In vitro study on the regulation of microglial autophagy by Liuwei Dihuang Pill for the treatment of Alzheimer disease

To explore the mechanism of action of the Liuwei Dihuang Pill in the intervention of Alzheimer disease (AD).

Twenty 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.

The 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).

Liuwei Dihuang Pill improves autophagy function of microglia by regulating GPNMB expression, thereby clearing Aβ deposition and exerting its effect on improving AD.