Effect of Yiqi Bushen prescription on hippocampal neuronal apoptosis in diabetic rats＊＊＊☆

Deshan Liu, Weiwei Lin, Wei Gao, Ping Chang, Wei Li

Department of Traditional Chinese Medicine, Qilu Hospital of Shandong University, Jinan 250012, Shandong Province, China

lNTRODUCTlON

Studies have shown that impaired learning and memory, brain evoked potential abnormalities and hippocampal neurodegenerative diseases caused by diabetes can be improved by traditional Chinese medicine[1-5]. It has been reported that cognitive dysfunction in diabetes mellitus is linked to apoptosis of hippocampal neurons, and that apoptosis is closely related to neuronal hypoxia[4-5].

Previous studies have demonstrated that hypoxia-inducible factor-1α (HIF-1α) plays an important role in diabetic complications[6-12], and that gene expression of HIF-1α rapidly increases after exposure to hypoxia, along with the target gene vascular endothelial growth factor (VEGF); however,this increase was suppressed by local administration of exogenous nerve growth factors[10-11]. Considering the relationship between diabetic neuropathy and microangiopathy, we assumed that a hippocampal hypoxic microenvironment may exist in diabetes mellitus, which induces abnormal HIF-1α expression,effects VEGF expression, and effects the oxidative stress system and apoptotic protein expression, resulting in neuronal apoptosis and cognitive dysfunction[11-12].

Yiqi Bushen prescription (YQBS), a Chinese medicine prescription that can supplement qi and tonify kidney, is mainly composed of crude Radix Astragali, prepared Rhizome of Rehmannia, Rhizoma Polygonati, Rhizoma Chuanxiong, Fructus Lycii, Herba Epimedii,Rhizoma Atracylodis, Radix Puerariae and Rhizoma Coptidis. Clinical studies have demonstrated that YQBS can significantly improve cognitive dysfunction in diabetic patients[13-14]. To verify whether a hypoxic microenvironment co-exists with cognitive dysfunction in diabetes mellitus, the present study first investigated the effects of YQBS on the learning and memory functions of diabetic rats and the association with hippocampal neuronal apoptosis.Subsequently, hippocampal neurons were cultured in high-glucose/hypoxic conditions to explore the relationship between apoptosis, HIF-1α expression and YQBS.

RESULTS

In vivo experiments

Quantitative analysis of experimental animals

Hippocampal neurons were isolated from 8 newborn Wistar rats that were less than 24 hours old. In addition, 30 of the 64 Wistar rats, aged 8 weeks, were used to prepare drug-containing serum, and the remaining 34 were randomly assigned to control(n = 10), model (n = 12; streptozotocin) and treatment (streptozotocin + YQBS; n = 12)groups. The model and treatment groups were intraperitoneally injected with streptozotocin to establish a diabetic model[3], and the treatment group was additionally intragastrically perfused with YQBS. Thirty-four rats were included in the final analysis.

YQBS increased body mass and decreased blood glucose levels in diabetic rats

Following streptozotocin injection, blood glucose levels were significantly elevated (P ＜ 0.01), and the high levels were maintained for up to 6 weeks, demonstrating that the models were successfully established. Following YQBS treatment, blood glucose levels were significantly reduced (P ＜ 0.01; supplementary Figure 1 online).

Over prolonged periods of time, the body mass of all rats increased, but this increase was slower in diabetic rats.

Following YQBS treatment, the body mass of diabetic rats rapidly increased and reached control group levels after 6 weeks (supplementary Figure 1 online).

YQBS improved the learning and memory capacity of diabetic rats

The Y-type maze was used to test learning and memory capacity. Frequent errors represented low learning and memory capacity. Results showed more errors in learning and memory in diabetic rats compared with control rats (P ＜ 0.05). YQBS significantly decreased the frequency of errors in learning and memory in diabetic rats(P ＜ 0.01; Figure 1).

Figure 1 Learning and memory changes in diabetic rats and treatment effects of Yiqi Bushen prescription. The Y-type maze was used to assess learning and memory capacity. Frequent errors represented low learning and memory capacity. Results are expressed as mean ± SD.There were 10, 12 and 12 rats in the control, model and treatment groups, respectively. aP ＜ 0.05, vs. control group; bP ＜ 0.01, vs. model group (LSD-t test).

YQBS increased hippocampal neuron survival in diabetic rats

Hematoxylin-eosin (HE) staining revealed that the number of hippocampal neurons significantly decreased in diabetic rats. After a 6-week treatment with YQBS,hippocampal neurons appeared to be arranged in an orderly manner, and the number of neurons remained at normal levels, with the addition of a few darkly stained neurons. The number of hippocampal neurons significantly decreased in the model group compared to the control group (P ＜ 0.01), and significantly increased in the treatment group when compared to the model group (P ＜ 0.01; Figure 2).

Figure 2 Hippocampal morphology and neuronal quantity observed by hematoxylin-eosin staining following light microscopy (× 200). Arrows represent neurons.

(A) In normal rats, the hippocampus was clearly outlined,neurons arranged orderly, neuronal cytoplasm was lightly stained, and the nucleoli and nuclear membrane were clearly visible.

(B) Diabetic rats exhibited obscure cell layers in the hippocampus, a significantly reduced number of neurons with a disorderly arrangement, swollen cell bodies, and a darkly stained cytoplasm in some neurons. Nucleoli were not observed.

(C) After Yiqi Bushen prescription treatment for 6 weeks,diabetic rats presented orderly arranged cells in the hippocampus and few darkly stained neurons. The number of neurons was similar to the control group.

(D) The quantity of hippocampal neurons are expressed as mean ± SD. There were 10, 12 and 12 rats in the control,model and treatment groups, respectively.aP ＜ 0.01, vs.control group;bP ＜ 0.01, vs. model group (LSD-t test).

YQBS inhibited hippocampal apoptosis protein expression in diabetic rats

Immunohistochemical staining was used to determine Bcl-2 and Bax expression in the hippocampus.

Quantitative analysis revealed that Bcl-2 protein expression and the ratio of Bcl-2/Bax were significantly elevated, but Bax protein expression significantly declined in the treatment group compared to the model group (P ＜ 0.01; Figure 3).

In vitro experiments

YQBS inhibited hippocampal neuronal apoptosis and promoted neuronal survival under high-glucose/hypoxic conditions

The high-glucose/hypoxic environment was simulated using 1 mmol/L xanthine, 20 U/L xanthine oxidase and 25 mmol/L glucose. Results revealed that survival of neurons significantly reduced and that apoptotic cell death significantly elevated following high-glucose and(or) xanthine/xanthine oxidase treatment (P ＜ 0.01).

Drug-containing serum (300, 600, or 1 200 mg/kg YQBS,40 mg/kg ligustrazine hydrochloride) increased the survival and decreased apoptosis of neurons exposed to a high-glucose/hypoxic environment (P ＜ 0.05). In particular, drug-containing serum with 600 mg/kg YQBS was the most effective (P ＜ 0.05; Table 1).

Figure 3 Hippocampal Bcl-2 and Bax protein expression in each group (immunohistochemical staining, × 400).Arrows = Bcl-2/Bax expression.Results showed that positive cells presented with a brown yellow, evenly stained cytoplasm. The percentage of positive cells out of the total number of cells counted was determined and results were grouped according to the following criteria: 0-5% (-, no staining), 5-25% (+, weak staining), 26-50% (++, moderate staining), 51-75% (+++,strong staining) and ＞75% (++++, very strong staining).(A) Bcl-2 staining was not present (-) in the control group(A1), the model group exhibited a few Bcl-2-posiitve cells(+) (A2), and the treatment group exhibited an increase in immunoreactivity (++) (A3).(B) Bax staining was not present (-) in the control group(B1), very strong immunoreactivity (++++) was observed in the model group (B2), and strong immunoreactivity (+++)was observed in the treatment group (B3).Leica Qwin V3 image analysis software was used to quantitatively analyze hippocampal Bcl-2, Bax and Bcl-2/Bax (C). Results are expressed as mean ± SD.There were 10, 12 and 12 rats ian the control, model and treatmebnt groups, respectively. P ＜ 0.01, vs. control group, P ＜ 0.01, vs. model group (LSD-t test). The darker the immunohistochemical products stained, the smaller the gray scale, and the greater the expression.

YQBS regulated HIF-1α, Bcl-2 and Bax mRNA expression in hippocampal neurons exposed to high-glucose/hypoxic conditions

Real-time PCR showed that Bcl-2 mRNA expression and the ratio of Bcl-2/Bax significantly reduced, but HIF-1α mRNA and Bax mRNA expression was significantly increased in high-glucose and (or)xanthine/xanthine oxidase-treated neurons (P ＜ 0.05).

Following treatment with drug-containing serum (300,600, or 1 200 mg/kg YQBS, 40 mg/kg ligustrazine hydrochloride), Bcl-2 mRNA expression and the ratio of Bcl-2/Bax significantly increased, while HIF-1α mRNA and Bax mRNA expression was significantly reduced in neurons exposed to a high-glucose/hypoxic environment. In particular, drug-containing serum with 600 mg/kg YQBS was the most effective (P ＜ 0.05;Table 2, Figure 4).

Table 1 Neuronal survival and apoptosis as detected by methyl thiazolyl tetrazolium and Annexin/PI double staining, respectively (%)

Table 2 Hypoxia-inducible factor-1α (HIF-1α) mRNA,Bcl-2, Bax mRNA and Bcl-2/Bax ratio in hippocampal neurons of each group

aP ＜ 0.05, vs. control group;bP ＜ 0.05, vs. high glucose group;cP ＜0.05, vs. xanthine/xanthine oxidase group;dP ＜ 0.05, vs. model group;eP ＜ 0.05, vs. low-dose YQBS group;fP ＜ 0.05, vs. moderate-dose YQBS (LSD-t test). Data are expressed as mean ± SD of 10 independent experiments. YQBS: Yiqi Bushen prescription. Gel electrophoresis images were analyzed using the gel analysis syste to determine absorbance. The absorbance ratio of target gene HIF-1α to GAPDH, and target gene Bcl-2 and Bax to β-actin were regarded as the relative content of target genes.

DlSCUSSlON

Hyperglycemia has been regarded as a direct cause of cognitive impairment in diabetes mellitus[3,6,15-16]. In this study, diabetic rats presented with learning and memory dysfunction after 6 weeks, and hippocampal neurodegeneration was evident following light microscopy. After diabetic rats were treated with YQBS,blood glucose levels significantly declined, learning and memory performance significantly improved, and neurodegeneration was ameliorated, indicating YQBS can effectively prevent and treat diabetic mellitus and associated cognitive dysfunction, consistent with a previous clinical report[5].

Figure 4 Agarose gel electrophoresis analysis of RT-PCR products of HIF-1α, Bcl-2 and Bax mRNA in the hippocampal neuron of rats.

Hippocampal neuronal apoptosis can damage learning and memory function, and hyperglycemia can trigger the apoptotic process[17]. Bcl-2 and Bax expression correlate with apoptosis, and the ratio of Bcl-2/Bax governs the induction ofapoptosis[18-20]. Results from the present study showed that YQBS can inhibit neuronal apoptosis by inhibiting Bax expression and upregulating the ratio of Bcl-2/Bax, thereby improving learning and memory performance of diabetic rats.

As hyperglycemia can induce hypoxia, hippocampal neuronal apoptosis and increase the expression of HIF-1α[5,21], we assumed that hypoxia occurs in the hippocampus after diabetes mellitus, which affects apoptosis protein expression through an increase in HIF-1α expression, resulting in neuronal apoptosis and learning and memory damage. In hypoxia, HIF-1α expression is elevated in a time-dependent and hypoxic level-dependent manner, but HIF-1α protein expression and transcriptional activity are mainly regulated by intracellular oxygen concentrations[22-24]. Neural cells in brain tissue have been shown to decrease as a result of apoptosis induced by HIF-1α[25-27]. In this study, high glucose concentrations induced HIF-1α mRNA expression and hippocampal neuronal apoptosis, and contributed to xanthine/xanthine oxidase-induced oxidative injury. YQBS upregulated Bcl-2 mRNA expression and the ratio of Bcl-2/Bax, downregulated Bax mRNA and HIF-1α mRNA expression, and reduced xanthine/xanthine oxidase-induced neuronal apoptosis under high glucose conditions, thereby protecting against neuronal injury. These findings support the hypothesis that hypoxia exists in the hippocampus in diabetes mellitus, and demonstrates that YQBS can reduce blood glucose and improve the hypoxic microenvironment.

Radix Astragali and prepared rhizome of rehmannia in YQBS can reduce blood glucose levels, promote learning and memory capacity, dilate blood vessels,improve microcirculation, enhance the anti-hypoxic capacity, prevent lipid peroxidation and regulate immunity[28-29]. Ligustrazine, extracted from Rhizoma Chuanxiong, can inhibit xanthine oxidase activity and inhibit HIF-1α expression[30], further supporting our results. In summary, YQBS can suppress high-glucose/hypoxia-induced neuronal apoptosis in the hippocampus, which can improve brain function of patients with diabetes mellitus.

MATERlALS AND METHODS

Design

A randomized, controlled, animal experiment and comparative observation of cytology.

Time and setting

The experiments were performed at the Animal Laboratory, Clinical Basic Institute, Qilu Hospital of Shandong University and Institute of Basic Medicine,Shandong Academy of Medical Sciences from 2007 to 2010.

Materials

Eight newborn Wistar rats less than 24 hours old and 64 adult male Wistar rats, aged 8 weeks, weighing 200-250 g,were provided by the Laboratory Animal Center of Shandong University (No. SCXK (Shandong) 20030004).The experimental procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China[31].

YQBS was composed of crude Radix Astragali, prepared Rhizome of Rehmannia, Rhizoma Polygonati, Rhizoma Chuanxiong, Fructus Lycii, Herba Epimedii, Rhizoma Atracylodis, Radix Puerariae and Rhizoma Coptidis.

YQBS powder was prepared by the Manufacturing Laboratory of Qilu Hospital, Shandong University (one gram was equal to 3.564 g of crude drug) and dissolved in distilled water to prepare a 5% (w/v) water solution.

Preliminary quality control of YQBS was established as described previously[32].

Methods

In vivo experiments

Model establishment and intervention: Rats were housed for 1 week for adaptation. Model and treatment groups were subjected to single intraperitoneal injection of 1%(w/v) streptozotocin (60 mg/kg; Sigma, St. Louis, MO,USA) to induce diabetes mellitus. The control group was injected with the same volume of citrate buffer solution.

After model establishment, the treatment group rats were administrated 5% (w/v) water solution containing 600 mg/kg YQBS, 10 mL/kg daily, while the control and model groups were intragastrically perfused with the same volume of normal saline, once a day for 6 consecutive weeks. During experimentation, the activity of rats, food and water intake, and urination and defecation were observed; body mass and random blood glucose were measured every 3 weeks.

Learning and memory determination by Y-type maze: At the end of the sixth week of administration, rats were tested using the Y-type maze (Zhangjiagang Sanxing Teaching Instrument, Zhangjiagang, Jiangsu, China).

According to a previously published method[33], learning and memory results were represented by error number.

Hippocampal neuronal morphology and quantity as observed by HE staining: The rats were sacrificed after

maze testing. The brain was harvested and immediately,immersed in 10% (v/v) formalin fixation solution, dehydrated, immersed in wax, embedded, sectioned, HE stained and observed by light microscopy (Olympus,Tokyo, Japan). Ten complete typical 200 × fields of view were randomly selected from each section (avoiding edge effect) to quantify neurons.

Hippocampal Bcl-2 and Bax protein expression as determined by immunohistochemistry: Hippocampal sections were determined using a ready-to-use streptavidin-biotin complex (SABC) immunohistochemical staining kit (Wuhan Boster, Hubei, China). The sections were dewaxed and treated with 3% (v/v) H2O2to deactivate endogenous peroxidase activity. Antigen retrieval was performed using a microwave, and sections were blocked with 5% (w/v) BSA at 37°C for 20 minutes.

Sections were incubated with rabbit anti-rat Bax and Bcl-2 monoclonal antibodies (1: 100; Boster) at 4°C overnight, followed by biotinylated goat anti-rabbit IgG (1:100; Boster) at 37°C for 20 minutes and SABC at 37°C for 20 minutes. A PBS wash was performed between each step. The sections were colorized with 3,3'-diaminobenzidine (Boster), dehydrated in gradient alcohol, permeabilized with xylene, mounted with neutral gum and observed by microscopy (DM4000, Leica,Heidelberg, Germany). Quantitative analysis was performed using Leica Qwin V3 image analysis software(Leica, Nussloch, Germany). Ten complete typical 200 ×fields of view were randomly selected from each section(avoiding edge effect) to determine mean gray values of positive immunoreactivity.

In vitro experiments

Preparation of YQBS and drug-containing serum: 30 adult rats were randomly assigned to five groups (n = 6),and administrated 300, 600 and 1 200 mg/kg YQBS,40 mg/kg ligustrazine hydrochloride (No. 0805062; King York, Tianjin, China) or normal saline. Blood was harvested after 3 days to prepare drug-containing serum,packaged and stored at -20°C.

Neuronal isolation, culture and identification: The bilateral hippocampus of newborn rats was isolated in a sterile fashion, cut into blocks (1 mm3), and digested in PBS containing 0.125% (w/v) trypsin (Sigma) at 37°C for 15 minutes. The reaction was terminated by DMEM/F12 culture medium containing 10% (v/v) fetal bovine serum.

A single cell suspension was prepared and filtrated with a 200-mesh screen. Cells were seeded onto 0.1 mg/mL polylysine (Sigma)-pretreated 6-well culture plates at a density of 1 × 106cells/L and incubated in 5% CO2at 37°C. Cytarabine (10 μg/mL; Sigma) was added on the third day for 24 hours to inhibit glial cell proliferation. Half of the culture medium was replaced every 3 days. Cell morphology and growth were observed by inverted phase contrast microscopy (Olympus; supplementary Figure 3 online). Microtubule-associated protein 2(MAP-2) immunofluorescent staining was performed on the seventh day (rabbit anti-rat MAP-2 antibody,TRITC-labeled goat anti-rabbit IgG; Sigma) to identify cultured cells (supplementary Figure 4 online).

Establishment of the high-glucose oxidative damage model and intervention: Cells were treated with xanthine(mmol/L)/xanthine oxidase (U/L; Sigma; 1.0/10, 1.0/20,2.0/20, 3.0/30). Cell survival was determined by the MTT assay. The 1.0/20 group was selected as the appropriate damage concentration to establish the oxidative damage model, whose survival rate was 61±10%. Primary neurons were cultured with 5.5, 10, 25, 50 mmol/L glucose,and cells cultured with mannitol using the same concentrations served as a control. According to results from cell survival experiments, 25 mmol/L glucose was identi-fied as the appropriate incubation concentration. Cell grouping and intervention are as follows:

A: Control group; B: high-glucose group; C: xanthine/xanthine oxidase group; D: model group, treated with drug-containing serum (normal saline); E: ligustrazine group treated with drug-containing serum (40 mg/kg ligustrazine hydrochloride); F, G, H: YQBS groups respectively treated with drug-containing serum (300, 600,and 1 200 mg/kg YQBS). X: Xanthine; XO: xanthine oxidase. DMEM: Dulbecco's modified Eagle's medium.

Hippocampal neuronal survival after high-glucose oxidative damage as determined by MTT: Cells (100 μL; 1 ×106cells/mL) were seeded on 96-well culture plates. 10 μL of MTT (0.5 mg/mL; Sigma) was added to each well(control wells were not treated) for 4 hours. The supernatant was discarded, and 100 μL of dimethyl sulfoxide was added to each well and left to shake for 10 minutes.

The absorbance (A) value (490 nm) was determined using an automatic microplate reader (Bio-Rad, Hercules,CA, USA). Neuronal survival rate = Aexperimentalgroup/Acontrolgroup× 100%.

Hippocampal neuronal apoptosis after high-glucose oxidative damage as determined by Annexin/propidium iodide (PI) staining: The Annexin V-FITC apoptosis kit(Keygen Biotech, Nanjing, Jiangsu, China) was used.

Briefly, cells were trypsinized (0.25% (w/v)), washed with PBS twice and centrifuged at 2 000 r/min for 5 minutes.

Cells of 1 × 105were collected, suspended using 500 μL binding buffer, mixed with 5 μL Annexin V-FITC and 5 μL PI at room temperature for 15 minutes, and analyzed using flow cytometry (Becton Dickinson, Franklin Lakes,NJ, USA) within 1 hour.

Hippocampal neuronal Bcl-2, Bax, HIF-1α mRNA expression after high-glucose oxidative damage as determined by real-time PCR: The trizol RNA kit was used (Sangon, Shanghai, China) to extract total RNA.

RNA concentration and purity were determined using an ultraviolet spectrophotometer (UV-2450PC; Shimadzu Suzhou, Jiangsu, China). The TaKaRa reverse transcription kit (Takara, Dalian, China) was used to reverse transcribe RNA into cDNA. GAPDH and β-actin served as internal references. PCR primers were synthesized by Invitrogen, Shanghai, China (Table 3).

PCR products were detected using 1.5% (w/v) agarose gel electrophoresis. Gel electrophoresis images were analyzed using the gel analysis system (Jieda Gel Analysis, Jiangsu, China) to determine absorbance. The absorbance ratio of target gene HIF-1α to GAPDH, and target gene Bcl-2 and Bax to β-actin were regarded as the relative content of target genes. Samples with a single product (no dimmer), were selected for real-time PCR. A standard curve for each detected gene and internal reference gene was prepared. The target gene and internal reference gene were amplified to determine the standard curve and melting curve. RT-PCR conditions were as follows: predenaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds,annealing at 60°C (Bax, GAPDH at 58°C) for 10 seconds,extension at 72°C for 15 seconds, for 55 cycles in total.

Melting curve conditions were 65°C for 15 seconds and 40°C for 30 seconds. The amplification and melting curve were analyzed using Lightcycler Fit point (Roche, Basel,Switzerland).

Table 3 Primer sequences for real-time PCR quantitative analysis

Statistical analysis

Results were analyzed using SPSS 16. 0 (SPSS,Chicago, IL, USA) and expressed as mean ± SD.

Intergroup differences were compared using one-way analysis of variance, and paired comparison was performed by LSD-t test. A value of P ＜ 0.05 was considered statistically significant.

Author contributions:Deshan Liu and Wei Gao conceived and designed this study, collected, integrated and analyzed data, and contributed to the critical revision and final approval of the manuscript. Deshan Liu, Wei Gao and Ping Chang per-formed animal experiments. Weiwei Lin and Wei Li performed cell culture experiments and data analysis. Deshan Liu was in charge of funds.

Conflict of interest:None declared.

Funding:This study was supported by the Science and Technology Development Program of Shandong Province, No.032050116; the Natural Science Foundation of Shandong Province, No. ZR2010HM077; the 20 Staff Foundation of 1020 Project of Shandong Province.

Ethical approval:This study received permission from the Animal Ethics Committee of Qilu Hospital of Shandong University.

Supplementary information:Supplementary data associated with this article can be found, in the online version, by visiting www.nrronline.org, and entering Vol. 6, No. 21, 2011 after selecting the “NRR Current Issue” button on the page.

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