CD44通过影响猪流行性腹泻病毒复制调节钠氢交换体3活性

doi:10.11843/j.issn.0366-6964.2024.05.035

摘要/Abstract

摘要： 本研究旨在研究CD44(cluster of differentiation 44)对感染猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)的猪小肠上皮细胞(IPEC-J2)中钠氢交换体3(NHE3)表达及膜转移的影响。以IPEC-J2为细胞模型，采用RT-qPCR和Western blot检测感染PEDV后不同时间点IPEC-J2细胞中NHE3和PEDV N表达量变化；转染质粒调控IPEC-J2中CD44表达后，采用TCID50和Western blot检测PEDV感染后不同时间点PEDV复制水平和NHE3蛋白表达变化，采用火焰原子吸收法检测IPEC-J2细胞内外Na⁺浓度变化。转录组数据和细胞试验结果显示，与对照组相比，PEDV感染后IPEC-J2细胞中CD44蛋白表达水平和mRNA表达量均呈上调趋势，24～48 h内上升显著(P<0.05)，而PEDV N蛋白表达水平在12～48 h内则呈显著下降趋势(P<0.05)。此外，CD44重组质粒转染试验结果显示，与PEDV感染组相比，过表达CD44后感染PEDV组细胞中病毒滴度和PEDV N蛋白表达水平显著降低(P<0.05)，而干扰CD44后感染PEDV组细胞中病毒滴度和PEDV N蛋白表达水平则显著上升(P<0.05)。以上结果表明，高表达CD44具有抑制PEDV复制的作用，干扰CD44后PEDV复制增多。同时，为研究PEDV感染情况下，CD44是否参与了IPEC-J2细胞中NHE3表达的调节，采用Western blot和火焰原子吸收法检测了调节CD44后膜NHE3蛋白的表达水平和细胞内外Na⁺浓度。结果表明，过表达CD44显著促进了膜NHE3蛋白的表达和活性(P<0.05)，细胞内外Na⁺浓度逐渐恢复正常水平。相反，干扰CD44显著降低了膜NHE3蛋白的表达和活性(P<0.05)，细胞内外Na⁺浓度呈现失衡状态。结果提示，CD44可能是缓解PEDV引发仔猪腹泻的潜在治疗靶点，它通过抑制IPEC-J2细胞中PEDV的复制来增加转移至质膜上的NHE3数量，从而维持细胞内外Na⁺运转平衡。

关键词: CD44, 猪流行性腹泻病毒, 钠氢交换体3, 钠离子

Abstract: This study aimed to investigate the effect of CD44 (cluster of differentiation 44) on the expression and membrane transfer of Na⁺/H⁺ exchanger 3 (NHE3) in porcine intestinal epithelial cells (IPEC-J2) infected with porcine epidemic diarrhea virus (PEDV). Using IPEC-J2 as the cell model, the expression of NHE3 and PEDV N in IPEC-J2 cells at different time points after infection with PEDV was detected by RT-qPCR and Western blot. After the transfection plasmid regulated the expression of CD44 in IPEC-J2, TCID₅₀ and Western blot were used to detect the changes of PEDV replication level and NHE3 protein expression at different time points after PEDV infection, and the changes of Na⁺ concentration inside and outside IPEC-J2 cells were detected by flame atomic absorption method. Transcriptome data and cell experimental results showed that compared with the control group, the expression level of CD44 protein and mRNA expression in IPEC-J2 cells after PEDV infection showed an upward trend, and increased significantly within 24-48 h (P<0.05), while the expression level of PEDV N protein decreased significantly within 12-48 h (P<0.05). In addition, the results of CD44 recombinant plasmid transfection experiments showed that the viral titer and PEDV N protein expression levels in cells infected with PEDV group after overexpression of CD44 were significantly reduced (P<0.05), while the viral titer and PEDV N protein expression levels in cells infected with PEDV group after interference with CD44 were significantly increased (P<0.05). These results showed that overexpression of CD44 had the effect of inhibiting PEDV replication, and PEDV replication increased after interfering with CD44. At the same time, in order to investigate whether CD44 participated in the regulation of NHE3 expression in IPEC-J2 cells under PEDV infection, Western blot and flame atomic absorption spectrometry were used to detect the expression level of surface NHE3 protein and the concentration of Na⁺ inside and outside the cell after regulating CD44. The results showed that overexpression of CD44 significantly promoted the expression of surface NHE3 protein and enhanced its activity (P<0.05), and the concentration of Na⁺ inside and outside the cell gradually returned to normal levels. In contrast, interference with CD44 significantly reduced the expression and activity of surface NHE3 protein (P<0.05), and the concentration of Na⁺ inside and outside cells was higher than normal, showing an imbalance. The results suggest that CD44 may be a potential therapeutic target for alleviating PEDV-induced piglet diarrhea, and it increases the amount of NHE3 transferred to the plasma membrane by inhibiting the replication of PEDV in IPEC-J2 cells, thereby maintaining the balance of intracellular and extracellular Na⁺.

Key words: CD44, porcine epidemic diarrhea virus, NHE3, Na⁺

中图分类号:

S852.659.6

王静, 张淑娟, 胡霞, 刘向阳, 张兴翠, 宋振辉. CD44通过影响猪流行性腹泻病毒复制调节钠氢交换体3活性[J]. 畜牧兽医学报, 2024, 55(5): 2176-2185.

WANG Jing, ZHANG Shujuan, HU Xia, LIU Xiangyang, ZHANG Xingcui, SONG Zhenhui. CD44 Regulates Na⁺/H⁺ Exchanger 3 Activity by Influencing Porcine Epidemic Diarrhea Virus Replication[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2176-2185.

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