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Zhen-Nian Zhang,1,* Zhen Hui,1,* Chang Chen,1 Yan Liang,1 Li-Li Tang,1 Su-Lei Wang,1 Cheng-Cheng Xu,1 Hui Yang,1 Yang Zhao,1 Jing-Si Zhang2 1Department of Neurology, Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, Nanjing, 210022, People’s Republic of China; 2Department of Neurology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jing-Si ZhangDepartment of Neurology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, No. 528 of Zhangheng Road, Pudong New Area, Shanghai, 201203, People’s Republic of ChinaTel/Fax +86 13127823579Email zhangzn88_dr@163.comObjective: The present study aimed to investigate the effect of echinacoside on autophagy-related indicators through the mTOR signaling pathway, especially the effect on the clearance of autophagy substrate P62 and α-synuclein, the core pathological products of Parkinson’s disease (PD), to provide new strategies for the treatment of PD.Methods: A mouse model of subacute PD was established by the intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). First, the neurobehavioral symptoms in mice of each group were evaluated, and the monoamine neurotransmitters in the striatum in each group were measured with a high-performance liquid phase. Immunofluorescence double staining was adopted to observe the expression of tyrosine hydroxylase (TH), α-synuclein, and LC3. The transmission electron microscope was used to observe the changes of ultrastructure in substantia nigra and the formation of autophagosomes. Then, the expressions of TH, α-synuclein, Beclin 1, LC3, P62, mTOR, and the up-stream protein AKT were detected by Western blot.Results: When compared with the model group, the neurobehavioral function significantly improved in the echinacoside group (P < 0.01), together with increased expression of TH, DA, and DOPAC in the brain (P < 0.01). In the echinacoside group, while the expressions of Beclin 1 and LC3-II increased (P < 0.01), the expression levels of P62 and α-synuclein decreased significantly (P < 0.01). Echinacoside could up-regulate the expression level of the survival signal p-AKT/AKT and decrease the expression of mTOR.Conclusion: Echinacoside could increase autophagy by inhibiting the expression of mTOR, thereby promoting the clearance of α-synuclein and the degradation of the autophagy substrate P62 and exerting the neuroprotective effect.Keywords: Parkinson’s disease, MPTP, echinacoside, α-synuclein, P62, autophagy, mTOR

Objective The research is to elucidate the mechanism of programmed neuronal necrosis induced by aluminum exposure. The mechanism of programmed neuronal cell necrosis caused by aluminum exposure is due to the key death proteins RIP1, RIP3, MLKL and CaMKⅡ in vivo and vitro experiments. Methods In vivo study, mice were intraperitoneally injected with freshly prepared maltol aluminum for two months to establish aluminum-induced cognitive impairment model. The key death related proteins were measured by Western blot and the relationships between those proteins were detected by IP. LDH release was used to show the cell death, and ROS indicated the damage of brain. In Al-exposed SH-SY5Y cells, GSK'872 was interfered to observe the expression of key death related proteins by WB and IP, as well as PI dyeing. Besides, NSA and KN-93 were administered to detect the LDH release and ROS change, including mitochondrial membrane potential variation in the Al-exposed cells. Results Expression of RIP1, RIP3, MLKL and CaMKⅡ proteins were increased in vivo and vitro study after aluminum exposure. Inhibition of RIP3, MLKL and CaMKⅡ protein expression could reduce PI stained cells and LDH release. ROS declined after administration of GSK'872 and NSA in Al-exposed cells, while mitochondrial membrane potential elevated after intervention of KN-93 in Al-exposed cells with decreased mitochondrial membrane potential. Conclusion Programmed neuronal necrosis was one of the main causes though that aluminum lead to neuron reduction. The signal pathways involved in programmed necrosis were RIP1-RIP3-MLKL and RIP1-RIP3-CaMKⅡ pathways, one same initiating molecule – RIP1, two downstream pathways: RIP3-MLKL pathway and RIP3-CaMKII pathway.

Zhen-Nian Zhang,1,* Zhen Hui,1,* Chang Chen,1 Yan Liang,1 Li-Li Tang,1 Su-Lei Wang,1 Cheng-Cheng Xu,1 Hui Yang,1 Jing-Si Zhang,2 Yang Zhao1 1Department of Neurology, Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, Nanjing, 210022, People’s Republic of China; 2Department of Neurology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yang ZhaoDepartment of Neurology, Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, No. 157 of Daming Road, Qinhuai District, Nanjing, 210022, People’s Republic of ChinaTel/Fax +86 13851498418Email zhao_dr66@163.comObjective: To explore the neuroprotective effect and the related mechanisms of echinacoside (ECH) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson’s disease (PD) mice.Methods: Parkinson’s disease is induced in mice by MPTP and the neurobehaviors of mice in different groups are observed. Then, immunohistochemistry and Western blot analysis are adopted to measure the expression of tyrosine hydroxylase (TH) and α-synuclein in the substantia nigra (SN). The content of dopamine (DA) and other neurotransmitters in the brain is detected by high-performance liquid chromatography. The expression of nerve growth factors and inflammatory factors in SN in mice in each group is measured by quantitative polymerase chain reaction. Finally, the expression of oxidative stress-related parameters in each group is measured.Results: Compared with the model group, the pole-climbing time among mice in the moderate and high-dose ECH groups is significantly reduced (P < 0.01). The rotarod staying time, as well as fore and hind-limb strides, shows a significant increase (P < 0.01), as does spontaneous activity (P < 0.01). Moreover, the expression levels of TH, DA, glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor in SN in mice show significant increases in these two groups (P < 0.01). The content of superoxide dismutase, catalase, and glutathione peroxidase indicates significant increases in the low, moderate, and high-dose ECH groups (P < 0.01), and the content of MDA was reduced (P < 0.01). In the high-dose ECH group, the expression of interleukin (IL) 6 and tumor necrosis factor-α is significantly reduced (P < 0.01), while the expression of IL-10 shows a marked increase (P < 0.01) alongside a decrease in the expression of α-synuclein (P < 0.01).Conclusion: Echinacoside improves neurobehavioral symptoms in PD mice and significantly increases the expression of TH and DA. The neuroprotective effect potentially correlates with anti-inflammation and anti-oxidation actions, promotes the expression of nerve growth factor, and reduces the accumulation of α-synuclein.Keywords: Parkinson’s disease, MPTP, echinacoside, α-synuclein, oxidative stress, neuroprotection, BDNF, GDNF, IL-6, TNF-α, IL-10

Stroke is a leading reason of death and long-term disability, and most studies mainly focus on efforts to protect neurons. However, failed clinical trials suggest that therapies against single target in neurons may not be sufficient and the involvement of endothelial cells and glial cells have been underestimated. Astrocytes are the major source of ApoE in the brain and endothelial cells express high level of ApoE receptors. Thus, ApoE may mediate the interaction between astrocytes and endothelial cells. To address whether and how ApoE-mediated astrocytes–endothelial cells interaction contributes to the pathogenesis of stroke, we used oxygen and glucose deprivation-reoxygenation (OGD-R) as a stroke model and investigated the effects of OGD-R on astrocytes-endothelial cell co-cultures in the current study. We find that OGD-R leads to various damages to endothelial cells, including compromised cell viability, increased ROS level, enhanced caspase activity, and higher apoptotic rate. Meanwhile, mouse astrocytes could secrete ApoE to activate PI3K/eNOS signaling in endothelial cells to prevent OGD-R induced injuries. In addition, OGD-R induces down-regulation of ApoE in astrocyte–endothelial cell co-cultures while melatonin restores astrocytic ApoE expression via pCREB pathway and protects endothelial cell in OGD-R treated co-cultures. Our study provides evidence that astrocytes could protect endothelial cells via ApoE in OGD-R condition and Melatonin could induce ApoE expression to protect endothelial cells.

Stroke is a leading reason of death and long-term disability, and most studies mainly focus on efforts to protect neurons. However, failed clinical trials suggest that therapies against single target in neurons may not be sufficient and the involvement of endothelial cells and glial cells have been underestimated. Astrocytes are the major source of ApoE in the brain and endothelial cells express high level of ApoE receptors. Thus, ApoE may mediate the interaction between astrocytes and endothelial cells. To address whether and how ApoE-mediated astrocytes–endothelial cells interaction contributes to the pathogenesis of stroke, we used oxygen and glucose deprivation-reoxygenation (OGD-R) as a stroke model and investigated the effects of OGD-R on astrocytes-endothelial cell co-cultures in the current study. We find that OGD-R leads to various damages to endothelial cells, including compromised cell viability, increased ROS level, enhanced caspase activity, and higher apoptotic rate. Meanwhile, mouse astrocytes could secrete ApoE to activate PI3K/eNOS signaling in endothelial cells to prevent OGD-R induced injuries. In addition, OGD-R induces down-regulation of ApoE in astrocyte–endothelial cell co-cultures while melatonin restores astrocytic ApoE expression via pCREB pathway and protects endothelial cell in OGD-R treated co-cultures. Our study provides evidence that astrocytes could protect endothelial cells via ApoE in OGD-R condition and Melatonin could induce ApoE expression to protect endothelial cells.

Background/Aims: Angiotensin II (Ang II) mediated signaling plays a key role in the development of chronic kidney damage that contributes to renal fibrosis. However, the gene expression changes regulated by Ang II in the early stage of acute renal injury remain unclear. Methods: C57BL/6 wild-type (WT) mice were injected with Ang II (1500 ng/kg/min) for 1, 3 and 7 days. A time series analysis of microarrays was performed to evaluate Ang II-induced differentially gene expression in the kidneys. The data of gene expression in the kidney was further dissected by ANOVA analysis, gene expression profiles, gene network construction and quantitative real-time RT-PCR. Ang II-induced renal inflammation and fibrosis in mice were confirmed by pathological examination. Results: Our microarray data showed that a total of 1,511 differentially expressed genes were identified in the kidneys at 1, 3 and 7 days after Ang II infusion. These genes function in multiple biological processes, including response to stimuli, immune response, cell adhesion, metabolic process, kidney development, regulation of blood pressure, and ion transport, which may play critical roles in the pathobiology of Ang II-induced acute renal injury at the early stage. Furthermore, among these genes, 20 genes were further selected for final investigation. The dynamic gene network analysis demonstrated that fatty acid binding protein 1 (Fabp1) localized in the core of the network. Conclusions: Our data suggests that genes involved in lipid metabolic process, especially Fabp1, may play a central role in the development of Ang II-induced acute renal injury at the early stage.