Effects of sericin on heme oxygenase-1 expression in the hippocampus and cerebral cortex of type 2 diabetes mellitus rats＊＊☆

Zhihong Chen, Yaqiang He, Wenliang Fu, Jingfeng Xue

Department of Human Anatomy, Chengde Medical University, Chengde 067000, Hebei Province, China

lNTRODUCTlON

Diabetes mellitus can accelerate brain aging,including the impairment of cognitive function, dementia, and neurodegeneration[1]. A drug used to treat diabetes mellitus is expected to be safe,have no side effects, be easily administrable,and have significant curative effects. Sericin possesses precisely these characteristics.

Sericin resists oxidation and reduces blood glucose[2]. Our previous studies have confirmed that sericin effectively decreased blood glucose, protected islet cells, gonads and kidneys, and sericin pretreatment also had similar effects[3-5]. Few studies have described the protective effects of sericin on nervous system injury following diabetes mellitus.

Heme oxygenase-1 catabolizes heme to biliverdin, carbon monoxide, and free iron,and serves as a sensitive index of cell stress[6]. Biliverdin is subsequently converted to bilirubin by biliverdin reductase.Bilirubin accumulation can lead to nervous system toxicity[7-8]. With excessive expression of heme oxygenase-1, a large amount of Fe2+accumulates, which causes neurodegeneration and disruption of neural cell function in the central nervous system[9-10]. Simultaneously, carbon monoxide, one of the byproducts of heme degradation through heme oxygenase, can destroy mitochondria and mitochondrial membranes in neural cells, finally leading to necrosis and apoptosis of neural cells[11-12].

Therefore, effective inhibition of heme oxygenase-1 expression can relieve nervous system injury. The present study sought to investigate heme oxygenase-1 expression in the hippocampus and cerebral cortex of diabetic rats following sericin treatment, and explored the protective effects of sericin on the central nervous system of diabetic rats.

RESULTS

Quantitative analysis of experimental animals

A total of 30 male Sprague-Dawley rats were included, and equally and randomly assigned to control, model, and treatment groups. Streptozotocin was intraperitoneally injected into rats of the model and treatment groups for 3 consecutive days to establish a model of diabetes mellitus. One week following model induction, the blood glucose of the rats was found to be ≥ 16.7 mmol/L,indicating successful establishment of the model[13]. Rat models in the treatment group were intragastrically administered sericin. 30 rats were included in the final analysis.

Blood glucose levels in rats of each group

Blood glucose levels were significantly elevated in the diabetic rats (29.00 ± 5.39 mmol/L vs. 11.12 ±2.22 mmol/L, P ＜ 0.01). The blood glucose levels were significantly decreased following 35 days of sericin treatment (14.03 ± 3.98 mmol/L, P ＜ 0.01)(supplementary Table 1 online).

Heme oxygenase-1 protein and mRNA expression in the rat hippocampus and cerebral cortex

Heme oxygenase-1 protein and mRNA expression were determined using western blot assay and reverse transcription (RT)-PCR, respectively. Heme oxygenase-1 protein and mRNA expressions were significantly increased in the hippocampus and cerebral cortex of diabetic rats (P ＜ 0.01). Heme oxygenase-1 protein and mRNA expression in the hippocampus and cerebral cortex were significantly decreased following 35 days of sericin treatment (P ＜ 0.01 or P ＜ 0.05; Figures 1–2,Table 1).

Figure 1 Heme oxygenase-1 (HO-1) protein expression in the rat hippocampus and cerebral cortex (western blot assay). 1: Control group; 2: model group; 3: treatment group.

Figure 2 Heme oxygenase-1 (HO-1) mRNA expression in the rat hippocampus (A) and cerebral cortex (B)(reverse transcription-PCR). 1: Control group; 2: model group; 3: treatment group.

Table 1 Heme oxygenase-1 (HO-1) expression in the hippocampus and cerebral cortex of rats in each group(x ±s, n = 10, absorbance ratio of HO-1 to β-actin)

Pathological changes in the rat hippocampus and cerebral cortex

Morphological changes in the rat hippocampus and cerebral cortex were observed using hematoxylin-eosin staining. In the hippocampal CA1 region, cells were arranged closely, regularly, with round and large nuclei.

The nucleoli were clear in the rats in the control group(Figure 3A). Disordered pyramidal cells, dispersed neural cells, chromatolysis, and nucleus displacement were visible in the model group. Moreover, some cells became vacuole-shaped. There was partial cell loss, and the remaining cells were small in size. Cells also appeared pyknotic, and polygonal or irregular cell bodies were evident. The cytoplasm was concentrated, and there was an unclear boundary of the nuclear membrane, and the nucleoli disappeared (Figure 3B). In the treatment group,the cells were arranged closely and orderly, and there was little cell loss, the nuclei appeared round and large,and the nucleoli appeared clear (Figure 3C).

Figure 3 Pathological changes in the rat hippocampal CA1 region (hematoxylin-eosin staining, × 400). (A) Neural cells were arranged orderly in the control group. (B)Neural cells were arranged disorderly; chromatolysis,nucleus displacement, partial cell loss and pyknosis were visible in the model group. (C) Pathological changes were less pronounced in the treatment group compared with the model group, showing loss of a few cells.

Neural cells in the rat cerebral cortex presented with a triangular shape, and the cell morphology was normal in the control group (Figure 4A). Swelling of the neural cell body, nucleus displacement, chromatolysis, nucleolus disappearance, partial cell loss, and some vacuole-shaped cells were visible in the model group(Figure 4B). Pathological changes in the neural cells of the rat cerebral cortex were significantly lessened, with an integrated morphology, sometimes with abnormal neural cells in the treatment group (Figure 4C).

Figure 4 Pathological changes in the rat cerebral cortex(hematoxylin-eosin staining, × 400). (A) Normal morphology was visible in the control group. (B) Swelling of the neural cell body, nucleus displacement, and some vacuole-shaped cells were observed in the model group.(C) Pathological changes were significantly diminished,with integrated morphology, sometimes with abnormal neural cells in the treatment group.

DlSCUSSlON

Diabetes mellitus-induced changes in the structure and function of the central nervous system can lead to cognitive impairment[14]. A suitable animal model of diabetes mellitus is very important to the exploration of drugs that could potentially yield positive therapeutic effects with little or no toxicity and side effects.

Conventional animal models of diabetes mellitus are mainly comprised of four types: the chemical-induced animal model, the spontaneous hereditary animal model,the partial pancreatectomy animal model, and transgenic animal models. The streptozotocin-induced animal models of diabetes mellitus are commonly used for the study of diabetes mellitus, and these models are characterized by high stability, slight damage to the liver and kidney, and a high success rate of model establishment[13,15-18]. However, the injection dose of streptozotocin is controversial[19]. In accordance with a previous study[13], 25 mg/kg streptozotocin was consecutively injected intraperitoneally three times to establish the animal model of diabetes mellitus,demonstrating a high success rate of model induction and a low mortality rate.

Maines[20]isolated, purified, and obtained heme oxygenase-1 from animals and humans. The heme oxygenase system consists of three isozymes, of which heme oxygenase-1, which is also known as heat shock protein 32, can be induced when cells and tissues are in a stressed state[21]. Heme oxygenase-1 is mainly distributed in the liver, spleen, the reticuloendothelial system, bone marrow, and brain tissues where erythrocyte metabolism is active[22]. Following onset of diabetes mellitus, chronic hyperglycemia can induce the increase in reactive oxygen species production in the mitochondrial electron transport chain[23-24]. Excessive reactive oxygen species results in progressive angiopathy and atherosclerosis. Reactive oxygen species affect blood supply in the heart, brain, and limbs by activation of the vascular injury pathway and through direct effects on the vascular endothelial cells[23-24]. Ischemia/hypoxia in brain tissue can induce excessive expression of heme oxygenase-1[23-24].

Results from this study demonstrated that morphological structures of the hippocampus and cerebral cortex of the diabetic rats undergo significant change, and heme oxygenase-1 expression in the hippocampus and cerebral cortex was significantly increased, consistent with the above-mentioned results.

The silkworm cocoon, the cocoon shell of bombyx mori,has been used to treat diabetes mellitus[25]. Sericin is derived from bombyx mori. The present study verified that sericin diminished blood glucose, improved the morphological structure of the hippocampus and cerebral cortex, and reduced heme oxygenase-1 expression in the hippocampus and cerebral cortex in diabetic rats.

Results from this study suggest that sericin improves ischemia/hypoxia in the brain tissue, downregulates heme oxygenase-1 expression in the brain tissue, and mitigates the toxic effects of biliverdin, carbon monoxide,and free iron in the nervous system by diminishing blood glucose levels. This results in a protective effect on the hippocampus and cerebral cortex of diabetic rats.

In summary, sericin markedly decreased blood glucose,improved pathological changes in the hippocampus and cerebral cortex, reduced heme oxygenase-1 expression in the hippocampus and cerebral cortex in diabetic rats.

However, the mechanism of inhibition of heme oxygenase-1 expression in the hippocampus and cerebral cortex requires further investigation.

MATERlALS AND METHODS

Design

Randomized controlled animal study.

Time and setting

Experiments were performed at the Institute of Basic Medical Sciences, Chengde Medical University, China from June 2009 to April 2010.

Materials

A total of 30 healthy, clean, male, Sprague-Dawley rats,aged 3 months, weighing 200–250 g (license No. 712024)were supplied by the Experimental Animal Center, Hebei Medical University. Protocols were conducted 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[26].

Sericin was made from the color silkworm cocoon(Institute of Sericulture, Chengde Medical University,China) by immersion, water decoction, filtration, and condensation.

Methods

Model induction and intervention

The rat model of diabetes mellitus was established by intraperitoneal injection of 2% streptozotocin (25 mg/kg)(prepared with pH 4.4 sodium citrate-citric acid buffer;Sigma, St. Louis, MO, USA) for 3 consecutive days in the model and treatment groups. Following successful establishment, rats from the treatment group daily received intragastric administration of sericin (2.4 g/kg)for 35 consecutive days. Rats in the model group did not receive any treatment.

Determination of blood glucose level

After drug withdrawal, rats were fasted for over 12 hours and were intraperitoneally anesthetized with 4% chloral hydrate. Approximately 3 mL blood was collected from the inner canthus, and centrifuged for 20 minutes at 3 000 r/min. The blood serum was collected. Blood glucose level was detected by the glucose oxidase method with a Boehringer Mannheim/Hitachi 717 automated clinical biochemistry analyzer (Hitachi, Tokyo,Japan).

Sample preparation

Following blood collection, the rats were decapitated and the brain was dissected. The hippocampus and cerebral cortex from one hemisphere were rapidly dissociated and stored in liquid nitrogen, whereas the brain tissues from the other hemisphere were fixed in Bouins fluid.

Hematoxylin-eosin staining

Brain tissues were fixed in Bouins fluid, embedded in paraffin, and serially sliced into 5-μm-thick sections.Sections from the hippocampal CA1 region and cerebral cortex of rats were subjected to hematoxylin-eosin staining, observed and photographed under a microscope (Olympus, Tokyo, Japan).

RT-PCR

100 mg of hippocampal tissue and 100 mg of cerebral cortex tissue were triturated in liquid nitrogen. Total RNA was extracted from the hippocampus and cerebral cortex in accordance to the instructions for Trizol reagent(Invitrogen, Carlsbad, CA, USA). A one-step method RT-PCR kit (TAKARA Biotechnology, Dalian, Liaoning Province, China) and 600-bp DNA ladder (Beijing Taigemei Technology, Beijing, China) were utilized in the present study. 5 μL RNA was electrophoresed in a 1%agarose gel. 28 S, 18 S, and 5 S bands were detectable,which indicated integration of total RNA. Using an ultraviolet spectrophotometer (DU800; Beckman Coulter,Fullerton, CA, USA), the value of absorbance at 260 nm/absorbance at 280 nm was 1.9–2.1, which suggested that the RNA was not contaminated. 3 μg of total RNA served as a template for PCR. The first step in the RT-PCR assay was reverse transcription of the RNA template into cDNA. PCR primer was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequence was as follows:

Primer Sequence Product size(bp)Heme oxygenase-1 Forward: 5’-CAG TCT ATG CCC CAC TCT AC-3’406 Reverse: 5’-AAG GCG GTC TTA GCC TCT TC-3’β-actin Forward: 5’-GAG GGA AAT CGT GCG TGA C-3’445 Reverse: 5’-CTG GAA GGT GGA CAG TGA G-3’

PCR reaction conditions were as follows: 94 °C for 2 minutes, 94 °C for 30 seconds, 50–65 °C for 30 seconds,72 °C for 1 minute. Cycles were conducted from the second step. PCR products were electrophoresed in a 2% agarose gel (containing 0.5 mg/L Goldview). ZF ultraviolet transmission reflection analyzer (Shanghai Jiapeng Technology, Shanghai, China) was used for photography. Quantity One-4.6.2 software (BIO-RAD,Hercules, CA, USA) was employed for quantitative analysis. The ratio of the absorbance of the target band to the absorbance of the β-actin band served as the relative level of the target mRNA expression[27].

Western blot assay

100 mg of hippocampal tissue and 100 mg of cerebral cortex tissue were homogenized in ice-cold 0.01 mmol/L,pH 7.4 PBS. Tissues were centrifuged, and then the supernatant was discarded. 200 μL lysate (radio immunoprecipitation assay: phenylmethyl sulfonyl fluoride was 100: 1) was added for 40 minutes at 4 °C, followed by centrifugation at 12 000 r/min for 15 minutes. The collected supernatant was the extracted protein. Protein concentration was quantified using a bicinchoninic acid protein kit (Beijing Solarbio Science &Technology, Beijing, China). Protein extracts (50 μg)were electrophoresed in a 12% sodium dodecyl sulfate polyacrylamide gel at 80–120 V for 2 hours, and transferred onto a 2 mA/cm2membrane stained with ponceau red for 2 hours. Subsequently, the membranes were blocked with 5% skimmed milk. Tissues were incubated in rabbit anti-heme oxygenase-1 (1: 200),β-actin (1: 1 000) polyclonal antibodies (Beijing Biosynthesis Biotechnology, Beijing, China) at 4 °C overnight, and then incubated with goat anti-rabbit IgG(1: 5 000; KPL, Gaithersburg, Maryland, USA) at room temperature for 1 hour, and developed by Super ECL Plus luminescence fluid (Applygen Technologies Inc.,Beijing, China). Following film scanning, visualized bands were analyzed utilizing the Quantity One-4.6.2 software (BIO-RAD). β-actin served as the internal reference. The absorbance ratio of heme oxygenase-1 to β-actin served as the relative level of heme oxygenase-1 protein[28].

Statistical analysis

The data were expressed as Mean ± SD, and were analyzed using SPSS 11.5 software (SPSS, Chicago, IL,USA). The differences between the groups were compared using one-way analysis of variance, followed by pairwise comparison and q-test. A value of P ＜ 0.05 was considered statistically significant.

Author contributions:Zhihong Chen obtained funding,participated in the study concept and design, data analysis, and manuscript drafting. Yaqiang He participated in the study analysis and statistical analysis. Wenliang Fu participated in the experimental animal breeding and disposal, and provided data support. Jingfeng Xue participated in the study concept and design, article authorization and study instruction.

Conflicts of interest:None declared.

Funding:This study was supported by the Grant of Department of Education of Hebei Province (GH/IGF-1 action mechanism in diabetes mellitus-induced gonadal axis injury and protective effects of sericin), No. 2006301; the Grant of the Department of Technology of Hebei Province (Protective effects of sericin on testicular dysfunction following diabetes mellitus), No.08276101D-19.

Ethical approval:This study was approved by the Animal Ethics Committee of Hebei Province in China.

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. 6, 2011 item after selecting the “NRR Current Issue” button on the page.

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