The results of this study show that IH, a flavonoid, can significantly improve the cognitive impairment of APP/PS1 transgenic mice, reduce the deposition of senile plaques in the brain, up-regulate the expression of ADAM10 by activating SIRT1/Akt /ERK/CREB signal pathway, and promoting the non-amyloid pathway of APP. And IH also reduced the production of ROS in the brain, increased the expression and activity of antioxidant enzymes SOD1, GSH and the expression of Nrf2, inhibited inflammation, and improved the pathological process of AD.
Aβ is released from APP through sequential cleavages by BACE-1 and γ-secretase. In nonamyloidogenic pathway, APP can be cleaved by α-secretase and γ-secretase to inhibit Aβ production . In neurons, ADAM10 (metalloprotease) is considered the major α-secretases . Up-regulating ADAM10 can suppress Aβ production , suggesting that ADAM10 may serve as a potential target in arresting AD. Previous study showed that CREB as ADAM10 promoter was induced could increase the expression of ADAM10 to preclude Aβ production . The ERK-CREB signal pathway has been studied for its role in AD . Previous studies have shown that the neuroprotective effect of AD may be caused by CREB activation mediated by ERK activation, which promotes the expression of ADAM10 . Inhibiting ERK could lead to decrease phosphorylation level of CREB. In addition, artemisinin regulated the protective effects against Aβ1-42-induced damage by ERK/CREB pathway . According to these results, IH plays the protective effect in AD via promoting ADAM10 by activating ERK/CREB pathway.
Studies suggest that activation of phosphoinositide AKT may protect against neuronal cell death in AD . It has been found that activating the AKT signaling pathway could upregulate HO-1 expression and enhance nuclear translocation of Nrf2 . Upregulated phosphatidylinositide 3-kinase (PI3K)/AKT signaling decreased Aβ levels and Aβ deposition in brain and ameliorated toxicity in Aβ-treated primary neuronal culture . Moreover, AKT as an upstream kinase regulating ERK activation and AKT inhibitor or AKT siRNA could decrease the phosphorylation of ERK , suggesting that ERK phosphorylation is dependent on AKT. Sirt1, one of the sirtuin family of NAD(+)-dependent deacetylases, has recently been shown to attenuate amyloidogenic processing of amyloid-β protein precursor (APP) in AD by increasing α-secretase production and activity . Sirt1 expression activated the AKT signaling pathway . In our study, we found IH could activate AKT via increasing Sirt1 expression and then lead to upregulate the ERK phosphorylation.
Oxidative stress, considered to be a key factor in the pathophysiology of stroke, is caused by the excessive production of reactive oxygen species (ROS) and the disorder of antioxidant capacity . GP91 had been recognized as biomarkers of oxidative stress [10–11]. Our results showed that IH treatment could reverse the up-regulation of ROS and GP91 expression. Moreover, the activities and expression of antioxidant enzymes SOD and GSH were increased. Nuclear factor erythroid-related factor 2 (Nrf2) has been implicated in antioxidant defense processes and plays a key role against oxidative stress . Previous studies have shown that up-regulation Nrf2 can alleviate H2O2-induced oxidative stress . It has shown that activation of Nrf2 restored the decreased activities of GSH, CATs and SODs [25–27]. Our results illustrated that IH treatment enhanced the Nrf2 expression, thereby improving the activity of antioxidant enzymes.
Inflammation is also associated with AD and that could have a role in contributing to the pathogenesis of AD . Astrocytes are key regulators of inflammatory responses in the central nervous system . Astrocyte activity may exacerbate inflammatory reactions by releasing numerous molecules, including interleukins, tumor necrosis factor alpha, etc . Our results show that IH can inhibit the activation of astrocytes and the release of inflammatory factors and reduce the inflammatory response.
In conclusion, IH promotes the expression of ADAM10 by activating SIRT1/AKT/ERK/CREB signaling pathway, thereby reducing the deposition of senile plaques. In addition, IH can also inhibit oxidative stress and inflammatory response. finally, improve the cognitive impairment of AD.
Figure 1. IH improves cognitive impairment of APP/PS1 mice. Four-month-APP/PS1 mice were treated with IH (i.p., 50 mg/kg/d) for 3 months. Morris water maze was used to test cognitive ability including 2 days of visible platform training, 5 days of hidden platform testing, and a probe trial. (A) In the visible platform and the hidden platform, the escape latency time of mice found the platform. (B) In the probe trial, the times of crossing the platform were recorded. Data were presented as the mean ± SD; n = 6, * P < 0.05; ** P < 0.01.
Figure 2. IH inhibites senile plaque depostion in APP/PS1 mice. Four-month-old APP/PS1 mice were treated with IH for 3 months. Aβ plaque in the cortex and hippocampus of APP/PS1 mice were detected by immunofluorescent.
Figure 3. IH upregulates the expression of ADAM10 in APP/PS1 mice. Four-month-old APP/PS1 mice were treated with IH for 3 months. Western blot detect the expression levels of ADAM10, BACE1 and the subunits of γ-secretase, including NCT, PS2 and APH-1. Data were presented as the mean ± SD; n = 6, * P < 0.05; ** P < 0.01; *** P < 0.001.
Figure 4. IH reduce the oxidative stress in APP/PS1 mice. Four-month-old APP/PS1 mice were treated with IH for 3 months. (A,) Western blot detect the expression levels of SOD1, Nrf2 and GP91 in the cortex. (B) ROS production in the cortex was detected with the dichlorofluorescein diacetate probe. Data were presented as the mean ± SD; n = 6, * P < 0.05; ** P < 0.01.
Figure 5. IH alleviated the neuroinflammatory response in APP/PS1 mice. Four-month-old APP/PS1 mice were treated with IH for 3 months. (A-B) Immunoblot analysis showed the expression levels of IL-1β and TNF-α. (C) IH treatment suppressed the activation of astrocytes around the Aβ plaque. Data were presented as the mean ± SD; n = 6, ** P < 0.01.
Figure 6. IH activated the Sirt1/AKT/ERK/CREB signaling pathways in the APP/PS1 mice. Four-month-old APP/PS1 mice were treated with IH for 3 months. (A-B) Immunoblot analysis showed the expression levels of Sirt1, p-AKT, AKT, p-ERK, ERK, p-CREB and CREB. Data were presented as the mean ± SD; n = 6, * P < 0.05; * P < 0.05; ** P < 0.01
All experimental procedures performed using animals were approved by the Laboratory of Animal Ethical Committee of China Medical University.