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Siegesbeckia orientalis L. (SO) is a traditional Chinese herbal medicine commonly used for inflammatory diseases. In ancient, SO is applied for stroke, but mechanisms have not been clear. The purpose is to investigate whether SO exerts neuroprotective effects by reducing microglia-related inflammatory injury after focal cerebral ischemia/reperfusion (I/R).
SO was extracted using 50% ethanol and quality control was performed by Waters ACQUITY-UPLC CLASS system. The focal cerebral I/R model was established in mice, neurological functions, weight loss and infarct volume was evaluated. Microglial status in penumbra was monitored by immunofluorescence staining and morphology. Mouse primary microglia was subjected to lipopolysaccharide stimulation for triggering inflammatory response, meanwhile microglia-neuron co-culture model was established in vitro. Apoptosis and pyroptosis for neurons were demonstrated using TUNEL assay, western blot and ELISA. Inflammatory cytokines were detected by qPCR and ELISA.
SO treatment reduced infarct volume, improved neurological functions, lessened neuronal apoptosis and pyroptosis shown by increased Bcl-2/Bax ratio, decreased cleaved Caspase-3 and suppressed NLRP3/Caspase-1/GSDMD expression after I/R. Moreover, microglial status from pro-inflammation to anti-inflammation was characterized by morphological change and increasing percentage of CD206/Iba1 positive cells after SO treatment. Additionally, SO decreased TLR4 and nuclear-located p65 expression, suppressed IL-1β, IL-6, IL-18, and TNF-α, but increased IL-10 secretion both in vivo and vitro.
SO reverses pro-inflammatory status of microglia as evidenced by suppression of TLR4/NF-κB cascade, down-regulation of pro-inflammatory cytokines and up-regulation of anti-inflammatory cytokines, which contributes to its neuroprotective against neuronal pyroptosis and apoptosis after ischemic stroke.
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