@article{LIU2026, 
author = {Yuxi LIU and Zhongkang ZHU and Songnan WANG and Shunuo HE and Ye YIN and Jiarui MIAO and Xu WANG and Danyu ZHAO},
title = {In vitro study on the regulation of microglial autophagy by Liuwei Dihuang Pill for the treatment of Alzheimer disease},
year = {2026},
journal = {Journal of Beijing University of Traditional Chinese Medicine},
volume = {49},
number = {5},
pages = {668-680},
keywords = {Liuwei Dihuang Pill, glycoprotein non-metastatic melanoma protein B, autophagy, microglia, Alzheimer disease},
url = {https://www.sciopen.com/article/10.3969/j.issn.1006-2157.2026.05.009},
doi = {10.3969/j.issn.1006-2157.2026.05.009},
abstract = {ObjectiveTo explore the mechanism of action of the Liuwei Dihuang Pill in the intervention of Alzheimer disease (AD).MethodsTwenty SD rats were randomly divided into a blank serum group and a drug-containing serum group, with 10 rats in each group. The rats in the drug-containing serum group were administered Liuwei Dihuang Pill (1.18 g/kg) by gavage, whereas the rats in the blank serum group were administered an equal volume of normal saline by gavage, twice daily for 5 consecutive days, to prepare the Liuwei Dihuang Pill drug-containing serum and blank serum as the control condition. BV2 cells (mouse microglia cell line) and N2a/APP695swe cells (mouse glioma cell line, transfected with APP695swe gene) were co-cultured and divided into: vector control, glycoprotein non-metastatic melanoma protein B (GPNMB) overexpression, negative control, and the GPNMB knockdown group to investigate the effect of GPNMB on the co-culture system, whereas the blank serum, drug-containing serum, negative control-drug-containing serum, and GPNMB knockdown-drug-containing serum group were used to explore the effects and mechanisms of Liuwei Dihuang Pill drug-containing serum on the co-culture system. The lentivirus transfection method was used to infect BV2 cells with the corresponding lentivirus for 48 h. Subsequently, BV2 cells were treated with the corresponding serum for 24 h and then co-inoculated with N2a/APP695swe cells in a co-culture system. The main components of the serum containing Liuwei Dihuang Pill were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry. The optimal volume fraction of the serum containing Liuwei Dihuang Pill was screened using the CCK-8 method. The transfection effect of GPNMB knockdown lentivirus was detected by real-time PCR and western blotting. The content of amyloid β-protein (Aβ) in the supernatant of N2a/APP695swe cells was measured by an enzyme-linked immunosorbent assay. The proliferation rate of N2a/APP695swe cells was determined by the CCK-8 method. The expression of GPNMB, a disintegrin and metalloprotease 10 (ADAM10), Beclin1, the ubiquitin-binding protein p62, and the microtubule-associated protein 1 light chain 3-Ⅱ/Ⅰ (LC3-Ⅱ/Ⅰ) in BV2 cells was detected by western blotting.ResultsThe main components of the serum containing Liuwei Dihuang Pill were trijuganone C, cornuside, rhein, D(-)-tartaric acid, fabacein, 7-dehydroxypyrogallin-4-carboxylic acid, goshonoside F1, cyclo (phe-leu), 6-methyladenosine, and Danshensu. The optimal volume fraction of the serum containing Liuwei Dihuang Pill was 10%. Compared with the negative control lentivirus, the GPNMB mRNA and protein expressions were decreased in the GPNMB knockdown lentivirus (P<0.05). Compared with the vector control group, the Aβ content in the supernatant of N2a/APP695swe cells was decreased, the proliferation rate of N2a/APP695swe cells was increased, the protein expression of ADAM10, Beclin1, and LC3-Ⅱ/Ⅰ in BV2 cells was increased, whereas the protein expression of p62 was decreased in the GPNMB overexpression group (P<0.05). Compared with the negative control group, the Aβ content in the supernatant of N2a/APP695swe cells was increased, the proliferation rate of N2a/APP695swe cells was decreased, the protein expression of ADAM10, Beclin1, and LC3-Ⅱ/Ⅰ in BV2 cells was decreased, and the protein expression of p62 was increased in the GPNMB knockdown group (P<0.05). Compared with the blank serum group, the Aβ content in the supernatant of N2a/APP695swe cells was decreased, the proliferation rate of N2a/APP695swe cells was increased, the protein expression of GPNMB, ADAM10, Beclin1, and LC3-Ⅱ/Ⅰ in BV2 cells was increased, and the protein expression of p62 was decreased in the drug-containing serum group and in the negative control-drug-containing serum group (P<0.05). Compared with the drug-containing serum group and the negative control-drug-containing serum group, the Aβ content in the supernatant of N2a/APP695swe cells was increased, the proliferation rate of N2a/APP695swe cells was decreased, the protein expression of GPNMB, ADAM10, Beclin1, and LC3-Ⅱ/Ⅰ in BV2 cells was decreased, and the protein expression of p62 was increased in the GPNMB knockdown-drug-containing serum group (P<0.05).ConclusionLiuwei Dihuang Pill improves autophagy function of microglia by regulating GPNMB expression, thereby clearing Aβ deposition and exerting its effect on improving AD.}
}