Hypoglycemic benefit and potential mechanism of a polysaccharide from Hericium erinaceus in streptozotoxin-induced diabetic rats
Journal Pre-proof Hypoglycemic benefit and potential mechanism of a polysaccharide from Hericium erinaceus in streptozotoxin-induced diabetic rats Wu-D...
Journal Pre-proof Hypoglycemic benefit and potential mechanism of a polysaccharide from Hericium erinaceus in streptozotoxin-induced diabetic rats Wu-Dan Cai, Zhi-Chao Ding, Yao-Yao Wang, Yan Yang, He-Nan Zhang, Jing-Kun Yan
The in vivo hypoglycemic activity of HEP-C from Hericium erinaceus were evaluated.
HEP-C could attenuate the deteriorated hepatic lesions in STZ-induced diabetic rats.
HEP-C showed hypoglycemic activity via activating the PI3K/Akt signaling pathway.
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Abstract
In this study, the hypoglycemic effect and possible mechanism of a polysaccharide, HEP-
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C, isolated from the fruit body of Hericium erinaceus were evaluated in streptozotoxin (STZ)induced diabetic rats. Compared with the untreated STZ-induced diabetic rats, the supplements
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with HEP-C (150 and 300 mg/kg body weight [BW]) could significantly and dose-dependently relieve BW loss and organ injures, reduce fasting blood glucose, enhance glucose tolerance, alleviate hepatic function and serum lipid metabolism, elevate antioxidant enzyme activities, and suppress lipid peroxidation, which contributed to its potent hypoglycemic benefit. Liver histopathological observation revealed that HEP-C could effectively attenuate the deteriorated 2
hepatic lesions in STZ-induced diabetic rats. HEP-C with potent hypoglycemic effect positively mediated glycogen synthesis by activating the phosphatidylinositol-3-kinase/protein kinase B signaling pathway. In summary, these results suggested that HEP-C, as a new dietary functional food or therapeutic agent, exhibited great potential for the prevention and treatment of diabetes mellitus and its complications.
Diabetes mellitus (DM), also known as “Xiaoke disease” in traditional Chinese medicine, is a global endocrine and metabolic disease. It is manifested in the metabolic disorders of sugar,
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fat, and protein caused by insufficient insulin secretion and imbalance of acid and base in the
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human body, with the increase in blood glucose level as the main feature [1]. DM, cancer, and cardiovascular disease are regarded as the world’s top three major diseases [2]. The morbidity
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and mortality of DM increase yearly, and the ages of those afflicted with the decreased over the past decade [3]. DM is harmful not only because of the elevation of blood glucose level but
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also because it causes the occurrence of chronic complications of multiple systems and organs [4]. At present, the treatment of DM mainly focuses on controlling blood glucose elevation, preventing the occurrence of complications by regular injections of insulin combined with oral hypoglycemic agents (e.g., glibenclamide, metformin, sulfonylureas, and α-glucosidase inhibitors), and giving increasing attention to dietary adjustment and other measures. Although 3
these conventional methods can rapidly improve the clinical symptoms of patients with diabetes, long-term use of these drugs causes drug resistance. Such drugs severely damage the liver, kidney, and other organs [5,6]. As a result, the search for new non-toxic hypoglycemic drugs from natural sources for the prevention and treatment of DM and its complications in clinical settings has been extensively conducted. Polysaccharides (PSs) and their derivatives derived from edible and medicinal fungi (mushrooms) exhibit low or no toxicity and stable
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therapeutic effect in the treatment of DM. Moreover, they can considerably reduce blood glucose level and effectively prevent and control the occurrence of complications, and some products have entered the clinical treatment stage [7-10]. Therefore, edible and medicinal PSs
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have provided a novel method for searching effective hypoglycemic drugs in the treatment of
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DM and its complications.
Hericium erinaceus (Bull.) Pers., also called Houtou, Lion’s Mane, and Yamabushitake,
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is a large edible and medicinal fungus that belongs to the Aphyllophorales, Hydnaecase, and
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Hericium families [11]. In recent years, H. erinaceus, as a traditional folk medicine and tonic food, has attracted increasing interest in the development of functional foods and biological
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medicines due to its excellent physiological and health benefits, including fortifying the spleen, nourishing the stomach, tranquilizing the mind, and fighting cancer. H. erinaceus is rich in
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proteins, fats, celluloses, PSs, and various amino acids. Among them, the PSs of H. erinaceus are one of the major bioactive ingredients and possess a broad spectrum of pharmacological and biological activities, such as immunomodulatory, antitumor, antioxidant, hepatoprotective, hypoglycemic, hypolipidemic, antifatigue, and antiaging functions [12]. At present, no sufficient evidence has been observed to draw definitive conclusions about the effectiveness of 4
the PSs of H. erinaceus in DM. Our research group has successfully prepared four watersoluble PSs, namely, HEP-W, HEP-S, HEP-C, and HEP-A, from the fruit body of H. erinaceus by using different solvent extractions. HEP-C extracted using citric acid solution exhibited stronger radical-scavenging abilities, antioxidant capacities, and α-amylase and α-glucosidase inhibitory activities than the other PSs [13]. Our results implied that HEP-C with prominent antioxidant activities showed hypoglycemic potential in vitro. However, no or few studies are
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available regarding the hypoglycemic activity and mechanism of action of HEP-C in vivo.
Therefore, the objective of this study was to evaluate the in vivo hypoglycemic activity of HEP-C obtained from the fruit body of H. erinaceus by using streptozotoxin (STZ)-induced
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diabetic rats as a model. The potential hypoglycemic mechanism of HEP-C via the
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phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was also explored. This study will provide a scientific basis to improve the exploration and utilization
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of H. erinaceus in functional foods and clinical medicines and promote the prevention and
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treatment of DM and its complications.
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2. Materials and methods
2.1. Materials and chemicals
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The fruiting body of H. erinaceus was supplied by Shanghai Guosen Biotechnology Co.,
Ltd. (Shanghai, China). The strain used was H0605, which was obtained from the Herbarium of Edible Fungi Culture Collection Center Branch of the China Culture Collection of Agricultural Microorganisms. STZ (with a purity of ≥98%) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, 5
MO, USA). Metformin (with a purity of ≥97%) was obtained from Shanghai Yuanye Biotechnology Co. Ltd. (Shanghai, China). Commercial assay kits for alanine transaminase (ALT), aspartate transaminase (AST), triacylglycerides (TG), total cholesterol (TC), superoxide
dismutase
(SOD),
glutathione
peroxidase
(GSH-Px),
catalase
(CAT),
malondialdehyde (MDA), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). PI3K, Akt,
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phospho-Akt (p-Akt), glycogen synthase (GS), glycogen synthesis kinase-3β (GSK-3β), βactin and secondary antibodies were purchased from Beijing CWBIO (CWBIO, Beijing, China). All other chemicals and solvents were of laboratory grade and used without further
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purification.
2.2. Extraction, isolation and physicochemical properties of HEP-C
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Citric acid extraction (pH 3.0) was used to extract polysaccharide (HEP-C) from the fruit
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body of H. erinaceus according to a previous study [13]. Briefly, 50 g of pretreated powder was extracted twice with 500 mL of citric acid (pH 3.0) aqueous solution at 95 °C for 8 h. After
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centrifugation at 4000 rpm for 20 min, the supernatant was neutralized with addition of 0.5 M NaOH, concentrated under the reduced pressure, and precipitated with 4 volumes of 95%
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ethanol at 4 °C overnight. Subsequently, the precipitate obtained by centrifugation was subjected to deproteinization, dialysis, and lyophilization to yield a partially purified polysaccharide, named HEP-C. The freeze-dried HEP-C was sealed in a plastic bag and then stored in a desiccator at 25 °C prior to use. Total carbohydrate, protein, uronic acid and β-1,3-glucan contents, monosaccharide 6
composition, and molecular weight and molecular weight distribution of HEP-C were determined. The details of operation conditions and methods were described as previously reported [13].
2.3. Animals and experimental design Forty male SD rats (150 ± 30 g, five weeks old) were purchased from the Experimental
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Animal Center of Jiangsu University (Zhenjiang, China). All the animals were acclimatized to standard conditions under a constant 12 h light/dark cycle, ambient temperature of 23 °C ± 0.5 °C, and environmental humidity of 50% ± 5% for 1 week prior to the experiment. After
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acclimation, eight rats were randomly selected and regarded as the normal control (NC) group
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and intragastrically administered with 6 mL/kg body weight (BW) physiological saline solution (PSS, 0.9%, w/v) per day. All the other rats were fasted for 12 h with free access to tap water
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and then intraperitoneally injected with freshly prepared STZ solution in 0.1 M citrate buffer
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(pH 4.5) at 65 mg/kg BW. After 72 h, the fasting blood glucose (FBG) levels of the rats were determined using a one-touch glucometer (Johnson & Johnson) from the tail vein. The rats with
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FBG levels > 11.1 mmol/L and < 33.3 mmol/L were regarded as the STZ-induced diabetic rats and selected for subsequent experiments. The STZ-induced diabetic rats were randomly
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divided into four groups (n=8) and intragastrically administered with PSS (6 mL/kg BW), metformin (75 mg/kg BW), and HEP-C (150 and 300 mg/kg BW) per day, which were assigned to the model control (MC), positive control (PC), HEP-C low dose (HEP-C-L), and HEP-C high dose (HEP-C-H) groups, respectively. During the experiment, the rats were given free access to commercial standard chow and tap water. 7
After 4 weeks of treatment, the rats were fasted overnight, weighed, and sacrificed. Blood samples were obtained and centrifuged at 3000 rpm for 10 min at 4 °C, and the serum was collected and stored at -80 °C for further biochemical analyses. The liver, kidney, spleen, and thymus were excised and weighed after washing with ice-cold PSS to calculate the organ indexes (each organ weight as a percentage of BW). A portion of the liver was fixed with 10% paraformaldehyde for histopathological observation, and the other parts were homogenized in
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ice-cold PSS and centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was then collected and stored at −80 °C for further analysis.
All animals used in this study were cared in accordance with the National Institutes of
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Health guide for the care and use of Laboratory animals (NIH, revised 1978). All experimental
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Committee of Jiangsu University, China.
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procedures involving the use of animals were approved by the Laboratory Animal Management
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2.4. Determination of FBG and oral glucose tolerance test (OGTT) During the experiment, the FBG levels of the fasted rats were measured weekly using the
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glucometer described in Section 2.2. At the final week of feeding, OGTT was conducted following the reported method [14]. In brief, all the rats were fasted for 12 h and orally
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administered with 2.0 g/kg BW glucose. The blood samples were withdrawn from the tail vein at intervals of 0, 30, 60, 90, and 120 min, and the FBG levels were immediately determined using the glucometer. Glucose tolerance was expressed as the area under the blood glucose curve (AUC, mmol/(L·h)), which was equal to (1/4 FBG at 0 h + 1/2 FBG at 0.5 h + 3/4 FBG at 1.0 h + 1/2 FBG at 2.0 h). 8
2.5. Biochemical analysis The ALT, AST, SOD, CAT, and GSH-Px activities and the serum contents of MDA, TG, TC, LDL, and HDL, as well as the liver contents of SOD, CAT, GSH-Px, and MDA were determined according to the corresponding manufacturer’s protocols of commercial assay kits
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supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
2.6. Histopathological observation
The fixed liver samples were embedded with paraffin wax and cut into 5 μm sections
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through a rotary microtome. Subsequently, the resultant sections were stained with
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hematoxylin-eosin (H&E) and examined by using a DM6000B light microscope (Leica,
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Germany).
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2.7. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from the liver by using TRIzol reagent, and the RNA
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concentration was quantified. Reverse transcription was conducted in 10 μL reaction mixtures containing 5 μL 100 ng/μL total RNA, 0.5 μL 5×primer script buffer, 0.5 μL PrimeScript RT
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Enzyme Mix, 0.5 μL 50 μM Oligo dT primer, 0.5 μL 100 μM Random 6 mers, and 1.5 μL RNase free dH2O at 37 °C for 15 min and 85 °C for 5 s. Then, RT-PCR amplification experiment was performed using the ABI 7500 Fast Real-time PCR system (Applied Biosystems, USA) in 20 μL reaction mixtures containing 2 μL reversed-transcribed cDNA, 10 μL 2×UltraSYBR mixture, 0.5 μL forward primer, 0.5 μL reverse primer, and 7 μL Rnase-free 9
water. The thermal cycling conditions were as follows: 95 °C for 3 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The specific primers were listed as follows: PI3K, F: 5’ACTGTGCAGACGAACCCATA-3’, R: 5’- GCAATCTCAGCTGCCTTCTC-3’; Akt, F: 5’GCCTCTGCTTTGTCATGGAG-3’, R: 5’-AGCATGAGGTTCTCCAGCTT-3’; GSK-3β, F: 5’-ACTACCAAATGGGCGAGACA-3’, R: 5’-GGCGTTATTGGTCTGTCCAC-3’; β-actin, F: 5’-CAGGCATTGCTGACAGGATG-3’, R: 5’-TGCTGATCCACATCTGCTGG-3’. The β-
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actin mRNA signals were used to normalize the relative expression levels of target mRNAs.
2.8. Western blot analysis
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Total protein was extracted by cell lysis buffer (Beyotime Biotechnology, Beijing, China)
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containing 10 mM phenylmethanesulfonyl fluoride (PMSF, Sigma, USA), EDTA-free protease inhibitor Cocktail（Roche, Germany) and PhosSTOP（Roche, Germany) for 30 min on ice,
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and then centrifuged at 12,000 rpm for 15 min at 4 °C. Then, protein concentration was
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quantified using the BCA protein assay kit (Tiangen Biotech Ltd., Beijing, China) to ensure equal amounts of protein of each sample were loaded and separated by sodium dodecyl sulfate-
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polyacrylamide gel electrophoresis (SDS-PAGE). After full separation, proteins in gel were transferred onto polyvinylidene fluoride (PVDF) membranes (0.45 μm, Millipore, USA) and
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then blocked with 5% Difco Skim Milk (BD, USA) for 2 h at room temperature. These membranes were incubated with PI3K-p85 (85 kDa), PI3K-p110 (110 kDa), Akt (60 kDa), pAkt (60 kDa), GSK-3β (46 kDa) and GS (81-85 kDa) at 4 °C overnight. Subsequently, the membranes were washed with Tris-buffered saline buffer containing 0.1% Tween 20 (TBST, pH 7.4) and incubated with horseradish peroxidase-conjugated anti-rabbit, anti-mouse or anti10
goat IgG antibodies at room temperature for 1 h. Immunoblots were detected by enhanced chemiluminescence (ECL, Beyotime Biotechnology, China) , and the grey level was analyzed using ImageQuant LAS 4000 system(GE Healthcare, USA).
2.8. Statistical analysis All experiments are conducted in three replicates and the mean ± standard deviation (SD)
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is used in the analysis. The statistical analysis was performed by Student’s t-test and analysis of variance (ANOVA) using OriginPro Software Version 8.0 (OriginLab Corp., MA, USA).
3.1. Effects of HEP-C on BW and organ indexes in STZ-induced diabetic rats
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PSs, as the main active substance of H. erinaceus, have attracted increasing attention due
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to their multiple physiological effects and health benefits. They can reduce blood glucose and lipid, promote stomach health, and improve immunity [12]. In our research, a new partially
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purified polysaccharide, HEP-C, was obtained from the fruit body of H. erinaceus through citric acid extraction (pH 3.0), and the results of physicochemical properties demonstrated that
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the HEP-C with 81.51% carbohydrate, 14.99% uronic acid, and 1.97% protein as well as 1.26 mg/g fruit body β-1,3-glucan was mainly composed of rhamnose, arabinose, mannose, glucose, and galactose at a molar ratio of 9.0:2.0:1.0:40.7:7.5, with weight-average molecular weights of 263.6 and 229.8 kDa, which suggested the HEP-C was an acidic heteropolysaccharide. Furthermore, we also found that the HEP-C possessed excellent free radical scavenging ability 11
and inhibitory effects on α-glucosidase and α-amylase activities in vitro, revealing its potential hypoglycemic activity [13]. To validate this finding, we evaluated the in vivo hypoglycemic benefits and possible underlying mechanisms of HEP-C on STZ-induced diabetic rats. BW loss is common in DM, probably due to the inefficient use of most proteins and carbohydrates [15]. In the present study, the effect of HEP-C on weight gain rate in STZinduced diabetic rats after 4 weeks of treatment was investigated, and the results are listed in
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Table 1. The weight gain rate in the NC group was evidently higher than those in the STZinduced diabetic groups (MC, PC, HEP-C-L, and HEP-C-H) after 4 weeks of feeding. This result may be because STZ causes BW loss or the degradation of structural proteins in rats with
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diabetes. By contrast, the weight gain rates in the HEP-CL-L, HEP-C-H, and PC groups were
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significantly higher than that in STZ-induced diabetic rats in the MC group (47.38%) (P<0.0.5) but lower than that in normal rats in the NC group (81.65%). This finding implied that the HEP-
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C treatments could significantly and dose-dependently alleviate BW loss in STZ-induced
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diabetic rats, which was consistent with previous studies [16-19]. As presented in Table 1, we noted that organ indexes except for the spleen significantly
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increased in the MC group compared with that in the NC group (P<0.01), which indicated the occurrence of organ lesions in STZ-induced diabetic rats. After 4 weeks of oral administrations
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of HEP-C or metformin, the organ indexes, including liver, kidney, and thymus, apparently decreased (P<0.05), but the spleen remarkably increased compared with that in the MC group (P<0.01). Thus, HEP-C could effectively improve organ health of STZ-induced diabetic rats at different degrees. Chen et al. demonstrated that when diabetic mice were treated with metformin and MFP (1000 mg kg-1 day-1), the spleen weight in the mice increases to near12
normal values [16], this result was consistent with our present study. PSs obtained from Talinum triangulare [20], Inonotus obliquus [21], and Huidouba [17] ameliorated organ damage in STZ-induced diabetic mice. These results suggested that HEP-C, especially at a high dose of 300 mg/kg BW, exhibited a positive effect on alleviating BW loss and organ injures in STZ-induced diabetic rats.
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3.2. Effects of HEP-C on FBG and OGTT in STZ-induced diabetic rats
Hyperglycemia is one of the main characteristics of DM. In this work, the FBG levels were determined weekly to evaluate the hypoglycemic benefits of HEP-C in STZ-induced
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diabetic rats during the experimental period. The obtained results are displayed in Fig. 1. The
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FBG levels in the NC group were always significantly lower than those in the STZ-induced diabetic groups. At the beginning of the experiment (0 week), compared with the NC group,
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the FBG levels in the MC, PC, HEP-C-L, and HEP-C-H groups were evidently greater than 20
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mmol/L (P<0.01), which further suggested the successful establishment of STZ-induced diabetic rats. After receiving 1 week of treatment with HEP-C (150 and 300 mg/kg BW) and
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metformin (75 mg/kg BW), we found that the FBG levels sharply decreased compared with the MC group (P<0.01), but no statistically significant difference was observed in the HEP-C-
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treated groups. However, significant differences were observed in the steady decrease in FBG levels of the HEP-C-treated groups from the 2nd week to the 4th week compared with the MC group (P<0.05). After 4 weeks of continuous oral administration of HEP-C at the high dose of 300 mg/kg BW, the FBG value was 14.79 ± 5.98 mmol/L, which was close to the PC group (12.94 ± 2.16 mmol/L). These results demonstrated that HEP-C could reduce the FBG levels 13
in STZ-induced diabetic rats, thereby exerting potent hypoglycemic activity. In clinical practice, OGTT is widely used for the diagnosis of DM. It is a glucose tolerance test used to determine the function of islet β cells and the body’s ability to regulate blood glucose level, and it is currently recognized as the gold standard for the diagnosis of DM [22]. Table 2 presented the FBG levels in normal and STZ-induced diabetic rats after oral administration of glucose (2.0 g/kg BW) at different intervals. In all the tested groups, the
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maximum FBG levels occurred at 30 min and then gradually slowed down until 120 min. Within the measured time range, the FBG levels in the MC group were significantly higher than those in the NC group (P<0.001), which suggested that the injection of STZ apparently
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increased the FBG levels and then reduced oral glucose tolerance ability. By contrast, HEP-C
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supplementation significantly and dose-dependently decreased FBG levels in the STZ-induced diabetic rats compared with the MC group (P<0.001). As shown in Table 2, the AUG for
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glucose in the MC group was remarkably higher than that in the NC group (P<0.001),
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suggesting that the STZ-induced diabetic rats showed significantly impaired glucose tolerance ability. However, HEP-C supplements, especially at the dose of 300 mg/kg BW, could
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markedly lower the AUC values of the STZ-induced diabetic rats compared with the MC group (P<0.001), which was almost close to the PC group. Our results suggested that HEP-C
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effectively enhanced the glucose tolerance of STZ-induced diabetic rats, accelerated the consumption of exogenous glucose, and reduced the concentration of blood glucose. Similar results have also been observed in previous studies [17, 18, 23-25].
3.3. Effects of HEP-C on hepatic function and lipid metabolism in STZ-induced diabetic rats 14
DM causes hepatic damage or deteriorated liver function, such as nonalcoholic liver disease, steatohepatitis, and cirrhosis [26, 27]. In general, the activities of ALT and AST in the serum as two important bioindicators are used to evaluate hepatocyte injury and liver function [27]. As shown in Table 3, the activities of ALT and AST in the MC group were markedly higher than those in the NC group (P<0.01), which suggested a significant impairment on liver function in STZ-induced diabetic rats. By contrast, after 4 weeks of treatment of HEP-C, the
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activities of ALT and AST in the STZ-induced diabetic rats were evidently and dosedependently reduced compared with those in the MC group (P<0.05). The activities of ALT and AST in the HEP-C-H group (300 mg/kg BW) were 14.38 ± 6.94 and 22.19 ± 3.69 U/L,
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respectively, which were slightly higher than those of metformin at 10.90 ± 1.16 and 17.65 ±
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1.98 U/L, respectively. These result indicated that HEP-C could ameliorate impaired liver function in STZ-induced diabetic rats by reducing the activities of serum ALT and AST. Zhu
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et al. reported that treatment with 200 and 400 mg/kg BW PSG-1 from Ganoderma atrum
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significantly decreased ALT or AST activities, thereby showing that PSG-1 may improve liver function in diabetic rats [28]; this finding was consistent with our results.
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Dyslipidemia is one of the most important characteristics of DM and the major cause of cardiovascular disease in DM [29]. Hyperlipidemia is characterized by high levels of TC, TG,
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and LDL and low level of HDL [30]. Table 3 presented the levels of serum lipids, such as TC, TG, LDL, and HDL, in normal and STZ-induced diabetic rats after 4 weeks of treatment. The levels of TC, TG, and LDL in the MC group were significantly higher (P<0.05) than those in the NC group, and the HDL level in the MC group was remarkably lower than that in the NC group (P<0.001). These findings further implied that STZ caused dyslipidemia in rats with 15
diabetes. The supplements of HEP-C in the STZ-induced diabetic rats for 4 weeks evidently reduced the levels of TC, TG, and LDL (P<0.05), but a significant improvement in HDL level was observed (P<0.001) compared with that of the MC group. However, no significant differences appeared in the TC levels among the PC, HEP-C-L, and HEP-C-H groups. Thus, HEP-C played a beneficial role in antihyperlipidemic activity by ameliorating abnormal serum
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lipid metabolism in rats with diabetes.
3.4. Effects of HEP-C on oxidative stress in STZ-induced diabetic rats
Oxidative stress is usually caused by the increased free radicals and weakened defenses
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in the body. In DM, persistent hyperglycemia increases the reactive oxygen species content in
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the body and remarkably enhances oxidative stress, which causes increased level of peripheral tissue inflammation, impaired insulin secretion, reduced glucose utilization, and deteriorated
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diabetic lesions. Increasing oxidative stress is one of the most important participants in the
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occurrence and development of DM and its complications [31]. At present, PSs derived from edible and medicinal mushrooms (or fungi) exert beneficial hypoglycemic effects by
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alleviating oxidative stress in DM [8, 10, 28]. Furthermore, our previous study proved that HEP-C isolated from the fruit body of H. erinaceus via citric acid extraction can effectively
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scavenge free radicals in vitro [13]. The determination of antioxidant activities, such as SOD, CAT, and GSH-Px, can be regarded as a standard to evaluate the antioxidant potentials of animals. MDA is a final by-product of lipid peroxidation, so the content of MDA can be utilized as an evaluation index of lipid peroxidation degree in vivo [32]. In the present study, the activities of SOD, CAT, and GSH-Px and MDA level in the serum and livers of normal and 16
STZ-induced diabetic rats were investigated. The obtained results are summarized in Tables 4 and 5. The activities of SOD, CAT, and GSH-Px in the serum and livers of STZ-induced diabetic rats in the MC group significantly decreased, but the MDA level sharply increased compared with the NC group (P<0.01). These findings further revealed that the occurrence of DM was closely associated with oxidative stress, which coincided with the literature [33]. After 4 weeks of oral supplements with HEP-C or metformin, a significant increase in the activities
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of SOD, CAT, and GSH-Px were associated with an evident diminution of MDA level in the serum and livers of STZ-induced diabetic rats compared with the MC group. Thus, HEP-C could attenuate oxidative stress in STZ-induced diabetic rats by elevating the activities of
3.5. Histopathological changes in liver
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antioxidant enzymes and suppressing lipid peroxidation.
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Fig. 2 depicted the histopathological examination of liver slices after H&E staining. As
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shown in Fig. 2A, the hepatic cell architecture in the NC group was normal and clear, with neat cell arrangement, clear cell boundary, uniform cytoplasm, and no nuclear atrophy. Compared
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with the NC group, the liver morphology in the MC group exhibited localized necrosis, fuzzy cell structure, peripheral hepatic cord disorder, no evident cytoplasm, and severe cell atrophy
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(Fig. 2B). This result indicated that STZ-induced diabetes caused severe hepatic lesions. By contrast, metoformin supplement showed a positive effect on attenuating liver histopathological changes in STZ-induced diabetic rats (Fig. 2C). Treatments with HEP-C for 4 weeks also alleviated hepatic damage to different degrees (Figs. 2D and 2E). HEP-C at the high dose of 300 mg/kg BW presented relatively orderly arranged hepatic cell cords and a 17
diminution of lipid accumulation in hepatic cells (Fig. 2E), which was almost similar to that of normal hepatic architecture (Fig. 2A). Therefore, these observations demonstrated that HEP-C played a protective role in the liver against lesions in STZ-induced diabetic rats.
3.6. Effect of HEP-C on the PI3K/Akt signaling pathway in STZ-induced diabetic rats In this work, RT-PCR and Western blot analysis were performed to investigate the possible
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corresponding hypoglycemic mechanism of HEP-C on STZ-induced diabetic rats. Fig. 3 displayed the effect of HEP-C on the relative mRNA expression levels of PI3K, Akt, and GSK3β in STZ-induced diabetic rats. Compared with the NC group, the relative expression levels
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of PI3K and Akt at the gene levels were markedly reduced in the MC group (P<0.05),
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accompanied with a significant enhancement in the mRNA expression of GSK-3β (P<0.01). However, HEP-C treatments evidently and dose-dependently reversed the mRNA expression
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levels of PI3K, Akt, and GSK-3β in STZ-induced diabetic rats compared with the MC group
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(P<0.05), which was almost similar to the PC group. For example, compared with the MC group, the relative mRNA expression levels of PI3K and Akt in the HEP-C-H group (300
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mg/kg BW) increased by 36.03% and 43.29%, respectively, whereas the GSK-3β mRNA expression level decreased by 33.08%. Hence, HEP-C might regulate glucose metabolism to
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exert hypoglycemic benefits in STZ-induced diabetic rats through a PI3K/Akt signaling pathway.
The PI3K/Akt signaling pathway is a major effector of insulin and is closely responsible for glycogen synthesis in the liver [9, 34]. To further clarify the influence of HEP-C on the underlying PI3K/Akt signaling pathway in STZ-induced diabetic rats, we evaluated several 18
critical proteins, including PI3K-p85, PI3K-p110, Akt, p-Akt, GSK-3β, and GS, by using Western blot analysis. The results were summarized in Fig. 4. As shown in Figs. 4A–4D, the supplements of HEP-C for 4 weeks significantly up-regulated the relative protein expression levels of PI3K-p85, PI3K-p110, Akt, and p-Akt in the livers of STZ-induced diabetic rats compared with that of the MC group in a concentration-dependent manner (P<0.05). However, the up-regulated levels were slightly lower than that of metformin. These findings were similar
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to the changes in mRNA expression levels of PI3K and Akt (Figs. 3A and 3B), which suggested that HEP-C could mediate glucose metabolism by improving glycogen synthesis. PI3K is an important intermediate of insulin signaling and comprises two subunits: an adaptor subunit
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(PI3K-p85) and catalytic subunit (PI3K-p110). The PI3K-p110 subsequently activated Akt,
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which was the main downstream target of PI3K. Once Akt was activated, p-Akt, as a functional protein, could regulate various downstream signaling pathways, such as glucose transport,
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glycogen synthesis, and glycogenesis inhibition [35]. p-Akt also caused the inactivation of a
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specific isoform of GSK-3, GSK-3β, which positively regulated the expression of GS and potentially ameliorated glucose transport activity [36]. As presented in Figs. 4E and 4F, we
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found that the administration of HEP-C, especially at the high dose of 300 mg/kg BW, evidently reversed the relative protein expression of GSK-3β and GS compared with the MC group,
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which was similar to that of metformin (75 kg/mg BW). Thus, HEP-C exhibited a positive effect on glucose transport in STZ-induced diabetic rats. However, no statistically significant differences were observed among the PC, HEP-C-L, and HEP-C-H groups. Combined with the results of RT-PCR and Western blot analyses, HEP-C could not only elevate the expression levels of PI3K, Akt, and GS but also suppress the expression of GSK-3β, resulting in the 19
activation of the PI3K/Akt signaling pathway, which further contributed to its potent hypoglycemic activity in vivo.
4. Conclusions In conclusion, HEP-C isolated from H. erinaceus exhibited excellent hypoglycemic potential on STZ-induced diabetic rats by restoring BW loss and organ health; alleviating FBG
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level; ameliorating glucose tolerance, hepatic function, and serum lipid metabolism; and attenuating oxidative stress and lipid peroxidation, which was further visually confirmed by liver histopathological observation. The results of RT-PCR and Western blot analyses showed
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that HEP-C activated the PI3K/Akt signaling pathway by reversing the expression levels of
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PI3K, Akt, GSK-3β, and GS in the livers of STZ-induced diabetic rats. Therefore, HEP-C could be explored as a promising functional food ingredient or antidiabetic agent for the prevention
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and treatment of DM and its complications. Further investigations on the purification, structural
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characterization, chain conformation, and the structure–activity relationship of HEP-C are
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currently underway in our laboratory.
Conflict of interest
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The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
Acknowledgements This work was supported financially by the National Natural Science Foundation of China 20
(31671812), the Key Research & Development Program (Modern Agriculture) of Jiangsu Province (BE2017362), Jiangsu Overseas Research & Training Program for University Prominent Young & Middle-aged Teachers and Presidents and the Priority Academic Program
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Development (PAPD) of Jiangsu Higher Education Institutions.
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Fig. 1. Effects of HEP-C on FBG levels in STZ-induced diabetic rats for 4 weeks of experiment. Each value is expressed as mean ± SD (n = 8). * P<0.05, ** P<0.01, and *** P<0.001, compared with MC group. # P<0.05, ## P<0.01, and ### P<0.001, compared with NC group.
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Fig. 2. Effect of HEP-C on liver histophatological changes stained with H&E. (A) NC group, (B) MC group, (C) PC (metformin, 75 mg/kg BW) group, (D) HEP-C-L (HEP-C, 150 mg/kg
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BW) group, and (E) HEP-C-H (HEP-C, 300 mg/kg BW) group.
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Fig. 3. Effect of HEP-C on the mRNA expression levels of (A) PI3K, (B) Akt, and (C) GSK3β in STZ-induced diabetic rats. Values are expressed as mean ± SD (n = 8). * P<0.05, ** P<0.01, ***
P<0.001, compared with MC group. # P<0.05,
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and
##
P<0.01, and
###
P<0.001, compared
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with NC group. β-actin was used as internal control, and the values normalized to β-actin.
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Fig. 4. Effect of HEP-C on the protein expression levels of (A) PI3K-p85, (B) PI3K-p110, (C) Akt, (D) p-Akt, (E) GSK-3β, and (F) GS in STZ-induced diabetic rats. Values are expressed as
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mean ± SD (n = 8). * P<0.05, ** P<0.01, and *** P<0.001, compared with MC group. # P<0.05, ##
P<0.01, and ### P<0.001, compared with NC group.
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Table 1. Effect of HEP-C on weight gain rate and organ indexes of STZ-induced diabetic rats after 4 weeks of treatment. Weight gain rate (%)
Groups
Liver (%)
Kidney (%)
Spleen (%)
Thymus (%)
81.65 ± 4.69***
29.39 ± 4.12**
6.72 ± 0.66
2.10 ± 0.21**
1.42 ± 0.26
MC
47.38 ± 4.12##
40.24 ± 4.74##
9.98 ± 1.62
1.94 ± 0.25 ##
1.71 ± 0.30
PC
61.45 ± 3.27**
33.57 ± 7.35*
7.35 ± 2.83
2.39 ± 0.25**
1.48 ± 0.23
HEP-C-L
49.28 ± 6.04##
37.88 ± 8.49#
8.33 ± 2.39#
2.22 ± 0.21**
1.64± 0.37
HEP-C-H
55.33 ± 4.79#*
34.54 ± 7.06*
7.88 ± 1.46*
2.45 ± 0.33**
1.57 ± 0.27**
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NC
Values are expressed as mean ± SD (n = 8). * P<0.05, **P<0.01, and ***P<0.001, compared with MC group. P<0.05, ## P<0.01, and ### P<0.01, compared with NC group.
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#
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Table 2. Effect of HEP-C on OGTT and AUC in STZ-induced diabetic rats. FBG (mmol/L)
AUC
Groups 0 min
30 min
60 min
90 min
120 min
(mmol/L·h)
NC
7.23 ± 0.60***
12.53± 0.55***
11.40 ± 1.37***
8.97± 0.31***
6.83± 0.31***
20.04± 1.06***
MC
27.77 ± 1.91###
28.33 ± 1.16###
28.23 ± 0.97###
26.93 ± 0.84###
26.60±1.15###
55.58± 1.60###
PC
8.07 ± 0.75***
19.47±1.70***###
7.47±1.22***
25.40± 1.64***###
18.80±0.40***###
17.70±1.11***###
40.67± 1.08***###
14.37±0.31***###
12.17±0.40***###
32.68± 1.55***###
13.23
± 11.47
0.40***### 19.20±0.82***###
1.11***### ±
23.73± 0.80***###
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20.20 HEP-C-L
0.87***### 16.53 HEP-C-H
15.47±1.20***###
±
±
20.67± 0.95***### 1.31***###
P<0.05, ## P<0.01, and ### P<0.01, compared with NC group.
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#
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Values are expressed as mean ± SD (n = 8). * P<0.05, **P<0.01, and ***P<0.001, compared with MC group.
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Table 3. Effect of HEP-C on the activities of ALT, AST, TC, TG, LDL and HDL in serum in STZ-induced diabetic rats. Groups
ALT (U/L)
AST (U/L)
TC (mmol/L)
TG (mmol/L)
LDL (mmol/L)
HDL (mmol/L)
NC
2.41 ± 1.03 ***
15.30 ± 3.98**
1.97 ± 0.35*
0.77 ± 0.26**
0.24 ± 0.07**
11.95 ± 0.81***
MC
33.10 ± 4.45###
29.95 ± 3.01##
2.55 ± 0.27#
1.90 ± 0.20 ##
0.51 ± 0.06##
2.82 ± 1.42###
PC
10.90 ± 1.16**
17.65 ± 1.98**
2.19 ± 0.52
1.15 ± 0.18*
0.27 ± 0.09**
9.38± 1.82*** ##
HEP-C-L
22.82 ± 3.76 ##
28.63 ± 3.53##
1.90 ± 0.25
1.30± 0.65* #
0.33 ± 0.14**#
7.82 ± 1.03***##
HEP-C-H
14.38 ± 6.94 *
22.19 ± 3.69*
1.93 ± 0.41
1.13 ± 0.33*
0.28 ± 0.14**
8.80 ± 2.39***##
P<0.05, ## P<0.01, and ### P<0.01, compared with NC group.
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#
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Values are expressed as mean ± SD (n = 8). * P<0.05, ** P<0.01, and *** P<0.001, compared with MC group.
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Table 4. Effect of HEP-C on the activities of SOD, CAT, and GSH-Px and MDA level in serum in STZ-induced diabetic rats. SOD (U/mL)
CAT (U/mL)
GSH-Px (U/mL)
MDA (nmol/mL)
NC
755.59 ± 79.59 **
81.74 ± 5.55***
1761.86 ± 30.64***
3.83 ± 0.99***
MC
623.16 ± 82.25##
50.15 ± 6.66###
1378.67 ± 40.71###
8.58 ± 1.30###
PC
719.41 ± 123.67**
71.91 ± 5.79***
1596.40 ± 84.62*
3.83 ± 0.99***
HEP-C-L
666.06 ± 135.95#
47.05 ± 7.54###
1587.26 ± 32.70
5.51 ± 1.47 **
HEP-C-H
681.71 ± 164.32#
65.69 ± 5.68*
1642.69± 77.91**
4.27 ± 1.36***
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Groups
Values are expressed as mean ± SD (n = 8). * P<0.05, **P<0.01, and ***P<0.001, compared with MC group. P<0.05, ## P<0.01, and ### P<0.01, compared with NC group.
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#
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Table 5. Effect of HEP-C on the activities of SOD, CAT, and GSH-Px and MDA level in livers in STZ-induced diabetic rats. SOD (U/mg prot)
CAT (U/mg prot)
GSH-Px (U/mg prot)
MDA (nmol/mg prot)
NC
421.16 ± 2.22***
47.15 ± 5.33***
1194.36 ± 35.46**
0.55 ± 0.20***
MC
373.76 ± 29.55###
25.00 ± 0.73###
790.78 ± 56.41##
1.18 ± 0.16###
PC
417.99 ± 18.62**
34.38 ± 6.40** #
1036.26 ± 80.94*
0.63 ± 0.08**
HEP-C-L
406.67 ± 25.91*
25.83± 0.77* ##
714.30 ± 25.08##
0.78 ± 0.23**
HEP-C-H
412.19 ± 22.18**
29.97 ± 4.31** #
1013.85± 21.40*
0.75 ± 0.23**
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Groups
Values are expressed as mean ± SD (n = 8). * P<0.05, ** P<0.01, and ***P<0.001, compared with MC group. P<0.05, ## P<0.01, and ### P<0.01, compared with NC group.
