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⁺