Harpagide alleviate neuronal apoptosis and blood-brain barrier leakage by inhibiting TLR4/MyD88/NF-κB signaling pathway in Angiotensin II-induced microglial activation in vitro

Angiotensin II, the effector peptide of the renin-angiotensin system, is not only a pivotal peptide implicated in the regulation of blood pressure but also a key mediator of the inflammatory processes that play an important role in the pathology of hypertension-related cSVD. Harpagide is the major bioactive constituent of Scrophulariae Radix widely used in traditional Chinese medicine for numerous diseases including hypertension. The present study aimed to investigate the effect of harpagide on Ang II-induced neuroinflammation and the potential mechanism. Pretreated with harpagide or resatorvid (the TLR4 pathway inhibitor), BV2 cells were treated with Ang II or LPS (the TLR4 activator). NO, pro-inflammatory cytokines, the proteins on TLR4/MyD88/NF-κB signaling pathway and the expression of CD86, CD206, TREM2 in BV2 cells were detected respectively. Subsequently, the effects of harpagide on neurotoxicity and BBB destruction triggered by Ang II-induced neuroinflammation were investigated in the co-cultures of BV2 microglia/HT22 hippocampal neurons, BV2 microglia/bEnd.3 endotheliocyte and BV2 microglia/BBB monolayer model. We found that Ang II converted microglia into M1 state and resulted in neuroinflammation through activating TLR4/MyD88/NF-κB signaling pathway. It also triggered the imbalance of TLR4/TREM2 in microglia. Ang II-mediated inflammation microglia further led to neuronal apoptosis and BBB damage. Harpagide showed the effect of alleviating Ang II-mediated neuroinflammation as well as the resulting neurotoxicity and BBB destruction through inhibiting the TLR4/MyD88/NF-κB pathway. The anti-inflammatory and neuroprotective effect of harpagide suggested that it might be a potential therapeutic strategy in hypertensive cSVD.