六味地黄丸调控小胶质细胞自噬干预阿尔茨海默病的体外研究OA

Objective To explore the mechanism of action of the Liuwei Dihuang Pill in the intervention of Alzheimer disease(AD).Methods 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.Results 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).Conclusion Liuwei Dihuang Pill improves autophagy function of microglia by regulating GPNMB expression,thereby clearing Aβ deposition and exerting its effect on improving AD.