A total of 48 specific-pathogen-free 6-week-old male Sprague-Dawley rats (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were used in the current study. The rats were housed with a natural light cycle at 18–26°C and relative humidity of 40–70%. They were randomly divided into four groups: Control group, diabetic model group, high-dose sericin group and low-dose sericin group, with 12 rats in each group. The type 2 diabetes rat model was established as follows: by high-fat and high-glucose diet combined with streptozotocin (35 mg/kg, twice) intraperitoneal injection. The rats were fed with high-fat and high-glucose diets as previously described (18) for 4 weeks and streptozotocin (35 mg/kg) was administered twice in succession by intraperitoneal injection. After 72 h, blood glucose was measured and rats with a fasting blood glucose of 11.1 mmol/l were deemed to have diabetes (17,19). Model rats were fed with high-fat and high-glucose diet for another 3 weeks and the diet was changed to normal diet when they were fed with sericin. The control group was fed with normal diet throughout the experiment. For the high- and low-dose sericin groups, the rats were continuously administered with 2.4 and 1.8 g/kg/day sericin for 35 days, respectively, while the control rats were administered with the same volume of saline for 35 days. All animal experiments were approved by the Ethics Committee of Chengde Medical University (Chengde, China) and conducted according to the ethical guidelines of Chengde Medical University.

The rats in each group were fasted for 12 h following treatment and anesthetized by intraperitoneal injection of 10% chloral hydrate (300 mg/kg body weight) (20–22). Then, the thoracic cavity was opened and venous blood samples were collected from the right atrium of the rats. During blood sample collection, rats were in deep anesthesia and no signs of peritonitis were observed. Then, the rats were sacrificed by decapitation and the left hepatic lobe was collected following opening of the abdominal cavity. The blood was centrifuged at 800 × g for 20 min at 4°C, and the serum was isolated. The liver tissues were kept in liquid nitrogen until further analysis.

The blood glucose level of rats in each group was measured using a glucose oxidase method (23) on a Beckman Coulter AU5800 Clinical Chemistry Analyzer (Beckman Coulter, Inc., Brea, CA, USA).

The liver morphology of each group was observed under an Olympus BH-2 light microscope (Olympus Corporation, Tokyo, Japan) following H&E staining. Briefly, the liver tissues were fixed with Bouins fixative [a mixture of picric acid saturated liquid (1.22%), formaldehyde and glacial acetic acid at a ratio of 15:5:1] for 24 h at room temperature and embedded in paraffin, and continuously sliced into 5-µm-thick sections. At room temperature, the sections were stained with hematoxylin for 7 min, differentiated with 1% HCl in 70% alcohol for 5 sec, stained with eosin for 1 min and dehydrated in a serial ethanol solution of increasing concentrations. Then, the sections were deparaffinized with xylene and sealed.

The hepatic glycogen content was determined by periodic acid-Schiff staining. The liver tissues were fixed as above described and embedded in paraffin, and continuously sliced into 5-µm-thick sections. The sections were oxidized with periodic acid for 10 min and stained with Schiff reagent for 15 min at room temperature. Cell nuclei were re-stained with hematoxylin for 5 min at room temperature. Then, sections were deparaffinized with xylene and sealed. Cells with red or magenta particles in the cytoplasm were defined as positive for glycogen. Tissue sections digested with amylase were used as negative controls.

For the quantification of glycogen content, six rat livers were randomly selected from each group, three sections were selected from each rat liver, and three views were observed in each section. The liver lobules with intact tissue structure were selected for observation using an light microscope (BH-2; Olympus Corporation, Tokyo, Japan; magnification, ×200). Image-Pro Plus 6.0 image analysis software (Media Cybernetics, Inc., Rockville, MD, USA) was used to calculate the integral optical density of glycogen in hepatocyte cytoplasm, and the mean value was determined as the glycogen content.

Protein expression of IR, IRS-1, PI3K and AKT in the liver was analyzed. Briefly, the sections were dewaxed and incubated in 3% H₂O₂-methanol at 37°C for 30 min. Following antigen retrieval by microwaving, the sections were blocked with 10% goat serum (OriGene Technologies, Inc., Beijing, China) at 37°C for 30 min. The sections were then incubated with primary antibodies against IR (1:100; cat. no. ab131238), IRS-1 (1:100; cat. no. ab131487; both Abcam, Cambridge, MA USA), PI3K (1:100; cat. no. 611398; BD Biosciences, San Jose, CA, USA) and AKT (1:200; cat. no. ab179463; Abcam) at 4°C overnight. Next, the sections were incubated with goat anti-rabbit IgG secondary antibodies, which was provided by the rabbit streptavidin-biotin assay system (cat. no. SP-9001; Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) according to the manufacturer's protocol, and then counterstained with DAB for 5–8 min at room temperature. Cell nuclei were re-stained with hematoxylin for 10 min at room temperature. PBS was used to substitute the primary antibody as the negative control. Cells with brownish yellow and/or brown particles were defined as positive staining.

For quantification, six rat livers were randomly selected from each group, three sections were selected from each rat liver, and three views were observed in each section. The liver lobules with intact tissue structure were selected for observation by an Olympus BH-2 microscope (magnification, ×200). Image-Pro Plus 6.0 image analysis software was used to calculate the integral optical density of each protein, and the mean value was determined as the corresponding protein expression level.

Total protein was extracted by RIPA Tissue/Cell Lysates (Beijing Solarbio & Technology Co., Ltd., Beijing, China) from 100 mg liver tissues, and the protein concentration was determined using a BCA protein kit (Kangwei Shiji Biotechnology Co., Ltd., Beijing, China). Proteins (108 µg/lane) were separated by 10% SDS-PAGE and transferred on to a PVDF membrane. Following blocking with 5% skim milk overnight at 4°C, the membrane was incubated with primary antibodies [IR, IRS-1, AKT (all 1:1,000), PI3K (1:500) and β-actin (1:1,000; cat no. AF7018; Affinity Biosciences, Cincinnati, OH, USA)] at room temperature for 2 h. Then, the membrane was incubated with goat anti-rabbit IgG (1:5,000; cat. no. 074-1506) or goat anti-mouse IgG (1:5,000; cat. no. 074-1806; both KPL, Inc., Gaithersberg, MD, USA) for a further 1.5 h at room temperature. The membrane was developed with Super ECL Plus ultra-sensitive luminescent liquid (Applygen Technologies, Inc., Beijing, China). The protein bands were analyzed with Quantity One v4.6.2 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The density of each target band was calculated relative to the β-actin band to determine relative expression levels.

Total RNA was extracted from 100 mg liver tissues with a TaKaRa MiniBEST Universal RNA Extraction kit (cat. no. 9767) and reverse transcribed into cDNA using a PrimeScript™ RT Master mix (cat. no. RR036A; both Takara Biotechnology Co., Ltd., Dalian, China). The reverse transcription protocol was as follows: 37°C for 15 min and 85°C for 5 sec. The sample was put on ice and the obtained cDNA was stored at −20°C. Then, the mRNA expression of IR, IRS-1, PI3K, AKT and GAPDH was determined by qPCR with a SYBR Premix Ex Taq™ II kit (cat. no. RR820A; Takara Biotechnology Co., Ltd.). The primer sequences are listed in Table I. The PCR procedure was as follows: Pre-denaturation at 98°C for 1 min, followed by 40 cycles of denaturation at 98°C for 7 sec, annealing and polymerization at 60°C for 30 sec, then final polymerization at 60°C for 5 min. A relative standard curve method (24) was used to quantify the mRNA and the relative mRNA expression level of each target gene was determined relative to the corresponding GAPDH.

Statistical analysis was performed with the statistical software SPSS 20.0 (IBM Corp., Armonk, NY, USA). All data are expressed as mean ± standard deviation. One-way analysis of variance was used to compare multiple groups, followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To observe the effect of sericin on the liver morphology of type 2 diabetic rats, H&E staining was performed. In the control group, the structure of the hepatic lobules was clear and the hepatocytes were arranged in a cord-like manner around the central vein (Fig. 1). The cell nuclei were large and round, located in the center of cells, and the cytoplasm was stained uniformly. The liver sinus was clear. In the diabetic model group, the hepatocytes were basically arranged in a cord-like manner around the central vein, but the liver cells were swollen, the volume increased, and obvious vacuolar structure appeared in the cytoplasm. A number of the liver cells exhibited soluble necrosis, and the hepatic sinus exhibited stenosis or atresia. Compared with the diabetic model group, the pathological changes of the rat liver in the high- and low-dose sericin groups were markedly reduced (Fig. 1). Rat liver lobular structures in these two sericin treatment groups were clear, and the hepatocytes were arranged in a cord-like manner around the central vein, with large round nuclei in the center of the cells. A small number of hepatocytes exhibited vacuolar structure in the cytoplasm, and the liver sinus was clear. This indicates that sericin may improve the liver morphological structure of type 2 diabetic rats.

To determine the effect of sericin on the glycogen content in type 2 diabetic rat livers, periodic acid-Schiff staining was performed. Hepatic glycogen positive expression was observed in the liver sections of all groups, indicated by red and purple particles in the cytoplasm. As indicated in Fig. 2A, the number of positive cells in the control group was high, and the staining was dark purple. In the diabetic model group, there were fewer positively stained cells, and the staining was a lighter reddish color. In the high-dose sericin group, there was a higher number of positively stained cells compared with the low-dose sericin group, and the staining was darker. As indicated in Fig. 2B, compared with the control group, the glycogen content in the rat liver of the diabetic model group was significantly decreased (P<0.05). Compared with the diabetic model group, the glycogen content in the liver of the high- and low-dose sericin groups was significantly increased (P<0.01). Furthermore, the glycogen content in the rat liver of the high-dose sericin group was significantly higher compared with the low-dose sericin group (P<0.01). This indicates that sericin may significantly increase the liver glycogen content of type 2 diabetic rats.

To determine the effect of sericin on the blood glucose level of the type 2 diabetic rats, the rat blood glucose level was measured. As indicated in Fig. 2C, the blood glucose level in the diabetic model group was significantly increased compared with the control group (P<0.05). Compared with the diabetic model group, the blood glucose levels of the rats in the high- and low-dose sericin groups were significantly decreased (P<0.01). However, there was no significant difference in the blood glucose levels between the high- and how-dose sericin groups. This indicates that sericin may significantly decrease the blood glucose levels of type 2 diabetic rats.

The protein levels of IR, IRS-1, PI3K, and AKT were detected with immunohistochemical staining and western blot analysis. Compared with the control group, the protein expression levels of IR, IRS-1, PI3K and AKT were significantly decreased in the diabetic model group (P<0.05; Figs. 3 and 4). Compared with the diabetic model group, the expression levels of these proteins in the liver of the high- and low-dose sericin groups were significantly increased (P<0.05; Figs. 3 and 4). Furthermore, the expression levels of AKT (Fig. 3) and IR (Figs. 3 and 4) in the high-dose sericin group were significantly higher compared with the low-dose sericin group (P<0.05).

To further verify these results, RT-qPCR was conducted to detect the mRNA levels of IR, IRS-1, PI3K and AKT. As indicated in Fig. 5, the mRNA levels of IR, IRS-1, PI3K and AKT in the diabetic model group were significantly lower compared with the control group (P<0.05). However, following sericin treatment in the high- and how-dose sericin groups, mRNA expression was significantly higher compared with the diabetic model group (P<0.05). No significant difference in mRNA levels of these genes was identified between the high- and low-dose sericin groups.

In summary, these results indicate that sericin may promote the insulin/PI3K/AKT signaling pathway in type 2 diabetic rat liver by upregulating IR, IRS-1, PI3K and AKT expression, and thus may reduce blood glucose levels.