Background Atherosclerosis (AS) is a pathological vascular disorder responsible for the majority of cardiovascular deaths. Sarsasapogenin (Sar) is a natural steroidal compound which has been extensively applied to multiple human diseases due to its pharmacological properties. In the present paper, the impacts of Sar on oxidized low-density lipoprotein (ox-LDL)-treated vascular smooth muscle cells (VSMCs) and its possible action mechanism were investigated. Methods Firstly, Cell Counting Kit-8 (CCK-8) estimated the viability of VSMCs following treatment with ascending doses of Sar. Then, VSMCs were treated by ox-LDL to stimulate an in vitro cell model of AS. CCK-8 and 5-Ethynyl-2’-deoxyuridine (EDU) assays were used to assess cell proliferation. Wound healing and transwell assays were applied to measure the migratory and invasive capacities, respectively. The expression of proliferation-, metastasis-, and stromal interaction molecule 1 (STIM1)/Orai signaling-associated proteins was measured by western blot. Results The experimental data illuminated that Sar treatment noticeably protected against ox-LDL-elicited VSMCs proliferation, migration, and invasion. Besides, Sar lowered the elevated STIM1 and Orai expression in ox-LDL-treated VSMCs. Further, STIM1 elevation partially abrogated the impacts of Sar on the proliferation, migration, and invasion of VSMCs challenged with ox-LDL. Conclusions In conclusion, Sar might reduce STIM1 expression to impede the aggressive phenotypes of ox-LDL-treated VSMCs.
Su Pan,Li Zibo,Yan Xiangli,Wang Baoying,Bai Ming,Li Yucheng,Xu Erping
Objective and design Mast cells (MCs), as the fastest immune responders, play a critical role in the progression of neuroinflammation-related diseases, especially in depression. Quercetin (Que) and kaempferol (Kae), as two major diet-derived flavonoids, inhibit MC activation and exhibit significant antidepressant effect due to their anti-inflammatory capacity. The study aimed to explore the mechanisms of inhibitory effect of Que and Kae on MC activation, and whether Que and Kae suppress hippocampal mast cell activation in LPS-induced depressive mice. Subjects and treatment In vitro assays, human mast cells (HMC-1) were pretreated with Que or Kae for 1 h, then stimulated by phorbol 12-myristate 13-acetate (PMA) and 2,5-di-t-butyl-1,4-benzohydroquinone (tBHQ) for 3 h or 12 h. In vivo assays, Que or Kae was administered by oral gavage once daily for 14 days and then lipopolysaccharide (LPS) intraperitoneally injection to induce depressive behaviors. Methods The secretion and expression of TNF-α were determined by ELISA and Western blotting. The nuclear factor of activated T cells (NFAT) transcriptional activity was measured in HMC-1 stably expressing NFAT luciferase reporter gene. Nuclear translocation of NFATc2 was detected by nuclear protein extraction and also was fluorescently detected in HMC-1 stably expressing eGFP-NFATc2. We used Ca 2+ imaging to evaluate changes of store-operated calcium entry (SOCE) in HMC-1 stably expressing fluorescent Ca 2+ indicator jGCamP7s. Molecular docking was used to assess interaction between the Que or Kae and calcium release-activated calcium modulator (ORAI). The hippocampal mast cell accumulation and activation were detected by toluidine blue staining and immunohistochemistry with β-tryptase. Results In vitro assays of HMC-1 activated by PtBHQ (PMA and tBHQ), Que and Kae significantly decreased expression and secretion of TNF-α. Moreover, NFAT transcriptional activity and nuclear translocation of NFATc2 were remarkably inhibited by Que and Kae. In addition, the Ca 2+ influx mediated by SOCE was suppressed by Que, Kae and the YM58483 (ORAI inhibitor), respectively. Importantly, the combination of YM58483 with Que or Kae had no additive effect on the inhibition of SOCE. The molecular docking also showed that Que and Kae both exhibit high binding affinities with ORAI at the same binding site as YM58483. In vivo assays, Que and Kae significantly reversed LPS-induced depression-like behaviors in mice, and inhibited hippocampal mast cell activation in LPS-induced depressive mice. Conclusions Our results indicated that suppression of SOCE/NFATc2 pathway-mediated by ORAI channels may be the mechanism of inhibitory effect of Que and Kae on MC activation, and also suggested Que and Kae may exert the antidepressant effect through suppressing hippocampal mast cell activation.
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