Network Pharmacology Analysis and Experimental Validation of Kaempferol in the Treatment of Ischemic Stroke by Inhibiting Apoptosis and Regulating Neuroinflammation Involving Neutrophils

Abstract

Kaempferol, a natural plant flavonoid compound, has a neuroprotective effect on ischemic stroke, while the specific mechanism remains unclear. In the current study, we applied the comprehensive strategy that combines network pharmacology and experimental evaluation to explore the potential mechanism of kaempferol in the treatment of cerebral ischemia. First, network pharmacology analysis identified the biological process of kaempferol, suggesting that kaempferol may partly help in treating ischemic stroke by regulating apoptosis and inflammatory response. Then, we evaluated the efficacy of kaempferol in the acute stage of ischemic stroke and elucidated its effects and possible mechanisms on cell apoptosis and neuroinflammation involved by neutrophils. The results showed that kaempferol could significantly reduce the modified neurological severity score (mNSS), and reduce the volume of cerebral infarction and the degree of cerebral edema. In terms of anti-apoptosis, kaempferol could significantly reduce the number of TUNEL-positive cells, inhibit the expression of pro-apoptotic proteins and promote the expression of anti-apoptotic proteins. Kaempferol may play an anti-apoptotic role by up-regulating the expression level of the BDNF-TrkB-PI3K/AKT signaling pathway. In addition, we found that kaempferol inhibited neuron loss and the activation of glial cells, as well as the expression level of the inflammatory protein COX-2 and the classic pro-inflammatory signaling pathway TLR4/MyD88/NF-κB in the ischemic brain, reduced MPO activity and neutrophil counts in peripheral blood, and down-regulated neutrophil aggregation and infiltration in the ischemic brain. Western blot revealed that kaempferol down-regulated the activation of the JAK1/STAT3 signaling pathway in neutrophils and ischemic brains. Our study showed that kaempferol inhibited the activation and number of neutrophils in the rat peripheral blood and brain, which may be related to the down-regulation of the JAK1/STAT3 pathway.