关键词: 淋巴细胞, 伤口愈合, 白细胞介素类, 肿瘤坏死因子α, 受体, 骨化三醇, 受体, Notch1, 固有淋巴细胞

Abstract: 【Abstract】 Objective To investigate the role of group 3 innate lymphoid cells （ILC3） in skin wound healing, and to explore the underlying mechanisms. Methods Twenty-four 5-week-old male C57BL/6 mice were randomly and equally allocated into 3 groups: the skin wound + ILC3 inhibitor group （referred to as ILC3 inhibitor group）, the skin wound group, and the control group, with 8 mice in each group. Four days before the establishment of the wound model, mice in the ILC3 inhibitor group were intraperitoneally injected with 1 μg of ILC3 inhibitor every 2 days for a total of 2 doses, mice in the skin wound group were injected with an equal volume of physiological saline solution, and mice in the control group were fed normally. To establish a mouse skin wound model, a full-thickness circular incision with a diameter of 0.6 cm was made around the midpoint of the dorsal midline using a biopsy punch after the intraperitoneal injection of anesthetics, which was histologically confirmed to be a full-thickness injury. The size of the wounds was observed and recorded, photographs of the wounds were taken on days 0, 1, 3, 5, 7, and 9 after wounding, and corresponding wound healing rates were calculated. On day 9 after wounding, tissue samples were collected from the wound edges, and subjected to flow cytometry analysis to quantify ILC3 infiltrating around the skin wound, and hematoxylin and eosin （HE） staining was performed to assess the healing status of the skin wounds. Real-time quantitative polymerase chain reaction （qRT-PCR） was conducted to determine the mRNA expression of vitamin D receptor （VDR）, Notch1, tumor necrosis factor-alpha （TNF-α）, interleukin （IL）-17A, IL-17F, and IL-22 in the wound-edge tissues, and Western blot analysis to determine their protein expression. Statistical analysis was carried out by using one-way analysis of variance and t test. Results On day 9 after wounding, the skin wound group showed an increased number of ILC3 in the wound-edge tissues（5.31% ± 1.47% vs. 3.10% ± 0.54%, P < 0.01）, increased mRNA and protein expression of TNF-α, IL-22, IL-17A, and IL-17F （all P < 0.05）, but decreased mRNA and protein expression of VDR （both P < 0.05） compared with the control group; the protein expression of Notch1 was significantly higher in the skin wound group than in the control group（P < 0.05）, but there was no significant difference in its mRNA expression between the two groups （P > 0.05）. On days 1, 3 and 5, the wound healing rates were significantly higher in the ILC3 inhibitor group （45.17% ± 9.90%, 61.58% ± 11.61%, 75.61% ± 9.12%, respectively） than in the skin wound group （25.87% ± 10.96%, 47.78% ± 13.81%, 64.55% ± 10.29%, respectively, all P < 0.05）. On day 9, the ILC3 inhibitor group showed a decreased number of ILC3 around the wound （2.69% ± 0.95%, P < 0.01）, decreased mRNA and protein expression of TNF-α, IL-22, IL-17A, and IL-17F in the wound-edge tissues （all P < 0.05）, but increased mRNA and protein expression of Notch1 and VDR in the wound-edge tissues （all P < 0.05） compared with the skin wound group. On day 9 after wounding, histopathological examination with HE staining revealed continuous and intact epithelial structure, as well as dense and neatly arranged collagen fibers in the ILC3 inhibitor group, and the structures of hair follicles, blood vessels, and sebaceous glands were similar to those in the control group. Conclusions Skin ILC3 infiltrated local wounds and were involved in the skin wound healing process through inflammatory factors such as TNF-α, IL-17A, IL-17F, and IL-22. Downregulating the number of ILC3 may promote skin wound healing by activating VDR and Notch1, as well as inhibiting the TNF-α signaling pathway and the expression of downstream inflammatory factors.

Key words: Lymphocytes, Wound healing, Interleukins, Tumor necrosis factor-alpha, Receptors, calcitriol, Receptor, Notch1, Innate lymphoid cells