基于 RNA-seq 的转录组分析西红花乙醇提取物对皮质酮诱导的 PC12 细胞损伤的保护作用。
RNA-seq based transcriptome analysis of ethanol extract of saffron protective effect against corticosterone-induced PC12 cell injury.
机构信息
Department of Pharmacy, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, China.
Department of Pharmacology, Capital Medical University School of Basic Medical Sciences, Beijing, 100069, China.
出版信息
BMC Complement Med Ther. 2022 Jan 31;22(1):29. doi: 10.1186/s12906-022-03516-1.
BACKGROUND
At present, oral antidepressants are commonly used in the clinical treatment of depression. However, the current drug treatment may lead to more serious adverse reactions. Therefore, we focus on Chinese traditional medicine, trying to find an effective and safe alternative or complementary medicine. Crocus sativus (saffron) is a traditional Chinese herbal medicine, which is typically used in the clinic to regulate anxiety, insomnia, amnesia, and other mental disorder. The study aimed to explore the neuroprotective effect of ethanol extract of saffron (EES) on corticosterone (CORT)- induced injury in PC12 cells and further explored its potential mechanism.
METHODS
The authenticity of saffron and the active components of EES were identified by a water test and ultra-performance liquid chromatography-time of flight mass spectrometry system. The screening of cytotoxicity for PC12 cells was incubated with EES in different concentrations for 24 h, and the protective efficacy of EES on CORT (500 μM) -induced PC12 cell injury, cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay. The differentially expressed genes (DEGs) of EES-protected PC12 cells were analyzed using the RNA-seq method, and the results were analyzed for GO and KEGG enrichment. The results of RNA-seq were verified by qPCR analysis.
RESULTS
The saffron was initially identified as authentic in the water test and 10 compounds were identified by Ultra Performance Liquid Chromatography (UPLC)- Mass Spectrometry (MS). The results of CCK-8 demonstrated that EES at concentrations above 640 μg/mL exerted a certain cytotoxic effect, and PC12 cells pretreated with EES (20, 40, and 80 μg/mL) significantly reversed the 500 μM CORT-induced cell death. RNA-seq analysis showed that EES regulated 246 differential genes, which were mainly enriched in the MAPK signaling pathway. Dusp5, Dusp6, Gadd45b, Gadd45G, and Pdgfc were further validated by qPCR. Experimental data showed that the results of qPCR were consistent with RNA-seq.
CONCLUSIONS
These findings provide an innovative understanding of the molecular mechanism of the protective effect of EES on PC12 cells at the molecular transcription level, and Dusp5, Dusp6, Gadd45b, Gadd45g, and Pdgfc may be potential novel targets for antidepressant treatment.
背景
目前，临床治疗抑郁症常使用口服抗抑郁药。但目前的药物治疗可能会导致更严重的不良反应。因此，我们专注于中药，试图寻找一种有效且安全的替代药物或补充药物。藏红花（西红花）是一种传统的中草药，临床上通常用于调节焦虑、失眠、健忘等精神障碍。本研究旨在探讨藏红花乙醇提取物（EES）对皮质酮（CORT）诱导的 PC12 细胞损伤的神经保护作用，并进一步探讨其潜在机制。
方法
通过水试验和超高效液相色谱-飞行时间质谱系统鉴定藏红花的真伪和 EES 的活性成分。将 EES 以不同浓度孵育 24 h 后，筛选对 PC12 细胞的细胞毒性，通过细胞计数试剂盒-8（CCK-8）法评估 EES 对 CORT（500 μM）诱导的 PC12 细胞损伤、细胞活力的保护作用。采用 RNA-seq 方法分析 EES 保护的 PC12 细胞的差异表达基因（DEGs），并对 GO 和 KEGG 富集进行分析。通过 qPCR 分析验证 RNA-seq 的结果。
结果
水试验初步鉴定藏红花为真品，超高效液相色谱-质谱联用仪鉴定出 10 种化合物。CCK-8 结果表明，EES 浓度高于 640μg/mL 时会产生一定的细胞毒性，EES（20、40 和 80μg/mL）预处理可显著逆转 500μM CORT 诱导的细胞死亡。RNA-seq 分析表明，EES 调节了 246 个差异基因，主要富集在 MAPK 信号通路中。进一步通过 qPCR 验证 Dusp5、Dusp6、Gadd45b、Gadd45G 和 Pdgfc。实验数据表明，qPCR 结果与 RNA-seq 一致。
结论
这些发现为 EES 对 PC12 细胞的保护作用在分子转录水平上的分子机制提供了新的认识，Dusp5、Dusp6、Gadd45b、Gadd45g 和 Pdgfc 可能是抗抑郁治疗的潜在新靶点。
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