Acupuncture improves transplanted neural stem cells in dementia mice by modulating hippocampal microenvironment via microRNA-124 mediated Notch and Wnt pathways

Figure 1 Schematic diagram of the mechanism by which acupuncture regulates the downstream targets of microRNA-124. A: Notch signaling pathway; B: Wnt signaling pathway (canonical); C: Wnt signaling pathway (noncanonical); D: Notch and Wnt interaction; E: Cell cycle. AD: Alzheimer’s disease; NSCs: Neural stem cells; ADAM10: A disintegrin and metalloproteinase 10; Notch1: Notch homolog 1; HERP: Hairy/enhancer of split related protein; Hes1: Hairy and enhancer of split 1; Hes5: Hairy and enhancer of split 5; LRP5/6: Lipoprotein receptor-related protein 5/6; GSK3β: Glycogen synthase kinase 3β; TCF: T-cell factor; C-myc: Myc proto-oncogene; Cyclin D1: Cell cycle protein D1; Cylin D2: Cell cycle protein D2; PLC: Phospholipase C; PKC: Protein kinase C.

Figure 2 Experiment operational flowchart: Timing of cell culture and transplantation as well as acupuncture intervention. SAMR1: Senescence-accelerated mice resistant 1; SAMP8: Senescence-accelerated mouse prone 8; RC: Senescence-accelerated mice resistant 1 control group; PC: Senescence-accelerated mouse prone 8 control group; PT: Senescence-accelerated mouse prone 8-neural stem cells transplantation group; PS: Senescence-accelerated mouse prone 8-sham transplantation group; PTA: Senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint group; PTN: Senescence-accelerated mouse prone 8-neural stem cells transplantation with non-acupoint group; PTAH: Senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint and microRNA-124 high expression group; PTAL: Senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint and microRNA-124 low expression group; PTAC: Senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint and microRNA-124 control group.

Figure 3 Results of the water maze hidden platform test, Alzheimer’s disease pathological marker expression, and microRNA-124 gene expression. A-C: Water maze test results. The time from entering the water to finding the platform was recorded as the escape latency. If the mice did not find the platform within 90 seconds, the escape latency was recorded as 90 seconds; D-G: Alzheimer’s disease pathological marker expression; H: MicroRNA-124 gene expression. ^aP < 0.05, compared with the senescence-accelerated mice resistant 1 control group; ^bP < 0.05, compared with the senescence-accelerated mouse prone 8 control group; ^cP < 0.05, compared with the senescence-accelerated mouse prone 8-sham transplantation group; ^dP < 0.05, compared with the senescence-accelerated mouse prone 8-neural stem cells transplantation with non-acupoint group; ^eP < 0.05, compared with the senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint and microRNA-124 low expression group. RC: Senescence-accelerated mice resistant 1 control group; PC: Senescence-accelerated mouse prone 8 control group; PT: Senescence-accelerated mouse prone 8-neural stem cells transplantation group; PS: Senescence-accelerated mouse prone 8-sham transplantation group; PTA: Senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint group; PTN: Senescence-accelerated mouse prone 8-neural stem cells transplantation with non-acupoint group; PTAH: Senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint and microRNA-124 high expression group; PTAL: Senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint and microRNA-124 low expression group; PTAC: Senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint and microRNA-124 control group; Aβ: Amyloid β; APP: Amyloid precursor protein; P-Tau: Phosphorylated tau.

Figure 4 Expression of nestin, neuronal nuclear antigen, glial fibrillary acidic protein, Notch and Wnt signaling pathway proteins and genes. A: Notch homolog 1 and Delta-like 1 gene expression; B: Hairy and enhancer of split 1 and hairy and enhancer of split 5 gene expression; C: Glycogen synthase kinase 3β, Wingless-related integration site 1 (Wnt1), Wnt3a and β-catenin gene expression; D: Notch homolog 1 and Delta-like 1 protein expression; E: Hairy and enhancer of split 1 and hairy and enhancer of split 5 protein expression; F: Hairy/enhancer of split related protein expression; G: Hairy/enhancer of split related protein gene expression; H: Glycogen synthase kinase 3β, Wnt1, Wnt3a and β-catenin protein expression; I: Myc proto-oncogene, cell cycle protein D1 and cell cycle protein D2 protein expression; J: Myc proto-oncogene, cell cycle protein D1 and cell cycle protein D2 gene expression; K: Wnt5a, phospholipase C, protein kinase C and calmodulin-dependent protein kinase II protein expression; L: Wnt5a, phospholipase C, protein kinase C and calmodulin-dependent protein kinase II gene expression; M: Nestin, neuronal nuclear antigen, glial fibrillary acidic protein, Notch and Wnt signaling pathway protein expression; N: Nestin, neuronal nuclear antigen and glial fibrillary acidic protein expression. ^aP < 0.05, compared with the senescence-accelerated mice resistant 1 control group; ^bP < 0.05, compared with the senescence-accelerated mouse prone 8 control group; ^cP < 0.05, compared with the senescence-accelerated mouse prone 8-sham transplantation group; ^dP < 0.05, compared with the senescence-accelerated mouse prone 8-neural stem cells transplantation with non-acupoint group; ^eP < 0.05, compared with the senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint and microRNA-124 low expression group. RC: Senescence-accelerated mice resistant 1 control group; PC: Senescence-accelerated mouse prone 8 control group; PT: Senescence-accelerated mouse prone 8-neural stem cells transplantation group; PS: Senescence-accelerated mouse prone 8-sham transplantation group; PTA: Senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint group; PTN: Senescence-accelerated mouse prone 8-neural stem cells transplantation with non-acupoint group; PTAH: Senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint and microRNA-124 high expression group; PTAL: Senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint and microRNA-124 low expression group; PTAC: Senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint and microRNA-124 control group; Notch1: Notch homolog 1; Delta1: Delta-like 1; Hes1: Hairy and enhancer of split 1; Hes5: Hairy and enhancer of split 5; GSK3β: Glycogen synthase kinase 3β; Wnt1: Wingless-related integration site 1; Wnt3a: Wingless-related integration site 3a; HERP: Hairy/enhancer of split related protein; C-myc: Myc proto-oncogene; Cyclin D1: Cell cycle protein D1; Cyclin D2: Cell cycle protein D2; Wnt5a: Wingless-type integration site family member 5a; PLC: Phospholipase C; PKC: Protein kinase C; CamkII: Calcium/calmodulin-dependent protein kinase II; NeuN: Neuronal nuclear antigen; GFAP: Glial fibrillary acidic protein.

Figure 5 Expression of the interacted protein cyclin D1 and cell proliferation results. A: Cell cycle protein D1 flow cytometry results; B: Cell cycle protein D1 expression; C: Cell cycle flow cytometry results; D: Cell cycle proliferation index; E: G0/G1 results; F: G2/M results; G: S results; H: Nucleotides percentage; I: Cell apoptosis flow cytometry results; J: Apoptosis rate. ^aP < 0.05, compared with the senescence-accelerated mice resistant 1 control group; ^bP < 0.05, compared with the senescence-accelerated mouse prone 8 control group; ^cP < 0.05, compared with the senescence-accelerated mouse prone 8-sham transplantation group; ^dP < 0.05, compared with the senescence-accelerated mouse prone 8-neural stem cells transplantation with non-acupoint group; ^eP < 0.05, compared with the senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint and microRNA-124 low expression group. RC: Senescence-accelerated mice resistant 1 control group; PC: Senescence-accelerated mouse prone 8 control group; PT: Senescence-accelerated mouse prone 8-neural stem cells transplantation group; PS: Senescence-accelerated mouse prone 8-sham transplantation group; PTA: Senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint group; PTN: Senescence-accelerated mouse prone 8-neural stem cells transplantation with non-acupoint group; PTAH: Senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint and microRNA-124 high expression group; PTAL: Senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint and microRNA-124 low expression group; PTAC: Senescence-accelerated mouse prone 8-neural stem cells transplantation with acupoint and microRNA-124 control group.