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ZiBu PiYin recipe prevents diabetes-associated cognitive decline in rats: possible involvement of ameliorating mitochondrial dysfunction, insulin resistance pathway and histopathological changes
© The Author(s). 2016
Received: 18 November 2015
Accepted: 15 June 2016
Published: 8 July 2016
Disturbance in energy metabolism, as a key factor in diabetes-associated cognitive decline (DACD), has become a promising therapeutic target of Chinese medicine ZiBu PiYin Recipe (ZBPYR). However, it is still not clear how ZBPYR affects the mitochondrial function in DACD rats’ brains, which is considered as the crucial cell organelle to supply energy for the brain.
Type 2 diabetes mellitus (T2DM) rat models were established by using high fat diet and streptozotocin (STZ) (30 mg/kg, ip). The evaluation of insulin sensitivity was performed by oral glucose tolerance and insulin tolerance test. After 7 weeks, the T2DM rats were treated with vehicle or ZBPYR for 11 weeks and morris water maze (MWM) test were used to evaluate memory function. The ultra structural changes of prefrontal cortex (PFC) and hippocampus were examined by transmission electron microscopy (TEM). The mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) were measured with JC-1 and DCFDA assay. The levels of insulin proteins were quantified by Western Blot analysis and the markers of histopathological changes were detected by immunohistochemistry.
ZBPYR could alleviate learning and memory impairment of DACD rats. TEM showed that ZBPYR prevented mitochondrial ultra-structural alterations and number changes in the PFC and hippocampus of the DACD rats. In addition, ZBPYR significantly increased ΔΨm and lowered the levels of ROS. Further investigation indicated that ZBPYR suppressed the release of cytochrome c from mitochondria, strengthened insulin signaling and inhibited GSK3β over-expression. These positive effects were associated with reduced Aβ1-42 deposition and restored expression levels of microtubule-associated protein MAP2.
ZBPYR showed excellent protective effect against DACD via ameliorating mitochondrial dysfunction, insulin resistance and histopathological changes.
Diabetes-associated cognitive decline (DACD) is diabetic complication, due to changes in the central nervous system (CNS) . Considerable attention has been given to DACD in the last decade . Previous studies have shown that cerebrovascular changes, oxidative stress, increased expression of advanced glycation end products (AGEs) and impaired cerebral insulin signaling are the underlying causes of DACD [3–6]. Our recent data have demonstrated that defection in energy metabolism of hippocampus might affect the function of the brain. It might be a key determinant of DACD .
Mitochondria are considered as crucial cell organelle that produces energy for regulating the cellular metabolism. It has been reported that brain mitochondria regulate energy-demanding neuro-transmission and calcium homeostasis, which are important mechanisms for the learning and memory process [8, 9]. In recent years, it has become increasingly clear that there is correlation between brain mitochondrial dysfunction and several neurodegenerative diseases . Mitochondrial dysfunction is an important contributor to the damage and loss of neurons . It has been also found that there was significant decrease in neuron numbers in the cerebral cortex or hippocampus in diabetic cognitive decline rats [12, 13]. Therefore, preventing mitochondrial dysfunctions is taken as a new therapeutic target for DACD.
Traditional Chinese medicine (TCM) has a long history in China. The ZiBu PiYin recipe (ZBPYR), derived from a modification of the Zicheng Decoction, is a TCM recorded in the book of Bujuji by Cheng Wu in the Qing Dynasty . Early reports demonstrated that ZBPYR protected glutamate induced neuron injury and improved learning and memory of pi-yin deficiency diabetic rats and type 2 diabetic rats [7, 14, 15]. Recent findings have proved that ZBPYR could protect db/db mice from reducing dendritic spine density, attenuated brain leptin and insulin signaling pathway injury . The potential effects of ZBPYR on the mitochondrial function of the brain in DACD model have not been investigated yet. In the present work, we used high-fat diet combined with Streptozotocin (STZ) rats as DACD model, aiming to test the hypothesis that ZBPYR could reverse the impairment of DACD mainly though alleviating brain mitochondrial dysfunction.
Tested drug and preparation
The ingredients of Zibu Piyin Recipe
Botanical Latin name
Plant part used
Panax ginseng C.A.Mey.
Red Ginseng Root
Dioscorea polystachya Turcz.
Common Yam Rhizome
Wolfiporia extensa (Peck) Ginns
Paeonia lactiflora Pall.
White Peony Root
Salvia miltiorrhiza Bunge
Dan shen Root
Lablab purpureus (L.) Sweet
White Hyacinth Bean
Nelumbo nucifera Gaertn.
Acorus gramineus Sol. ex Aiton
Grassleaf Sweetflag Rhizome
Polygala tenuifolia Willd.
Thinleaf Milkwort Root
Santalum album L.
Citrus maxima ‘Tomentosa’
Tangerine Red Epicarp
Glycyrrhiza uralensis Fisch. ex DC.
Male Sprague–Dawley (SD) rats (Six week-old, 200 ± 20 g) were purchased from the Experimental Animal Center of Dalian Medical University (Dalian, China). They were maintained at 22 ± 3 °C with 65 ± 5 % humidity on a 12 h light/dark cycle during the experiments. All surgery was performed under anesthesia with ether, and all efforts were made to minimize suffering.
Morris water maze test
Spatial learning and memory performance was assessed by morris water maze (MWM) test, which consisted of 5-day training (invisible plat training sessions and a probe trial) and visible plat training session on day 6. The apparatus consisted of a circular pool (120 cm in diameter, 50 cm in height and filled to a depth of 30 cm with water maintained at 28 ± 1 °C) and a transparent platform (9 cm in diameter and 29 cm in height), which was hidden under water. An automatic photographic was used to record the data (Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China). On the first day, rats were permitted to swim freely in the tank for 60 s without the platform to adapt to the new condition. Over the following 4 days, rats were trained with four trials per day at intervals of 120 s until they reached the platform and were allowed to rest for 20 s. If the rat failed to reach the platform within 120 s, it was gently guided to the platform and stayed for 20 s. The escape latency and swimming distance taken to reach the platform were measured automatically. After hidden platform test, the platform was removed. Rats had to swim for 120 s and the data referring % of total time in target quadrant. Time in searching original platforms and times across the platform were recorded. On the 6th day, a visible-platform test was undertaken. The platform was located 2 cm over the water surface and placed at a position different from the previous test. During this test, the escape latency was recorded.
Ultrastructural examination by transmission electron microscopy
4 % paraformaldehyde fixed prefrontal cortex (PFC) and hippocampus samples (3 mm × 3 mm) of Control, Model and ZBPYR groups were collected and fixed in 2 % glutaraldehyde in 0.1 mol/L sodium phosphate buffer (pH 7.4) overnight at 4 °C. After dehydration with a graded ethanol series, the sample was embedded in Epon812 and sectioned using a Leica EM UC6 ultramicrotome (Leica Co., Vienna, Austria). Samples were viewed and photographed using Transmission Electron Microscopy (TEM) (JEM-2000EX, JEDL, Japan).
Preparation mitochondria of prefrontal cortex and hippocampus
The rats were sacrificed by an animal expert in accordance with approved ministerial procedures appropriate to the species. The PFC and hippocampus were quickly separated and the tissue blocks were finely minced in 8 μm thickness . 50 pieces of sections were collected and homogenized in 1 mL isolation buffer according to the Tissue Mitochondria Isolation Kit (MitoSciences, Abcam, USA). The homogenate was centrifuged at 1000 g for 10 min at 4 °C and the supernatant was collected and further centrifuged at 12,000 g for 15 min at 4 °C. Then, mitochondrial pellets were collected, resuspending in 1 mL isolation buffer supplemented with 10 μL protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and further centrifuged at 12,000 g for 15 min at 4 °C. Finally, the mitochondrial pellets were resuspended with 500 μL isolation buffer supplemented with protease inhibitor. The concentration of mitochondrial protein was measured by the BCA protein assay kit (cwbiotech, Beijing, China).
Mitochondrial membrane potential assay
Changes in mitochondrial membrane potential (ΔΨm) were analyzed using a fluorescence spectrophotometer (PARADIGM, Molecular Devices, USA) and a mitochondria-selective dye (JC-1; Beyotime Institute of Biotechnology, Bejing, China). JC-1 was a lipophilic cation, driven by ΔΨm in normal polarized mitochondria assembled into a red fluorescence-emitting dimer forming J-aggregates. However, the monomeric form present in cells with depolarized mitochondrial membranes emited only green fluorescence.
Isolated mitochondria from PFC and hippocampus were stained with JC-1 staining solution (5 μg/mL) for 15 min at 37 °C and rinsed twice with assay buffer. The fluorescent intensity was analyzed on a spectrofluorometer as described to detect green fluorescence at excitation/emission wavelengths of 485/530 nm and red fluorescence at excitation/emission wavelengths of 550/595 nm. The ratio of red to green fluorescence intensity was determined for each sample as a measure of ΔΨm.
Measurement of reactive oxygen species production
Reactive oxygen species (ROS) Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to quantify ROS levels of PFC and hippocampus tissues to detect the changes of mitochondrial mass. The fluorescent intensity of the tissue suspensions was measured by a fluorescence spectrophotometer (PARADIGM, Molecular Devices, USA) at excitation wavelength of 485 nm and emission wavelength of 520 nm.
Protein extraction and western blotting analysis
The tissues of PFC and hippocampus were homogenized in ice cold lysis buffer . Then, the homogenate was centrifuged at 15,000 g for 5 min at 4 °C prior to collect the supernatants. Protein concentrations in the supernatants were measured using the bicinchoninic acid (BCA) protein assay kit (cwbiotech, Beijing, China). 50 μg of the sample proteins was separated by electrophoresis in 10 % sodium dodecylsulfate-polycrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidenedi fluoride membrance (Amersham, Buckinghamshire, UK). The membrance was blocked with 5 % skimmed milk in TBS-T (10 mM Tris-Cl, PH 8.0, 150 mM NaCl and 0.5 % Tween 20) at 4 °C overnight. It was rinsed three times (10 min/time) with TBS-T, followed by 2 h incubation with the first antibodies in the appropriate concentrations and then 2 h incubation with HRP-conjugated secondary antibody. The immuno-labeling was detected using the enhanced chemiluminesence system (Roche, GmbH, Mannheim, Germany) and visualized using the UVP Bio-spectrum Imaging System (UVP, Inc, Upland, CA, USA). Primary antibodies used from Cell Signaling Technologies (Danvers, MA, USA) were: protein kinase B (Akt) (#9272, 1:800), p-Ser473Akt (#4058, 1:800), GSK3β (#9315, 1:800), p-Ser9GSK3β (#9322, 1:800) and microtubule-associated protein 2 (MAP2) (#4542, 1:800). Primary antibody directed against cytochrome c (Abcam, Cambridge, UK, #ab110325, 1:800), insulin receptor substrate 2 (IRS2) (Millipore Corporation, Billerica, MA, USA, #MABS15, 1:2000) and p-Ser731 IRS-2 (Abcam plc, Cambridge, UK, #ab3690, 1:200) were also used in the experiments. Goat anti-rabbit (#NA9340, 1:2000) or Goat anti-mouse (#NA9310, 1:2000) (GE Healthcare, Buckinghamshire, UK) were used as secondary antibodies. β-actin (Sigma-Aldrich, St Louis, MO, USA, #A2228, 1:2000) and Voltage-dependent anion channel (VDAC) (Cell Signaling Technologies, Danvers, MA, USA, #4866, 1:800) were used as total internal quantitative control and mitochondrial internal quantitative, respectively.
Immunohistochemistry staining for Aβ1-42 and MAP2
Rats from each group were deeply anesthetized with pentobarbital sodium and perfused with 0.1 M PBS followed by 4 % paraformaldehyde. Brains were removed immediately and transferred to 15 %, 30 % sucrose solution (in PB) and then fixed in 4 % paraformaldehyde overnight at 4 °C. Sections of PFC and hippocampus were isolated and frozen on a cold stage, which was then cut at 6 μm according to the routine procedure . Immunohistochemical staining was performed using rabbit anti-(Rat MAP2 IgG) at dilutions of 1:80 (Sigma-Aldrich, St Louis, MO, USA) and mouse anti-(Rat Aβ1-42 IgG) at dilutions of 1:80 (Abcamplc, Cambridge, UK), respectively. The sections without first antibody incubation were used as background control. The images of PFC and the CA1, CA3 and DG regions of the hippocampus were captured by an upright microscope (Leica, Wetzlar, Germany). The quantitative analysis was used by the “integrated optical density” (IOD) function of Image Pro-Plus version 6.0.
Statistical analysis was performed using either an ANOVA (equal variance) or a Welch’s ANOVA (unequal variance) test. Data from the MWM test was analyzed using a repeated-measure analysis of variance for comparisons among trials. Student’s t test was used for comparison in two groups. The difference was considered to be statistically significant when p < 0.05.
Effects of ZBPYR on type 2 diabetes models
Effects of ZBPYR on diabetes-induced cognitive decline
In the probe trial, the percentage of the total time that the model rats spent in the target quadrant was significantly lower than control group (Fig. 3c, p < 0.01). However, ZBPYR group performed differently compared with model group (Fig. 3c, p < 0.05). Time in searching original platform was also acquired by the probe trial test. We found that the seconds that the model rats cost during the test was not significantly different between the control group and ZBPYR group (Fig. 3d). In addition, the number of times that the model group rats across the original platform was lower than that of control group (Fig. 3e, p < 0.05). However, there was no significant difference between ZBPYR group and model group. Performance in the visible platform version was similar in the three groups in terms of escape latency (Fig. 3f). All these findings suggest that ZBPYR could attenuate the impairment of learning and memory behaviors of the diabetic model rats.
Effects of ZBPYR on mitochondrial ultrastructure changes
Effects of ZBPYR on mitochondrial transmembrane potential alterations
Effects of ZBPYR on ROS Level
As shown in Fig. 5b, the increased ROS levels detected by DCFH assay were observed in the PFC and hippocampus of model groups (p < 0.05), and ZBPYR significantly inhibited the elevation of ROS levels with p < 0.01 in the PFC and p < 0.05 in the hippocampus.
Effects of ZBPYR on neuron cytoskeleton proteins and biomarker of inner mitochondrial membrane proteins
Effects of ZBPYR on insulin signaling and GSK3β
GSK3β was a well known down-stream target of insulin signaling [20, 21] and we tested the expression of GSK3β in our model. It was shown that there was no change in the total level of GSK3β in PFC and hippocampus (Fig. 7a and b). However, a down-regulation of p-GSK3β expression level at p-Ser9 was found in the PFC and hippocampus of model groups (p < 0.05) and ZBPYR increased the p-GSK3β level compared with the model groups (p < 0.05).
Effects of ZBPYR on the expressions of Aβ1-42 and MAP2
DACD is considered as a common neurodegenerative disease, which lacks an efficient therapeutic regimen [23–25]. The high efficiency and low toxicity of TCM, which has the advantage of providing multiple therapeutic effects on multiple targets, suggest the feasibility in the treatment of DACD. TCM ZBPYR plays crucial roles in learning and memory impairment in the diabetic rats through inhibiting endoplasmic reticulum stress, maintaining the morphology of dendritic spines and ameliorating DACD in proteomic analysis [7, 14, 16]. The individual herbs used in ZBPYR exhibited some pharmacological effects. For example, ginsenosides in Panax ginseng C.A.Mey reduced cerebral ischemia-induced tau phosphorylation and attenuated the symptoms of DACD [26, 27]. Additionally, senegenin from Polygala tenuifolia Willd processed neuroprotective potential against Aβ-induced neurotoxicity . It was also found that β-asarone in Acorus gramineus Sol. ex Aiton had the therapeutic potential against high-fat diet induced obesity in rats . These compounds may contribute to the effects of ZBPYR in preventing diabetes or neuroprotection. In spite of that, the mechanism of ZBPYR in regulating energy metabolism is still unknown. Mitochondria play a central role in energy metabolism. In the brain neurons, glucose is metabolized by mitochondria to produce cellular energy. It has been reported that mitochondrial dysfunction existed in multiple peripheral tissues of diabetic rats [30–32]. However, the mitochondrial functions in DACD animals were still unclear. The aim of this study was to investigate the function of brain mitochondria in high fat diet combined with STZ induced DACD rats and its related mechanisms of the protective effects of ZBPYR.
In our study, high fat diet combined with STZ injection rat model showed hyperinsulinaemia and hyperglycaemia, indicating that the diabetic model was successfully built. In addition, the rat model registered cognitive decline by MWM. ZBPYR treatment could restore the learning and memory retrieval behaviors induced by diabetes.
The energy of brain mainly depends on mitochondria. Maintaining mitochondrial function is essential for neuron survival and offers protection against neurodegeneration [33–36]. Here, we investigated the amount, structure, and function of mitochondria in the DACD rat brain. Our data indicated that the changes in mitochondrial density and structure by TEM were observed in the region of PFC and hippocampus of DACD rats. In addition, loss of ΔΨm and increased production of ROS were also found in the model groups, which suggested that mitochondria dysfunction existed. ZBPYR could alleviate the changes in the structure and increase the number of the mitochondria in per area. It was also found that mitochondrial membrane potential was pronounced and the level of ROS decreased in the same region of brain after ZBPYR treatment. Taken together, these results proved that mitochondria dysfunction was the mean target of ZBPYR. Accumulative evidence suggests that alterations in mitochondrial structure and function are associated with insulin signaling and related complications [37–41]. Moreover, recent epidemiological evidence suggests that CNS insulin resistance is a risk factor for cognitive decline [42–44]. Downstream targets of insulin signaling pathway, e.g., insulin receptor substrate serine phosphorylation 2 was up-regulated in the brains of obese rats . Akt, a key marker protein of insulin signaling, mediates the effect of insulin via important intracellular signaling cascades including the PI3K/Akt pathway [46, 47]. This work identified increase in p-IRS2 and decrease in p-Akt in the PFC and hippocampus of model rats, suggesting that central insulin signaling was impaired. ZBPYR could correct CNS insulin resistance by regulating p-IRS2 and p-Akt. In addition, ZBPYR exhibited strong activity in the inhibition of GSK3β. GSK3β is a downstream substrate of Akt and is the bridge connecting insulin signaling and Aβ, since the phosphorylation states of GSK3β in the brain were found to be involved in fluctuations of Aβ deposition [48–50]. In this study, we found that GSK3β activity was inhibited after ZBPYR treatment. Taken together, the present findings provide molecular biological evidence for the preventive effects of ZBPYR on DACD.
Amyloid β was reported to be the predictor of dementia and Aβ1-42 accumulation was found more obvious in the brain of cognitive decline patients [51–53]. Our previous study indicated that Aβ1-42 was down-regulated in db/db mice after ZBPYR treatment . In the present results, Aβ1-42 accumulation was more apparent in model group in the region of PFC and hippocampus (CA1 and CA3) and less detectable in ZBPYR group. Then, the density of dendritic marker MAP2, which reflected the changes of the neuronal morphology, was significantly decreased in either brain region of the models. ZBPYR was efficacious for alleviating the decreases in MAP2 levels, particularly in the PFC and hippocampus (CA1 and CA3). We proposed that Aβ1-42 accumulation and low expression of MAP2 in brain were the reason for DACD. ZBPYR significantly enhanced the learning ability of the DACD rats, which is largely due to the decrease in Aβ1-42 level and increase in MAP2 expression.
The findings of this study suggested that ZBPYR could prevent the brain impairment of DACD rats. The related mechanisms might be associated with improving mitochondrial dysfunction, insulin resistance and pathological changes including expressions of Aβ and MAP2.
Akt, protein kinase B; BCA, bicinchoninic acid; CNS, central nervous system; DACD, diabetes associated cognitive decline; FSI, fasting serum insulin; IOD, integrated optical density; IRS, Insulin receptor substrate; ITT, insulin tolerance test; MAP2, microtubule-associated protein 2; MWM, morris water maze; OGTT, oral glucose tolerance test; PB, sucrose solution; PFC, prefrontal cortex; RBG, red blood glucose; ROS, reactive oxygen species; STZ, Streptozocin; T2DM, type 2 diabetes mellitus; TCM, traditional Chinese medicine; TEM, transmission electron microscopy; VDAC, Voltage-dependent anion channel; ZBPYR, ZiBu PiYin recipe; ΔΨm, mitochondrial membrane potential
We thank Dr. Shengbo Yu at the department of anatomy in Dalian Medical University for offering the kind help of ultrastructural examination by transmission electron microscopy.
This work was supported by the Key Project of National Natural Science Foundation of China (No. 81230084), the Specialized Research Fund for the Doctoral Program of Higher Education of China (No.20132105130001) and Chinese Postdoctoral Science Foundation (No.2013 M541232).
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.
LBZ conceived and designed the study and involved in modifying the manuscript; ZS, LNL, HS, LPZ, XXS and WX performed the experiments and analyzed the data; LBZ and ZS had the primary responsibility for final content. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
This information is not relevant.
Ethics approval and consent to participate
All experiments were approved by the Committee on the Ethics of Animal Experiments of Dalian Medical University (Permit Number: SYXK (Liao) 2008-0002). All animal experiments were conducted in accordance with the NIH Principles of Laboratory Animal Care and the institutional guidelines for the care and use of laboratory animals at Dalian Medical University.
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