Scutellarin, an herbal agent, is known to possess anti-oxidant and anti-in ammatory properties. In activated microglia, it has been reported that this is achieved through acting on the MAPKs, a key pathway that regulates microglia activation. This study sought to determine if scutellarin would affect the commonly described microglia phenotypes, namely, M1 and M2, thought to contribute to pro-and anti-in ammatory roles, respectively. This is in consideration of its potential effect on the polarization of microglia phenotypes that are featured prominently in cerebral ischemia. For this purpose, we have used an experimentally induced cerebral ischemia rat model and LPS-stimulated BV-2 cell model. Thus, by Western blot and immuno uorescence, we show here a noticeable increase in expression of M2 microglia markers, namely, CD206, Arg1, YM1/2, IL-4 and IL-10 in activated microglia both in vivo and in vitro. Remarkably, scutellarin treatment markedly augmented the increased expression of the respective markers in activated microglia. It is therefore suggested scutellarin can exert the polarization of activated microglia from M1 to M2 phenotype. Because M1 microglia are commonly known to be proin ammatory, while M2 microglia are anti-in ammatory and neuroprotective effect, it stands to reason therefore that with the increase of M2 microglia which became predominant by scutellarin, the local in ammatory response is ameliorated. More importantly, we have found that scutellarin promotes the M2 polarization through inhibiting the JNK and p38 signaling pathways, and concomitantly augmenting the ERK1/2 signaling pathway. This lends its strongly support from observations in LPS activated BV-2 microglia treated with p38 and JNK inhibitors in which expression of M2 markers was increased; on the other hand, in cells subjected to ERK1/2 inhibitor treatment, the expression was suppressed. In light of the above, MAPKs pathway is deemed to be a potential target of scutellarin in mitigating microglia mediated neuroin ammation in activated microglia.

Objective This study elucidated the neuroprotective mechanisms of scutellarin in microglia and astrocytes during the progression of neuropathology in cerebral ischemia. Methods Network pharmacology was first used to filtrate the core targets and pathways. Arising from this, JAK2/STAT3 signaling pathway was specifically identified and experimentally verified. Expression of JAK2/STAT3 signaling related proteins in TNC-1 astrocytes subjected to different treatments with BV-2 microglia conditioned medium (CM) was then analyzed by western blot and immunofluorescence staining. Along with the above, expression of the various biomarkers was also evaluated in astrocytes given pretreatment with AG490, the JAK2/STAT3 signaling inhibitor. In tandem, middle cerebral artery occlusion (MCAO) in rats was performed in different experimental groups to detect the expression of the above biomarkers in the cerebral astrocytes. Results Network pharmacology suggests that JAK2/STAT3 signaling pathway is one of the mechanisms by which scutellarin can mitigate the cerebral ischemia damage. In TNC-1 astrocytes, p-JAK2 and p-STAT3 expression was significantly up-regulated in microglia CM group. Scutellarin promoted the up-regulation of various markers, and of note, AG490 neutralized the effect of scutellarin. In vivo at 1, 3, and 7d after MCAO, p-JAK2 and p-STAT3 expression was significantly increased. Consistent with in vitro results, the rise in expression was augmented by scutellarin. Conclusion It is concluded from this study that scutellarin exerts its neuroprotective effects via activated microglia by activating the astrocyte JAK2/STAT3 signaling pathway. This has given the herbal compound a firm experimental basis for its clinical application.

Scutellarin is used to treat brain ischaemia. However, its underlying mechanism of action remains unclear. This study aimed to elucidate the potential mechanism of action of scutellarin in brain ischaemia through network pharmacology and experimental verification. The JAK2/STAT3 signalling pathway was identified and experimentally verified. Expression of JAK2/STAT3 signalling related proteins in TNC-1 astrocytes with BV-2 microglia-conditioned medium (CM), CM + lipopolysaccharide (LPS) (CM + L), and CM pretreated with scutellarin + LPS (CM + SL) was analysed by Western Blot and immunofluorescence staining. Expression levels of JAK2, p-JAK2, STAT3, and p-STAT3 were evaluated in astrocytes pre-treated with AG490. Middle cerebral artery occlusion (MCAO) in rats was performed in different experimental groups to detect expression of the above biomarkers. Network pharmacology suggested that the JAK2/STAT3 signalling pathway is one of the mechanisms by which scutellarin mitigates cerebral ischaemic damage. In TNC-1 astrocytes, p-JAK2 and p-STAT3 expression were significantly up-regulated in the CM + L group. Scutellarin promoted the up-regulation of various markers and AG490 neutralised the effect of scutellarin. In vivo, up-regulation of p-JAK2 and p-STAT3 after ischaemia is known. These results are consistent with previous reports. Scutellarin further enhanced this upregulation at 1, 3, and 7 d after MCAO. Scutellarin exerts its therapeutic effects on cerebral ischaemia by activating the astrocyte JAK2/STAT3 signalling, which provides a firm experimental basis for its clinical application.

Scutellarin, an herbal agent, is known to possess anti-oxidant and anti-inflammatory properties. In activated microglia, it has been reported that this is achieved through acting on the MAPKs, a key pathway that regulates microglia activation. This study sought to determine if scutellarin would affect the commonly described microglia phenotypes, namely, M1 and M2, thought to contribute to pro- and anti-inflammatory roles, respectively. This is in consideration of its potential effect on the polarization of microglia phenotypes that are featured prominently in cerebral ischemia. For this purpose, we have used an experimentally induced cerebral ischemia rat model and LPS-stimulated BV-2 cell model. Thus, by Western blot and immunofluorescence, we show here a noticeable increase in expression of M2 microglia markers, namely, CD206, Arg1, YM1/2, IL-4 and IL-10 in activated microglia both in vivo and in vitro. Remarkably, scutellarin treatment markedly augmented the increased expression of the respective markers in activated microglia. It is therefore suggested scutellarin can exert the polarization of activated microglia from M1 to M2 phenotype. Because M1 microglia are commonly known to be proinflammatory, while M2 microglia are anti-inflammatory and neuroprotective effect, it stands to reason therefore that with the increase of M2 microglia which became predominant by scutellarin, the local inflammatory response is ameliorated. More importantly, we have found that scutellarin promotes the M2 polarization through inhibiting the JNK and p38 signaling pathways, and concomitantly augmenting the ERK1/2 signaling pathway. This lends its strongly support from observations in LPS activated BV-2 microglia treated with p38 and JNK inhibitors in which expression of M2 markers was increased; on the other hand, in cells subjected to ERK1/2 inhibitor treatment, the expression was suppressed. In light of the above, MAPKs pathway is deemed to be a potential target of scutellarin in mitigating microglia mediated neuroinflammation in activated microglia.