CAI Xiang, QIU Bai-yi, DUAN Yao, WANG Wei, HE Ya-nan
Current Immunology. 2024, 44(6): 465-473.
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To investigate the mechanism by which salidroside improves keloid on regulating JAG1/Notch3 signaling pathway through miR-26a-5p, 10 keloid patients diagnosed by histopathology in Wuhan Hospital of Traditional Chinese Medicine were enrolled as the research objects from October 2019 to January 2021. The keloid tissues removed by surgery were used in the keloid group, and the adjacent healthy skin tissues were used in the normal group. Primary keloid fibroblasts were isolated and cultured, and the cells of generation 4~8 were divided into 8 groups: control group (normal culture), salidroside low-dose group (10 μmol/L salidroside), salidroside medium-dose group (20 μmol/L salidroside), salidroside high-dose group (40 μmol/L salidroside), miR-NC group (40 μmol/L salidroside+miR-NC), miR-26a-5p inhibitor group (40 μmol/L salidroside+miR-26a-5p inhibitor), sh-RNA group (40 μmol/L salidroside+miR-26a-5p inhibitor+sh-RNA) and sh-JAG1 group (40 μmol/L salidroside+miR-26a-5p inhibitor+sh-JAG1). Cells in each group were treated with salidroside for 24 h after transfection or directly treated with the corresponding dosage of salidroside for 24 h. The relative mRNA expressions of miR-26a-5p and JAG1 in tissues and cells were quantified using qRT-PCR. Cell proliferating viability was measured by the MTT assay and cell cloning capacity was detected using the plate cloning method. Cell apoptosis was detected by FACS and cell migration and invasion were assessed by Transwell method. Luciferase reporter system was utilized to examine the targeting interaction between miR-26a-5p and JAG1. Western blotting was used to detect the expressions of tissue hyperplasia-related proteins. The results showed that, compared to that of the normal group, the expression of miR-26a-5p in keloid group was significantly decreased (P＜0.05), while the JAG1 expression significantly increased (P＜0.05). Compared to the 0 μmol/L salidroside group, 10, 20, 40, 80, and 160 μmol/L salidroside treatment inhibited the survival rate of keloid fibroblasts in a concentration-dependent manner (all with P＜0.05). To ensure both the survival of keloid fibroblasts and the effect of salidroside, subsequent experiments were conducted with salidroside concentrations of 10, 20, and 40 μmol/L (It was proved that miR-26a-5p combines to JAG1). In the three salidroside groups, compared to those of the control group, the expression of miR-26a-5p and the apoptosis rate increased dependently, while the expression of JAG1, the numbers of cell clones, cell migrations, and invasions, and the expression of α-SMA, collagen Ⅰ, collagen Ⅲ, JAG1, and Notch3 proteins decreased significantly (all with P＜0.05). Compared to those of the salidroside high-dose group and the miR-NC group, the expression of miR-26a-5p and the cell apoptosis rate in the miR-26a-5p inhibitor group reduced significantly, while the expression of JAG1, the numbers of cell clones, migration and invasion, and the expression of α-SMA, collagen Ⅰ, collagen Ⅲ, JAG1 and Notch3 proteins increased significantly (all with P＜0.05). Inhibition of JAG1 expression suppressed the effect of miR-26a-5p inhibitor on promoting cell proliferation, migration and invasion, and on inhibiting cell apoptosis. In conclusion, the expression of miR-26a-5p is down-regulated and JAG1 is up-regulated in keloid tissue, salidroside can inhibit the JAG1/Notch3 signaling pathway and the proliferation, migration and invasion of keloid fibroblasts, while promoting cell apoptosis, which in turn improves keloid by up-regulating the expression of miR-26a-5p.
