基于Keap1/Nrf2/HO-1信号通路探讨四君子汤改善阿尔茨海默病的作用机制OA

Objective:To explore the mechanisms through which Si Junzitang(SJZD)ameliorates Alzheimer's disease(AD)induced by scopolamine(SCOP)in mice and the PC12 cell model induced by H2O2 based on the Kelch-like ECH-associated protein 1(Keap1)/nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)signaling pathway.Methods:In the animal experiments,an AD model was established in mice by intraperitoneal injection of SCOP(3 mg·kg-1).Morris water maze and open field tests(OFT)were conducted to assess learning and memory abilities.Hematoxylin-eosin(HE)staining and Nissl staining were performed to observe pathological changes in neurons.Immunofluorescence was used to detect amyloid β-protein1-42(Aβ1-42)expression,and immunohistochemistry was employed to detect phosphorylated(p)-Tau expression.Transmission electron microscopy(TEM)was employed to observe ultrastructural changes in hippocampal neurons and synapses.Biochemical methods were used to measure the levels of acetylcholine(ACh),acetylcholinesterase(AChE),superoxide dismutase(SOD),malondialdehyde(MDA),catalase(CAT),and lactate dehydrogenase(LDH).Real-time PCR and Western blot were employed to measure the mRNA and protein levels of molecules in the Keap1/Nrf2/HO-1 pathway in the hippocampus.In the cell experiments,a PC12 cell model of oxidative damage model was established with H2O2.Cell count kit-8(CCK-8)assays and flow cytometry were adopted to measure cell viability and apoptosis rates,and Western blot was employed to quantify the expression levels of proteins in the Keap1/Nrf2/HO-1 pathway.Results:The animal experiments showed that compared with the model group,the SJZD and donepezil groups showed shortened escape latency(P<0.01),increased time in the target quadrant and platform crossings,and increased movement distance and duration in the central area of the open field(P<0.05,P<0.01).HE and Nissl staining showed more organized neurons and increased Nissl bodies in the drug intervention groups(P<0.05,P<0.01),and the Aβ1-42 and p-Tau expression levels were downregulated(P<0.05,P<0.01).TEM revealed reduced ultrastructural damage in hippocampal neurons and synapses in the drug intervention groups.In addition,the drug intervention groups showed declined levels of MDA,LDH,and AChE(P<0.05,P<0.01),elevated levels of SOD,CAT,and ACh(P<0.05,P<0.01),reduced Keap1 expression and increased Nrf2,HO-1,and NQO1 expression in the hippocampus(P<0.05,P<0.01).The cell experiments showed that compared with the model group,the SJZD-containing serum increased the cell viability(P<0.05,P<0.01),and decreased total apoptosis rates(P<0.05,P<0.01).The drug intervention groups showed upregulated protein levels of Nrf2 and HO-1 and downregulated protein level of Keap1(P<0.05,P<0.01).Conclusion:SJZD demonstrates protective effects against SCOP-induced AD in mice and H2O2-induced damage in PC12 cells through antioxidant mechanisms mediated by the Keap1/Nrf2/HO-1 pathway.