盐酸小檗碱对鸡成肌细胞增殖分化的影响及机制解析

任文佳; 赵鹏飞; 李畅; 李莹莹; 唐铎; 刘屹森; 王守伟

doi:10.13386/j.issn1002-0306.2025040062

盐酸小檗碱对鸡成肌细胞增殖分化的影响及机制解析

Effect of Berberine Hydrochloride on the Proliferation and Differentiation of Chicken Myoblasts and the Mechanism Analysis

摘要

摘要: 为探究盐酸小檗碱对鸡成肌细胞增殖分化的影响。本研究通过CCK-8法、EdU染色法以及肌间线蛋白（Desmin）免疫荧光染色检测盐酸小檗碱对细胞增殖和分化的影响，通过实时荧光定量PCR实验（RT-qPCR）对生肌因子5（Myogenic Factor 5，MYF5）和生肌分化因子（Myogenic Differentiation 1，MYOD）基因表达水平进行测定，同时通过转录组测序技术探究盐酸小檗碱对鸡成肌细胞基因表达谱的影响。结果表明，盐酸小檗碱处理显著促进了鸡成肌细胞增殖，同时抑制MYOD（P<0.01）和MYF5（P<0.05）表达，Desmin染色在实验组胞质内荧光信号略弱于对照组，表明盐酸小檗碱在一定程度上抑制鸡成肌细胞的分化。此外，基因本体论富集分析（Gene Ontology，GO）表明，盐酸小檗碱处理后差异基因主要富集在细胞外空间与基质、细胞粘附、细胞迁移调节等方面；京都基因与基因组百科全书富集分析（Kyoto Encyclopedia of Genes and Genomes，KEGG）表明，差异基因主要富集在酪氨酸代谢、转化生长因子-β（TGF-β）信号通路、促分裂素原活化蛋白激酶（Mitogen-activated Protein Kinases，MAPK）信号通路以及细胞粘附分子相关信号通路；RT-qPCR结果显示在TGF-β通路中DNA结合抑制因子1（Inhibitor of DNA Binding 1，ID1）和垂体同源盒转录因子2（Paired-like Homeodomain Transcription Factor 2，PITX2）表达上调，而卵泡抑素（Follistatin，FST）表达下调，在MAPK通路中，促增殖基因胰岛素样生长因子2（Insulin-like Growth Factor 2，IGF2）、成纤维细胞生长因子7（Fibroblast Growth Factor 7，FGF7）、FBJ骨肉瘤癌基因转录因子（FBJ Murine Osteosarcoma Viral Oncogene Homolog，FOS）、血管内皮生长因子受体1（Fms-related Tyrosine Kinase 1，FLT1）和MAPK活化蛋白激酶3（MAPK-activated Protein Kinase 3，MAPKAPK3）表达均上调，而钙通道基因电压门控钙通道α1E亚基（Calcium Voltage-gated Channel Subunit Alpha1 E，CACNA1E）下调，表明盐酸小檗碱可能通过TGF-β/MAPK通路的交互作用，协同调控成肌细胞的增殖-分化平衡。本研究发现盐酸小檗碱促进鸡成肌细胞增殖并对其中的分子机制进行了解析和验证，同时为体外高效培养鸡成肌细胞提供了理论支持。

Abstract: To explore the effect of berberine hydrochloride on the proliferation and differentiation of chicken myoblasts. In this study, the effects of berberine hydrochloride on cell proliferation and differentiation were detected by CCK-8 assay, EdU staining and Desmin immunofluorescence staining. The gene expression levels of myogenic factor 5 (MYF5) and myogenic differentiation 1 (MYOD) were determined by real-time quantitative PCR (RT-qPCR). The effects of berberine hydrochloride on the gene expression profiles of chicken myoblasts were also investigated by RNA sequencing analysis. The results showed that berberine hydrochloride treatment significantly promoted the proliferation of chicken myoblasts while inhibiting the expression of MYOD (P<0.01) and MYF5 (P<0.05). Desmin staining showed slightly weaker fluorescence signals in the cytoplasm of the experimental group than that of the control group, indicating that berberine hydrochloride inhibited the differentiation of chicken myoblasts to a certain extent. In addition, Gene Ontology enrichment analysis (GO) showed that the differential genes were mainly enriched in extracellular space and matrix, cell adhesion, and cell migration regulation after berberine hydrochloride treatment; Kyoto Encyclopedia of Genes and Genomes enrichment analysis (KEGG) showed that the differential genes were mainly enriched in tyrosine metabolism, TGF-β signaling pathway, MAPK signaling pathway and cell adhesion molecule-related signaling pathway. The RT-qPCR results showed that in the TGF-β pathway, the expression of Inhibitor of DNA Binding 1(ID1) and Paired-like Homeodomain Transcription Factor 2(PITX2) was upregulated, while Follistatin (FST) was downregulated. In the MAPK pathway, the pro-proliferative genes Insulin-like Growth Factor 2 (IGF2), Fibroblast Growth Factor 7 (FGF7), FBJ Murine Osteosarcoma Viral Oncogene Homolog (FOS), Fms-related Tyrosine Kinase 1 (FLT1), and MAPK-activated Protein Kinase 3 (MAPKAPK3) were all upregulated, whereas the calcium channel gene Calcium Voltage-gated Channel Subunit Alpha1 E (CACNA1E) was downregulated. These findings suggested that berberine hydrochloride might synergistically regulate the proliferation-differentiation balance of myoblasts through the interaction between the TGF-β and MAPK signaling pathways. In this study, we found that berberine hydrochloride promoted the proliferation of chicken myoblasts and analyzed the molecular mechanism and provided theoretical support for the efficient culture of chicken myoblasts in vitro.

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