Ischemic brain disease and MCAO
Ischemic stroke has become one of the most devastating diseases, which cause high rates of disability and mortality in aged people.160–162 Acute excitotoxicity, oxidative stress, and inflammation are the three primary mechanisms involved in cell death during ischemic stroke.163 Cerebral edema is a detrimental feature after ischemic stroke and is one of the impact factors of clinical deterioration within the first 24 hours after stroke onset. Cerebral ischemia and reperfusion triggers a cascade of cellular events including cell death, oxidative stress, and inflammation, which all contribute to the breakdown of blood–brain barrier (BBB).160–162 Neuronal cell apoptosis plays an important role in the development of ischemic injury in the brain tissue. Mitochondrial apoptotic pathway is a major apoptotic pathway, and a large number of apoptosis-related proteins in mitochondria play an important role in the initiation and development of neuronal apoptosis.164 Pro-apoptotic and anti-apoptotic Bcl-2 family proteins play important roles in mitochondrial apoptotic pathway. Bax is a pro-apoptotic and Bcl-2 is an anti-apoptotic protein in the Bcl-2 family. Cytochrome C binds and activates apoptotic protease-activating factor-1 as well as procaspase-9, forming an apoptosome together with ATP. Apoptosome then activates caspase-9, leading to caspase-3 activation and eventually cellular apoptosis. Caspase-3 has been identified as a key mediator of apoptosis and cleaves the substrate PARP-1, which is a multifunctional nuclear enzyme whose activity is rapidly stimulated by DNA breaks.
The protective effect of LBPs was investigated in primary cultured rat hippocampal neurons subject to oxygen–glucose deprivation/reperfusion by Rui et al.153 Cultured hippocampal neurons were exposed to oxygen–glucose deprivation for 2 hours followed by a 24-hour re-oxygenation. Treatment with LBPs (10–40 mg/L) significantly attenuated neuronal damage and inhibited LDH release in a dose-dependent manner.153
Yang et al155 investigated the protective effect of LBP pre-treatment in an experimental stroke (MCAO) model in male C57BL/6N mice. To gain LBPs, dry L. barbarum residues were dissolved in water at 70°C, and the supernatant was concentrated, precipitated with 95% ethanol, and then vacuum dried to produce the extracts. The mice were administered 1 mg/kg or 10 mg/kg LBPs daily for 7 days, and then subjected to 2-hour transient MCAO by the intraluminal method followed by 22-hour reperfusion upon filament removal. LBP pre-treatment dose-dependently improved neurological deficits; decreased infarct size, apoptotic neurons in ischemic penumbra area, and cerebral edema; and protected the brain from BBB disruption as indicated by reduced Evans Blue dye leakage into the ipsilateral hemispheres and an upregulation of occludin expression.155 Occludin, one of the proteins located at tight junctions, plays an important role in maintaining the integrity of BBB. Pre-treatment with 10 mg/kg LBPs for 7 days also profoundly suppressed the upregulation of AQP4 expression in ipsi-lateral penumbral areas.155 Furthermore, 10 mg/kg LBPs suppressed GFAP activation in ipsilateral penumbral areas. Pre-treatment with 10 mg/kg LBPs reduced both nitrosative stress and lipid peroxidation in cerebral ischemic penumbra after MCAO. LBPs at both doses attenuated the expression of matrix metalloproteinase-9 (MMP-9) in ipsilateral penumbral areas.155 These findings clearly demonstrate the beneficial prophylactic effects of LBPs against ischemic damage and cerebral edema in a murine experimental stroke model. The neuroprotective effects of LBPs on ischemic stroke include reduction of neuronal damage and infarct, maintenance of BBB integrity, and alleviation of cerebral edema through antioxidation, suppression of upregulated MMP-9 and AQP4, anti-apoptosis, and inhibition of glial activation.
In a study using male Kunming mice, Wang et al165 examined the effect of intragastric administration with LBPs on brain injuries in MCAO mice. The study demonstrated that LBPs at doses of 20 mg/kg and 40 mg/kg significantly decreased the neurological deficit scores and the infarct area in MCAO mice. LBPs also significantly decreased MDA content, and increased SOD, GPx, CAT, and LDH activities in the ischemic brain.165 These findings suggest that LBPs might act as potential neuroprotective agent against the cerebral reperfusion-induced brain injury through reducing lipid peroxides, scavenging free radicals, and improving the energy metabolism.
In a similar study, Wang et al156 used male Imprinting Control Region mice to make the model of MCAO and investigated the protective effect of intragastric administration of 10 mg/kg, 20 mg/kg, and 40 mg/kg body weight LBPs or 0.4 mg/kg nimodipine for 7 days on MCAO-induced brain injuries. The results showed that intragastric administration of 20 mg/kg and 40 mg/kg LBPs markedly decreased the neurological deficit scores and the infarct volume in MCAO mice.156 Administration of 10–40 mg/kg LBPs also reduced neuronal morphological damage and neuronal apoptosis in ischemic penumbra of the left cortex. About 40 mg/kg LBPs significantly suppressed cortex overexpression of Bax, cytochrome C, caspase-3, -9, and cleaved PARP-1, and reduced the downregulated Bcl-2 expression in MCAO mice.156
In summary, the protective effects of LBPs on MCAO-induced brain injuries are mainly attributed to the reduction of oxidative stress, inhibition of apoptosis, and increase in the integrity of BBB. LBPs treatment reduces the oxidative stress via increasing the SOD, GPx, CAT, and LDH activities, but decreasing the content of MDA and lipid peroxidation. LBPs also inhibit the apoptosis via decreasing the expression of cytochrome C, cleave caspase-9, caspase-3, Bax, and cleaved PARP-1, but increasing the expression level of Bcl-2. In addition, LBPs increase the integrity of BBB expression through the upregulation of expression of occludin, but downregulation of the expression of MMP-9 and AQP4 (Figure 10).