关键词: 阿尔茨海默病, 积雪草酸, 海马神经元, 炎症, 氧化应激, 核因子E2相关因子2, 血红素氧合酶1

Abstract:

Objective To discuss the effect of asiatic acid （AA） on the inflammation and oxidative stress damage induced by lipopolysaccharide （LPS） in the primary cultured hippocampus neurons， and to clarify its mechanism. Methods The primary cultured rat hippocampus neurons （cell purity identified by immunofluorescence staining） were divided into control group， LPS （10 mg·L^-1） group， and LPS+AA group （10 mg·L^-1 LPS+10， 20， and 40 μmol·L^-1 AA）， AA group （20 μmol·L^-1 AA）， ML385 group ［10 μmol·L^-1 nuclear factor erythroid 2-related factor （Nrf2） inhibitor］， and LPS+ML385+AA group （10 mg·L^-1 LPS+10 μmol·L^-1 ML385+20 μmol·L^-1 AA）. After drug treatment， methylthiazolyldiphenyl-tetrazolium bromide （MTT） method was used to detect the survival rates of the hippocampus neurons in various groups； lactate dehydrogenase （LDH） kit was used to detect the LDH leakage rates of the hippocampus neurons in various groups； enzyme linked immunosorbent assay （ELISA） kit was used to detect the expression levels of inflammatory factors ［interleukin （IL）-1β and tumor necrosis factor （TNF）-α］ and the activities of superoxide dismutase （SOD） and malondialdehyde （MDA） levels in the hippocampus neurons in various groups； Griess method was used to detect the nitric oxide （NO） levels in supernatant of the hippocampus neurons in various groups； immunofluorescence staining was used to detect the expressions of Nrf2 and heme oxygenase-1 （HO-1） proteins in the hippocampus neurons in various groups； Western blotting method was used to detect the expression levels of Nrf2， HO-1， nuclear factor-kappa B（NF-κB）， and B-cell lymphoma 2 （Bcl-2） proteins in the hippocampus neurons in various groups. Results Compared with control group， the survival rate of the hippocampus neurons， SOD activity， and Bcl-2 expression level in the cells in LPS group were significantly decreased （P<0.01）， while the LDH leakage rate， expression levels of IL-1β and TNF-α， MDA level， and NO level， as well as the expression level of NF-κB protein， were significantly increased （P<0.01）； the fluorescence intensities and expression levels of Nrf2 and HO-1 proteins in hippocampus neurons were significantly decreased （P<0.01）. Compared with LPS group， the survival rates of hippocampus neurons， SOD activities， and expression levels of Bcl-2 in the cells in LPS+10 μmol·L^-1 AA group and 20 μmol·L^-1 AA group were significantly increased （P<0.01）， while the LDH leakage rates， expression levels of IL-1β and TNF-α， MDA levels， and NO levels， as well as expression levels of NF-κB protein， were significantly decreased （P<0.05 or P<0.01）， and the fluorescence intensities and protein expression levels of Nrf2 and HO-1 in the cells were significantly increased （P<0.01）. Compared with LPS+20 μmol·L^-1 AA group， the fluorescence intensities of Nrf2 and HO-1 in the cells in LPS+ML385+AA group were significantly decreased （P<0.01）， and the expression levels of Nrf2 protein in the nucleus and cytoplasm， the expression levels of HO-1 and Bcl-2 proteins in the cells were significantly decreased （P<0.01）， while the expression level of NF-κB protein was significantly increased （P<0.01）. Conclusion AA can improve LPS-induced inflammation and oxidative stress damage in the primary cultured rat hippocampus neurons， and its mechanism may be related to the activation of the Nrf2/HO-1 signaling pathway.

Key words: Alzheimer’s disease, Asiatic acid, Hippocampus neuron, Inflammation, Oxidative stress, Nuclear factor erythroid 2-related factor, Heme oxygenase-1