Qingxin kaiqiao fang ameliorates memory impairment and inhibits apoptosis in APP/PS1 double transgenic mice through the MAPK pathway

Abstract

Background: Qingxin kaiqiao fang (QKF) has been found to treat Alzheimer's disease (AD) through apoptosis inhibition. The mitogen-activated protein kinase (MAPK) pathway is closely related to apoptosis in the course of AD. This study aimed to investigate whether QKF-induced apoptosis depression is achieved through MAPK pathway.

Materials and methods: C57BL/6 J and APP/PS1 mice were used as control and model groups. APP/PS1 mice were treated with different dosages of QKF (4.75, 9.5, and 19 g⋅kg^-1⋅d^-1⋅ig, respectively) for 12 weeks as L-QKF, M-QKF, and H-QKF groups. The M-QKF-treated APP/ PS1 mice were administrated with 2 µg/kg of U46619 and saline, intra ventricular ventricle injection, as M-QKF+U46619 and M-QKF+saline groups and were injected with PD98059 0.3 mg/kg and the same volume of dimethyl sulfoxide (DMSO), intravenous, as M-QKF+PD98059 and M-QKF+DMSO groups. After 12 weeks treatment, Morris water maze was performed for behavior study. Pathological degeneration was examined by H&E staining, Nissl staining, and transmission electron microscope observation of hippocampus; immunohistochemistry and Western blot (WB) were tested for amyloid β (Aβ) expression. Apoptosis was measured through TUNEL assay; Bax, Bcl-2, and caspase-3 expression through WB; and cleaved caspase-3 expression through ELISA. MAPK pathway was detected via WB for the expressions of ERK1/2, JNK, and p38 MAPK and their phosphorylation patterns.

Results: QKF improved the learning and memory capability, as well as inhibited neuronal apoptosis and then reduced the pathological degeneration of APP/PS1 mice. M-QKF reduced neuron apoptosis by inhibiting p38 MAPK and activating ERK1/2 but had no significant effect on JNK.

Conclusion: QKF, especially at the middle dose, alleviated the learning and memory impairment and played an antiapoptotic role in AD through MAPK pathways.

Keywords: APP; Alzheimer’s disease; MAPK; PS1 mice; Qingxin kaiqiao; apoptosis.

Figure 1

QKF ameliorate learning and memory capability of model mice. Notes: (A) Representative path tracking in the navigation tests with hidden platform. (B) Average latencies curve of four trials per day in the four consecutive days. (C) Quantified and analyzed escape latency of each group. (D) Representative path tracking in the probe tests without hidden platform. (E) The average times that the mice crossed platform location in 60 seconds. (F) The percentage of searching time that the mice of individual groups spent in the target quadrant. Values are expressed as mean±SEM, n=10 per group. Significant differences between groups are indicated as *P<0.05 and **P<0.01. L-QKF, low-dose QKF group; M-QKF, middle-dose QKF group; H-QKF, high-dose QKF group. Abbreviations: QKF, qingxin kaiqiao fang; SEM, standard error of the mean; WT, wild type.

Figure 2

QKF alleviate pathological degeneration in the brain of APP/PS1 mice. Notes: (A) Nissl staining in CA1 region of the hippocampus (original magnification, ×400; scan bar, 20 µm). (B) Nissl staining in CA1 region of the hippocampus (original magnification, ×400; scan bar, 20 µm). (C) Immunohistochemistry for Aβ expression in the CA1 region of the hippocampus for each group (original magnification, ×400; scan bar, 20 µm). (D) Analysis of Aβ deposition levels by immunohistochemical staining. Values are expressed as the mean±SEM, n=30 per group. Significant differences between groups are indicated as *P<0.05 and **P<0.01. (E) WB for Aβ1-42 expression in the hippocampus of mice. (F) ODs indicative of Aβ1-42 protein expression. Values are expressed as mean±SEM, n=5 per group. Significant differences between groups are indicated as *P<0.05 and **P<0.01. (G) Ultrastructural observation of the CA1 area in the hippocampus of mice in each group using a transmission electron microscope (original magnification, ×18,500; scan bar, 2 µm). L-QKF, low-dose QKF group; M-QKF, middle-dose QKF group; H-QKF, high-dose QKF group. Abbreviations: QKF, qingxin kaiqiao fang; Aβ, amyloid β; SEM, standard error of the mean; WB, Western blot.

Figure 3

QKF alleviate neuronal apoptosis in the hippocampus of APP/PS1 mice. Notes: (A) TUNEL staining in the hippocampal CA1 area of each group of mice (original magnification, ×400). (B) Statistical analysis results of TUNEL staining. (C) Protein expression of Bax, Bcl-2, and Caspase-3 in the hippocampus. (D) ODs indicative of Bax protein expression. (E) ODs indicative of Bcl-2 protein expression. (F) ODs indicative of caspase-3 protein expression. OD values of Bax, Bcl-2, and caspase-3 expression in each group were quantified and analyzed from the WB results. (G) Levels of cleaved caspase-3 in mice brain were determined by an ELISA assay. Values are expressed as mean±SEM, n=5 per group. Significant differences between two groups are indicated as **P<0.01. M-QKF, middle-dose QKF group. Abbreviations: QKF, qingxin kaiqiao fang; WB, Western blot; SEM, standard error of the mean.

Figure 4

The effects of QKF on MAPK pathway. Notes: (A) Protein expression of p-ERK1/2 and ERK1/2 in the hippocampus. (B) ODs indicative of ERK1/2 protein expression. (C) ODs indicative of p-ERK1/2 protein expression. (D) Protein expression of p-JNK1, p-JNK2 and JNK1, JNK2 in the hippocampus. (E) ODs indicative of JNK1 protein expression. (F) ODs indicative of p-JNK1 protein expression. (G) ODs indicative of JNK2 protein expression. (H) ODs indicative of p-JNK2 protein expression. (I) Protein expression of p-p38 MAPK and p38 MAPK in the hippocampus. (J) ODs indicative of p38 MAPK protein expression. (K) ODs indicative of p-p38 MAPK protein expression OD values of p-ERK1/2, ERK1/2, p-JNK1, p-JNK2, JNK1, JNK2, p-p38 MAPK, and p38 MAPK expression in each group are quantified and analyzed from the WB results. Values are expressed as mean±SEM, n=5 per group. Significant differences between two groups are indicated as *P<0.05 and **P<0.01. M-QKF, middle-dose QKF group. Abbreviations: QKF, qingxin kaiqiao fang; WB, Western blot; SEM, standard error of the mean.

Figure 5

M-QKF alleviate neuronal apoptosis by activating ERK1/2. Notes: (A) Representative path tracking in the navigation tests with hidden platform. (B) Representative path tracking in the probe tests without hidden platform. (C) Average latencies curve of four trials per day in the four consecutive days. (D) Escape latency of each group was quantified and analyzed. (E) The average times that the mice crossed platform location in 60 seconds. (F) The percentage of searching time that the mice of individual groups spent in the target quadrant where the platform has been located in days 1–4. (G) Protein expression of Bax, Bcl-2, and caspase-3 in the hippocampus. (H) Protein expression of p-ERK1/2 and ERK1/2 in the hippocampus. (I) ODs indicative of Bax, Bcl-2, and caspase-3 protein expression. (J) ODs indicative of p-ERK1/2 and ERK1/2 protein expression. (K) Levels of cleaved caspase-3 in mice brain were determined by an ELISA assay. Values are expressed as mean±SEM, n=5 per group. Significant differences between two groups are indicated as *P<0.05 and **P<0.01. M-QKF, middle-dose QKF group. Abbreviations: QKF, qingxin kaiqiao fang; SEM, standard error of the mean; DMSO, dimethyl sulfoxide.

Figure 5

M-QKF alleviate neuronal apoptosis by activating ERK1/2. Notes: (A) Representative path tracking in the navigation tests with hidden platform. (B) Representative path tracking in the probe tests without hidden platform. (C) Average latencies curve of four trials per day in the four consecutive days. (D) Escape latency of each group was quantified and analyzed. (E) The average times that the mice crossed platform location in 60 seconds. (F) The percentage of searching time that the mice of individual groups spent in the target quadrant where the platform has been located in days 1–4. (G) Protein expression of Bax, Bcl-2, and caspase-3 in the hippocampus. (H) Protein expression of p-ERK1/2 and ERK1/2 in the hippocampus. (I) ODs indicative of Bax, Bcl-2, and caspase-3 protein expression. (J) ODs indicative of p-ERK1/2 and ERK1/2 protein expression. (K) Levels of cleaved caspase-3 in mice brain were determined by an ELISA assay. Values are expressed as mean±SEM, n=5 per group. Significant differences between two groups are indicated as *P<0.05 and **P<0.01. M-QKF, middle-dose QKF group. Abbreviations: QKF, qingxin kaiqiao fang; SEM, standard error of the mean; DMSO, dimethyl sulfoxide.

Figure 6

M-QKF alleviates neuronal apoptosis by inhibiting p38 MAPK. Notes: (A) Representative path tracking in the navigation tests with hidden platform. (B) Representative path tracking in the probe tests without hidden platform. (C) Average latencies curve of four trials per day in the four consecutive days. (D) Escape latency of each group were quantified and analyzed. (E) The average times that the mice crossed platform location in 60 seconds. (F) The percentage of searching time that the mice of individual groups spent in the target quadrant where the platform has been located in days 1–4. (G) Protein expression of Bax, Bcl-2, and caspase-3 in the hippocampus. (H) Protein expression of p-p38 MAPK and p38 MAPK in the hippocampus. (I) ODs indicative of Bax, Bcl-2, and caspase-3 protein expression. (J) ODs indicative of p-p38 MAPK and p38 MAPK protein expression. (K) Levels of cleaved caspase-3 in mice brain were determined by an ELISA assay. Values are expressed as the mean±SEM, n=5 per group. Significant differences between two groups are indicated as *P<0.05 and **P<0.01. M-QKF, middle-dose QKF group. Abbreviations: QKF, qingxin kaiqiao fang; SEM, standard error of the mean.

Figure 6

M-QKF alleviates neuronal apoptosis by inhibiting p38 MAPK. Notes: (A) Representative path tracking in the navigation tests with hidden platform. (B) Representative path tracking in the probe tests without hidden platform. (C) Average latencies curve of four trials per day in the four consecutive days. (D) Escape latency of each group were quantified and analyzed. (E) The average times that the mice crossed platform location in 60 seconds. (F) The percentage of searching time that the mice of individual groups spent in the target quadrant where the platform has been located in days 1–4. (G) Protein expression of Bax, Bcl-2, and caspase-3 in the hippocampus. (H) Protein expression of p-p38 MAPK and p38 MAPK in the hippocampus. (I) ODs indicative of Bax, Bcl-2, and caspase-3 protein expression. (J) ODs indicative of p-p38 MAPK and p38 MAPK protein expression. (K) Levels of cleaved caspase-3 in mice brain were determined by an ELISA assay. Values are expressed as the mean±SEM, n=5 per group. Significant differences between two groups are indicated as *P<0.05 and **P<0.01. M-QKF, middle-dose QKF group. Abbreviations: QKF, qingxin kaiqiao fang; SEM, standard error of the mean.