Inhibitory Effect of Crocin on Pituitary Adenomas via IRF7/NF-κB Signaling Pathway
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摘要:
目的 通过临床样本及垂体腺瘤HP75细胞的相关分子生物学实验探讨藏红花素（Crocin）在垂体腺瘤（PA）中的作用及其机制。 方法 收集2022年6月至2023年5月昆明医科大学第一附属医院神经外二科及耳鼻咽喉颅底外科16例PA样本，3例正常对照垂体组织样本来自于昆明医科大学法医学院人体解剖。通过对临床样本检测IRF7 mRNA表达量，敲低HP75细胞IRF7表达检测增殖、迁移、侵袭及凋亡能力；进一步检测HP75细胞中IRF7调控NF-κB表达，以及藏红花素调控PA细胞的生长及其对IRF7/NF-κB信号通路调控作用。 结果 RT-qPCR检测及免疫组化显示，与正常对照组相比，PA中IRF7 mRNA的表达量增加（P < 0.001）；si-IRF7组的IRF7蛋白表达量降低（P < 0.001）；CCK-8、Transwell及流式细胞术检测结果显示，与对照组相比，敲低IRF7降低HP75细胞的细胞活力（P < 0.001），抑制HP75细胞的迁移和侵袭（P < 0.001），促进HP75细胞凋亡（P < 0.001）。此外，敲低IRF7能抑制p-NF-κB p65/NF-κB p65的表达（P < 0.001），抑制p-NF-κB p65/NF-κB p65的表达（P < 0.001）；而过表达IRF7能部分逆转Crocin的作用（P < 0.001），回复p-NF-κB p65/NF-κB p65的部分表达（P < 0.01）；最后，HP75细胞的生物学行为检测结果显示，与Crocin组相比，过表达IRF7能提高HP75细胞的细胞活力，同时促进其迁移和侵袭，抑制细胞凋亡（P < 0.001）。 结论 Crocin处理能抑制PA细胞的增殖、迁移和侵袭，并促进细胞凋亡，缓解PA的发展进程。在机制上，IRF7在PA中高表达，敲低IRF7能抑制PA的恶性生长；Crocin抑制PA细胞增殖、迁移、侵袭，促进细胞凋亡的作用可通过抑制IRF7/NF-κB信号通路实现。 Abstract:Objective To explore the role and mechanism of crocin in pituitary adenoma （PA） through clinical samples and related molecular biology experiments of HP75 cells. Methods From June 2022 to May 2023, 16 PA samples were collected from the Second Department of Neurology and Otolaryngology skull base surgery of the First Affiliated Hospital of Kunming Medical University. Three normal control samples were from the human anatomy of the Forensic College of Kunming Medical University. The expression of IRF7 mRNA in clinical samples was detected, and the proliferation, migration, invasion and apoptosis of HP75 cells were detected by knocking down the expression of IRF7; the expression of NF-κB was regulated by IRF7 in HP75 cells, and crocin regulated the growth of PA cells and its regulatory effect on IRF7/NF-κB signaling pathway. Results RT-qPCR and immunohisto-chemistry showed that compared with the normal control group, the expression of IRF7 mRNA in PA was significantly increased （P < 0.001）; the expression of IRF7 protein in si-IRF7 group was significantly decreased （P < 0.001）; CCK-8, Transwell and flow cytometry results showed that compared with the control group, knockdown of IRF7 significantly decreased the cell viability of HP75 cells （P < 0.001）, inhibited the migration and invasion （P < 0.001）, and promoted the apoptosis of HP75 cells （P < 0.001）. In addition, knockdown of IRF7 could inhibit the expression of p-NF-κB p65/NF-κB p65 （P < 0.001） and p-NF-κB p65/NF-κB p65 （P < 0.001）. Overexpression of IRF7 partially reversed the effect of crocin （P < 0.001） and restored the expression of p-NF-κB p65/NF-κB p65 （P < 0.01）. Finally, the biological behavior of HP75 cells showed that compared with crocin group, overexpression of IRF7 could improve the cell viability of HP75 cells, promote their migration and invasion, and inhibit cell apoptosis （P < 0.001）. Conclusion Crocin treatment can inhibit the proliferation, migration and invasion of PA cells, promote cell apoptosis, and alleviate the development of PA. In the mechanism, IRF7 is significantly overexpressed in PA, and knockdown of IRF7 can inhibit the malignant growth of PA. Crocin can inhibit the proliferation, migration and invasion of PA cells, and promote apoptosis by inhibiting IRF7/NF-κB signaling pathway. -
Key words:
- Pituitary adenoma /
- Crocetin /
- IRF7/NF -κB signaling pathway /
- Migration /
- Invasion /
- Apoptosis
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图 1 IRF7在垂体腺瘤中高表达
A：RT-qPCR检测16例PA临床样本中IRF7 mRNA的表达情况（$\bar x \pm s $）；B：免疫组化检测临床样本中IRF7的表达情况（DAB染色，200×）。***P < 0.001。
Figure 1. High expression of IRF7 was observed in pituitary adenomas
图 2 敲低IRF7抑制垂体腺瘤细胞增殖、迁移、侵袭，促进凋亡（$\bar x \pm s $，n = 3）
A：Western blot检测IRF7的转染效率（A1：Western blot检测IRF7的蛋白表达电泳图，A2：IRF7蛋白表达的统计学分析）；B：CCK-8检测HP75细胞的细胞活力；C：Transwell检测HP75细胞迁移和侵袭（100×）（C1：Transwell检测HP75细胞迁移和侵袭，C2：HP75细胞迁移统计学分析，C3：HP75细胞侵袭统计学分析）；D：流式细胞术检测HP75细胞凋亡（D1：流式细胞术检测HP75细胞凋亡，D2：HP75细胞凋亡统计学分析）。***P < 0.001。
Figure 2. Knocking down IRF7 inhibited the proliferation, migration, and invasion of pituitary adenoma cells and promoted apoptosis（$\bar x \pm s $，n = 3）
图 3 敲低IRF7抑制NF-κB信号通路（$\bar x \pm s $，n = 3）
A：Western blot检测NF-κB p65和p-NF-κB p65的表达（A1：Western blot检测NF-κB p65和p-NF-κB p65的蛋白表达电泳图，A2：NF-κB p65和p-NF-κB p65蛋白表达的统计学分析）；B：免疫荧光检测NF-κB p65的表达（1000×）。**P < 0.01。
Figure 3. Knocking down IRF7 inhibited the NF-κB signaling pathway（$\bar x \pm s $，n = 3）
图 4 藏红花素抑制垂体腺瘤细胞恶性生长，且调控IRF7/NF-κB信号通路（$\bar x \pm s $，n = 3）
A：Crocin浓度梯度处理后CCK-8检测HP75细胞的细胞活力；B：CCK-8检测HP75细胞的细胞活力；C：Transwell检测HP75细胞迁移和侵袭（100×）（C1：Transwell检测HP75细胞迁移和侵袭，C2：HP75细胞迁移统计学分析，C3：HP75细胞侵袭统计学分析）；D：流式细胞术检测HP75细胞凋亡（D1：流式细胞术检测HP75细胞凋亡，D2：HP75细胞凋亡统计学分析）；E：Western blot检测IRF7/NF-κB信号通路相关蛋白的表达水平（E1：Western blot检测IRF7/NF-κB信号通路相关蛋白表达的电泳图，E2：IRF7/NF-κB信号通路相关蛋白表达的统计学分析）；F：Western blot检测IRF7的核转移。***P < 0.001。
Figure 4. Crocin inhibited the malignant growth of pituitary adenoma cells and regulated the IRF7/NF-κB signaling pathway（$\bar x \pm s $，n = 3）
图 5 藏红花素抑制垂体腺瘤细胞恶性生长，调控IRF7/NF-κB信号通路（$\bar x \pm s $，n = 3）
A：Western blot检测IRF7的过表达效率（A1：Western blot检测IRF7蛋白表达的电泳图，A2：IRF7蛋白表达的统计学分析）；B：Western blot检测NF-κB p65和p-NF-κB p65的表达（B1：Western blot检测NF-κB p65和p-NF-κB p65蛋白表达的电泳图，B2：NF-κB p65和p-NF-κB p65蛋白表达的统计学分析）；C：CCK-8检测HP75细胞的细胞活力；D：Transwell检测HP75细胞迁移和侵袭（100×）（D1：HP75细胞迁移统计学分析，D2：HP75细胞侵袭统计学分析，D3：Transwell检测HP75细胞迁移和侵袭）；E：流式细胞术检测HP75细胞凋亡（E1：流式细胞术检测HP75细胞凋亡，E2：HP75细胞凋亡统计学分析）。**P < 0.01、***P < 0.001。
Figure 5. Crocin inhibited the malignant growth of pituitary adenoma cells and regulated the IRF7/NF-κB signaling pathway（$\bar x \pm s $，n = 3）
表 1 引物序列
Table 1. Primer sequence
靶基因 序列（F：正向引物；R：反向引物）（5′-3′） IRF7 F：GCTGGACGTGACCATCATGTA R：GGGCCGTATAGGAACGTGC β-actin F：CATGTACGTTGCTATCCAGGC R：CTCCTTAATGTCACGCACGAT 
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