Naomaitai Ameliorated Brain Damage in Rats with Vascular Dementia by PI3K/PDK1/AKT Signaling Pathway

Abstract

Background/aims: Naomaitai can improve blood perfusion and ameliorate the damage in the paraventricular white matter. This study was focused on observing the neuroprotective effect of Naomaitai on the vascular dementia of rat and exploring the action mechanism of PI3K/PDK1/AKT signaling pathway.

Methods: A vascular dementia model of rats was established by permanent, bilateral common carotid artery occlusion. Rats' behavior was tested by Neurological deficit score and the Morris water maze. The pathology and apoptosis were detected through HE staining and TUNEL assay. Myelin sheath loss and nerve fiber damage were detected by LFB staining. Inflammatory factors, oxidative stress, and brain damage markers were detected through ELISA. The expression of apoptosis-related proteins and PI3K/PDK1/AKT signaling pathway related proteins were measured by western blot. The expressions of PI3K, PDK1, AKT, and MBP in paraventricular white matter cells were detected by immunofluorescence.

Results: Naomaitai treatment decreased neurological function score in rats with vascular dementia, ameliorated paraventricular white matter damage caused by long-term hypoxia, and hypoperfusion reduced the brain injury markers S-100β and NSE contents, suppressed inflammatory reaction and oxidative stress, reduced IL-1β, IL-6, TNF-α, and MDA contents, and remarkably increased IL-10 and SOD contents. TUNEL and western blot assay showed that Naomaitai treatment decreased neuronal cell apoptosis, increased Bcl-2 expression, and reduced caspase-3 and Bax expression. Furthermore, we found Naomaitai inhibited PI3K and PDK1 expression and activated phosphorylated AKT protein in rats with vascular dementia. However, the protective effect of Naomatai in rats with vascular dementia was inhibited, and expression of PI3K signaling pathway-related proteins was blocked after administration of PI3K inhibitor.

Conclusion: Naomaitai can ameliorate brain damage in rats with vascular dementia, inhibit neuronal apoptosis, and have anti-inflammatory and antioxidative stress effects, which may be regulated by the PI3K/PDK1/AKT signaling pathway.

Figure 1

NMT lessens neurological impairment in VD rats. Bederson scoring system was used to detect neurological impairment in rats. MWM test was conducted to determine the spatial cognition and memory ability of rats. (a) The neurological deficit score; (b) Morris water maze test. ∗P<0.05 vs. Sham group; #P<0.05 vs. VD group.

Figure 2

NMT improves white matter damage in VD rats. HE staining was utilized to observe the changes of brain tissue morphology in rats. ELISA was performed to determine the expression levels of brain damage markers. (a) Hematoxylin & eosin staining (Bar, 50μm); (b) ELISA was performed to determine the expression levels of NSE and S-100β. ∗P<0.05 vs. Sham group; #P<0.05 vs. VD group.

Figure 3

NMT suppresses oxidative stress and inflammation in the white matter of VD rats. ELISA was performed to determine the expression levels of oxidative stress factors and inflammation related factors contents in white matter. (a) The expression levels of oxidative stress factors (MDA and SOD) in white matter; (b) the expression levels of inflammation related factors (IL-1β, IL-6, TNF-α and IL-10) contents in white matter. ∗P<0.05 vs. Sham group; #P<0.05 vs. VD group.

Figure 4

Naomaitai can reduce myelin demyelination and improve white matter damage in VD rats. LFB staining was utilized to observe the damage of white matter nerve fibers and the loss of myelin sheath. Immunofluorescence method was used to determine the MBP expression in white matter of rats. (a) LFB staining (bar, 50μm); (b) the expression of MBP determined by Immunofluorescence method (bar, 50μm). ∗P<0.05 vs. Sham group; #P<0.05 vs. VD group.

Figure 5

NMT inhibits apoptotic factor expression in the white matter of VD rats. Apoptosis-positive cells were determined via Tunel assays. Apoptosis-related protein expression was determined via Western blot assay. (a) Tunel assays (bar, 50μm); (b) Bcl-2, Caspase-3 and Bax protein expression determined via Western blot assay. ∗P<0.05 vs. Sham group; #P<0.05 vs. VD group.

Figure 6

NMT mitigates brain injury in VD rats through the PI3K/PDK1/AKT signaling pathway. Immunofluorescence method and western blot assay were used to determine the protein expression of PI3K, PDK1, and AKT. (a) The protein expression of PI3K determined by Immunofluorescence method (bar, 50μm); (b) the protein expression of PDK1 determined by Immunofluorescence method (bar, 50μm); (c) the protein expression of Akt determined by Immunofluorescence method (bar, 50μm); (d) the protein expression of PI3K, PDK1, and AKT determined by Western blot assay. ∗P<0.05 vs. Sham group; #P<0.05 vs. VD group.

Figure 7

PI3K inhibitor reverses the therapeutic effect of NMT on VD rats. Bederson scoring system was used to detect neurological impairment in rats. MWM test was conducted to determine the spatial cognition and memory ability of rats. HE staining was utilized to observe the changes of brain tissue morphology in rats. ELISA was performed to determine the expression levels of brain damage markers, oxidative stress factors and inflammation related factors. Apoptosis-positive cells were determined via Tunel assays. Apoptosis-related protein expression was determined via Western blot assay. Immunofluorescence method and western blot assay were used to determine the protein expression of PI3K, PDK1, and AKT. LFB staining was utilized to observe the damage of white matter nerve fibers and the loss of myelin sheath. Immunofluorescence method was used to determine the MBP expression in white matter of rats. (a) The neurological deficit score and Morris water maze test; (b) hematoxylin & eosin staining (bar, 50μm); (c) ELISA was performed to determine the expression levels of NSE and S-100β; (d) the expression levels of oxidative stress factors (MDA and SOD) in white matter; (e) the expression levels of inflammation related factors (IL-1β, IL-6, TNF-α, and IL-10) contents in white matter; (f) Tunel assays (bar, 50μm); (g) the protein expression of PI3K, PDK1 and AKT determined by Western blot assay; (h) the protein expression of PDK1 and Akt determined by Immunofluorescence method (bar, 50μm); (i) LFB staining (bar, 50μm); (j) the expression of MBP determined by Immunofluorescence method (bar, 50μm). $P<0.05 vs. HNMT group.