Shen-Zhi-Ling oral liquid ameliorates cerebral glucose metabolism disorder in early AD via insulin signal transduction pathway in vivo and in vitro

Abstract

Background: Shen-Zhi-Ling oral liquid (SZL) is an herbal formula known for its efficacy of nourishing "heart and spleen", and is used for the treatment and prevention of middle- and early-stage dementia. This study investigated the effects of SZL on amelioration of AD, and examined whether the underlying mechanisms from the perspective of neuroprotection are related to brain glucose metabolism.

Methods: Firstly, LC-MS/MS was used to analysis the SZL mainly enters the blood component. Then, the effects of SZL on cognitive and behavioral ability of APP/PS1 double transgenic mice and amyloid protein characteristic pathological changes were investigated by behavioral study and morphological observation. The effects of SZL on the ultrastructure of mitochondria, astrocytes, and micrangium related to cerebral glucose metabolism were observed using transmission electron microscopy. Then, micro-PET was also used to observe the effects of SZL on glucose uptake. Furthermore, the effects of SZL on insulin signaling pathway InR/PI3K/Akt and glucose transporters (GLUT1 and GLUT3) were observed by immunohistochemistry, Western-blot and RT-qPCR. Finally, the effects of SZL on brain glucose metabolism and key enzyme were observed. In vitro, the use of PI3K and/or GSK3β inhibitor to observe the effects of SZL drug-containing serum on GLUT1 and GLUT3.

Results: In vivo, SZL could significantly ameliorate cognitive deficits, retarded the pathological damage, including neuronal degeneration, Aβ peptide aggregation, and ultrastructural damage of hippocampal neurons, improve the glucose uptake, transporters and glucolysis. Beyond that, SZL regulates the insulin signal transduction pathway the insulin signal transduction pathway InR/PI3K/Akt. Furthermore, 15% SZL drug-containing serum increased Aβ₄₂-induced insulin signal transduction-pathway related indicators and GLUT1 and GLUT3 expression in SH-SY5Y cells. The improvement of GLUT1 and GLUT3 in the downstream PI3K/Akt/GSK3β signaling pathway was reversed by the use of PI3K and/or GSK3β inhibitor.

Conclusions: In summary, our results demonstrated that improving glucose uptake, transport, and glycolysis in the brain may underlie the neuroprotective effects of SZL, and its potential molecular mechanism may be related to regulate the insulin signal transduction pathway.

Keywords: Glucolysis; Glucose metabolism; Glucose transporter; Insulin signal transduction; SH-SY5Y cells; Shenzhiling oral liquid.

Fig. 1

The research framework of the study

Fig. 2

Experimental program

Fig. 3

LC–MS/MS was used to analyze the main blood components of SZL. Total ion flow pattern in positive ion mode was detected by UHPLC-QE-MS (A–C). Total ion flow pattern in negative ion mode was detected by UHPLC-QE-MS (D–F). SZL serum containing drug (A, D), Blank serum without drug (B, E), SZL oral solution (C, F). Heat map of the main components of SZL into blood (G)

Fig. 4

Effect of SZL cognitive deficits and improved memory abilities in APP/PS1 mice. Simple pattern of MWM (A). The figures represent the first, second, third, and fourth quadrants, respectively. Each quadrant of the inner wall of the MWM has visual signs to help the mice remember route. The platform is in the third quadrant effect of SZL treatment on the moving track (B). The movement trajectories of mice in each group entering water in different quadrants on the fifth day were selected for comparison. The first quadrant is in the upper left corner of the moving track map, and the remaining quadrants are 2, 3, and 4 in the counterclockwise direction. The effect of SZL treatment on mean swimming distances (C), Escape latency (D) times of crossing platform (E) and target quadrant dwelling time (F) in the MWM test. CG: control group, MG: model group, DG: donepezil group, SG: SZL group. All data are presented as means ± SEM (n = 15). *P < 0.05, **P < 0.01 versus Control group, ^△P < 0.05, ^△△P < 0.01 versus Model group; One-way ANOVA was used to calculate the p-values. The average swimming distance and escape latency of mice in the Morris Water Maze experiment were repeated measurements, both of which were in line with the spherical test, and a repeated-measures ANOVA was applied

Fig. 5

Effect of SZL on the main structure and the expression of Aβ₄₂ in APP/PS1 mice. Statistical analysis of positive cells of HE (A1, A2; scale bar = 50 μm; n = 6). Statistical analysis of amyloid plaques area (B1–B3; scale bar = 200 μm; n = 3). Statistical analysis of protein expressions of Aβ₄₂ (C). All data are presented as means ± SEM.CG: control group, MG: model group, DG: donepezil group, SG: SZL group. *P < 0.05, **P < 0.01 versus Control group, ^△P < 0.05, ^△△P < 0.01 versus Model group; One-way ANOVA was used to calculate the p-values

Fig. 6

Effect of SZL on hippocampal ultrastructural in APP/PS1 mice. A Ultrastructure of neurons (× 1.5 k, scale = 2.0 μm, n = 3), B Ultrastructure of neuronal organelles (× 8 k, scale = 500 nm, n = 3), C Ultrastructure of astrocytes (× 2 k, scale = 2.0 μm, n = 3), D Ultrastructure of microvascular (× 4 k, scale = 1.0 μm, n = 3). Statistical analysis of Lipofuscin area (A1) and Number of damaged mitochondria (B1). All data are presented as means ± SEM.CG: control group, MG: model group, DG: donepezil group, SG: SZL group. *P < 0.05, **P < 0.01 versus Control group, ^△P < 0.05, ^△△P < 0.01 versus Model group; One-way ANOVA was used to calculate the p-values

Fig. 7

Effect of SZL on the expression of InR, p-InR, IRS2, p-IRS2 in hippocampus in APP/PS1 mice. Statistical analysis of positive cells of InR (A1, A2), IRS2 (B1, B2) (n = 6). Statistical analysis of mRNA expression of InR (C), IRS2 (D) (n = 3). Statistical analysis of protein expressions of p-InR/ InR(E). p-IRS2/ IRS2 (F) (n = 3).CG: control group, MG: model group, DG: donepezil group, SG: SZL group. All data are presented as means ± SEM.*P < 0.05, **P < 0.01 versus Control group, ^△P < 0.05, ^△△P < 0.01 versus Model group; One-way ANOVA was used to calculate the p-values

Fig. 8

The effect of SZL on PI3K/Akt/GSK3β pathway. Statistical analysis of positive cells of GSK3β (A1, A2), p-GSK3β (B1, B2) (n = 6). Statistical analysis of mRNA expression of GSK3β (C) (n = 3). Statistical analysis of protein expressions of p-PI3K/PI3K (D), p-Akt/Akt (E), GSK3β (F), p-GSK3β (G) (n = 3).CG: control group, MG: model group, DG: donepezil group, SG: SZL group. All data are presented as means ± SEM. *P < 0.05, **P < 0.01 versus Control group, ^△P < 0.05, ^△△P < 0.01 versus Model group; One-way ANOVA was used to calculate the p-values

Fig. 9

Effect of SZL on glucose transporter-related proteins and glycolysis-related genes. Statistical analysis of hippocampal glucose uptake (A1, A2): 1.Standard mouse brain template localization.2. Hippocampal positioning (n = 3). Statistical analysis of positive cells of GLUT3 (B1, B2), Statistical analysis of protein expressions of GLUT1 (D), GLUT3 (E) (n = 6). Statistical analysis of mRNA expression of GLUT1 (C), HK1 (F), COXIV (G), ATPase (H), AMPK (I) (n = 3).CG: control group, MG: model group, DG: donepezil group, SG: SZL group. All data are presented as means ± SEM.*P < 0.05, **P < 0.01 versus Control group, ^△P < 0.05, ^△△P < 0.01 versus Model group; One-way ANOVA was used to calculate the p-values

Fig. 10

Effect of SZL-containing serum on the expression of insulin signal transduction pathway. Statistical analysis of protein expressions of p-PI3K/PI3K (A), p-Akt/Akt (B), GSK3β (E), p-GSK3β (F). Statistical analysis of mRNA of PI3K (C), Akt (D), GSK3β (G). CG: control group. All data are presented as means ± SEM (n = 6). *P < 0.05, **P < 0.01 versus Control group, ^△P < 0.05, ^△△P < 0.01 versus Model group; One-way ANOVA was used to calculate the p-values

Fig. 11

Effect of SZL-containing serum on the expression of dysfunction of CLUTs. Statistical analysis of fluorescence intensity of GLUT1 (A1, A2). Statistical analysis of protein expressions of GLUT1 (B), GLUT3 (D). Statistical analysis of mRNA expression of GLUT1 (C), GLUT3 (E).CG: control group. All data are presented as means ± SEM (n = 6). *P < 0.05, **P < 0.01 versus Control group, ^△P < 0.05, ^△△P < 0.01 versus Model group, ^▼P < 0.05, ^▼▼P < 0.01 versus SZL-containing serum group; One-way ANOVA was used to calculate the p-values

Fig. 12

Effects of SZL-containing serum with PI3K and/or GSK3β inhibitor. Statistical analysis of protein expressions of p-Akt (A1), p-GSK3β (A2), GLUT1 (A3), GLUT3 (A4) with PI3K inhibitor. Statistical analysis of protein expressions of p-GSK3β (B1), GLUT1 (B2), GLUT3 (B3) with GSK3β inhibitor. Statistical analysis of protein expressions of GLUT1 (C1), GLUT3 (C2) with PI3K and GSK3β inhibitor. CG: control group. All data are presented as means ± SEM (n = 6). *P < 0.05, **P < 0.01 versus Control group, ^△P < 0.05, ^△△P < 0.01 versus Model group; ^▼P < 0.05, ^▼▼P < 0.01 versus SZL-containing serum group; One-way ANOVA was used to calculate the p-values