State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, China.
Collaborative Innovation Center of Marine Science and Technology, Hainan University, Haikou, China.
Front Immunol. 2023 Mar 16;14:1129800. doi: 10.3389/fimmu.2023.1129800. eCollection 2023.
B-cell lymphoma-2 (Bcl-2) is the first identified member of the Bcl-2 family that performs an anti-apoptotic function in mammals. However, its role in teleosts is not fully understood. In this study, Bcl-2 of (TroBcl2) was cloned, and its role in apoptosis was investigated.
In this study, Bcl-2 of (TroBcl2) was cloned by PCR. Quantitative real-time PCR (qRT-PCR) was used to detect its mRNA expression level in healthy condition and after LPS stimulation. Subcellular localization was performed by transfecting the pTroBcl2-N3 plasmid into golden pompano snout (GPS) cells and observed under an inverted fluorescence microscope DMi8 and further verified by immunoblotting. overexpression and RNAi knockdown method were performed to evaluate the role of TroBcl2 in apoptosis. The anti-apoptotic activity of TroBcl2 was detected by flow cytometry. The effect of TroBcl2 on the mitochondrial membrane potential (MMP) was measured by an enhanced mitochondrial membrane potential assay kit with JC-1. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was performed to evaluate the role of TroBcl2 in the DNA fragmentation. Immunoblotting was used to verify whether TroBcl2 inhibits the release of cytochrome c from mitochondria into the cytoplasm. The Caspase 3 and Caspase 9 Activity Assay Kits were used to investigate the effect of TroBcl2 on caspase 3 and caspase 9 activities. The effects of TroBcl2 on the expression of apoptosis-related and nuclear factor- κB (NF-κB) signaling pathway-related genes were evaluated by qRT-PCR and Enzyme linked immunosorbent assay (ELISA). Luciferase reporter assay was used to evaluate the activity in NF-κB signaling pathway.
The full-length coding sequence of TroBcl2 contains 687 bp and encodes a protein containing 228 amino acids. Four conserved Bcl-2 homology (BH) domains and one invariant "NWGR" motif located in BH1 were identified in TroBcl2. In healthy , TroBcl2 was widely distributed in the eleven tested tissues, and higher expression levels were found in immune-related tissues, such as spleen and head kidney tissues. After stimulation with lipopolysaccharide (LPS), the expression of TroBcl2 in the head kidney, spleen, and liver was significantly upregulated. In addition, subcellular localization analysis revealed that TroBcl2 was localized in both the cytoplasm and nucleus. Functional experiments showed that TroBcl2 inhibited apoptosis, possibly by reducing mitochondrial membrane potential loss, decreasing DNA fragmentation, preventing cytochrome c release into cytoplasm, and reducing the caspase 3 and caspase 9 activations. Moreover, upon LPS stimulation, overexpression of TroBcl2 suppressed the activation of several apoptosis-related genes, such as , and . Furthermore, knockdown of TroBcl2 significantly increased the expression of those apoptosis-related genes. In addition, TroBcl2 overexpression or knockdown induced or inhibited, respectively, the transcription of NF-κB and regulated the expression of genes (such as and ) in the NF-κB signaling pathway as well as the expression of the downstream inflammatory cytokine . Overall, our study suggested that TroBcl2 performs its conserved anti-apoptotic function via the mitochondrial pathway and may serve as an anti-apoptotic regulator in .
B 细胞淋巴瘤-2（Bcl-2）是哺乳动物中第一个被鉴定出具有抗细胞凋亡功能的 Bcl-2 家族成员。然而，其在硬骨鱼类中的作用尚不完全清楚。在本研究中，克隆了 （TroBcl2）的 Bcl-2，并研究了其在细胞凋亡中的作用。
本研究通过 PCR 克隆了 （TroBcl2）的 Bcl-2。采用定量实时 PCR（qRT-PCR）检测其在健康状态和 LPS 刺激后的 mRNA 表达水平。通过将 pTroBcl2-N3 质粒转染到金黄 pompano 鼻（GPS）细胞中，并在倒置荧光显微镜 DMi8 下观察，进行亚细胞定位，并用免疫印迹进一步验证。通过过表达和 RNAi 敲低方法评估 TroBcl2 在细胞凋亡中的作用。通过流式细胞术检测 TroBcl2 的抗凋亡活性。使用增强型线粒体膜电位测定试剂盒（JC-1）测量 TroBcl2 对线粒体膜电位（MMP）的影响。通过末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）法评估 TroBcl2 对 DNA 片段化的作用。免疫印迹用于验证 TroBcl2 是否抑制细胞色素 c 从线粒体释放到细胞质中。使用 Caspase 3 和 Caspase 9 活性测定试剂盒研究 TroBcl2 对 caspase 3 和 caspase 9 活性的影响。通过 qRT-PCR 和酶联免疫吸附测定（ELISA）评估 TroBcl2 对凋亡相关和核因子-κB（NF-κB）信号通路相关基因表达的影响。通过荧光素酶报告基因测定评估 NF-κB 信号通路的活性。
TroBcl2 的全长编码序列包含 687 bp，编码一个含有 228 个氨基酸的蛋白质。在 TroBcl2 中鉴定出四个保守的 Bcl-2 同源（BH）结构域和一个位于 BH1 中的不变“NWGR”基序。在健康的 中，TroBcl2 在 11 种检测组织中广泛分布，在免疫相关组织（如脾脏和头肾组织）中表达水平较高。在 LPS 刺激后，头肾、脾脏和肝脏中的 TroBcl2 表达明显上调。此外，亚细胞定位分析表明 TroBcl2 定位于细胞质和细胞核中。功能实验表明，TroBcl2 抑制细胞凋亡，可能是通过减少线粒体膜电位丧失、减少 DNA 片段化、阻止细胞色素 c 释放到细胞质中以及降低 caspase 3 和 caspase 9 的活性来实现的。此外，在 LPS 刺激下，TroBcl2 的过表达抑制了几个凋亡相关基因的激活，如 、 和 。此外，TroBcl2 的敲低显著增加了这些凋亡相关基因的表达。此外，TroBcl2 的过表达或敲低诱导或抑制了 NF-κB 信号通路中的 NF-κB 和调节基因（如 和 ）的转录以及下游炎症细胞因子 的表达。总体而言，我们的研究表明 TroBcl2 通过线粒体途径发挥其保守的抗细胞凋亡功能，并且可能作为 的抗凋亡调节剂发挥作用。
Zotero 插件
