L-苹果酸对猪卵巢颗粒细胞增殖及转录谱的影响

doi:10.11843/j.issn.0366-6964.2025.11.017

Abstract

Abstract:

The present study was designed to examine the effects of L-malic acid (L-MA) on the cell proliferation of porcine ovarian granulosa cells (PGCs) and to uncover the potential mechanism based on transcriptomic analysis. In this study, primary PGCs were isolated, cultured and randomly assigned to either a control group or L-MA treatment groups (10, 25, 50, 100, and 200 μmol·L^-1). CCK-8 assay, EdU incorporation assay (5-ethynyl-2'-deoxyuridine) and qPCR analysis were used to assess the cell viability and cell proliferation. Furthermore, RNA sequencing (RNA-seq) was employed to delineate L-MA-induced the transcriptional landscape alterations in PGCs. Compared with the control group, 25, 50, 100 and 200 μmol·L^-1 L-MA significantly increased cell viability of PGCs (P < 0.05). PGCs exhibited the highest cell viability at an L-MA final concentration of 50 μmol·L^-1. Treatment with 50 μmol·L^-1 L-MA markedly upregulated the mRNA expression of proliferating cell nuclear antigen (PCNA) (P < 0.01), a key proliferation marker, and enhanced the proportion of EdU-positive cells in PGCs. Transcriptomic analysis revealed that under the threshold of |FC|≥1.5 and P < 0.05, 169 significantly upregulated and 118 downregulated genes were identified in PGCs with L-MA exposure. GO enrichment analysis demonstrated that differentially expressed genes (DEGs) were predominantly enriched in cellular components related to cell cycle complexes and nucleases, with molecular functions and biological processes related to genetic material replication, epigenetic modification, substance transport and metabolism, as well as cell proliferation and differentiation. KEGG and GSEA analyses further indicated that DEGs were primarily involved in signaling pathways associated with cellular immunity, genetic material replication, substance metabolism, and reproductive functions. Notably, predictions of transcription factor (TF) and binding sequences between TF and TF's target genes suggested that ELF3 was identified as the most likely candidate transcriptional factor mediated by L-MA, with potential binding sites within the PCNA promoter region. In summary, the findings demonstrate that L-MA significantly enhances the proliferation of porcine ovarian granulosa cells. This proliferative effect may be attributed to L-MA-mediated regulation of gene expression involving cell cycle complexes and nucleases, enhancement of molecular functions and biological processes related to genetic material replication, epigenetic modification, and substance transport, as well as improvement of cell health. This study provides important scientific evidence for the potential application of L-malic acid-based products in modulating granulosa cell proliferation and ovarian function.

Key words: L-malic acid, porcine ovarian granulosa cells, cell proliferation, transcriptome analysis

CLC Number:

S828.3

ZHAO Xiaoyi, ZHU Longlong, LIU Hui, ZHANG Dongyan, CAI Long, WANG Yalei, WANG Jing, ZHAO Junxing, CHEN Meixia. Effects of L-Malic Acid on the Proliferation and Transcriptional Profile of Porcine Ovarian Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5563-5574.

Figures/Tables 7

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References 43

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基因 Gene	对照组 Control	处理组 Treatment	倍变化 FC	log₂FC	P值 P-value	校正P值 P-adjust	上调/下调 Regulation
ONECUT2	0.18	0.37	2.08	1.06	0.01	1.00	up
ELF3	0.65	1.30	2.00	1.00	＜0.01	0.01	up
SIM2	0.02	0.15	9.47	3.24	0.01	1.00	up
STAT4	0.16	0.04	0.27	-1.91	0.03	1.00	down
FOXA1	0.06	0.14	2.26	1.18	0.03	1.00	up
HEYL	0.09	0.19	2.26	1.17	0.05	1.00	up
POU2F2	0.10	0.24	3.08	1.62	0.01	1.00	up
ZBED1	1.92	3.93	2.06	1.05	＜0.01	＜0.01	up

转录因子 TF	检索基因 Query	起始位置 Start	终止位置 Stop	正链/负链 Strand	结合位点预测分数 Score	P值 P-value	校正P值 Q value	结合位点匹配序列 Mached sequence
ELF3	PCNA	1 049	1 060	-	13.020 4	2.25E-05	0.089 6	GAGGCGGAAGCC
ELF3	PCNA	660	673	+	11.485 7	4.88E-05	0.108 0	TCAAACTTCCTGTG
ELF3	PCNA	1 771	1 784	-	11.278 6	5.54E-05	0.108 0	CAACACTTCCTCAG

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Effects of L-Malic Acid on the Proliferation and Transcriptional Profile of Porcine Ovarian Granulosa Cells

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