Amer Johnny, Salhab Ahmad, Hussini Enas, Shweiki Rasha, Zahran Iman, Far Mohammad
Department of Allied and Applied Medical Sciences, Division of anatomy, Biochemistry and Genetics, An-Najah National University, Nablus, Palestine.
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, An-Najah National University, Nablus, Palestine.
Front Pharmacol. 2024 Nov 25;15:1484278. doi: 10.3389/fphar.2024.1484278. eCollection 2024.
Vitamin D has an immunomodulatory property influencing the activity of NKT cells. We aimed to study the impact of osteopontin (OPN), a key driver of fibrosis, on NKT cells' vitamin D receptor (VDR) and activity alterations.
Liver fibrosis was induced in BALB/C mice with carbon-tetrachloride (CCl) for 8 weeks with either vitamin D [100 ng/kg] or InVivoMAb anti-mouse OPN [100 μg/kg] 2X/week started at week-4 of CCl The liver injury profile of serum ALT, AST, and inflammatory cytokines were evaluated. Histopathological findings were assessed via H&E staining and Sirius-Red staining. Fibrotic genes of αSMA, CREBP, and collagen III were assessed using RT-PCR. Fast blood sugar, insulin, liver cholesterol, and triglyceride were evaluated. Liver tissue-resident (tr)-NKT cells were obtained for VDR expressions, molecular pathways of p-STAT1 and P-STAT-5, and activation markers of CD107a and NKp46 using flow cytometry.
Following vitamin D treatment, H&E staining revealed reduced microvascular and macrovascular steatosis, while Sirius-Red staining showed less fibrosis accumulation in liver fibrosis mice than in untreated counterparts. Results were associated with a significant decrease in serum cytokines of IL-β/IL-6/IL-4/OPN/TNF-α and serum AST and ALT by 2-fold and 3-fold, respectively. Fibrotic markers showed an average 1.3-fold decrease in αSMA, CREB, and Col-III in liver fibrosis mice following vitamin D treatment. Quantitated liver cholesterol and triglycerides, serum insulin, and fasting blood sugar ameliorated their levels following vitamin D treatment in liver fibrosis mice. OPN-neutralizing antibody over-expressed VDR on trNKT cells and increased CD107a and NKp46 activities of 3.1 and 3.5 folds, respectively, associated with increasing in p-STAT1 and p-STAT5 phosphorylation. These results were accompanied with a decrease in hepatic-stellate-cell activation markers of αSMA, Col-III, and desmin.
VDR expressions affect trNKT cells activity and could modulate progressions of liver fibrosis. Using an OPN-neutralizing antibody exhibited an antifibrotic effect by alleviating the liver injury profile through NKT cells. It is also suggested as an immunomodulatory target of liver fibrosis.
维生素D具有免疫调节特性，可影响自然杀伤T细胞（NKT细胞）的活性。我们旨在研究骨桥蛋白（OPN，纤维化的关键驱动因子）对NKT细胞维生素D受体（VDR）及活性改变的影响。
用四氯化碳（CCl）诱导BALB/C小鼠肝纤维化8周，从CCl处理第4周开始，每周两次给予维生素D[100 ng/kg]或抗小鼠OPN单克隆抗体[100 μg/kg]。评估血清谷丙转氨酶（ALT）、谷草转氨酶（AST）及炎性细胞因子的肝损伤情况。通过苏木精-伊红（H&E）染色和天狼星红染色评估组织病理学结果。采用逆转录-聚合酶链反应（RT-PCR）评估α平滑肌肌动蛋白（αSMA）、环磷腺苷反应元件结合蛋白（CREBP）和III型胶原的纤维化基因。评估空腹血糖、胰岛素、肝脏胆固醇和甘油三酯水平。利用流式细胞术获取肝组织驻留（tr）-NKT细胞，检测VDR表达、p-STAT1和P-STAT-5的分子途径以及CD107a和NKp46的激活标志物。
维生素D治疗后，H&E染色显示微血管和大血管脂肪变性减轻，而天狼星红染色显示肝纤维化小鼠的纤维化积累少于未治疗的小鼠。结果与血清细胞因子白细胞介素-β/白细胞介素-6/白细胞介素-4/OPN/肿瘤坏死因子-α以及血清AST和ALT显著降低相关，分别降低了2倍和3倍。维生素D治疗后，肝纤维化小鼠的纤维化标志物αSMA、CREB和III型胶原平均降低了1.3倍。肝纤维化小鼠经维生素D治疗后，定量的肝脏胆固醇和甘油三酯、血清胰岛素和空腹血糖水平均有所改善。OPN中和抗体使trNKT细胞上的VDR过表达，并使CD107a和NKp46活性分别增加3.1倍和3.块，这与p-STAT1和p-STAT5磷酸化增加有关。这些结果伴随着肝星状细胞激活标志物αSMA、III型胶原和结蛋白的减少。
VDR表达影响trNKT细胞活性，并可调节肝纤维化进程。使用OPN中和抗体通过减轻NKT细胞介导的肝损伤表现出抗纤维化作用。它也被认为是肝纤维化的免疫调节靶点。
