关键词: 人参健智片, 认知障碍, D-半乳糖, SH-SY5Y细胞, 氧化应激, 炎症

Abstract:

Objective To discuss the improvement effect of Renshen Jianzhi Tablets on cognitive function and protective effect of Penshen Jiouzhi Tablets on hippocampal neurons in cognitive impairment model rats， and to clarify its potential mechanism. Methods Forty-two SD rats were randomly divided into control group， model group， donepezil hydrochloride group （0.45 mg·kg^-1 donepezil hydrochloride）， Kaixin San group （1.35 g·kg^-1 Kaixin San）， low and high dose of Renshen Jianzhi Tablets groups （2.17 and 10.85 g·kg^-1 Renshen Jianzhi Tablets）， with 7 rats in each group. Except for control group， the rats in the other groups were subcutaneously injected with 500 mg·kg^-1 D-galactose daily to establish cognitive impairment model for 10 consecutive weeks. Morris water maze test was used to evaluate the learning and memory abilities of the rats in various groups； HE staining was used to observe the pathol morphology of hippocampus tissue of the rats in various groups. In in vitro experiments， human neuroblastoma SH-SY5Y cells were divided into control group， hydrogen peroxide （H₂O₂） group， lipopolysaccharide （LPS） group， 62.500 0， 125.000 0 and 250.000 0 mg·L^-1 Renshen Jianzhi Tablets groups. H₂O₂ and LPS were used to treat the SH-SY5Y cells to establish oxidative stress and inflammatory models， respectively. Methylthiazolyldiphenyl-tetrazolium bromide （MTT） method was used to detect the survival rate of the cells in various groups； 2'，7'-dichlorodihydrofluorescein diacetate （DCFH-DA） fluorescence method was used to detect the level of reactive oxygen species （ROS） in the cells in various groups； enzyme linked immunosorbent assay （ELISA） method was used to detect the levels of malondialdehyde （MDA）， glutathione （GSH）， tumor necrosis factor α （TNF-α）， interleukin-6 （IL-6）， interleukin-1β （IL-1β） and the activity of superoxide dismutase （SOD） in the cell supernatant in various groups. Ultra high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS） technology combined with chemical structure characteristics， secondary mass spectrometry fragment ion information and literature comparison was used to identify the compound components in Renshen Jianzhi Tablets. Results The Morris water maze test results showed that compared with control group， the number of platform crossings of the rats in model group was significantly decreased （P<0.01）， and the retention time in the target quadrant was significantly shortened （P<0.01）； compared with model group， the number of platform corssings of rats in Donepezil HCl group was significantly increased（P<0.01）， the number of platform crossings of the rats in low dose of Renshen Jianzhi Tablets groups was significantly increased （P<0.01）， and the retention time in the target quadrant was significantly prolonged （P<0.01）. The HE staining results showed that compared with control group， the number of hippocampal neurons of the rats in model group was decreased， the arrangement was disordered， and the intercellular space was enlarged； compared with model group， the number of hippocampal neurons of the rats in donepezil hydrochloride group， Kaixinsan group， low and high doses of Renshen Jianzhi Tablets groups was increased， the morphology was relatively regular， and the arrangement was relatively neat. The MTT assay results showed that compared with control group （P<0.01）， the survival rates of the cells in 0.625 mmol·L^-1 H₂O₂ group and 500 mg·L^-1 LPS were decreased and closest to 50%； compared with control group， reached， the cell survival rate of the cells in 500 mg·L^-1 Renshen Jianzhi Tablets group was significantly decreased （P<0.01）. The ROS fluorescence probe assay results showed that compared with control group， the ROS level in the cells in H₂O₂ group was significantly increased （P<0.01）； compared with H₂O₂ group， the ROS levels in the cells in 62.500 0， 125.000 0 and 250.000 0 mg·L^-1 Renshen Jianzhi Tablets groups were significantly decreased （P<0.01）， and the effect was most obvious in 125.000 0 mg·L^-1 Renshen Jianzhi Tablets group. The ELISA results showed that compared with control group， the MDA level in the cell supernatant in H₂O₂ group was significantly increased （P<0.01）， while the GSH level and SOD activity were significantly decreased （P<0.01）； compared with H₂O₂ group， the MDA levels in the cell supernatant in 62.500 0， 125.000 0 and 250.000 0 mg·L^-1 Renshen Jianzhi Tablets groups were significantly decreased （P<0.05 or P<0.01）， and the GSH levels and SOD activities were significantly increased （P<0.05 or P<0.01）. Compared with control group， the levels of TNF-α， IL-6 and IL-1β in the cell supernatant in LPS group were significantly increased （P<0.01）； compared with LPS group， the levels of TNF-α， IL-6 and IL-1β in the cell supernatant in 62.500 0， 125.000 0 and 250.000 0 mg·L^-1 Renshen Jianzhi Tablets groups were significantly decreased （P<0.01）. The UHPLC-MS/MS analysis results showed that a total of 73 compounds were identified in Renshen Jianzhi Tablets. Conclusion Renshen Jianzhi Tablets can effectively improve the learning and memory abilities of cognitive impairment model rats and protect the structural integrity of hippocampal neurons. Its mechanism may be related to inhibiting oxidative stress damage of nerve cells and reducing inflammatory response.

Key words: Renshen Jianzhi tablets, Cognitive impairment, D-galactose, SH-SY5Y cells, Oxidative stress, Inflammation