	稀释比 Dilution ratio	货号 Identifier
AKR1B1 Rabbit mAb	Abclonal	1∶5 000	A22132
β-Tubulin Mouse mAb	Abclonal	1∶5 000	AC021
HRP-conjugated PCNA Monoclonal antibody	Proteintech	1∶5 000	10D10E11
Rabbit Polyclonal Anti-MYH3	Proteintech	1∶1 000	22287-1-AP
mTOR Monoclonal antibody	Proteintech	1∶5 000	81670-1-RR
Phospho-mTOR (Ser2448) Recombinant antibody	Proteintech	1∶5 000	67778-1-Ig
AMPK Alpha Polyclonal antibody	Proteintech	1∶5 000	10929-2-AP
Phospho-AMPK Beta 1 (Ser182) Recombinant antibody	Proteintech	1∶5 000	83924-1-RR
S6 Ribosomal protein Recombinant antibody	Proteintech	1∶5 000	80208-1-RR
p70(S6K) Polyclonal antibody	Proteintech	1∶5 000	14485-1-AP
Tuberin/TSC2 Polyclonal antibody	Proteintech	1∶5 000	24601-1-AP
Raptor Polyclonal antibody	Proteintech	1∶5 000	20984-1-AP
Rabbit Monoclonal Anti-Cyclin D1	Proteintech	1∶5 000	60186-1-lg
Mouse Polyclonal Anti-Myogenin	Santa	1∶1 000	sc-52903
Rabbit Monoclonal Anti-Ki67	Abmart	1∶1 000	TW0001
HRP-conjugated Affnipure Goat Anti-Rabbit lgG (H+L)	Proteintech	1∶5 000	SA00001-1
HRP-conjugated Affnipure Goat Anti-Mouse lgG (H+L)	Proteintech	1∶5 000	SA00001-2
Cy3 Goat Anti-Rabbit lgG (H+L)	Abclonal	1∶200	AS007
Cy3 Goat Anti-Mouse lgG (H+L)	Abclonal	1∶200	AS008

名称 Name	引物序列(5′→3′) Primer sequence	产物长度/bp Product length
sus-AKR1B1	F: GAGGAGCTCCGCAGTCATGG；R: GTTCTCGGCAATGCGTTCAG	176
sus-PCNA	F: ATCUGGTGTGACCCGGACT；R: CTGGCATCACCGAAGAGCAGTT	163
sus-Ki67	F: AGCCCGTATCTGGTGCAAAA；R: CCTGCATCTGTGTAAGGGCA	267
sus-ccnd1	F: ATCAGGTGTGACCCGGACT；R: CGCCTCAAATGTTCACGTCG	184
sus-MyH3	F: GAAGAGTACGCCAAGGGGAAA；R: GTGTACCGGTCCTTGAGGTT	200
sus-MyOG	F: AGGCTACGAGCGGACTGA；R: GCAGGGTGCTCCTCTTCA	230
sus-GADPH	F: ACCCAGAAGACTGTGGATGG；R: AAGCAGGGATGATGTTCTGG	79

氨基酸 Amino acid	氨基酸数量/个 Number	占比/% Percent
丙氨酸Ala (A)	18	57
精氨酸Arg (R)	10	3.2
天冬氨酸Asn (N)	15	4.7
天门冬氨酸Asp (D)	18	5.7
半胱氨酸Cys (C)	6	1.9
谷氨酰胺Gln (Q)	11	3.5
谷氨酸Glu (E)	24	7.6
甘氨酸Gly (G)	18	5.7
组氨酸His (H)	9	2.8
异亮氨酸Ile (I)	16	5.1
丙氨酸Ala (A)	18	57
精氨酸Arg (R)	10	3.2
亮氨酸Leu (L)	34	10.8
赖氨酸Lys (K)	26	8.2
苯丙氨酸Phe (F)	10	3.2
甲硫氨酸Met (M)	6	1.9
脯氨酸Pro (P)	22	7.0
丝氨酸Ser (S)	14	4.4
苏氨酸Thr (T)	14	4.4
色氨酸Trp (W)	6	1.9
酪氨酸Tyr (Y)	13	4.1
缬氨酸Val (V)	26	8.2

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AKR1B1介导AMPK/mTOR/S6通路调控猪骨骼肌卫星细胞增殖和分化

AKR1B1 Regulates Proliferation and Differentiation of Porcine Skeletal Muscle Satellite Cells via the AMPK/mTOR/S6 Signaling Pathway

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