Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, 210009, China.
School of Pharmacy, Bengbu Medical College, Bengbu, 233030, China; Anhui Province Engineering Technology Research Center of Biochemical Pharmaceutical, Bengbu, 233030, China.
J Ethnopharmacol. 2023 Nov 15;316:116729. doi: 10.1016/j.jep.2023.116729. Epub 2023 Jun 3.
ETHNOPHARMACOLOGICAL RELEVANCE: Saikosaponins B2 (SSB2) is one of the main active components isolated from Radix Bupleuri (Bupleurum chinense DC.), a herb widely used of traditional Chinese medicine. It has been used for the treatment of depression for more than two thousand years. However, the molecular mechanisms remain to be determined. AIM OF THE STUDY: In this study, we evaluated the anti-inflammatory effect and elucidated underlying molecular mechanisms of SSB2 in LPS-induced primary microglia and CUMS-induced mice model of depression. METHOD: The effects of SSB2 treatment were investigated both in vitro and in vivo. The chronic unpredictable mild stimulation (CUMS) procedure was applied to establish the animal model of depression. Behavioural tests were used to evaluate the depressive-like behaviors in CUMS-exposed mice, including sucrose preference test, open field test, tail suspension test, and forced swimming test. The GPX4 gene of microglia was silenced using shRNA, and inflammatory cytokines were determined by Western Blot and immunofluorescence analysis. Endoplasmic reticulum stress and ferroptosis-related markers were detected by qPCR, flow cytometry and confocal microscopy. RESULT: SSB2 reversed depressive-like behaviours in CUMS-exposed mice and relieved central neuroinflammation and ameliorated hippocampal neural damage. SSB2 alleviated LPS-induced activation of microglia through the TLR4/NF-κB pathway. LPS-induced ferroptosis, with increased levels of ROS, intracellular Fe, mitochondrial membrane potential, lipid peroxidation, GSH, SLC7A11, FTH, GPX4 and Nrf2, and decreased transcription levels of ACSL4 and TFR1, was attenuated with SSB2 treatment in primary microglia cells. GPX4 knockdown activated ferroptosis, induced endoplasmic reticulum (ER) stress, and abrogated the protective effects of SSB2. Further, SSB2 attenuated ER stress, balanced calcium homeostasis, reduced lipid peroxidation and intracellular Fe content by regulating the level of intracellular Ca. CONCLUSIONS: Our study suggested that SSB2 treatment can inhibit ferroptosis, maintain calcium homeostasis, relieve endoplasmic reticulum stress and attenuate central neuroinflammation. SSB2 exhibited anti-ferroptosis and anti-neuroinflammatory effects through the TLR4/NF-κB pathway in a GPX4-dependent manner.
民族药理学相关性：柴胡皂苷 B2（SSB2）是从柴胡（Bupleurum chinense DC.）中分离得到的主要活性成分之一，柴胡是一种广泛应用于传统中药的草药。它已被用于治疗抑郁症超过两千年。然而，其分子机制仍有待确定。
研究目的：本研究评估了 SSB2 在 LPS 诱导的原代小胶质细胞和 CUMS 诱导的抑郁小鼠模型中的抗炎作用，并阐明其潜在的分子机制。
研究方法：采用体内外方法研究 SSB2 的作用。采用慢性不可预知性轻度刺激（CUMS）程序建立抑郁动物模型。行为测试用于评估 CUMS 暴露小鼠的抑郁样行为，包括蔗糖偏好试验、旷场试验、悬尾试验和强迫游泳试验。使用 shRNA 沉默小胶质细胞的 GPX4 基因，通过 Western Blot 和免疫荧光分析测定炎性细胞因子。通过 qPCR、流式细胞术和共聚焦显微镜检测内质网应激和铁死亡相关标志物。
研究结果：SSB2 逆转了 CUMS 暴露小鼠的抑郁样行为，减轻了中枢神经炎症，改善了海马神经损伤。SSB2 通过 TLR4/NF-κB 通路逆转 LPS 诱导的小胶质细胞激活。LPS 诱导的铁死亡，表现为 ROS 水平升高、细胞内 Fe 增加、线粒体膜电位降低、脂质过氧化、GSH 降低、SLC7A11、FTH、GPX4 和 Nrf2 转录水平降低，以及 ACSL4 和 TFR1 转录水平降低，这些现象均被 SSB2 处理减轻。在原代小胶质细胞中，GPX4 敲低激活铁死亡，诱导内质网（ER）应激，并消除 SSB2 的保护作用。此外，SSB2 通过调节细胞内 Ca2+水平，减轻 ER 应激，平衡钙稳态，减少脂质过氧化和细胞内 Fe 含量。
研究结论：本研究表明，SSB2 治疗可抑制铁死亡、维持钙稳态、减轻内质网应激和中枢神经炎症。SSB2 通过 TLR4/NF-κB 通路、依赖 GPX4 发挥抗铁死亡和抗炎作用。
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