球状脂联素通过影响尾加压素Ⅱ诱导的血管外膜来源的肿瘤坏死因子-α的分泌介导血管重构。
Globular adiponectin-mediated vascular remodeling by affecting the secretion of adventitial-derived tumor necrosis factor-α induced by urotensin II.
机构信息
Department of Cardiology, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.
Division of Cardiology, Department of Internal Medicine, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, China.
出版信息
J Zhejiang Univ Sci B. 2022 Dec 15;23(12):1014-1027. doi: 10.1631/jzus.B2200346.
OBJECTIVES
In this study, we explored how adiponectin mediated urotensin II (UII)-induced tumor necrosis factor-α (TNF-α) and α-smooth muscle actin (α-SMA) expression and ensuing intracellular signaling pathways in adventitial fibroblasts (AFs).
METHODS
Growth-arrested AFs and rat tunica adventitia of vessels were incubated with UII and inhibitors of signal transduction pathways for 1‒24 h. The cells were then harvested for TNF-α receptor (TNF-α-R) messenger RNA (mRNA) and TNF-α protein expression determination by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Adiponectin and adiponectin receptor (adipoR) expression was measured by RT-PCR, quantitative real-time PCR (qPCR), immunohistochemical analysis, and cell counting kit-8 (CCK-8) cell proliferation experiments. We then quantified TNF-α and α-SMA mRNA and protein expression levels by qPCR and immunofluorescence (IF) staining. RNA interference (RNAi) was used to explore the function of the adipoR genes. To investigate the signaling pathway, we applied western blotting (WB) to examine phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK). In vivo, an adiponectin ()-knockout (APN-KO) mouse model mimicking adventitial inflammation was generated to measure TNF-α and α-SMA expression by application of qPCR and IF, with the goal of gaining a comprehensive atlas of adiponectin in vascular remodeling.
RESULTS
In both cells and tissues, UII promoted TNF-α protein and TNF-α-R secretion in a dose- and time-dependent manner via Rho/protein kinase C (PKC) pathway. We detected marked expression of adipoR1, T-cadherin, and calreticulin as well as a moderate presence of adipoR2 in AFs, while no adiponectin was observed. Globular adiponectin (gAd) fostered the growth of AFs, and acted in concert with UII to induce α-SMA and TNF-α through the adipoR1/T-cadherin/calreticulin/AMPK pathway. In AFs, gAd and UII synergistically induced AMPK phosphorylation. In the adventitial inflammation model, deficiency up-regulated the expression of α-SMA, UII receptor (UT), and UII while inhibiting TNF-α expression.
CONCLUSIONS
From the results of our study, we can speculate that UII induces TNF-α protein and TNF-α-R secretion in AFs and rat tunica adventitia of vessels via the Rho and PKC signal transduction pathways. Thus, it is plausible that adiponectin is a major player in adventitial progression and could serve as a novel therapeutic target for cardiovascular disease administration.
目的
在这项研究中，我们探讨了脂联素如何介导尾加压素 II（UII）诱导的肿瘤坏死因子-α（TNF-α）和α-平滑肌肌动蛋白（α-SMA）表达，并进一步探讨了随之发生的细胞内信号通路在血管外膜成纤维细胞（AFs）中的作用。
方法
用 UII 和信号转导通路抑制剂孵育生长停滞的 AFs 和大鼠血管外膜 1-24 小时。然后通过逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）分别检测 TNF-α 受体（TNF- α-R）信使 RNA（mRNA）和 TNF-α 蛋白的表达。通过 RT-PCR、实时定量 PCR（qPCR）、免疫组化分析和细胞计数试剂盒-8（CCK-8）细胞增殖实验检测脂联素和脂联素受体（adipoR）的表达。然后通过 qPCR 和免疫荧光（IF）染色定量检测 TNF-α 和 α-SMA mRNA 和蛋白的表达水平。采用 RNA 干扰（RNAi）技术探讨 adipoR 基因的功能。通过 Western blot（WB）检测腺苷酸 5'-单磷酸（AMP）激活的蛋白激酶（AMPK）的磷酸化来研究信号通路。在体内，通过应用 qPCR 和 IF 来测量 TNF-α 和 α-SMA 的表达，构建脂联素（）敲除（APN-KO）小鼠模型来模拟外膜炎症，以获得脂联素在血管重塑中的全面图谱。
结果
在细胞和组织中，UII 通过 Rho/蛋白激酶 C（PKC）通路以剂量和时间依赖的方式促进 TNF-α 蛋白和 TNF-α-R 的分泌。我们在 AFs 中检测到脂联素受体 1、T-钙粘蛋白和钙网蛋白的显著表达，以及脂联素受体 2 的中等表达，但未检测到脂联素。球形脂联素（gAd）促进 AFs 的生长，并通过 adipoR1/T-钙粘蛋白/钙网蛋白/AMPK 通路与 UII 协同诱导 α-SMA 和 TNF-α 的表达。在 AFs 中，gAd 和 UII 协同诱导 AMPK 磷酸化。在外膜炎症模型中，缺乏脂联素导致 α-SMA、UII 受体（UT）和 UII 的表达上调，同时抑制 TNF-α 的表达。
结论
根据我们的研究结果，我们可以推测 UII 通过 Rho 和 PKC 信号转导途径诱导 AFs 和大鼠血管外膜 TNF-α 蛋白和 TNF-α-R 的分泌。因此，脂联素可能是外膜进展的主要参与者，并可能成为心血管疾病治疗的新靶点。
Zotero 插件