Mechanism of Linggui Zhugantang in Repairing Blood-brain Barrier Injury of Alzheimer's Disease / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo observe the effect of Linggui Zhugantang （LG） on the blood-brain barrier （BBB） model of Alzheimer's disease （AD） in vitro and to explore the mechanism of LG in repairing the BBB injury in AD. MethodA total of 50 male SPF rats were randomized into five groups： high-dose （4.8 g·kg-1）， medium-dose （2.4 g·kg-1）， and low-dose （1.2 g·kg-1） LG groups， western medicine （0.5 g·kg-1 donepezil hydrochloride） group， and normal group （normal saline of equivalent volume）. They received （ig） corresponding drugs twice a day for 7 d. Drug-containing serum was respectively collected from the abdominal aorta 1 h after the last administration. The BBB injury of AD in vitro was induced with the cell co-culture method， and 6 groups were designed： normal group， model group， high-， medium-， and low-dose LG groups， and western medicine group. The model group was added with 100 μL amyloid β1-42 （Aβ1-42， final concentration： 5 μmol·L-1）， and high-dose， medium-dose， and low-dose LG groups and the western medicine group were added with corresponding 10% drug-containing serum in addition to the 100 μL Aβ1-42 （final concentration： 5 μmol·L-1）. Cell survival rate was detected by methyl thiazolyl tetrazolium （MTT） assay， expression of BBB-related skeleton proteins （claudin-5， ZO-1， occludin）， matrix metalloproteinase-2 （MMP-2）， and matrix metalloproteinase-9 （MMP-9） by Western blot， and content of inflammatory factors interleukin-1β （IL-1β）， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α） by enzyme-linked immunosorbent assay （ELISA）. BBB Aβ transporter low-density lipoprotein receptor-related protein 1 （LRP-1） and advanced glycation end product receptor （RAGE） at different time points in high-dose， medium-dose， and low-dose LG groups were determined by Real-time PCR and Western blot. ResultCell survival rate of the model group was lower than that of the normal group （P<0.05） and the survival rates of the western medicine group and high-dose LG group was higher than that in the model group （P<0.05）. The skeleton proteins were down-regulated and MMP-2 and MMP-9 were up-regulated in the model group compared with those in the normal group （P<0.05）. The expression of skeleton proteins was higher （P<0.05） and that of MMP-2 and MMP-9 was lower （P<0.05） in the western medicine group and high-dose LG group than in the model group. Compared with the model group， only the medium-dose LG group showed the up-regulation （P<0.05） of claudin-5 （P<0.05） and the decrease （P<0.05） of MMP-2. IL-1β， IL-6， and TNF-α in the model group were up-regulated （P<0.05） compared with those in the normal group， and those inflammatory factors in the western medicine group and high-dose and medium-dose LG groups were lower （P<0.05） than those in the model group. LRP-1 expression was up-regulated and RAGE expression was down-regulated at 3 h compared with those at 0 h （P<0.05）， while the expression of the two became stable at 6， 12， 24， 36 h. At 3 h， LRP-1 expression was down-regulated and RAGE expression was up-regulated in model group compared with those in the normal group at 3 h （P<0.05）. Moreover， the LRP-1 content was higher and RAGE content was lower in the western medicine group and high-dose LG group than in the model group. ConclusionLG can repair the BBB injury in vitro by inhibiting the expression of inflammatory factors and MMP-2， MMP-9， promoting the expression of skeletal proteins， and regulating the balance of transporters.