IH treatment significantly improve cognitive impairment in APP/PS1 mice
To illustrate whether IH improves cognitive impairment, APP/PS1 mice were subjected to Morris water maze. The results showed that after IH treatment, the latency time for mice to find the platform was significantly reduced (Fig 1A). The number of mice crossing the platform was significantly more than that of the vehicle group (Fig 1B). The results suggested that IH could significantly improve the cognitive impairment of mice.
IH treatment alleviated Aβ deposition in APP/PS1 mice
In order to explore the mechanism of IH improving behavior, we detected the generation of senile plaques by immunofluorescence. The results showed that the senile plaques in the brain of mice decreased significantly after 3 months of IH treatment (Fig2).
IH up-regulated the expression of ADAM10
Aβ is produced by the cleavage of APP through amyloid pathway, BACE1 and γ-secretase are the main cleavage enzymes. The results showed that IH had no effect on the expression of BACE1 and γ-secretase (Fig 3A-B). However, in this study, we found that ADAM10, as the main enzyme in the non-amyloid pathway, can be significantly up regulate its expression by IH (Fig 3A-B) and then reduce the production of Aβ.
IH inhibited oxidative stress in APP/PS1 mice
Oxidative stress plays an important role in the pathogenesis of AD. Over-production of reactive oxygen species (ROS) and disorder of antioxidant capacity result in subsequent oxidative damage . To illustrate the effects of IH on oxidative stress, we investigated the levels of ROS, SOD and GSH in brain tissue of mice. The results showed that the fluorescence intensity of DCFH-DA was significantly decreased after IH treatment (Fig 4A). The level of SOD and GSH was enhanced compared to the vehicle group (Fig 4B).
gp91 has been recognized as biomarkers of oxidative stress . In this study, IH significantly decreased the level of gp91 (Figure 4C). Taken together, these results illustrated that IH could alleviate oxidative stress in APP/PS1 mice.
A large number of studies have shown that the transcriptional factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) plays an important defensive role in the antioxidant response of the brain by promoting the expression of several antioxidant enzymes . In our paper, we found that IH increased the expression of Nrf2 to against oxidative stress (Fig 4C).
Isorhamnetin reduced inflammatory response in APP/PS1 mice
To confirm if IH could inhibit inflammatory response in AD, we detected the release of inflammatory factors and astrocytes activation. The results found that after IH treatment, the expression of TNF-α and IL-1β were decreased (Fig 5A-B). Western blotting and immunofluorescence results showed that IH could inhibit astrocytes activation induced by Aβ (Fig 5B).
Isorhamnetin activated Sirt1/AKT/ERK/CREB signaling pathways
Previous study showed that CREB, the promoter of ADAM10, could up- regulate CREB and promote the expression of ADAM10 . ERK is the upstream of CREB, it can be activated by Akt and its activation can promote the phosphorylation of CREB . Our results showed the IH significantly promoted phosphorylation level of Akt, ERK and CREB (Fig 6A-B). Notably, expression of Sirt1 activated the Akt signaling pathway, and IH could increase the Sirt1 expression (Fig 6A-B).