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Okra polysaccharides can reverse the metabolic disorder induced by high-fat diet and cognitive function injury in Aβ1–42 mice
Experimental Gerontology 130 (2020) 110802
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Okra polysaccharides can reverse the metabolic disorder induced by high-fat diet and cognitive function injury in Aβ1–42 mice Tingxu Yana, Tingting Nianb, Bo Wua, Feng Xiaoa, Bosai Hea, Kaishun Bic, Ying Jiaa,
T
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a
School of Functional Food and Wine, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China c School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China b
A R T I C LE I N FO
A B S T R A C T
Section Editor: Holly M Brown-Borg
Epidemiological studies showed that a high-fat diet threatened human health seriously. It can induce various diseases, such as obesity, metabolic disturbance and cognitive dysfunction which also related to insulin signaling. In the present study, Aβ1–42 induced AD model mice and normal mice were given a standard diet and high-fat diet, respectively. Meanwhile, Okra polysaccharides were used to treat AD mice to explore the possible mechanism between Alzheimer's disease and insulin signals. Weight and blood glucose of mice were measured weekly. Through the Morris water maze and the novel object recognition test, the Okra polysaccharides could improve the cognitive impairment of the AD mice. In addition, we also performed the serum chemistry analysis of mice, studied the histopathological changes in the hippocampal CA1 region by HE staining and detected the expressions of AKT, PI3K, ERK1/2, and GSK3β in the hippocampus by western blot. These results suggested that a high-fat diet can aggravate the metabolic disorder in AD mice and Okra polysaccharides can significantly reverse the metabolic disorder induced by high-fat diet and cognitive function injury in AD mice.
Keywords: Okra polysaccharides Alzheimer's disease High-fat diet PI3K/AKT/GSK3β ERK1/2
1. Introduction In recent years, epidemiological and clinical studies have revealed a close relationship between T2D and Alzheimer's disease (Zoe Arvanitakis et al., 2004). Aβ oligomers may also interfere with insulin signaling in hippocampus neurons (Zhao et al., 2008), and insulin may regulate Aβ levels by modulation of β and γ secretases (Farris et al., 2003). Moreover, the central nervous system insulin receptors are highly expressed in regions relevant for cognition, such as the cortex and hippocampus. Insulin signaling is a key factor in brain homeostasis, metabolism, synaptic plasticity, and memory, imploring the need for therapeutic interventions that target the insulin signaling axis, in particular the common convergence point, ERK (Dineley et al., 2014). In the hippocampus, the ERK cascade is critical in the induction and maintenance of long term potentiation (LTP) and memory consolidation as it converges with a number of other signaling cascades, including PI3K/AKT, CaMKII, PKA, and PKC (Sweatt, 2004; Kelleher III, et al., 2004; Sweatt, 2001). Insulin receptor substrate activates PI3K activation which then leads to AKT activation (Williams, 2011). Obesity is often caused by and is associated with, consumption of diets that are high in fat (Grant, 1999). There has been solid evidence that long-term high-fat causes an increased incidence of metabolic
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diseases such as obesity, diabetes, and hyperinsulinemia. Additionally, epidemiological and clinical data have suggested that high-fat diet may also contribute to the development of Alzheimer's disease (AD) (Cordner and Tamashiro, 2015; Eskelinen et al., 2008; Francis and Stevenson, 2013). Recently, with the research of Okra (Abelmoschus esculentus L. Moench), we found it has huge potential. Okra, an annual vegetable belonging to the malvaceae family, is also known as lady's finger. Immature okra pods are consumed in most areas of the world, supplying carbohydrates, minerals, and vitamins and are also a source of dietary medicines (Adelakun et al., 2009). The peel and seed powders of Okra have been reported to play antidiabetic and antihyper lipidemic roles in STZ-induced diabetic rats (Sabitha et al., 2011). Okra is full of polysaccharides and polysaccharides have the ability to regulating metabolism and improving Alzheimer's disease (Sabitha et al., 2011; Huang et al., 2017a; Fan et al., 2013). In the present study, to explore the relationship between AD and obesity. We are focusing on the insulin signaling pathway as a starting point. Since insulin signaling plays an important role in the pathological changes of AD, and obesity can also mediate changes in insulin signaling pathways. Although some potential mechanisms have been proposed, there is still a considerable knowledge gap by identifying roles and molecular mechanisms that underlie the association between
Corresponding author at: School of Functional Food and Wine, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China. E-mail address: [email protected] (Y. Jia).
https://doi.org/10.1016/j.exger.2019.110802 Received 8 October 2019; Received in revised form 12 November 2019; Accepted 28 November 2019 Available online 30 November 2019 0531-5565/ © 2019 Elsevier Inc. All rights reserved.
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1
4
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5
3
A 4
2
5 1
3
B Fig. 1. Monosaccharides analysis of OP by RP-HPLC. (A) (1) Mannose, (2) Rhamnose, (3) Glucose, (4) Galactose, (5) Arabia sugar were analyzed as standards by using HPLC with a C-18 reverse phase column at 250 nm; (B) The major components of the OP were detected for comparison with the retention times of the standard compounds. Table 1 The body weight of control or AD mice response to HFD or OP.
⁎
Group
Week 1
CON Aβ CON+HFD Aβ + HFD Aβ + OPL Aβ + OPH Aβ + HFD + OPL Aβ + HFD + OPH
34.05 33.21 32.48 34.81 33.97 33.24 34.81 33.67
± ± ± ± ± ± ± ±
Week 2 0.91 0.96 0.82 0.58 0.82 0.67 0.86 1.30
37.19 36.44 36.48 38.51 36.88 36.07 37.61 33.58
± ± ± ± ± ± ± ±
Week 4 0.82 0.84 0.77 0.44 0.96 0.35 0.53 1.76
39.23 38.87 43.30 44.02 39.32 39.16 42.95 43.14
± ± ± ± ± ± ± ±
Week 6 0.72 0.63 0.55 1.15⁎ 0.79 1.30 1.14 0.77
44.84 42.71 52.40 53.98 42.91 44.63 50.07 49.60
± ± ± ± ± ± ± ±
Week 8 1.13 1.19 0.49⁎⁎⁎ 1.04⁎⁎⁎ 0.57 0.59 0.66$ 0.71$
47.73 47.37 58.77 59.31 46.70 47.65 56.59 55.14
± ± ± ± ± ± ± ±
0.61 0.61 0.32⁎⁎⁎ 0.39⁎⁎⁎ 0.32 0.42 0.41$$ 0.49$$$
p < 0.05, ⁎⁎⁎p < 0.001 compared with the control group, $p < 0.05, $$p < 0.01, $$$p < 0.001 compared with the AD mice fed with HFD group (Aβ + HFD).
province, China) in August 2018. High-fat diet (SN10060) was provided by Ruidi Biotec (Shanghai, China). Aβ1–42 was purchased from SigmaAldrich. Donepezil (≥98%), Serum insulin kit was provided by Melone Pharmaceutical Co. Ltd., The following antibodies were supplied by CST (USA): anti-ERK1/2, anti-GSK3β, anti-PI3K p85, anti-AKT, antiphosphor-AKT (Ser473), anti-phosphor-ERK1/2 (T202/Y204), antiphosphor- PI3K p85 (Y607), anti-phosphor- GSK3β (Ser9) and β-actin.
obesity and AD. 2. Experimental section 2.1. Materials The fruits of Okra were purchased from Laiyang (Shandong 2
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Table 2 The blood glucose of control or AD mice response to HFD or OP.
⁎⁎
Group
Week 1
CON Aβ CON+HFD Aβ + HFD Aβ + OPL Aβ + OPH Aβ + HFD + OPL Aβ + HFD + OPH
6.60 6.85 6.59 6.69 6.91 6.68 6.96 6.54
± ± ± ± ± ± ± ±
Week 2 0.23 0.33 0.18 0.09 0.19 0.27 0.24 1.13
6.78 6.54 6.73 7.33 6.70 6.61 6.58 6.61
± ± ± ± ± ± ± ±
Week 4 0.20 0.24 0.17 0.16 0.24 0.17 0.14 0.23
6.38 6.74 6.84 7.49 6.80 6.73 6.89 6.81
± ± ± ± ± ± ± ±
Week 6 0.35 0.11 0.23 0.12⁎⁎ 0.15 0.22 0.16 0.17
6.71 6.80 6.99 8.09 6.91 6.59 6.74 6.63
± ± ± ± ± ± ± ±
Week 8 0.14 0.08 0.14 0.27⁎⁎ 0.30 0.21 0.28$ 0.32$$
6.81 6.41 7.45 9.31 6.77 6.55 6.79 6.90
± ± ± ± ± ± ± ±
0.17 0.17 0.15 0.10⁎⁎⁎ 0.21 0.30 0.15$$$ 0.20$$$
p < 0.01, ⁎⁎⁎p < 0.001 compared with the control group, $p < 0.05, $$p < 0.01, $$$p < 0.001 compared with the AD mice fed with HFD group (Aβ + HFD). 2.0
3
1.5
TC (mmol/L)
TG (mmol/L)
*
1.0 0.5 0.0
$ 2
1
Fig. 2. The level of serum lipids in mice. (A) Serum triglyceride (TG), (B) Total cholesterol (TC), (C) High-density lipoprotein cholesterol (HDL-c), (D) Low-density lipoprotein cholesterol (LDL-c). Values indicated the mean ± S.E.M. and were analyzed by one-way analysis of variance (ANOVA) followed by Tukeyʼs multiple comparison test (n = 8). ⁎ p < 0.05 compared with the control group, $ p < 0.05 compared with the AD mice fed with HFD group (Aβ + HFD).
0
A
B 0.50
2.5 2.0
$
1.5 1.0 0.5 0.0
LDL-c (mmol/L)
HDL-c (mmol/L)
*
0.25
0.00
C
D room temperature, and vacuum rotary evaporation was repeated until TFA was fully vaporized and then 2 mL water was added to the resolution. 800 μL of the solution was taken to put in a tube to derivative treatment, 800 μL of NaOH solution (0.3 mol/L) and 800 μL of PMP (0.5 mol/L) were added in solution. Mixed well and then react in a water bath of 70 °C for 2 h. 0.3 mol/L HCl (800 μL) was added after the solution restored at room temperature. 1 mL chloroform was added for extraction and water layer solution was collected. Repeat extraction three times. The standard compounds were also treated with the same derivatization procedure. The multi-components of hydrolyzate were analyzed by using an Agilent 1260 HPLC with an UV-DAD detector at a λ max of 250 nm. Chromatographic separation was performed on an Agilent C-18 reverse phase column (2.1 mm × 100 mm, 1.8 μm) with an injection volume of 20 μL. The mobile phase consisted of a mixture of distilled water (A): acetonitrile (B) 84:16. (flow rate 1 mL/min).
All other chemicals and solvents were of analytical reagent grade. 2.2. Isolation and purification of polysaccharides from Okra The polysaccharide was extracted with water (95 °C, mass ratio1:15) three times (6 h each time). The aqueous filtrates were combined and concentrated, then 95% ethanol (volume ratio 1:4) was added to the aqueous filtrates, 4 °C for overnight. The precipitate collected by centrifugation was dissolved in distilled water, after that, the supernatant was treated with Sevagreagent [V(n-butano1): V(chloroform)1: 4] to remove free proteins. Then the remaining were combined and concentrated. 2.3. Monosaccharides analysis of Okra polysaccharides by HPLC The Okra polysaccharides (10 mg) was dissolved with 2 mL of 2 M trifluoroacetic acid (TFA) at 110 °C for 8 h. The reaction solutions were filled with N2. 2 mL methanol was added after the solution restored at 3
Experimental Gerontology 130 (2020) 110802
The time in the target quadrant (s)
T. Yan, et al.
100 #
RI (%)
80 60
$
$
*
*
40 20 0
Fig. 3. Effects of OP on improving memory injury in novel object recognition. Values indicated the mean ± S.E.M. and were analyzed by one-way analysis of variance (ANOVA) followed by Tukeyʼs multiple comparison test (n = 8). ⁎ p < 0.05 compared with the control group, #p < 0.05 compared with the AD mice, $p < 0.05 compared with the AD mice fed with HFD group (Aβ + HFD).
50 40
#
$$
30
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***
20 10 0
Fig. 4. Time spent in the target quadrant on the 6th day. Values indicated the mean ± S.E.M. and were analyzed by one-way analysis of variance (ANOVA) followed by Tukeyʼs multiple comparison test (n = 8). ⁎⁎⁎p < 0.001 compared with the control group, #p < 0.05 compared with the AD mice, $$p < 0.01 compared with the AD mice fed with HFD group (Aβ + HFD).
2.4. Animals and diet administrated Okra polysaccharides 600 mg/kg (OPH) for 8 weeks].
Male 12-week-old Kunming (KM) mice, weighing 22–30 g, were purchased from the Central Animal House of University. The experiment was carried out in compliance with the National Institutes of Health and institutional guidelines for the humane care of animals and was approved by the Animal Care Committee of Shenyang Pharmaceutical University. Mice were housed in groups of four per cage, and maintained at a constant temperature (23 ± 1 °C) and humidity (55 ± 5%) under a 12-h light/dark cycle. All studies were carried out in accordance with the Animal Ethics Committee of the institution (approved on January 20, 2016, NO. SYPU-IACUCC2016–0120). All animals had free access to food and water. The normal group was fed with a standard diet (SD), the obesity group was fed with high-fat diet (HFD). Aβ1–42 was dissolved in DMSO (≥99.9%, 2 μL) and diluted in physiological saline to a stock concentration of 1.0 mg/mL. The solution of Aβ1–42 was incubated at 37 °C for 5 days to obtain the fibrillated form of Aβ1–42. The mice were allowed to acclimate for seven days prior to their use in the studies. The mice were randomly assigned to eight groups (n = 8 mice/group); Group 1 [CON; consumed SD], Group 2 [CON+HFD; consumed HFD for 8 weeks], Group 3 [Aβ; Aβ1–42 injections and then consumed SD for 8 weeks], Group 4 [Aβ + HFD; Aβ1–42 injections and then consumed HFD for 8 weeks], Group 5 [Aβ + OPL; Aβ1–42 injections, then consumed SD and oral administrated Okra polysaccharides 300 mg/kg (OPL) for 8 weeks], Group 6 [Aβ + OPH; Aβ1–42 injections, then consumed SD and oral administrated Okra polysaccharides 600 mg/kg (OPH) for 8 weeks], Group 7 [Aβ + HFD + OPL; Aβ1–42 injections, then consumed HFD and oral administrated Okra polysaccharides 300 mg/kg (OPL) for 8 weeks], Group 8 [Aβ + HFD + OPH; Aβ1–42 injections, then consumed HFD and oral
2.5. Measurements of body weight, blood glucose During the experimental period, body weight and blood glucose were examined every week. Blood glucose was measured with Jin-Wen glucose meter (Sinocare Inc). 2.6. Serum chemistry analysis After overnight (12 h) fasting, the mice were drawn blood from the eyeball. Serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) levels were measured by Shenyang Red Cross Hospital. Serum insulin was measured by ELISA assay kit. 2.7. Novel object recognition test In the novel object recognition test, it contains two phases: acquisition phase and trial phase. For the acquisition phase, mice were transferred to the arena in the presence of two identical objects located in two corners. After 10 min of object exploration, the mice were returned back to the home cage. For the trial phase, 1 h after initial exposure, one of the objects was replaced with a novel object, after which the mice were transferred again to the arena for 5 min of object exploration in the presence of one familiar (A) and one novel (B) object. Time spent exploring the novel object (B) was expressed as the recognition index (RI), where RI = tB / (tA + tB) × 100 normalizing all data for statistical analysis.
Table 3 Escape latency of the trial days. Group
Day 1
CON Aβ CON+HFD Aβ + HFD Aβ + OPL Aβ + OPH Aβ + HFD + OPL Aβ + HFD + OPH
71.16 67.32 74.57 71.54 69.01 64.41 58.68 65.30
Day 2 ± ± ± ± ± ± ± ±
5.00 5.39 5.35 3.37 4.08 5.62 5.20 4.53
53.35 72.79 69.50 70.49 65.51 57.03 65.59 61.30
Day 3 ± ± ± ± ± ± ± ±
3.91 3.10 1.99 4.05 3.99 4.54 3.79 5.34
47.37 60.41 54.10 56.41 60.87 53.52 59.66 52.38
Day 4 ± ± ± ± ± ± ± ±
4.53 4.35 3.20 3.90 4.37 3.11 3.49 1.08
36.87 59.39 44.35 59.70 46.52 40.31 45.87 37.70
Day 5 ± ± ± ± ± ± ± ±
5.18 2.17⁎ 3.85 4.20⁎⁎ 2.74 1.82# 5.77 6.10$
24.70 46.11 26.43 44.46 31.91 23.18 26.23 23.00
± ± ± ± ± ± ± ±
3.70 5.34⁎ 3.04 2.23⁎ 5.98 3.29## 5.35$ 1.02$
⁎ p < 0.05, ⁎⁎p < 0.01 compared with the control group, #p < 0.05, ##p < 0.05 compared with the AD mice, $p < 0.05 compared with the AD mice fed with HFD group (Aβ + HFD).
4
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pPI3K/PI3K ratio (% Control)
Fig. 5. Effects of Okra polysaccharide on the histopathological changes in the hippocampus of Aβ1–42 and HFD treated mice. Control mice (A), AD mice (B), Control mice fed with HFD (C), AD mice fed with HFD (D), AD mice treated with OPL (E), AD mice treated with OPH (F), AD mice treated with HFD and OPL (G), AD mice treated with HFD and OPH (H). The magnification was 100×.
120 ##
100
##
$
*
80
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***
60 40 20 0
120 ##
100
##
pGSK3 /GSK3 ratio (% of Control)
pAKT/AKT ratio (% of Control)
A $$$ $
80 60
***
***
***
40 20 0
120 100 80 60
** ***
40 20 0
Fig. 6. Effects of OP on the PI3K/AKT/GSK3β signaling. (A) Western blot analysis of protein extracts from the hippocampus of mice in each group. The Ratio of densitometric analysis of the pPI3K/PI3K, (B) pAKT/AKT, (C) pGSK3β/GSK3β. ⁎⁎p < 0.01, ⁎⁎⁎p < 0.001 compared with the control group, ##p < 0.01 compared with the AD mice, $p < 0.05, $$$p < 0.001 compared with the AD mice fed with HFD group (Aβ + HFD).
5
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A 2.5
###
2
### $$$
pERK1/2/ -actin ratio
ERK1/2/ -actin ratio
3
$$$
*** 1
***
0
2.0
***
1.5 *** ###
1.0
###
$$$ $$$
0.5 0.0
B
C
Fig. 7. Effects of OP on ERK1/2 and pERK1/2. (A) ERK1/2/β-actin, (B) pERK1/2/β-actin. ⁎⁎⁎p < 0.001 compared with the control group, ###p < 0.001 compared with the AD mice, $$$p < 0.001 compared with the AD mice fed with HFD group (Aβ + HFD).
solution for 48 h, and then transferred to 30% sucrose in 0.1 mol/L PBS (pH 7.4) for at least 16 h until they sank for cryoprotection. The tissues were subsequently kept in the final sucrose solution until sectioning. Serial (neighboring) sections of 10-μm thickness was cut and stained with H&E. The histological evaluation was scored blindly. CA1 regions of the hippocampus were examined (the magnification was 100).
2.8. Morris water maze test In the MWM test, the place navigation test was performed as two trials daily for 5 consecutive days, and the mice were placed in the water facing the pool wall from two different starting points every day and allowed to swim freely to seek the hidden platform positioned in the center of the IV quadrant. If the mice found the platform within 90 s and remained on it for at least 2 s, then the track was terminated. If the mice did not find the platform within 90 s, the mice were guided to the platform and were allowed to stay on it for 20 s. After the whole group was finished, the mice were prepared to start the next track. On the 6th day, the platform was removed and the mice were allowed to swim freely in the pool for 90 s. The time spent by the animal in the target quadrant was evaluated.
2.11. Statistical analysis The statistical analyses were performed using SPSS software, version 19.0, and data were obtained by applying the analysis of variance (ANOVA) followed by Tukey's multiple comparison test. All of the data are expressed as the mean ± S.E.M. The data were considered to be statistically significant if the probability had a value of 0.05 or less.
2.9. Western blot 3. Results
Protein concentrations of detergent-soluble fractions from mice hippocampus were determined by the western blot protein assay, mice hippocampus slices were flash-frozen on dry ice after drug treatment, followed by the standard procedure for western blot. All antibodies were diluted in blocking buffer. The following antibodies were used: anti-ERK1/2 (1:1000), anti-GSK3β (1:1000), anti-PI3K p85 (1:1000), anti-AKT (1:1000), anti-phosphor-AKT (Ser473) (1:2000), anti-phosphor-ERK1/2 (T202/Y204) (1:1000), anti-phosphor- PI3K p85 (Y607) (1:1000), anti-phosphor- GSK3β (Ser9) (1:1000) and β-actin (1:1000). Bound antibodies were visualized using the enhanced chemiluminescence detection system.
3.1. Monosaccharides profiling of Okra polysaccharides To determine the monosaccharides profile of the Okra polysaccharides, it was analyzed by using Reverse Phase High-Performance Liquid Chromatography (RP-HPLC). It has been reported that Okra is rich in polysaccharide (Fan et al., 2013; Liu et al., 2018). We used Mannose, Rhamnose, Glucose, Galactose, and Arabia sugar as standard controls (Fig. 1A). The chromatographic profile of OP revealed that Mannose, Rhamnose, Glucose, Galactose, and Arabia sugar could all be detected as components by comparing the Okra polysaccharides profile to the retention time of the standard compounds (Fig. 1B).
2.10. Histopathological examination For histopathology, the entire brains were postfixed in 4% PFA 6
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3.8. Effects of OP on ERK1/2 and pERK1/2
3.2. Okra polysaccharides lower body weight and blood glucose in HFD induced AD mice
As shown in Fig. 7, compared with the control mice, the expression of ERK1/2 in AD mice (fed with SD or HFD) was remarkably higher and the expression of pERK1/2 was significantly lower. Meanwhile, OP treatment could dramatically restore the above changes in the hippocampus of AD mice (fed with SD or HFD).
As showed in Table 1, for all groups of mice at 2 weeks, there was no significant difference in body weight. From week 4, we found that obesity AD mice (AD mice fed with HFD) were weighted considerably more than other mice. Moreover, from week 6, the AD mice and control mice after the HFD, were significantly heavier than mice on the standard diet or OP treatment mice. From week 6 to week 8, obesity AD mice treated with OPL/OPH, compared to the AD obesity mice, there was a significantly decrease in body weight. As showed in Table 2, from week 4, the obesity AD mice were significantly higher than other mice in the test of blood glucose. However, the blood glucose was significantly decreased by OP treatment in the obesity AD mice during week 6 to week 8.
4. Discussion Okra is consumed as a vegetable, supplying carbohydrates, minerals, and vitamins. In some areas, Okra is also a source of dietary medicine for the treatment of gastric irritation and the prevention of cancers (Kahlon et al., 2007; L.C.T.F.F.G.H. A., 2004). Okra is rich in polysaccharides and polyphenolic compounds, which have various biological activities (Wittschier 1 and Lengsfeld, 2007; Deters et al., 2005; Arapitsas, 2008), and for example, it is used as a dietary therapy for blindness, cataracts, and glaucoma development in type 2 diabetic patients (Augustin, 2012). In combination with previous studies, many medicinal plants rich in polysaccharides have a variety of activities, such as, promoting cognitive function and ameliorating metabolic stress-exacerbated oral glucose intolerance (Huang et al., 2017a; Huang et al., 2017b). Therefore, the purpose of this study is to explore the therapeutic effect of Okra polysaccharides on AD and the mechanism between them. AD is a devastating neurodegenerative disease with no effective treatment as yet. Current US Food and Drug Administration-approved AD drugs such as acetylcholinesterase inhibitors and NMDA receptor antagonists only alleviate disease symptoms in about half of the patients for approximately 6–12 months (Winslow et al., 2011). Thus, there is a huge need to search for a novel way of AD treatment. Impaired insulin signaling has been demonstrated in both postmortem human AD brains and in mouse models of AD (Hoyer, 2004; Steen et al., 2005). In addition to that, HFD can also damage insulin signaling and damage metabolic function (Zhao et al., 2017; O'Neill and O'Driscoll, 2015). In this study, we found that after the HFD, AD mice blood glucose，TC，HDL-C level had been elevated (Table 2, Fig. 2B, C), the result of HE staining, the mice hippocampus neurons histopathological damaged more serious (Fig. 5). Insulin stimulation of PI3K/AKT, which is the classic insulin signaling cascade, is required for GLUT translocation and the inhibition of kinases, such as GSK3β. In the current study, western blot data revealed elevated expression levels of PI3K, phosphorylated PI3K, AKT and phosphorylated AKT, as well as decreased GSK3β levels, in the hippocampus of OP-treated mice compared with AD mice fed with HFD. These data suggest that OP improves metabolic dysfunction and cognitive impairment via the regulation of PI3K/AKT/GSK3β signaling proteins. Insulin signaling networks employ kinase-linked cascades including PI3K/AKT and MAP kinase or ERK (Ha and Kim, 2013; Yoon et al., 2015). The increase in phosphorylation of ERK in response to insulin may be caused by translocation of the enzyme from the cytoplasm to the membrane that requires tyrosine and threonine phosphorylation of the enzyme by MEK. Previously, significant increase in the amount of MEK2 in membranes with similar extent and kinetics of those of phosphorylation of membrane ERK2 by insulin was reported (Kim and Kahn, 1997; Kim and Kim, 1997). In addition, tau phosphorylation was increased in more advanced insulin-resistant models by a mechanism independent of GSK3β, which was phosphorylated and thus inhibited by hyperactivated AKT (Sajan et al., 2016). Moreover, ERK1/2 is relevant to lipid metabolism, Insulin-induced ERK signaling pathways are required for cell cycle progression during adipogenesis, as demonstrated in insulin receptordeficient mice (M et al., 2002; Bluher et al., 2004). Therefore, HFD can aggravate metabolic impairment is compared to SD. Amazingly, the expression of ERK1/2 and pERK1/2 had markedly different between AD mice and obesity AD mice (Fig. 7). Indicating that OP implored the need for therapeutic interventions that target the insulin signaling axis, in
3.3. Okra polysaccharides lower serum lipids in obesity AD mice As showed in Fig. 2A and D, TG and LDL-C were no significant differences in all groups. On the other hand (Fig. 2B and C), levels of TC and HDL-C in AD mice fed with HFD were significantly higher than control mice. Under feeding HFD and treatment of OP, only in OPH treatment mice, the TC and HDL-C had a significant downward trend. 3.4. Okra polysaccharides improve memory injury in AD mice novel object recognition Fig. 3 showed that there was no significant difference in exploring time of the novel object in control mice fed with SD or HFD. However, AD mice with SD or HFD spent significantly much longer time to explore the novel object compared to the control group. Moreover, OPH showed an approving effect on the memory injury alleviation in AD mice. 3.5. Morris water maze As showed in Table 3, from day 1 to day 3, there were no significant differences in escape latency among the eight groups. From the fourth day, escape latency of AD mice was significantly longer than control mice, whatever fed with SD or HFD. On the other hand, in the fourth and fifth day, OP treatment reversed the phenomenon in the AD mice. On the sixth day, AD mice exhibited a smaller percent time in the target quadrant than other mice (Fig. 4). Moreover, OPH administration markedly prolonged the time spent in the target quadrant of AD mice or AD mice fed with HFD, which indicated that OP could mitigate the cognition impairment. 3.6. The histopathological changes in the hippocampus Hematoxylin and eosin (H&E) staining was performed to examine the neuronal integrity and orderliness in the hippocampus. The neuronal layers in the CA1 region of the hippocampus showed rarefaction, disorder, pronounced shrinkage of nuclei, and swollen neuronal bodies in the AD mice (Fig. 5B, D) compared with the control mice (Fig. 5A, C), but the worsen shrinkage of nuclei and neuron rarefaction in obesity AD mice. OPL and OPH significantly inhibited histopathological damage (Fig. 5F–H). 3.7. Effects of OP on the PI3K/AKT/GSK3β signaling Western blotting results (Fig. 6A) showed that PI3K (Fig. 6B) and AKT (Fig. 6C) were reduced in AD mice (fed with SD or HFD) and this tendency was reversed by OP treatment significantly. However, the data of GSK3β (Fig. 6D) indicated that OP has no influence on it. 7
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particular the common convergence point, ERK. In the present study, we chose a high-fat diet for two months, because at that time, the body weight and blood glucose values of mice had produced a significant difference. However, the control mice fed with HFD didn't found metabolic disorder and memory/neuron injury, because the time of feeding HFD was short, but the AD mice fed with HFD had markedly metabolic disorder. It is consistent with previous reports, short-term high-fat-diet intake did not significantly cause cognitive impairment and brain injury in wild-type mice (Mielke et al., 2006), on the other hand, the same short-term consumption of high-fat diet significantly enhanced cognitive impairment in 5XFAD mice (Lin et al., 2016). In the result of western blot, the OP can indeed reverse the changes in Aβ1–42 and HFD, especially OPH. For sure, the detail and specific mechanisms of OP reverse the metabolic disorder induced by high-fat diet and cognitive function injury in Alzheimer's disease need to be studied further.
Fan, S., Guo, L., Zhang, Y., Sun, Q., Yang, B., Huang, C., 2013. Okra polysaccharide improves metabolic disorders in high-fat diet-induced obese C57BL/6 mice. Mol. Nutr. Food Res. 57 (11), 2075–2078. Farris, W., Mansourian, S., Chang, Y., Lindsley, L., Eckman, E.A., Frosch, M.P., Eckman, C.B., Tanzi, R.E., Selkoe, D.J., Guenette, S., 2003. Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo. Proc. Natl. Acad. Sci. U. S. A. 100 (7), 4162–4167. Francis, H., Stevenson, R., 2013. The longer-term impacts of Western diet on human cognition and the brain. Appetite 63, 119–128. Grant, W.B., 1999. Dietary links to Alzheimer s disease 1999 update. J. Alzheimers Dis. 1 (4–5), 197–201. Ha, H.J., Kim, S.J., 2013. Association of insulin receptor and syndecan-1 by insulin with activation of ERK I/II in osteoblast-like UMR-106 cells. J. Recept. Signal Transduct. Res. 33 (1), 37. Hoyer, S., 2004. Glucose metabolism and insulin receptor signal transduction in Alzheimer disease. Eur. J. Pharmacol. 490 (1–3), 115. Huang, S., Mao, J., Ding, K., Zhou, Y., Zeng, X., Yang, W., Wang, P., Zhao, C., Yao, J., Xia, P., Pei, G., 2017a. Polysaccharides from Ganoderma lucidum promote cognitive function and neural progenitor proliferation in mouse model of Alzheimer’s disease. Stem Cell Rep. 8 (1), 84–94. Huang, Y.C., Tsay, H.J., Lu, M.K., Lin, C.H., Yeh, C.W., Liu, H.K., Shiao, Y.J., 2017b. Astragalus membranaceus-polysaccharides ameliorates obesity, hepatic steatosis, neuroinflammation and cognition impairment without affecting amyloid deposition in metabolically stressed APPswe/PS1dE9 mice. Int. J. Mol. Sci. 18 (12). Kahlon, T.S., Chapman, M.H., Smith, G.E., 2007. In vitro binding of bile acids by okra, beets, asparagus, eggplant, turnips, green beans, carrots, and cauliflower. Food Chem. 103 (2), 676–680. Kelleher III, 2., R.J., Govindarajan, A. 1, Jung, H.-Y. 1, Kang, H. 1, Tonegawa, S. 1, 2004. Translational control by MAPK signaling in long-term synaptic plasticity and memory. Cell 116 (3), 467–480. Kim, S.J., Kahn, C.R., 1997. Insulin regulation of mitogen-activated protein kinase kinase (MEK), mitogen-activated protein kinase and casein kinase in the cell nucleus: a possible role in the regulation of gene expression. Biochem. J. 323 (Pt 3), 621 ( Pt 3). Kim, S.J., Kim, K.H., 1997. Insulin rapidly stimulates ERK2 in the membrane of osteoblast-like UMR-106 cell. IUBMB Life 43 (5), 1023–1031. L.C.T.F.F.G.H. A, 2004. Glycosylated compounds from okra inhibit adhesion of Helicobacter pylori to human gastric mucosa. J. Agric. Food Chem. 52 (6), 1495–1503. Lin, B., Yu, H., Koki, T., Nobutaka, K., Cao, C., Shokei, K.M., 2016. High-fat-diet intake enhances cerebral amyloid angiopathy and cognitive impairment in a mouse model of Alzheimer’s disease, independently of metabolic disorders. J. Am. Heart Assoc. 5 (6), e003154. Liu, J., Zhao, Y., Wu, Q., John, A., Jiang, Y., Yang, J., Liu, H., Yang, B., 2018. Structure characterisation of polysaccharides in vegetable “okra” and evaluation of hypoglycemic activity. Food Chem. 242, 211–216. M, B., Michael, M.D., Peroni, O.D., Ueki, K., Carter, N., Kahn, B.B., Kahn, C.R., 2002. Adipose tissue selective insulin receptor knockout protects against obesity and obesity-related glucose intolerance. Dev. Cell 3 (1), 25–38. Mielke, J.G., Nicolitch, K., Avellaneda, V., Earlam, K., Ahuja, T., Mealing, G., Messier, C., 2006. Longitudinal study of the effects of a high-fat diet on glucose regulation, hippocampal function, and cerebral insulin sensitivity in C57BL/6 mice. Behav. Brain Res. 175 (2), 374–382. O’Neill, S., O’Driscoll, L., 2015. Metabolic syndrome: a closer look at the growing epidemic and its associated pathologies. Obes. Rev. 16 (1), 1–12. Sabitha, V., Ramachandran, S., Naveen, K.R., Panneerselvam, K., 2011. Antidiabetic and antihyperlipidemic potential of Abelmoschus esculentus (L.) Moench. in streptozotocin-induced diabetic rats. J. Pharm. Bioallied Sci. 3 (3), 397–402. Sajan, M., Hansen, B., Robert Ivey, I., Sajan, J., Ari, C., Song, S., Braun, U., Leitges, M., Faresehiggs, M., Farese, R.V., 2016. Brain insulin signaling is increased in insulinresistant states and decreases in FOXOs and PGC-1α and increases in Aβ1–40/42 and phospho-tau may abet alzheimer development. Diabetes 65 (7), 1892. Steen, E., Terry, B.M., Rivera, E.J., Cannon, J.L., Neely, T.R., Tavares, R., Xu, X.J., Wands, J.R., Sm, D.L.M., 2005. Impaired insulin and insulin-like growth factor expression and signaling mechanisms in Alzheimer’s disease—is this type 3 diabetes? J. Alzheimers Dis. 7 (1), 63–80. Sweatt, J.D., 2001. The neuronal MAP kinase cascade a biochemical signalintegration system subserving synaptic plasticity and memory. J. Neurochem. 76 (1), 1–10. Sweatt, J.D., 2004. Mitogen-activated protein kinases in synaptic plasticity and memory. Curr. Opin. Neurobiol. 14 (3), 311–317. Vadas, O., Burke, J.E., Zhang, X., Berndt, A., Williams, R.L., 2011. Structural basis for activation and inhibition of class I phosphoinositide 3-kinases. Sci. Signal. 4, re2. Winslow, B.T., Onysko, M.K., Stob, C.M., Hazlewood, K.A., 2011. Treatment of Alzheimer disease. Am. Fam. Physician 83 (12), 1403. Wittschier, N., Lengsfeld, C., Vorthems, S., Stratmann, U., Ernst, J.F., Verspohl, E.J., Hensel, A., 2007. Large molecules as anti-adhesive compounds against pathogens. J. Pharm. Pharmacol. 59 (6), 777–786. Yoon, S.H., Ramalingam, M., Kim, S.J., 2015. Insulin stimulates integrin-linked kinase in UMR-106 cells: potential role of heparan sulfate on syndecan-1. J. Recept. Signal Transduction 35 (6), 613. Zhao, W.Q., De Felice, F.G., Fernandez, S., Chen, H., Lambert, M.P., Quon, M.J., Krafft, G.A., Klein, W.L., 2008. Amyloid beta oligomers induce impairment of neuronal insulin receptors. FASEB J. 22 (1), 246–260. Zhao, N., Liu, C.C., Van, A.I., Martens, Y.A., Linares, C., Knight, J.A., Painter, M.M., Sullivan, P.M., Bu, G., 2017. Apolipoprotein E4 impairs neuronal insulin signaling by trapping insulin receptor in the endosomes. Neuron 96 (1), 115–129.e5. Zoe Arvanitakis, R.S.W., Bienias, J.L., Evans, D.A., Bennett, D.A., 2004. Diabetes mellitus and risk of Alzheimer disease and decline in cognitive function. Arch. Neurol. 61 (5), 661–666.
5. Conclusion In summary, the treatment of Alzheimer's disease is considerable difficulty in medicine, accompany the rise of Alzheimer's patients, the treatment of the disease is increasingly urgent. This study explored the treatment of Alzheimer's disease by two aspects, on the one hand, Okra polysaccharides can reverse AD mice cognitive disorder, on the other hand, it also proves HFD can aggravate the process of Alzheimer's disease. In addition, this study takes the lead in confirming that the Okra polysaccharide can reverse the cognitive impairment and metabolic disorder by HFD through the insulin signaling pathway. Role of the funding source There is no funding supports this research. Declaration of competing interest The authors have no conflict of interest to declare. Acknowledgements This research was supported by National Natural Science Foundation of China (No. 81573580). Key Laboratory of polysaccharide bioactivity evaluation of TCM of Liaoning Province. Key techniques study of consistency evaluation of drug quality and therapeutic effect (18-400-4-08) and Liaoning Distinguished Professor Project for Ying Jia (2017). The Doctoral Scientific Research Foundation of Liaoning Province (2019-BS-233). References Adelakun, O.E., Oyelade, O.J., Ade-Omowaye, B.I.O., Adeyemi, I.A., Van de Venter, M., 2009. Chemical composition and the antioxidative properties of Nigerian Okra Seed (Abelmoschus esculentus Moench) flour. Food Chem. Toxicol. 47 (6), 1123–1126. Arapitsas, P., 2008. Identification and quantification of polyphenolic compounds from okra seeds and skins. Food Chem. 110 (4), 1041–1045. Moise, M.M., Benjamin, L.M., Doris, T.M., Dalida, K.N., Augustin, N.O., 2012. Role of Mediterranean diet, tropical vegetables rich in antioxidants, and sunlight exposure in blindness, cataract and glaucoma among African type 2 diabetics. Int. J. Ophthalmol. 5, 231–237. Bluher, M., Patti, M.S., Kahn, B.B., Kahn, C.R., 2004. Intrinsic heterogeneity in adipose tissue of fat-specific insulin receptor knock-out mice is associated with differences in patterns of gene expression. J. Biol. Chem. 279 (30), 31891–31901. Cordner, Z.A., Tamashiro, K.L., 2015. Effects of high-fat diet exposure on learning & memory. Physiol. Behav. 152 (Pt B), 363–371. Deters, A.M., Lengsfeld, C., Hensel, A., 2005. Oligo- and polysaccharides exhibit a structure-dependent bioactivity on human keratinocytes in vitro. J. Ethnopharmacol. 102 (3), 391–399. Dineley, K.T., Jahrling, J.B., Denner, L., 2014. Insulin resistance in Alzheimer's disease. Neurobiol. Dis. 72 (Pt A), 92–103. Eskelinen, M.H., Ngandu, T., Helkala, E.L., Tuomilehto, J., Nissinen, A., Soininen, H., Kivipelto, M., 2008. Fat intake at midlife and cognitive impairment later in life: a population-based CAIDE study. Int. J. Geriatr. Psychiatry 23 (7), 741–747.
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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Okra polysaccharides can reverse the metabolic disorder induced by high-fat diet and cognitive function injury in Aβ1–42 mice Tingxu Yana, Tingting Nianb, Bo Wua, Feng Xiaoa, Bosai Hea, Kaishun Bic, Ying Jiaa,
T
⁎
a
School of Functional Food and Wine, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China c School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China b
A R T I C LE I N FO
A B S T R A C T
Section Editor: Holly M Brown-Borg
Epidemiological studies showed that a high-fat diet threatened human health seriously. It can induce various diseases, such as obesity, metabolic disturbance and cognitive dysfunction which also related to insulin signaling. In the present study, Aβ1–42 induced AD model mice and normal mice were given a standard diet and high-fat diet, respectively. Meanwhile, Okra polysaccharides were used to treat AD mice to explore the possible mechanism between Alzheimer's disease and insulin signals. Weight and blood glucose of mice were measured weekly. Through the Morris water maze and the novel object recognition test, the Okra polysaccharides could improve the cognitive impairment of the AD mice. In addition, we also performed the serum chemistry analysis of mice, studied the histopathological changes in the hippocampal CA1 region by HE staining and detected the expressions of AKT, PI3K, ERK1/2, and GSK3β in the hippocampus by western blot. These results suggested that a high-fat diet can aggravate the metabolic disorder in AD mice and Okra polysaccharides can significantly reverse the metabolic disorder induced by high-fat diet and cognitive function injury in AD mice.
Keywords: Okra polysaccharides Alzheimer's disease High-fat diet PI3K/AKT/GSK3β ERK1/2
1. Introduction In recent years, epidemiological and clinical studies have revealed a close relationship between T2D and Alzheimer's disease (Zoe Arvanitakis et al., 2004). Aβ oligomers may also interfere with insulin signaling in hippocampus neurons (Zhao et al., 2008), and insulin may regulate Aβ levels by modulation of β and γ secretases (Farris et al., 2003). Moreover, the central nervous system insulin receptors are highly expressed in regions relevant for cognition, such as the cortex and hippocampus. Insulin signaling is a key factor in brain homeostasis, metabolism, synaptic plasticity, and memory, imploring the need for therapeutic interventions that target the insulin signaling axis, in particular the common convergence point, ERK (Dineley et al., 2014). In the hippocampus, the ERK cascade is critical in the induction and maintenance of long term potentiation (LTP) and memory consolidation as it converges with a number of other signaling cascades, including PI3K/AKT, CaMKII, PKA, and PKC (Sweatt, 2004; Kelleher III, et al., 2004; Sweatt, 2001). Insulin receptor substrate activates PI3K activation which then leads to AKT activation (Williams, 2011). Obesity is often caused by and is associated with, consumption of diets that are high in fat (Grant, 1999). There has been solid evidence that long-term high-fat causes an increased incidence of metabolic
⁎
diseases such as obesity, diabetes, and hyperinsulinemia. Additionally, epidemiological and clinical data have suggested that high-fat diet may also contribute to the development of Alzheimer's disease (AD) (Cordner and Tamashiro, 2015; Eskelinen et al., 2008; Francis and Stevenson, 2013). Recently, with the research of Okra (Abelmoschus esculentus L. Moench), we found it has huge potential. Okra, an annual vegetable belonging to the malvaceae family, is also known as lady's finger. Immature okra pods are consumed in most areas of the world, supplying carbohydrates, minerals, and vitamins and are also a source of dietary medicines (Adelakun et al., 2009). The peel and seed powders of Okra have been reported to play antidiabetic and antihyper lipidemic roles in STZ-induced diabetic rats (Sabitha et al., 2011). Okra is full of polysaccharides and polysaccharides have the ability to regulating metabolism and improving Alzheimer's disease (Sabitha et al., 2011; Huang et al., 2017a; Fan et al., 2013). In the present study, to explore the relationship between AD and obesity. We are focusing on the insulin signaling pathway as a starting point. Since insulin signaling plays an important role in the pathological changes of AD, and obesity can also mediate changes in insulin signaling pathways. Although some potential mechanisms have been proposed, there is still a considerable knowledge gap by identifying roles and molecular mechanisms that underlie the association between
Corresponding author at: School of Functional Food and Wine, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China. E-mail address: [email protected] (Y. Jia).
https://doi.org/10.1016/j.exger.2019.110802 Received 8 October 2019; Received in revised form 12 November 2019; Accepted 28 November 2019 Available online 30 November 2019 0531-5565/ © 2019 Elsevier Inc. All rights reserved.
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1
4
2
5
3
A 4
2
5 1
3
B Fig. 1. Monosaccharides analysis of OP by RP-HPLC. (A) (1) Mannose, (2) Rhamnose, (3) Glucose, (4) Galactose, (5) Arabia sugar were analyzed as standards by using HPLC with a C-18 reverse phase column at 250 nm; (B) The major components of the OP were detected for comparison with the retention times of the standard compounds. Table 1 The body weight of control or AD mice response to HFD or OP.
⁎
Group
Week 1
CON Aβ CON+HFD Aβ + HFD Aβ + OPL Aβ + OPH Aβ + HFD + OPL Aβ + HFD + OPH
34.05 33.21 32.48 34.81 33.97 33.24 34.81 33.67
± ± ± ± ± ± ± ±
Week 2 0.91 0.96 0.82 0.58 0.82 0.67 0.86 1.30
37.19 36.44 36.48 38.51 36.88 36.07 37.61 33.58
± ± ± ± ± ± ± ±
Week 4 0.82 0.84 0.77 0.44 0.96 0.35 0.53 1.76
39.23 38.87 43.30 44.02 39.32 39.16 42.95 43.14
± ± ± ± ± ± ± ±
Week 6 0.72 0.63 0.55 1.15⁎ 0.79 1.30 1.14 0.77
44.84 42.71 52.40 53.98 42.91 44.63 50.07 49.60
± ± ± ± ± ± ± ±
Week 8 1.13 1.19 0.49⁎⁎⁎ 1.04⁎⁎⁎ 0.57 0.59 0.66$ 0.71$
47.73 47.37 58.77 59.31 46.70 47.65 56.59 55.14
± ± ± ± ± ± ± ±
0.61 0.61 0.32⁎⁎⁎ 0.39⁎⁎⁎ 0.32 0.42 0.41$$ 0.49$$$
p < 0.05, ⁎⁎⁎p < 0.001 compared with the control group, $p < 0.05, $$p < 0.01, $$$p < 0.001 compared with the AD mice fed with HFD group (Aβ + HFD).
province, China) in August 2018. High-fat diet (SN10060) was provided by Ruidi Biotec (Shanghai, China). Aβ1–42 was purchased from SigmaAldrich. Donepezil (≥98%), Serum insulin kit was provided by Melone Pharmaceutical Co. Ltd., The following antibodies were supplied by CST (USA): anti-ERK1/2, anti-GSK3β, anti-PI3K p85, anti-AKT, antiphosphor-AKT (Ser473), anti-phosphor-ERK1/2 (T202/Y204), antiphosphor- PI3K p85 (Y607), anti-phosphor- GSK3β (Ser9) and β-actin.
obesity and AD. 2. Experimental section 2.1. Materials The fruits of Okra were purchased from Laiyang (Shandong 2
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Table 2 The blood glucose of control or AD mice response to HFD or OP.
⁎⁎
Group
Week 1
CON Aβ CON+HFD Aβ + HFD Aβ + OPL Aβ + OPH Aβ + HFD + OPL Aβ + HFD + OPH
6.60 6.85 6.59 6.69 6.91 6.68 6.96 6.54
± ± ± ± ± ± ± ±
Week 2 0.23 0.33 0.18 0.09 0.19 0.27 0.24 1.13
6.78 6.54 6.73 7.33 6.70 6.61 6.58 6.61
± ± ± ± ± ± ± ±
Week 4 0.20 0.24 0.17 0.16 0.24 0.17 0.14 0.23
6.38 6.74 6.84 7.49 6.80 6.73 6.89 6.81
± ± ± ± ± ± ± ±
Week 6 0.35 0.11 0.23 0.12⁎⁎ 0.15 0.22 0.16 0.17
6.71 6.80 6.99 8.09 6.91 6.59 6.74 6.63
± ± ± ± ± ± ± ±
Week 8 0.14 0.08 0.14 0.27⁎⁎ 0.30 0.21 0.28$ 0.32$$
6.81 6.41 7.45 9.31 6.77 6.55 6.79 6.90
± ± ± ± ± ± ± ±
0.17 0.17 0.15 0.10⁎⁎⁎ 0.21 0.30 0.15$$$ 0.20$$$
p < 0.01, ⁎⁎⁎p < 0.001 compared with the control group, $p < 0.05, $$p < 0.01, $$$p < 0.001 compared with the AD mice fed with HFD group (Aβ + HFD). 2.0
3
1.5
TC (mmol/L)
TG (mmol/L)
*
1.0 0.5 0.0
$ 2
1
Fig. 2. The level of serum lipids in mice. (A) Serum triglyceride (TG), (B) Total cholesterol (TC), (C) High-density lipoprotein cholesterol (HDL-c), (D) Low-density lipoprotein cholesterol (LDL-c). Values indicated the mean ± S.E.M. and were analyzed by one-way analysis of variance (ANOVA) followed by Tukeyʼs multiple comparison test (n = 8). ⁎ p < 0.05 compared with the control group, $ p < 0.05 compared with the AD mice fed with HFD group (Aβ + HFD).
0
A
B 0.50
2.5 2.0
$
1.5 1.0 0.5 0.0
LDL-c (mmol/L)
HDL-c (mmol/L)
*
0.25
0.00
C
D room temperature, and vacuum rotary evaporation was repeated until TFA was fully vaporized and then 2 mL water was added to the resolution. 800 μL of the solution was taken to put in a tube to derivative treatment, 800 μL of NaOH solution (0.3 mol/L) and 800 μL of PMP (0.5 mol/L) were added in solution. Mixed well and then react in a water bath of 70 °C for 2 h. 0.3 mol/L HCl (800 μL) was added after the solution restored at room temperature. 1 mL chloroform was added for extraction and water layer solution was collected. Repeat extraction three times. The standard compounds were also treated with the same derivatization procedure. The multi-components of hydrolyzate were analyzed by using an Agilent 1260 HPLC with an UV-DAD detector at a λ max of 250 nm. Chromatographic separation was performed on an Agilent C-18 reverse phase column (2.1 mm × 100 mm, 1.8 μm) with an injection volume of 20 μL. The mobile phase consisted of a mixture of distilled water (A): acetonitrile (B) 84:16. (flow rate 1 mL/min).
All other chemicals and solvents were of analytical reagent grade. 2.2. Isolation and purification of polysaccharides from Okra The polysaccharide was extracted with water (95 °C, mass ratio1:15) three times (6 h each time). The aqueous filtrates were combined and concentrated, then 95% ethanol (volume ratio 1:4) was added to the aqueous filtrates, 4 °C for overnight. The precipitate collected by centrifugation was dissolved in distilled water, after that, the supernatant was treated with Sevagreagent [V(n-butano1): V(chloroform)1: 4] to remove free proteins. Then the remaining were combined and concentrated. 2.3. Monosaccharides analysis of Okra polysaccharides by HPLC The Okra polysaccharides (10 mg) was dissolved with 2 mL of 2 M trifluoroacetic acid (TFA) at 110 °C for 8 h. The reaction solutions were filled with N2. 2 mL methanol was added after the solution restored at 3
Experimental Gerontology 130 (2020) 110802
The time in the target quadrant (s)
T. Yan, et al.
100 #
RI (%)
80 60
$
$
*
*
40 20 0
Fig. 3. Effects of OP on improving memory injury in novel object recognition. Values indicated the mean ± S.E.M. and were analyzed by one-way analysis of variance (ANOVA) followed by Tukeyʼs multiple comparison test (n = 8). ⁎ p < 0.05 compared with the control group, #p < 0.05 compared with the AD mice, $p < 0.05 compared with the AD mice fed with HFD group (Aβ + HFD).
50 40
#
$$
30
***
***
20 10 0
Fig. 4. Time spent in the target quadrant on the 6th day. Values indicated the mean ± S.E.M. and were analyzed by one-way analysis of variance (ANOVA) followed by Tukeyʼs multiple comparison test (n = 8). ⁎⁎⁎p < 0.001 compared with the control group, #p < 0.05 compared with the AD mice, $$p < 0.01 compared with the AD mice fed with HFD group (Aβ + HFD).
2.4. Animals and diet administrated Okra polysaccharides 600 mg/kg (OPH) for 8 weeks].
Male 12-week-old Kunming (KM) mice, weighing 22–30 g, were purchased from the Central Animal House of University. The experiment was carried out in compliance with the National Institutes of Health and institutional guidelines for the humane care of animals and was approved by the Animal Care Committee of Shenyang Pharmaceutical University. Mice were housed in groups of four per cage, and maintained at a constant temperature (23 ± 1 °C) and humidity (55 ± 5%) under a 12-h light/dark cycle. All studies were carried out in accordance with the Animal Ethics Committee of the institution (approved on January 20, 2016, NO. SYPU-IACUCC2016–0120). All animals had free access to food and water. The normal group was fed with a standard diet (SD), the obesity group was fed with high-fat diet (HFD). Aβ1–42 was dissolved in DMSO (≥99.9%, 2 μL) and diluted in physiological saline to a stock concentration of 1.0 mg/mL. The solution of Aβ1–42 was incubated at 37 °C for 5 days to obtain the fibrillated form of Aβ1–42. The mice were allowed to acclimate for seven days prior to their use in the studies. The mice were randomly assigned to eight groups (n = 8 mice/group); Group 1 [CON; consumed SD], Group 2 [CON+HFD; consumed HFD for 8 weeks], Group 3 [Aβ; Aβ1–42 injections and then consumed SD for 8 weeks], Group 4 [Aβ + HFD; Aβ1–42 injections and then consumed HFD for 8 weeks], Group 5 [Aβ + OPL; Aβ1–42 injections, then consumed SD and oral administrated Okra polysaccharides 300 mg/kg (OPL) for 8 weeks], Group 6 [Aβ + OPH; Aβ1–42 injections, then consumed SD and oral administrated Okra polysaccharides 600 mg/kg (OPH) for 8 weeks], Group 7 [Aβ + HFD + OPL; Aβ1–42 injections, then consumed HFD and oral administrated Okra polysaccharides 300 mg/kg (OPL) for 8 weeks], Group 8 [Aβ + HFD + OPH; Aβ1–42 injections, then consumed HFD and oral
2.5. Measurements of body weight, blood glucose During the experimental period, body weight and blood glucose were examined every week. Blood glucose was measured with Jin-Wen glucose meter (Sinocare Inc). 2.6. Serum chemistry analysis After overnight (12 h) fasting, the mice were drawn blood from the eyeball. Serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) levels were measured by Shenyang Red Cross Hospital. Serum insulin was measured by ELISA assay kit. 2.7. Novel object recognition test In the novel object recognition test, it contains two phases: acquisition phase and trial phase. For the acquisition phase, mice were transferred to the arena in the presence of two identical objects located in two corners. After 10 min of object exploration, the mice were returned back to the home cage. For the trial phase, 1 h after initial exposure, one of the objects was replaced with a novel object, after which the mice were transferred again to the arena for 5 min of object exploration in the presence of one familiar (A) and one novel (B) object. Time spent exploring the novel object (B) was expressed as the recognition index (RI), where RI = tB / (tA + tB) × 100 normalizing all data for statistical analysis.
Table 3 Escape latency of the trial days. Group
Day 1
CON Aβ CON+HFD Aβ + HFD Aβ + OPL Aβ + OPH Aβ + HFD + OPL Aβ + HFD + OPH
71.16 67.32 74.57 71.54 69.01 64.41 58.68 65.30
Day 2 ± ± ± ± ± ± ± ±
5.00 5.39 5.35 3.37 4.08 5.62 5.20 4.53
53.35 72.79 69.50 70.49 65.51 57.03 65.59 61.30
Day 3 ± ± ± ± ± ± ± ±
3.91 3.10 1.99 4.05 3.99 4.54 3.79 5.34
47.37 60.41 54.10 56.41 60.87 53.52 59.66 52.38
Day 4 ± ± ± ± ± ± ± ±
4.53 4.35 3.20 3.90 4.37 3.11 3.49 1.08
36.87 59.39 44.35 59.70 46.52 40.31 45.87 37.70
Day 5 ± ± ± ± ± ± ± ±
5.18 2.17⁎ 3.85 4.20⁎⁎ 2.74 1.82# 5.77 6.10$
24.70 46.11 26.43 44.46 31.91 23.18 26.23 23.00
± ± ± ± ± ± ± ±
3.70 5.34⁎ 3.04 2.23⁎ 5.98 3.29## 5.35$ 1.02$
⁎ p < 0.05, ⁎⁎p < 0.01 compared with the control group, #p < 0.05, ##p < 0.05 compared with the AD mice, $p < 0.05 compared with the AD mice fed with HFD group (Aβ + HFD).
4
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pPI3K/PI3K ratio (% Control)
Fig. 5. Effects of Okra polysaccharide on the histopathological changes in the hippocampus of Aβ1–42 and HFD treated mice. Control mice (A), AD mice (B), Control mice fed with HFD (C), AD mice fed with HFD (D), AD mice treated with OPL (E), AD mice treated with OPH (F), AD mice treated with HFD and OPL (G), AD mice treated with HFD and OPH (H). The magnification was 100×.
120 ##
100
##
$
*
80
***
***
60 40 20 0
120 ##
100
##
pGSK3 /GSK3 ratio (% of Control)
pAKT/AKT ratio (% of Control)
A $$$ $
80 60
***
***
***
40 20 0
120 100 80 60
** ***
40 20 0
Fig. 6. Effects of OP on the PI3K/AKT/GSK3β signaling. (A) Western blot analysis of protein extracts from the hippocampus of mice in each group. The Ratio of densitometric analysis of the pPI3K/PI3K, (B) pAKT/AKT, (C) pGSK3β/GSK3β. ⁎⁎p < 0.01, ⁎⁎⁎p < 0.001 compared with the control group, ##p < 0.01 compared with the AD mice, $p < 0.05, $$$p < 0.001 compared with the AD mice fed with HFD group (Aβ + HFD).
5
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A 2.5
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pERK1/2/ -actin ratio
ERK1/2/ -actin ratio
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Fig. 7. Effects of OP on ERK1/2 and pERK1/2. (A) ERK1/2/β-actin, (B) pERK1/2/β-actin. ⁎⁎⁎p < 0.001 compared with the control group, ###p < 0.001 compared with the AD mice, $$$p < 0.001 compared with the AD mice fed with HFD group (Aβ + HFD).
solution for 48 h, and then transferred to 30% sucrose in 0.1 mol/L PBS (pH 7.4) for at least 16 h until they sank for cryoprotection. The tissues were subsequently kept in the final sucrose solution until sectioning. Serial (neighboring) sections of 10-μm thickness was cut and stained with H&E. The histological evaluation was scored blindly. CA1 regions of the hippocampus were examined (the magnification was 100).
2.8. Morris water maze test In the MWM test, the place navigation test was performed as two trials daily for 5 consecutive days, and the mice were placed in the water facing the pool wall from two different starting points every day and allowed to swim freely to seek the hidden platform positioned in the center of the IV quadrant. If the mice found the platform within 90 s and remained on it for at least 2 s, then the track was terminated. If the mice did not find the platform within 90 s, the mice were guided to the platform and were allowed to stay on it for 20 s. After the whole group was finished, the mice were prepared to start the next track. On the 6th day, the platform was removed and the mice were allowed to swim freely in the pool for 90 s. The time spent by the animal in the target quadrant was evaluated.
2.11. Statistical analysis The statistical analyses were performed using SPSS software, version 19.0, and data were obtained by applying the analysis of variance (ANOVA) followed by Tukey's multiple comparison test. All of the data are expressed as the mean ± S.E.M. The data were considered to be statistically significant if the probability had a value of 0.05 or less.
2.9. Western blot 3. Results
Protein concentrations of detergent-soluble fractions from mice hippocampus were determined by the western blot protein assay, mice hippocampus slices were flash-frozen on dry ice after drug treatment, followed by the standard procedure for western blot. All antibodies were diluted in blocking buffer. The following antibodies were used: anti-ERK1/2 (1:1000), anti-GSK3β (1:1000), anti-PI3K p85 (1:1000), anti-AKT (1:1000), anti-phosphor-AKT (Ser473) (1:2000), anti-phosphor-ERK1/2 (T202/Y204) (1:1000), anti-phosphor- PI3K p85 (Y607) (1:1000), anti-phosphor- GSK3β (Ser9) (1:1000) and β-actin (1:1000). Bound antibodies were visualized using the enhanced chemiluminescence detection system.
3.1. Monosaccharides profiling of Okra polysaccharides To determine the monosaccharides profile of the Okra polysaccharides, it was analyzed by using Reverse Phase High-Performance Liquid Chromatography (RP-HPLC). It has been reported that Okra is rich in polysaccharide (Fan et al., 2013; Liu et al., 2018). We used Mannose, Rhamnose, Glucose, Galactose, and Arabia sugar as standard controls (Fig. 1A). The chromatographic profile of OP revealed that Mannose, Rhamnose, Glucose, Galactose, and Arabia sugar could all be detected as components by comparing the Okra polysaccharides profile to the retention time of the standard compounds (Fig. 1B).
2.10. Histopathological examination For histopathology, the entire brains were postfixed in 4% PFA 6
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3.8. Effects of OP on ERK1/2 and pERK1/2
3.2. Okra polysaccharides lower body weight and blood glucose in HFD induced AD mice
As shown in Fig. 7, compared with the control mice, the expression of ERK1/2 in AD mice (fed with SD or HFD) was remarkably higher and the expression of pERK1/2 was significantly lower. Meanwhile, OP treatment could dramatically restore the above changes in the hippocampus of AD mice (fed with SD or HFD).
As showed in Table 1, for all groups of mice at 2 weeks, there was no significant difference in body weight. From week 4, we found that obesity AD mice (AD mice fed with HFD) were weighted considerably more than other mice. Moreover, from week 6, the AD mice and control mice after the HFD, were significantly heavier than mice on the standard diet or OP treatment mice. From week 6 to week 8, obesity AD mice treated with OPL/OPH, compared to the AD obesity mice, there was a significantly decrease in body weight. As showed in Table 2, from week 4, the obesity AD mice were significantly higher than other mice in the test of blood glucose. However, the blood glucose was significantly decreased by OP treatment in the obesity AD mice during week 6 to week 8.
4. Discussion Okra is consumed as a vegetable, supplying carbohydrates, minerals, and vitamins. In some areas, Okra is also a source of dietary medicine for the treatment of gastric irritation and the prevention of cancers (Kahlon et al., 2007; L.C.T.F.F.G.H. A., 2004). Okra is rich in polysaccharides and polyphenolic compounds, which have various biological activities (Wittschier 1 and Lengsfeld, 2007; Deters et al., 2005; Arapitsas, 2008), and for example, it is used as a dietary therapy for blindness, cataracts, and glaucoma development in type 2 diabetic patients (Augustin, 2012). In combination with previous studies, many medicinal plants rich in polysaccharides have a variety of activities, such as, promoting cognitive function and ameliorating metabolic stress-exacerbated oral glucose intolerance (Huang et al., 2017a; Huang et al., 2017b). Therefore, the purpose of this study is to explore the therapeutic effect of Okra polysaccharides on AD and the mechanism between them. AD is a devastating neurodegenerative disease with no effective treatment as yet. Current US Food and Drug Administration-approved AD drugs such as acetylcholinesterase inhibitors and NMDA receptor antagonists only alleviate disease symptoms in about half of the patients for approximately 6–12 months (Winslow et al., 2011). Thus, there is a huge need to search for a novel way of AD treatment. Impaired insulin signaling has been demonstrated in both postmortem human AD brains and in mouse models of AD (Hoyer, 2004; Steen et al., 2005). In addition to that, HFD can also damage insulin signaling and damage metabolic function (Zhao et al., 2017; O'Neill and O'Driscoll, 2015). In this study, we found that after the HFD, AD mice blood glucose，TC，HDL-C level had been elevated (Table 2, Fig. 2B, C), the result of HE staining, the mice hippocampus neurons histopathological damaged more serious (Fig. 5). Insulin stimulation of PI3K/AKT, which is the classic insulin signaling cascade, is required for GLUT translocation and the inhibition of kinases, such as GSK3β. In the current study, western blot data revealed elevated expression levels of PI3K, phosphorylated PI3K, AKT and phosphorylated AKT, as well as decreased GSK3β levels, in the hippocampus of OP-treated mice compared with AD mice fed with HFD. These data suggest that OP improves metabolic dysfunction and cognitive impairment via the regulation of PI3K/AKT/GSK3β signaling proteins. Insulin signaling networks employ kinase-linked cascades including PI3K/AKT and MAP kinase or ERK (Ha and Kim, 2013; Yoon et al., 2015). The increase in phosphorylation of ERK in response to insulin may be caused by translocation of the enzyme from the cytoplasm to the membrane that requires tyrosine and threonine phosphorylation of the enzyme by MEK. Previously, significant increase in the amount of MEK2 in membranes with similar extent and kinetics of those of phosphorylation of membrane ERK2 by insulin was reported (Kim and Kahn, 1997; Kim and Kim, 1997). In addition, tau phosphorylation was increased in more advanced insulin-resistant models by a mechanism independent of GSK3β, which was phosphorylated and thus inhibited by hyperactivated AKT (Sajan et al., 2016). Moreover, ERK1/2 is relevant to lipid metabolism, Insulin-induced ERK signaling pathways are required for cell cycle progression during adipogenesis, as demonstrated in insulin receptordeficient mice (M et al., 2002; Bluher et al., 2004). Therefore, HFD can aggravate metabolic impairment is compared to SD. Amazingly, the expression of ERK1/2 and pERK1/2 had markedly different between AD mice and obesity AD mice (Fig. 7). Indicating that OP implored the need for therapeutic interventions that target the insulin signaling axis, in
3.3. Okra polysaccharides lower serum lipids in obesity AD mice As showed in Fig. 2A and D, TG and LDL-C were no significant differences in all groups. On the other hand (Fig. 2B and C), levels of TC and HDL-C in AD mice fed with HFD were significantly higher than control mice. Under feeding HFD and treatment of OP, only in OPH treatment mice, the TC and HDL-C had a significant downward trend. 3.4. Okra polysaccharides improve memory injury in AD mice novel object recognition Fig. 3 showed that there was no significant difference in exploring time of the novel object in control mice fed with SD or HFD. However, AD mice with SD or HFD spent significantly much longer time to explore the novel object compared to the control group. Moreover, OPH showed an approving effect on the memory injury alleviation in AD mice. 3.5. Morris water maze As showed in Table 3, from day 1 to day 3, there were no significant differences in escape latency among the eight groups. From the fourth day, escape latency of AD mice was significantly longer than control mice, whatever fed with SD or HFD. On the other hand, in the fourth and fifth day, OP treatment reversed the phenomenon in the AD mice. On the sixth day, AD mice exhibited a smaller percent time in the target quadrant than other mice (Fig. 4). Moreover, OPH administration markedly prolonged the time spent in the target quadrant of AD mice or AD mice fed with HFD, which indicated that OP could mitigate the cognition impairment. 3.6. The histopathological changes in the hippocampus Hematoxylin and eosin (H&E) staining was performed to examine the neuronal integrity and orderliness in the hippocampus. The neuronal layers in the CA1 region of the hippocampus showed rarefaction, disorder, pronounced shrinkage of nuclei, and swollen neuronal bodies in the AD mice (Fig. 5B, D) compared with the control mice (Fig. 5A, C), but the worsen shrinkage of nuclei and neuron rarefaction in obesity AD mice. OPL and OPH significantly inhibited histopathological damage (Fig. 5F–H). 3.7. Effects of OP on the PI3K/AKT/GSK3β signaling Western blotting results (Fig. 6A) showed that PI3K (Fig. 6B) and AKT (Fig. 6C) were reduced in AD mice (fed with SD or HFD) and this tendency was reversed by OP treatment significantly. However, the data of GSK3β (Fig. 6D) indicated that OP has no influence on it. 7
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particular the common convergence point, ERK. In the present study, we chose a high-fat diet for two months, because at that time, the body weight and blood glucose values of mice had produced a significant difference. However, the control mice fed with HFD didn't found metabolic disorder and memory/neuron injury, because the time of feeding HFD was short, but the AD mice fed with HFD had markedly metabolic disorder. It is consistent with previous reports, short-term high-fat-diet intake did not significantly cause cognitive impairment and brain injury in wild-type mice (Mielke et al., 2006), on the other hand, the same short-term consumption of high-fat diet significantly enhanced cognitive impairment in 5XFAD mice (Lin et al., 2016). In the result of western blot, the OP can indeed reverse the changes in Aβ1–42 and HFD, especially OPH. For sure, the detail and specific mechanisms of OP reverse the metabolic disorder induced by high-fat diet and cognitive function injury in Alzheimer's disease need to be studied further.
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5. Conclusion In summary, the treatment of Alzheimer's disease is considerable difficulty in medicine, accompany the rise of Alzheimer's patients, the treatment of the disease is increasingly urgent. This study explored the treatment of Alzheimer's disease by two aspects, on the one hand, Okra polysaccharides can reverse AD mice cognitive disorder, on the other hand, it also proves HFD can aggravate the process of Alzheimer's disease. In addition, this study takes the lead in confirming that the Okra polysaccharide can reverse the cognitive impairment and metabolic disorder by HFD through the insulin signaling pathway. Role of the funding source There is no funding supports this research. Declaration of competing interest The authors have no conflict of interest to declare. Acknowledgements This research was supported by National Natural Science Foundation of China (No. 81573580). Key Laboratory of polysaccharide bioactivity evaluation of TCM of Liaoning Province. Key techniques study of consistency evaluation of drug quality and therapeutic effect (18-400-4-08) and Liaoning Distinguished Professor Project for Ying Jia (2017). The Doctoral Scientific Research Foundation of Liaoning Province (2019-BS-233). References Adelakun, O.E., Oyelade, O.J., Ade-Omowaye, B.I.O., Adeyemi, I.A., Van de Venter, M., 2009. Chemical composition and the antioxidative properties of Nigerian Okra Seed (Abelmoschus esculentus Moench) flour. Food Chem. Toxicol. 47 (6), 1123–1126. Arapitsas, P., 2008. Identification and quantification of polyphenolic compounds from okra seeds and skins. Food Chem. 110 (4), 1041–1045. Moise, M.M., Benjamin, L.M., Doris, T.M., Dalida, K.N., Augustin, N.O., 2012. Role of Mediterranean diet, tropical vegetables rich in antioxidants, and sunlight exposure in blindness, cataract and glaucoma among African type 2 diabetics. Int. J. Ophthalmol. 5, 231–237. Bluher, M., Patti, M.S., Kahn, B.B., Kahn, C.R., 2004. Intrinsic heterogeneity in adipose tissue of fat-specific insulin receptor knock-out mice is associated with differences in patterns of gene expression. J. Biol. Chem. 279 (30), 31891–31901. Cordner, Z.A., Tamashiro, K.L., 2015. Effects of high-fat diet exposure on learning & memory. Physiol. Behav. 152 (Pt B), 363–371. Deters, A.M., Lengsfeld, C., Hensel, A., 2005. Oligo- and polysaccharides exhibit a structure-dependent bioactivity on human keratinocytes in vitro. J. Ethnopharmacol. 102 (3), 391–399. Dineley, K.T., Jahrling, J.B., Denner, L., 2014. Insulin resistance in Alzheimer's disease. Neurobiol. Dis. 72 (Pt A), 92–103. Eskelinen, M.H., Ngandu, T., Helkala, E.L., Tuomilehto, J., Nissinen, A., Soininen, H., Kivipelto, M., 2008. Fat intake at midlife and cognitive impairment later in life: a population-based CAIDE study. Int. J. Geriatr. Psychiatry 23 (7), 741–747.
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