Erzhi pills ameliorate cognitive dysfunction and alter proteomic hippocampus profiles induced by d-galactose and Aβ_1–40 injection in ovariectomized Alzheimer’s disease model rats

Figures & data

Figure 1. Schematic representation of the animal experimental design.

Table 1. The specnuezhenide content of Erzhi pills.

Number of experiment	m1 (mg)	Peak area	m2 (mg)	Content of Specnuezhenide (mg/g)
One	499.70	1785.80 ± 23.69	7.60	15.20
Two	500.60	1954.70 ± 4.53	8.50	16.90
Three	500.90	1852.20 ± 3.00	8.00	15.90

Note: m₁: The quality of Erzhi pills powder (precision weighing); m₂: The quality of specnuezhenide in Erzhi pills.

Figure 2. Effects of oral Erzhi pills on body weight in mice. (A) Male mice treated with Erzhi pills in acute toxicity test. (B) Female mice treated with Erzhi pills in acute toxicity test. Data are means ± SD, N = 10.

Figure 3. Effect of Erzhi pills on learning and memory ability in AD model rats induced by ovariectomy as well as d-galactose and Aβ_1–40 injection in Morris water test. (A) The effect of Erzhi pills on the escape latency in AD rats. (B) The effect of Erzhi pills on the times of crossing platform in AD rats. (C)The heatmap of the space exploration experiment in rats. The warmer the hue is in the heatmap, the longer the time will stay. ^#p < 0.05, vs. Sham-operated group; *p < 0.05, **p < 0.01, vs. model group. Mean ± SD, n = 10.

Figure 4. Effect of Erzhi pills on oestrogen level in AD model rats induced by ovariectomy as well as d-galactose and Aβ_1–40 injection. ^#p < 0.05, vs. Sham-operated group; *p < 0.05, vs. model group. Mean ± SD, n = 10.

Figure 5. Effect of Erzhi pills on the morphology and number of hippocampal neurons in AD model rats induced by ovariectomy as well as d-galactose and Aβ_1–40 injection. (A) The effect of the Erzhi pills on the CA1 cells in AD rats (HE staining, 200×). (B) The effect of Erzhi pills on neurons necrosis rate in hippocampal CA1 area of AD rats. (C) The effect of Erzhi pills on hippocampal CA1 region neurons of AD rats (Nissl staining, 200×). (D) The effect of Erzhi pills on the number of Nissl bodies in hippocampal CA1 area of AD rats. ^##p < 0.01, vs. Sham-operated group; *p < 0.05, **p < 0.01, vs. model group. Mean ± SD, n = 6.

Figure 6. Effects of Erzhi pills on the expression of ERβ in hippocampus of AD model rats induced by ovariectomy as well as d-galactose and Aβ_1–40 injection. (A) The effect of Erzhi pills on ERβ expression in hippocampus CA1 area of AD rats (IHC, 200×). (B) The effect of Erzhi pills on the number of ERβ positive cells in hippocampus CA1 area of AD rats. ^##p < 0.01, vs. Sham-operated group; *p < 0.05, **p < 0.01, vs. model group. Mean ± SD, n = 6.

Figure 7. Effects of Erzhi pills on the expression of Aβ_1–40 and p-tau⁴⁰⁴ in hippocampus of AD model rats induced by ovariectomy as well as d-galactose and Aβ_1–40 injection. (A) The effect of Erzhi pills on Aβ_1–40 expression in hippocampus CA1 area of AD rats (IHC, 200×). (B) The effect of Erzhi pills on p-tau⁴⁰⁴ expression in hippocampus CA1 area of AD rats (200×). (C) The expression of Aβ_1–40 and p-tau⁴⁰⁴ Positive cells in hippocampus CA1 region of AD rats. ^##p < 0.01, vs. Sham-operated group; **p < 0.01, vs. model group. Mean ± SD, n = 6.

Figure 8. Results of differential proteins by GO analysis and String network analysis. (A) The Cellular Component analysis result of differential proteins. (B) The Molecular Function analysis result of differential proteins. (C) The Biological Process analysis result from differential proteins. (D) The evidence wiring diagram of protein. (E) The Confidence wiring diagram of protein. (F) The molecular action wiring diagram of protein.

Table 2. The results of KEGG database signal path enrichment analysis (top 25).

Pathway	Count	Gene of participated protein
Synaptic vesicle cycle	6	Ap2b1, Cltc, Stx1a, Stx1b, Stxbp1, Vamp2
Hippo signalling pathway	6	Actb, Ctnnb1, Gsk3b, Ywhab, Ywhae, Ywhag
PI3K-Akt signalling pathway	5	Gsk3b, Ywhab, Ywhae, Ywhag, Akt1
Huntington’s disease	5	Ap2b1, Cltc, Ndufv2, Slc25a4, Sod1
Epstein–Barr virus infection	5	Gsk3b, Hspa1, Ywhab, Ywhae, Ywhag
Carbon metabolism	4	Aco2, Gpi, Mdh2, Ogdh
Metabolic pathways	4	Aco2, Gpi, Ndufv2, Ogdh
SNARE interactions in vesicular transport	4	Stx1a, Stx1b, Vamp2, Vamp3
Cell cycle	4	Gsk3b, Ywhab, Ywhae, Ywhag
Wnt signalling pathway	3	Gsk3b, CaMKII, β-catenin
MAPK signalling pathway	3	Cacna2d3, Hspa1, Mapt
Microbial metabolism in diverse environments	3	Gpi, Mdh2, Ogdh
Thyroid hormone signalling pathway	3	Actb, Ctnnb1, Gsk3b
Bacterial invasion of epithelial cells	3	Actb, Cltc, Ctnnb1
Influenza A	3	Actb, Gsk3b, Hspa1
AMPK signalling pathway	3	Rab10, Rab2a, Rab8a
Adherens junction	3	Actb, Baiap2, Ctnnb1
Arrhythmogenic right ventricular cardiomyopathy (ARVC)	3	Actb, Cacna2d3, Ctnnb1
Citrate cycle (TCA cycle)	2	Mdh2, Ogdh
Phagosome	2	Actb, Vamp3
Endocrine and other factor-regulated calcium reabsorption	2	Ap2b1, Cltc
Legionellosis	2	Hspa1, Rab1b
Parkinson s disease	2	Ndufv2, Slc25a4
Endocytosis	2	Ap2b1, Cltc
Alzheimer s disease	2	Gsk3b, Mapt

Figure 9. Effects of Erzhi pills on PI3K/Akt signalling pathway in hippocampal tissues of AD rats. (A) Erzhi pills enhance PI3K phosphorylation in hippocampal tissues of AD model rats induced by ovariectomy as well as d-galactose and Aβ_1–40 injection. (B) Erzhi pills enhance Akt phosphorylation in hippocampal tissues of AD model rats induced by ovariectomy as well as d-galactose and Aβ_1–40 injection. (C) Erzhi pills up-regulated 14-3-3 protein expressions and down-regulated GSK3β phosphorylation in hippocampal tissues of AD model rats induced by ovariectomy as well as d-galactose and Aβ_1–40 injection. (D) Erzhi pills up-regulated Bcl-2, Bcl-xl protein expressions and down-regulated Bad protein expressions in hippocampal tissues of AD model rats induced by ovariectomy as well as d-galactose and Aβ_1–40 injection. Data are presented as the mean ± SD. for at least five individual experiments. ^#p＜0.05, ^##p＜0.01, vs. Sham-operated group; *p < 0.05, **p＜0.01, vs. model group.