Glucose-lowering and hypolipidemic activities of polysaccharides from Cordyceps taii in streptozotocin-induced diabetic mice

Adult male Kunming mice (6 weeks old) were purchased from the Experimental Animal Center of Third Military Medical University, China (SCXKY 2012–0006). All procedures used in animal experiments were in compliance with the Zunyi Medical University Ethics Committee. The mice were housed in cages at 25 °C ± 2 °C in a 12-h dark–light cycle, with free access to standard pelleted feed and drinking water throughout the experimental period. After 7 days of adapted feeding, the mice were fasted for 12 h but given water ad libitum. Then, the mice were intraperitoneally (ip) injected with STZ (Sigma, USA) dissolved in an ice-cold 0.1 mol/L citrate buffer at pH 4.5 at a dose of 100 mg/kg body weight (bw) and after 4 h, gavaged with 0.2 mL of 5% glucose. Caudal vein blood samples (100 μL) were obtained from STZ-treated mice at 72 h. The mice with fasting blood glucose (FBG) values above 11.1 mmol/L were used as type 1 diabetic models.

After the diabetic state was confirmed, the STZ-induced diabetic mice were divided randomly into five groups (12 mice per group). Normal Kunming mice (12 mice) were served as the control group. All of the animals had free access to water and normal diet. The groups are described in detail as follows:

(i) Control group (CON): normal Kunming mice were treated with normal saline solution. (ii) Diabetic mellitus group (DM): diabetic mice were treated with normal saline solution. (iii) Metformin-treated group (MET): diabetic mice were treated with 100 mg/kg bw of metformin. (iv) Low-dose CTP-treated group (CTP-L): diabetic mice were treated with 100 mg/kg bw of CTP. (v) Medium-dose CTP-treated group (CTP-M): diabetic mice were treated with 200 mg/kg bw of CTP. (vi) High-dose CTP-treated group (CTP-H): diabetic mice were treated with 400 mg/kg bw of CTP.

CTP and metformin (Jingfeng, China) were dissolved in 1.0 mL of normal saline. The CON and DM groups received 1.0 mL normal saline respectively. All of the mice were dosed by gavage once daily for 28 days. The metformin and CTP doses were selected on the basis of literature and our previous work [19, 22, 23]. A schematic of the treatment schedule in this research is shown in Fig. 1.

Body weights and FBG levels of mice were measured at one week intervals after CTP was administered. After a 12 h overnight fast, the tail vein blood was used for FBG analysis. On the 28 day, blood samples were collected from the eye vein by quickly removing the eyeball. Then, serum was immediately harvested by centrifuging blood at 900×g for 15 min at room temperature. Samples were stored at − 80 °C for the following assay: serum insulin (R&D Systems, Minnesota, USA), triglyceride (TG), total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C-reactive protein (CRP) levels. The spleen and thymus were removed and weighed for the accession of immune organ indices. The pancreas was removed for histopathological examination.

Plasma glucose was measured using a Roche blood sugar test meter and test strips (Roche Inc., Basel, Switzerland). The insulin, TG, TC, HDL-C, LDL-C, TNF-α, IL-6, and CRP levels in serum were measured by corresponding commercial kits following the manufacturer’s instructions. The serum lipid kits and the inflammatory cytokine kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and Shanghai Jijin Chemistry Technology Co. (Shanghai, China), respectively. Insulin resistance (IR) was measured with the homeostasis model of assessment-insulin resistance (HOMA-IR) [24]. The HOMA-IR was estimated using the following equation: serum glucose (mmol/L) × serum insulin (mIU/L)/22.5.

Histopathological examination was performed using standard laboratory procedures. The tissues were embedded in paraffin blocks, and the 4 μm-thick slices were produced and placed onto glass slides. After hematoxylin–eosin (HE) staining, the slides were observed, and images were obtained using an optical microscope.

The voucher specimens of C. taii (strain GYYA 0601) were deposited at the Laboratory of Institute of Medicinal Biotechnology, Affiliated Hospital of Zunyi Medical University, Guizhou Province, China. The mycelia of C. taii were cultured and harvested as previously described [17, 25]. Subsequently, the mycelia were lyophilized and ground (60–100 mesh) for later experiments.

CTP was prepared as previously described with slight modifications [18, 19]. In brief, C. taii mycelia powder (2 kg) was repeatedly defatted with petroleum ether at 60 °C, and the residue was repeatedly extracted with chloroform and acetic ether (in that order). Subsequently, the residue was extracted with hot water (95 °C) six times (1:10, w/v). The water layers were combined and condensed to one-fifth of their total volume by using a rotary evaporator under reduced pressure at 50 °C and precipitated with three volumes of 95% (v/v) ethanol at 4 °C for 24 h. The precipitate was filtered and dried in an oven at 50 °C for 24 h. The dried crude polysaccharides were dissolved with distilled water, depigmented via the H₂O₂ method, deproteinized using the Sevag method, and dialyzed against distilled water for 3 days [19]. The non-dialyzable phase was re-concentrated and fractionally precipitated by ethanol to an 85% final concentration. The resulting precipitate was collected by centrifugation, washed three times with acetone, and dried to a constant weight by vacuum freeze-drying. Finally, CTP was obtained and stored at 4 °C before use. The polysaccharide content was determined using the phenol–sulfuric acid reaction with glucose as a standard. The yield and content of CTP were 3.8% (w/w) and 98.3% (w/w), respectively.

All data are reported as the mean ± standard deviation (SD). SPSS software version 21.0 (SPSS Inc.) was used for all analyses. Statistical analysis was performed by using one-way ANOVA, and post hoc comparisons were processed by Dunnett’s t-test. Statistical differences were considered significant at p < 0.05.

A decrease in body weight is one of the symptoms of DM. Therefore, the changes in body weight during the experimental period were investigated. As shown in Fig. 2a, the difference in body weights among all of the groups in the initial days was not significant. After 7 days, the body weights of the DM and MET groups were lower than that of the CON group by 10.71% (p < 0.05) and 10.13% (p < 0.05), respectively. The body weights of the CTP-L, CTP-M, and CTP-H groups were lower than that of the CON group by 5.62, 3.83, and 3.11%, respectively, although all of the results were not statistically different (p > 0.05). After 21 and 28 days, the body weights of the MET, CTP-L, CTP-M, and CTP-H groups were significantly higher (p < 0.05) than that of the DM group. Among all of the drug-treatment groups, CTP-H showed the greatest weight gain in DM. Thus, CTP treatment resulted in a clear dose-dependent increase in body weights from 100 mg/kg to 400 mg/kg in DM.

The FBG, insulin, and IR levels are important evaluation indices for the diagnostic and prognostic evaluation of DM. These indices for STZ-induced diabetic mice were evaluated after CTP treatment. As shown in Fig. 2b, compared with the CON group, all groups tested exhibited an evident increase in glucose over the complete period. However, compared with the DM group, all treatment groups exhibited a significant decrease (p < 0.05) in FBG levels at 7, 14, 21, and 28 days posttreatment. Furthermore, compared with the CTP-L and CTP-M groups, the CTP-H group showed a stronger glucose-lowering effect, which was similar to that of the MET group. Also, the effects of CTP on serum insulin levels and HOMA-IR values in diabetic mice are shown in Fig. 2c and d. The results revealed that the oral administration of different concentrations of CTP (CTP-L, CTP-M, and CTP-H) for 28 days to diabetic mice significantly increased the levels of serum insulin (p < 0.05) and significantly reduced the level of IR (p < 0.05) in a dose-dependent manner compared with the DM group. Compared with the MET group, CTP-H strongly reversed the HOMA-IR value. Thus, CTP reduced the FBG level, increased the insulin level, and decreased the IR of diabetic mice.

DM can also result in lipid metabolism dysfunction. Thus, we further assessed the serum levels of TC, TG, LDL-C, and HDL-C (Fig. 3) in STZ-induced diabetic mice after treatment with CTP for 28 days. As shown in Fig. 3, the TC, TG, and LDL-C levels in the DM group significantly increased (p < 0.05), whereas the HDL-C levels decreased (p < 0.05) compared with the CON group. Thus, a disorder in blood lipid metabolism occurred in STZ-induced diabetic mice. Similar to the positive control MET, the TC levels of all of the CTP treatment groups decreased in the STZ-induced diabetic mice, although the CTP-L and CTP-M groups did not show statistical differences (p > 0.05). The CTP-H group showed significant difference (p < 0.05) compared with the DM group (Fig. 3a). Likewise, medium and high doses of CTP decreased the TG levels (Fig. 3b) and significantly decreased the LDL-C levels (p < 0.05) compared with the DM group, whereas the positive control MET only exhibited a slight effect (Fig. 3c). Moreover, CTP at different doses enhanced the HDL-C levels, with the most marked effect by CTP-H compared with MET (Fig. 3d). Therefore, CTP could affect lipid metabolic parameters and effectively ameliorate lipid dysregulation in diabetic mice.

The cytoarchitectural changes in the pancreatic tissue were characterized by examining the pathological sections via HE staining (Fig. 4). In the pancreatic samples of normal mice (CON), a complete islet structure exhibiting regularly distributed and abundant pancreatic β-cells was observed. By contrast, the islet cytoarchitecture in STZ-induced mice (DM) showed pronounced endogenous destruction, such as cell rupture, cytolysis, and cavities. Furthermore, the number of pancreatic β-cells was reduced. After treatment with CTP, the abnormal changes in islet structure were rescued in the STZ lesions, contributing to architectural amelioration of islet structure and a gradual increase in β-cell counts.

DM is closely associated with inflammation and the immune system. As the primary immune organs, the spleen and thymus directly affect immune function among organisms [26]. In the current study, the effects of CTP on the spleen and thymus indices of STZ-induced diabetic mice were assessed, as shown in Fig. 5. STZ had no effect on the spleen index in STZ-induced diabetic mice, but the thymus index significantly decreased (p < 0.05). Compared with the CON group, the spleen index of all tested groups did not show an obvious statistical difference (Fig. 5a). Similar to the MET group, CTP reversed the thymus index in STZ-induced diabetic mice, and the CTP-H group reached an approximate level similar to that of CON group (Fig. 5b). As such, CTP could restore the thymus index in diabetic mice.

As shown in Fig. 6, we also measured the serum inflammatory cytokines TNF-α, IL-6, and CRP in STZ-induced diabetic mice. The TNF-α, IL-6, and CRP levels in the DM group were significantly increased compared with those in the CON group (p < 0.05). Similar to the MET group, medium- and/or high-dose CTP significantly decreased the pro-inflammatory cytokine levels (p < 0.05) compared with the DM group. Thus, CTP could inhibit the circulating levels of pro-inflammatory cytokines in diabetic mice.