Endocrine DOI 10.1007/s12020-014-0375-y
ORIGINAL ARTICLE
Protective effect of Schisandrae chinensis oil on pancreatic b-cells in diabetic rats Liping An • Yingping Wang • Chunmei Wang • Meizhen Fan • Xiao Han Guangyu Xu • Guangxin Yuan • Hongyu Li • Yu Sheng • Manli Wang • Jingbo Sun • Jinzhuo Zhan • Hui Sun • Na Li • Fuxiang Ding • Peige Du
•
Received: 29 January 2014 / Accepted: 29 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Islet cell dysfunction in type 2 diabetes is primarily attributed to increased apoptosis of pancreatic b-Cells. The aim of the present study was to investigate the effects of Schisandrae chinensis oil on pancreatic b-Cells in type 2 diabetes mellitus rats and the associated molecular mechanisms of action. Wistar rats were randomly divided into diabetic rats and control rats, diabetic rats treated with Schisandrae chinensis oil (1 mg/kg), and control rats treated with Schisandrae chinensis oil. The serum fasting blood glucose, insulin, total cholesterol, and triglyceride levels along with MDA content, SOD and CAT activities in pancreatic tissues were measured. TUNEL was used to observe the apoptosis of rat pancreatic cells. Western blot was used to determine specific protein expression. The results showed that the oil significantly decreased fasting blood glucose, total cholesterol, triglyceride levels as well as the pancreatic MDA, but increased SOD and CAT activities. The protein expression of Bcl-2, PDX-1, GLUT-2, and GCK but not caspase 3 was significantly enhanced in the oil-treated rats compared with diabetic rats. However, Bax content was not significantly
L. An C. Wang X. Han G. Xu G. Yuan H. Li Y. Sheng M. Wang J. Sun J. Zhan H. Sun N. Li P. Du (&) College of Pharmacy, Beihua University, Jilin 132013, Jilin, People’s Republic of China e-mail: [email protected] Y. Wang Institute of Special Economic Plants and Wildlives Utilization, Chinese Academy of Agricultural Sciences, Jilin 132019, People’s Republic of China M. Fan F. Ding (&) Affiliated Hospital, Beihua University, Jilin 132013, Jilin, People’s Republic of China e-mail: [email protected]
different between the control and DM groups. Schisandra chinensis oil improves pancreatic b-cell function by enhancing antioxidant potential of the pancreas, upregulating the expression of anti-apoptotic genes, increasing expression of glucose metabolism, and delaying islet cell apoptosis. Keywords Diabetes Schisandra chinensis oil Apoptosis Pancreatic b-Cells
Introduction Type 2 diabetes is characterized by impaired insulin secretion due to beta cell dysfunction and reduced beta cell mass. Beta cell mass is regulated by a balance of cell growth, including replication and neogenesis, and cell loss through apoptosis, necrosis, and senescence [1, 2]. The regulation of beta cell mass plays a pivotal role in the pathogenesis of type 2 diabetes [3]. Chronic hyperglycemia in diabetes leads to progressive loss of beta cell mass with a prolonged increase in the rate of beta cell apoptosis, despite new islet formation and normal beta cell replication. Therefore, increased beta cell apoptosis is regarded as one of the major factors in the pathogenesis of diabetes [4]. Therapeutic approaches designed to arrest apoptosis are a significant advance in the management of type 2 diabetes, to actually reverse the disease rather than just lower glycemia. Traditional Chinese medicine (TCM) has been used extensively to prevent and treat DM for thousands of years. The dried ripe fruit of Schisandra chinensis (Turcz.) Baill. is officially listed in the Chinese Pharmacopoeia as Wuweizi in Chinese and mainly used as a tonic and sedative [5]. Pharmacological studies have indicated that
123
Endocrine
Schisandra chinensis is associated with potent antiinflammatory and antioxidant function, with a protective role in central nervous system [6]. A new variety of Schisandra chinensis has been named as Optimal Red 01 by Chinese Academy of Agricultural Sciences. The Schisandrae chinensis oil is a low-temperature concentrate of the seed extract with ethyl acetate. Schisandra lignans is the main component of Schisandrae chinensis oil, which can lower the blood sugar, increase the serum insulin secretion, and decrease the secretion of glucagon [7]. However, the anti-diabetic mechanism of action is still unclear. In the present study, we observed the protective effects of Schisandrae chinensis oil on pancreatic b-cells in a type 2 diabetes mellitus rat model induced by high-fat diet plus multiple low dosages of streptozotocin.
Materials and methods Reagents Schisandrae chinensis oil was kindly provided by Chinese Academy of Agricultural Sciences (Changchun, China). Streptozotocin was purchased from Sigma Aldrich (USA). Kits to measure blood glucose, total cholesterol, and triglyceride were obtained from Beijing BHKT Clinical Reagent Co., Ltd (Beijing, China). Iodine [125I] insulin radioimmunoassay kit was purchased from Tianjing Nine Tripods Medical & Bioengineering Co., Ltd (Tianjing, China). Kits to measure SOD, MDA, GSH, and GSH-Px were provided by Nanjing Jiancheng Chemical Factory (Nanjing, China). Chemical agents for Western blot were obtained from Sigma Aldrich. All other chemical reagents were from a commercial source. Animals Six-week-old male Wistar rats were housed in an environmentally controlled breeding room (temperature: 20 ± 2 °C, humidity: 60 ± 5 %, 12 h light/dark cycle). All rats were provided with free access to tap water. All procedures were approved by the Ethics Committee for the Use of Experimental Animals of Jilin University. Animal model and experimental groups The diabetic rat model was developed using a high-fat diet plus multiple low doses of streptozotocin, as reported previously by Wang et al. [8]. The high-fat diet consisted of 22 % fat, 48 % carbohydrate, and 20 % protein (the total calorific value was 44.3 kJ/kg). The animals were housed at the Stoyer Center of Experimental Animal Holding, Changchun, China. The control rats were given
123
regular chow consisting of 5 % fat, 53 % carbohydrate, and 23 % protein (the total calorific value was 25 kJ/kg). Following 4 weeks of dietary intervention, the diabetic group was injected intraperitoneally (i.p.) with low doses of streptozotocin (30 mg/kg, dissolved in 0.1 M sodium citrate buffer, pH 4.4). One week later, blood samples were collected by tail incision for fasting blood glucose measurements by glucose oxidase peroxidase. Rats with fasting blood glucose of \7.8 mmol/L were injected with streptozotocin again (30 mg/kg). Control rats were administered vehicle citrate buffer (pH 4.4) in a matched volume (0.25 ml/kg) via intraperitoneal injections. The fasting blood glucose was measured again 4 weeks after the streptozotocin injections, and rats with fasting blood glucose of C7.8 mmol/L were considered diabetic. The diabetic rats were fed high-fat diet for another 4 weeks. The control and diabetic rats were then randomly divided into four groups: (1) control group (CON, rats treated with matched saline), (2) control Schisandrae chinensis oiltreated group (CON ? SCO, control rats treated with Schisandrae chinensis oil 1 mg/kg), (3) diabetic model group (DM, diabetic rats treated with saline in a matched volume), (4) diabetic Schisandrae chinensis oil-treated group (DM ? SCO, diabetic rats treated with 1 mg/kg of Schisandrae chinensis oil). Schisandrae chinensis oil was administered via oral gavage daily for 8 weeks. All the rats were allowed to feed on their respective diets until the end of the study. Measurement of glucose, insulin, and lipid metabolic parameters After a 12-h fast, rats were anesthetized with 20 % urethane (100 mg/kg). Blood samples were obtained from abdominal aorta, allowed to clot for 30 min at 4 °C, centrifuged (3,5009g, 10 min, 4 °C), and the supernatant was used for the measurement of glucose, insulin, and lipid metabolic parameters. Blood glucose was estimated by a commercially available glucose kit based on the glucose oxidase method. Insulin was measured by radioimmunoassay method. Total cholesterol and triglyceride levels were measured according to the manufacturer’s instructions, respectively. Determination of superoxide dismutase (SOD) and catalase (CAT) activities and lipid peroxidation levels (MDA) Pancreatic tissues were removed from each rat. The tail segment of pancreas was washed with saline and the homogenate was prepared using phosphate-buffered solution (pH 7.2) to obtain a 1:5 (w/v) homogenate. The homogenate was stored at -80 °C, and later thawed, to
Endocrine
determine the superoxide dismutase (SOD) and catalase (CAT) activities and lipid peroxidation levels (MDA). Immunohistochemistry Pancreata were rapidly fixed in 4 % paraformaldehyde in 0.1 M PBS at 4 °C for 24 h and embedded in paraffin. Paraffin-embedded implants were completely sectioned at 7 lm section thickness. Sections were stained using antiinsulin antibodies (1: 1000; Abcam). Secondary antibodies were biotin-linked anti-rabbit IgG (Fuzhou Maixin Biotechnology Development Co., Ltd.;1:200, CHN). The peroxidase reaction was developed with a DAB development kit (Fuzhou Maixin Biotechnology Development Co., Ltd., CHN). Western blot analysis Pancreatic islets were isolated by static digestion and discontinuous gradient centrifugation according to the method described by Zywert A [9]. Hanks’ solution (containing in mM: NaCl 137, KCl 5.36, MgSO4 0.81, Na2HPO4 0.34, KH2PO4. 0.44, CaCl2 1.26, NaHCO3 4.17) saturated was used during the isolation. Frozen pancreata were cut down with scissors and were incubated in Hanks’ solution with collagenase for 15 min. After the digestion with collagenase, islets were washed with Hanks’ solution without the enzyme. Islets homogenate was prepared at 4 °C. Bax, Bcl2, caspase 3, pancreatic duodenal homeobox-1(PDX-1), glucose transporter 2(GLUT-2), and glucokinase (GCK) protein levels were assessed by Western blot. Briefly, protein samples 80 lg were subjected to sodium dodecyl sulfate–PAGE using 10 % gels, followed by electrotransfer to polyvinylidene difluoride (PVDF) membranes (BioRad). Blots were probed with rabbit polyclonal antibodies (Bax, Bcl-2, caspase 3, PDX-1, GLUT-2, 1: 800; GCK, 1: 1,000; Cell Signaling Technology) and actin (1: 2,000; Santa Cruz Biotechnology) followed by incubation with horseradish peroxidase–conjugated secondary antibodies (goat Anti-rabbit IgG Horseradish Peroxidase Conjugate, 1: 2,000; Jackson ImmunoResearch). Protein bands were then radiographically visualized with enhanced chemiluminescence. An Imaging Densitometer was used to scan the protein bands and quantified using the Image Analysis Software.
from each rat. The same slice was taken from at least three islets and at least three different sections were taken in each group. After the rats were sacrificed, paraffin slices were obtained from 1-cm-thick pancreas tissues. The pancreas sections were deparaffinized by xylene, hydrated by graded ethanol, and stained using TUNEL assay kit (Roche, Applied Science, Basel, Switzerland), according to the manufacturer’s instructions. The Image-Plus medical image statistical analysis system was applied to measure the positive rate of apoptotic cells. Positive apoptotic cells were characterized by a brown nucleus. The percentage of apoptotic cells was determined with the Soft Imaging System (Image Pro Plus 5.02, Media Cybernetics, Inc., USA). Statistical analysis All data were expressed as mean ± S.E.M. ‘‘n’’ denotes the sample size in each group. Statistical analysis was performed using one-way analysis of variance (ANOVA) with post hoc test for multiple comparisons. SPSS software (version 13.0 for Windows) was used for statistical analysis. P \ 0.05 was considered statistically significant.
Results Body weight and biochemical parameters Mean values of the body weight and biochemical parameters from normal and diabetic rats were summarized in Table 1. Body weight was not significantly different among the four groups. Diabetic rats showed higher fasting blood glucose, total cholesterol, and triglyceride levels compared with those of control (P \ 0.05). The serum insulin levels were reduced slightly in diabetic group. The treatment with Schisandrae chinensis oil significantly decreased fasting blood glucose, triglyceride, and total cholesterol levels compared with the untreated diabetic group. Schisandrae chinensis oil did not affect these parameters in control rats. The data indicate that the oil improved the blood glucose and lipid metabolism in diabetic rats induced by a high-fat diet along with multiple low doses of streptozotocin.
TUNEL staining
Effect of Schisandrae chinensis oil on MDA, SOD, and CAT levels in the pancreatic tissues
The level of apoptosis in pancreatic tissues was assessed by TUNEL staining. The experimental procedures were carried out according to the manufacturer’s protocol. There were 8 rats in each group and three sections were taken
Schisandrae chinensis oil treatment significantly lowered MDA contents and upregulated SOD and CAT levels (P \ 0.05) in the pancreatic tissues compared with the untreated diabetic group, as shown in Table 2.
123
Endocrine Table 1 Body weight and biochemical parameters
Weight (g)
CON
CON ? SCO
DM
DM ? SCO
386.78 ± 15.12
388.61 ± 15.62
396.92 ± 13.52
392.15 ± 16.67
4.52 ± 0.42
4.97 ± 0.53
17.06 ± 1.57a
8.34 ± 0.46b
a
14.48 ± 1.23b
3.52 ± 0.43a
2.72 ± 0.56b
a
1.25 ± 0.34b
Glucose (mmol/L) Insulin (mIU/L)
17.58 ± 1.339
17.63 ± 1.102
2.53 ± 0.08
2.63 ± 0.16
Total Cholesterol (mmol/L) Triglyceride (mmol/L)
1.26 ± 0.09
1.14 ± 0.1
11.09 ± 0.93 1.56 ± 0.25
Data are expressed as mean ± S.E.M (n = 8) CON control group, CON ? SCO control rats treated with Schisandrae chinensis oil 1 mg/kg, DM diabetic group, DM ? SCO diabetic rats treated with Schisandrae chinensis oil 1 mg/kg a
Different from CON and CON ? SCO groups
b
Different from DM group (P \ 0.05)
Table 2 Pancreatic MDA levels and SOD and CAT activity
MDA (nmol/mgprot) SOD (U/mgprot) CAT (U/mgprot)
CON
CON ? SCO
DM
12.72 ± 0.59
12.42 ± 0.72
24.831 ± 1.67a
18.76 ± 0.71b
27.04 ± 0.27
12.87 ± 0.69
a
18.92 ± 0.54b
17.71 ± 0.38
a
22.25 ± 0.54b
27.24 ± 0.47 25.58 ± 0.46
24.67 ± 0.66
DM ? SCO
Data are expressed as mean ± S.E.M (n = 8) CON control group, CON ? SCO control rats treated with Schisandrae chinensis oil 1 mg/kg, DM diabetic group, DM ? SCO diabetic rats treated with Schisandrae chinensis oil 1 mg/kg, MDA malondialdehyde, SOD superoxide dismutase, CAT catalase a
Different from CON and CON ? SCO groups
b
Different from DM group (P \ 0.05)
Fig. 1 Insulin expression of rats pancreatic islet cells in all groups (9200). a CON: control group; b CON ? SCO: control rats treated with Schisandrae chinensis oil 1 mg/kg; c DM: diabetic group; d DM ? SCO: diabetic rats treated with Schisandrae chinensis oil 1 mg/kg
123
Endocrine
Fig. 2 TUNEL staining of rat pancreatic islet cells (9400). a CON: control group; b CON ? SCO: control rats treated with Schisandrae chinensis oil 1 mg/kg; c DM: diabetic group; d DM ? SCO: diabetic rats treated with Schisandrae chinensis oil 1 mg/kg
Immunohistochemistry Under a light microscope, the structure of rat islets in the control group and the control rats treated with Schisandrae chinensis oil group was integrated, the size of them was larger and the boundary was clear; insulin-positive granules after the staining showed a brown color and were widely distributed within the islets. Islets of rats in the diabetic model group presented various sizes, most of them were narrow and their boundaries were not clear, and insulin-positive granules after the staining showed a shallow color and there were a few of them. Compared with those in the model group, positively stained insulin granules significantly increased and the volume of islets was enlarged in the Schisandra chinensis oil-treated group (Figs. 1, 2).
Pancreatic cell apoptosis by TUNEL assay TUNEL assay revealed an increased number of apoptotic pancreatic cells in DM groups, whereas the treatment of Schisandrae chinensis oil dramatically decreased the cellular apoptosis (P \ 0.05). Schisandrae chinensis oil did not alter the rate of apoptosis in the control pancreatic tissues (Figs. 2, 3).
Effect of Schisandrae chinensis oil on protein expressions of Bax, Bcl-2, Caspase3, PDX-1, GLUT-2, and GCK by western blotting analysis Effect of Schisandrae chinensis oil treatment on Bax, Bcl-2, Caspase3, PDX-1, GLUT-2, and GCK protein expressions was observed and analyzed by Western immunoblotting (Fig. 3). Western blotting analysis showed an increase of Bcl-2 protein levels (P \ 0.05) in rats treated with the oil in contrast to those in diabetic rats, while Bax content was not significantly different between the control and DM groups (Fig. 4a). Caspase 3 expression was slightly decreased in the oil-treated rats compared with diabetic rats (Fig. 4b). The protein expression of PDX-1, GLUT-2, and GCK was significantly enhanced in treated rats compared with diabetic rats (P \ 0.05) (Fig. 4c, d). Further, the oil did not change the expression of Bax, Bcl-2, Caspase 3, PDX-1, GLUT-2, and GCK in the pancreatic tissues from normal rats.
Discussion The active ingredients in natural medicine extraction have a better hypoglycemic effect. Schisandra contains lignans, polysaccharides, volatile oils, fatty acids, vitamins, and
123
Endocrine
Fig. 3 TUNEL assay: Apoptosis rates. CON: control group; CON ? SCO: control rats treated with Schisandrae chinenesis oil 1 mg/kg; DM: diabetic group; DM ? SCO: diabetic rats treated with Schisandrae chinensis oil 1 mg/kg; Data are expressed as mean ± S.E. *P \ 0.05 versus CON and CON ? SCO groups; # P \ 0.05 versus DM group
amino acids etc. Schisandra lignans is one of the most important ingredients with pharmacological activities. Schisandra lignans from Schisandra can improve the sensitivity of cells to insulin at the molecular level, which has been demonstrated by some experiments. Schisandra lignans is also one of the main Schisandra Chinensis Oil [7, 10]. Schisandra chinensis oil reduced blood glucose levels in animal models, increased the serum insulin level and lowered levels of glucagon, and therefore, therapeutically effective in diabetes [11, 12]. The anti-diabetic effect of the oil was also confirmed, with markedly reduced blood glucose and lipid levels and improved pathological changes in pancreatic tissues of diabetic rats [13]. Type 2 diabetes mellitus is characterized by insulin resistance and pancreatic b-cell dysfunction [14]. In decompensated b-Cells, fasting blood glucose (FBG) was increased, further aggravating the pancreatic b-cell dysfunction. Uncontrolled pancreatic b-cell dysfunction leads to b-cell failure and the cessation of insulin secretion results in low hyperinsulinemia due to insulin resistance [15, 16]. In this study, serum FBG and FINS contents in diabetic model group were significantly decreased, indicating a severely impaired function of pancreatic b-cells. The impaired b-cells function might be closely associated with genetic and environmental factors, and was probably induced by oxidative stress. The declined antioxidant mechanisms elevated the oxidative stress and generated reactive oxygen species (ROS) [17, 18]. The results revealed that MDA level was significantly increased but the SOD and CAT activity was decreased in pancreatic tissues of the diabetic rats, suggesting that the increased ROS was due to the enhanced oxidative stress. The diminished activity of expression associated with antioxidant enzymes, such as SOD and GSH-Px in pancreatic
123
b-Cells, increases the damage by oxidative stress. High levels of ROS may cause large-scale non-specific oxidative damage including disruption of membrane integrity in pancreatic b-Cells, leading to apoptosis [17]. It also causes disorders in apoptosis-related gene expression, downregulates anti-apoptotic gene Bc1-2 expression, and upregulates pro-apoptotic genes Bad, Bid, and Bik [19, 20]. The TUNEL assay also demonstrated the increase in the rate of islet cell apoptosis and Bax/bcl-2 ratio, decrease in Bcl-2 expression of pancreatic tissue, no pronounced changes in Bax expression, and increase in the expression of apoptosis-related factor caspase 3 in the diabetic rats. Six weeks later, the blood glucose of rats in the oil-treated group returned to normal, the serum insulin levels were significantly increased, the MDA content was decreased, and the SOD and CAT activities were enhanced. The increase of apoptosis may be associated with the decreased expression of PDX-1-related protein. PDX-1 is a transcription factor expressed specifically in pancreatic b-Cells, and inhibits the apoptosis of pancreatic cells [21]. It is also a key transcription factor of insulin gene expression, and induces the differentiation of pancreatic b-Cells, promotes pancreatic the b-cell proliferation, and inhibits the apoptosis, playing a key role in the growth and function of the pancreatic b-Cells [22]. Studies demonstrated that in diabetic rats, the sensitivity of pancreatic b-Cells to apoptotic factors was increased. The activity of caspase 3 and pancreatic b-Cells apoptosis was increased. The anti-apoptotic Bcl-2 was abnormal. However, the number and volume of islets and the released insulin were decreased [21]. Sustained high blood sugar inhibited PDX1 gene expression. The transient spikes in blood sugar level promoted binding of PDX-1 and insulin gene in the pancreatic b-Cells, increased insulin mRNA levels, while the chronically elevated blood sugar level decreased both PDX-1 and insulin expressions. Alquobaili et al. [23] reported that sustained high blood sugar suppressed PDX-1 and insulin gene expressions, and inhibited the binding of PDX-1 protein and insulin gene promoters, thereby decreasing the synthesis and secretion of insulin. PDX-1 is also known to be a transcription factor for insulin releaserelated genes such as GLUT-2 and GCK. GLUT-2 is the major membrane transport protein that transports glucose into cells, and regulates synthesis and secretion of insulin in the glucose-stimulated islet cells. It is also one of the rate-limiting steps of glycolysis in pancreatic b-Cells. GCK is regulated by blood glucose, and is also the first ratelimiting enzyme in glucose metabolism. It stimulates the secretion of insulin and its activity determines the changes in glucose metabolism and insulin secretion, and regulates blood sugar levels. Thus, GCK plays an important role in maintaining glucose homeostasis [24]. GLUT-2 and GCK are important in glucose-stimulated GSIS, mediated by
Endocrine
Fig. 4 Western blotting analysis of the effects of Schisandrae chinensis oil on Bax, Bcl-2, Caspase3, PDX-1, GLUT-2, and GCK protein expression in rat pancreatic tissue. Expression and quantification of Bax, Bcl-2, Caspase3, PDX-1, GLUT-2, and GCK protein expression relative to the amount of actin; CON, control rat;
CON ? SCO, control rats treated with the oil 1 mg/kg; DM, diabetic rat; DM ? SCO, diabetic rats treated with Schisandrae chinensis oil 1 mg/kg. Representative bands are shown. Data are expressed as mean ± S.E.M. (n = 8). *P \ 0.05 versus CON, CON ? SC, DM ? SCO; #P \ 0.05 versus DM
PDX-1 binding to insulin gene promoter. The decrease in PDX-1 gene expression was associated with significantly downregulated GLUT-2 and GCK gene expression in our study. It reduced gene synthesis, resulting in a reduction of glucose-induced GSIS [25]. Chronically high blood sugar levels inhibit PDX-1 gene expression. Similar decreases in GLUT-2 expression, followed by downregulation of the PDX-1 and GCK expressions in pancreatic tissue of diabetic rats, were reported previously [26, 27]. The results showed that Schisandra chinensis oil increased the expression of PDX-1, GLUT-2, and GCK in diabetic rats to regulate glucose metabolism and promote insulin secretion. It is believed that Schisandra chinensis oil can enhance the antioxidant capacity of pancreas, upregulate the expression of anti-apoptotic gene, increase the expression
of glucose metabolism factors such as PDX-1, GLUT-2, and GCK, delay the apoptosis of islet cells, and further improve B cell functions. Acknowledgments This work was funded by project ‘‘2013-199’’ supported by the Education Department of Jilin Province, project ‘‘201262503’’ supported by Sci-tech Department of Jilin City and project ‘‘20122082’’ the Healthe Department of Jilin Province. Conflict of interest of interest.
The authors declare that they have no conflict
References 1. H. Sone, Y. Kagawa, Pancreatic beta cell senescence contributes to the pathogenesis of type 2 diabetes in high-fat diet-induced
123
Endocrine
2.
3.
4. 5. 6.
7.
8.
9.
10.
11.
12.
13.
14. 15.
diabetic mice. Diabetologia 48(1), 58–67 (2005). doi:10.1007/ s00125-004-1605-2 P. Marchetti, M. Bugliani, U. Boggi, M. Masini, L. Marselli, The pancreatic beta cells in human type 2 diabetes. Adv. Exp. Med. Biol. 771, 288–309 (2012) B. Thorens, The required beta cell research for improving treatment of type 2 diabetes. J. Intern. Med. 274(3), 203–214 (2013). doi:10.1111/joim.12096 M.O. Larsen, Beta-cell function and mass in type 2 diabetes. Dan. Med. Bull. 56(3), 153–164 (2009) C.P. Commission, China Pharmacopoeia. In vol. 1. (Chemical Industry Press, Beijing 2010), pp. 61–62 A. Panossian, G. Wikman, Pharmacology of Schisandra chinensis Bail.: an overview of Russian research and uses in medicine. J. Ethnopharmacol. 118(2), 183–212 (2008). doi:10.1016/j.jep. 2008.04.020 D.Y. Kwon, S. da Kim, H.J. Yang, S. Park, The lignan-rich fractions of Fructus Schisandrae improve insulin sensitivity via the PPAR-gamma pathways in in vitro and in vivo studies. J. Ethnopharmacol. 135(2), 455–462 (2011). doi:10.1016/j.jep. 2011.03.037 C. Wang, J. Li, X. Lv, M. Zhang, Y. Song, L. Chen, Y. Liu, Ameliorative effect of berberine on endothelial dysfunction in diabetic rats induced by high-fat diet and streptozotocin. Eur. J. Pharmacol. 620(1–3), 131–137 (2009). doi:10.1016/j.ejphar. 2009.07.027 A. Zywert, K. Szkudelska, T. Szkudelski, Inhibition of phosphodiesterase 3B in insulin-secreting cells of normal and streptozocin-nicotinamide-induced diabetic rats implications for insulin secretion. J. Physiol. Pharmacol. 65(3), 425–433 (2014) W. Xie, L. Du, Diabetes is an inflammatory disease: evidence from traditional Chinese medicines. Diabetes Obes. Metab. 13(4), 289–301 (2011). doi:10.1111/j.1463-1326.2010.01336.x K.F. Cai, Z.C. Qin, Y.L. Wang, Effects of Fructus Schisandrae oil on islet cell morphology and function of diabetic mice. Chin. J. Tradit. Med. Sci. Technol. 14(3), 177–178 (2007) K.F. Cai, Z.C. Qin, Y.L. Wang, Effects of Fructus Schisandrae oil on antioxidant and the expression of glucose transporter protein 4 mRNA of diabetic mice. Chin. Arch. Tradit. Chin. Med. 24(7), 1199–1200 (2006) L.P. An, Y.P. Wang, X.M. Liu, Effect of Fructus Schisandrae oil on blood glucose of type 2 diabetic rats induced by Streptozotocin. Chin. Tradit. Herb. Drugs 43(3), 52–556 (2012) J.H. Yki, Pathogenesis of noninsulin dependent diabetes mellitus. Lancet 343(8889), 91–95 (1994) W.H. Kim, J.W. Lee, Y.H. Suh, S.H. Hong, J.S. Choi, J.H. Lim, J.H. Song, B. Gao, M.H. Jung, Exposure to chronic high glucose
123
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
induces beta-cell apoptosis through decreased interaction of glucokinase with mitochondria: downregulation of glucokinase in pancreatic beta-cells. Diabetes 54(9), 2602–2611 (2005) M. Bensellam, D.R. Laybutt, J.C. Jonas, The molecular mechanisms of pancreatic beta-cell glucotoxicity: recent findings and future research directions. Mol. Cell. Endocrinol. 364(1–2), 1–27 (2012). doi:10.1016/j.mce.2012.08.003 T. van de Weijer, V.B. Schrauwen-Hinderling, P. Schrauwen, Lipotoxicity in type 2 diabetic cardiomyopathy. Cardiovasc. Res. 92(1), 10–18 (2011). doi:10.1093/cvr/cvr212 J.R. Zierath, A. Krook, H. Wallberg-Henriksson, Insulin action and insulin resistance in human skeletal muscle. Diabetologia 43(7), 821–835 (2000). doi:10.1007/s001250051457 U.K. Prospective Diabetes Study Group, U.K. prospective diabetes study 16. Overview of 6 years’ therapy of type II diabetes: a progressive disease. Diabetes 44(11), 1249–1258 (1995) G.C. Weir, D.R. Laybutt, H. Kaneto, S. Bonner-Weir, A. Sharma, Beta-cell adaptation and decompensation during the progression of diabetes. Diabetes 50(Suppl 1), S154–S159 (2001) J.D. Johnson, N.T. Ahmed, D.S. Luciani, Z. Han, H. Tran, J. Fujita, S. Misler, H. Edlund, K.S. Polonsky, Increased islet apoptosis in Pdx1 ± mice. J. Clin. Investig. 111(8), 1147–1160 (2003). doi:10.1172/JCI16537 W. Kim, J.M. Egan, The role of incretins in glucose homeostasis and diabetes treatment. Pharmacol. Rev. 60(4), 470–512 (2008). doi:10.1124/pr.108.000604 F. Al-Quobaili, M. Montenarh, Pancreatic duodenal homeobox factor-1 and diabetes mellitus type 2 (review). Int. J. Mol. Med. 21(4), 399–404 (2008) R. Singh, E.R. Pearson, P.M. Clark, A.T. Hattersley, The longterm impact on offspring of exposure to hyperglycaemia in utero due to maternal glucokinase gene mutations. Diabetologia 50(3), 620–624 (2007). doi:10.1007/s00125-006-0541-8 R.N. Kulkarni, U.S. Jhala, J.N. Winnay, S. Krajewski, M. Montminy, C.R. Kahn, PDX-1 haploinsufficiency limits the compensatory islet hyperplasia that occurs in response to insulin resistance. J. Clin. Investig. 114(6), 828–836 (2004). doi:10.1172/ JCI21845 H. Kaneto, T. Miyatsuka, D. Kawamori, T.A. Matsuoka, Pleiotropic roles of PDX-1 in the pancreas. Rev. Diabet. Stud. 4(4), 209–225 (2007). doi:10.1900/RDS.2007.4.209 H. Kaneto, T. Miyatsuka, T. Shiraiwa, K. Yamamoto, K. Kato, Y. Fujitani, T.A. Matsuoka, Crucial role of PDX-1 in pancreas development, beta-cell differentiation, and induction of surrogate beta-cells. Curr. Med. Chem. 14(16), 1745–1752 (2007)
ORIGINAL ARTICLE
Protective effect of Schisandrae chinensis oil on pancreatic b-cells in diabetic rats Liping An • Yingping Wang • Chunmei Wang • Meizhen Fan • Xiao Han Guangyu Xu • Guangxin Yuan • Hongyu Li • Yu Sheng • Manli Wang • Jingbo Sun • Jinzhuo Zhan • Hui Sun • Na Li • Fuxiang Ding • Peige Du
•
Received: 29 January 2014 / Accepted: 29 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Islet cell dysfunction in type 2 diabetes is primarily attributed to increased apoptosis of pancreatic b-Cells. The aim of the present study was to investigate the effects of Schisandrae chinensis oil on pancreatic b-Cells in type 2 diabetes mellitus rats and the associated molecular mechanisms of action. Wistar rats were randomly divided into diabetic rats and control rats, diabetic rats treated with Schisandrae chinensis oil (1 mg/kg), and control rats treated with Schisandrae chinensis oil. The serum fasting blood glucose, insulin, total cholesterol, and triglyceride levels along with MDA content, SOD and CAT activities in pancreatic tissues were measured. TUNEL was used to observe the apoptosis of rat pancreatic cells. Western blot was used to determine specific protein expression. The results showed that the oil significantly decreased fasting blood glucose, total cholesterol, triglyceride levels as well as the pancreatic MDA, but increased SOD and CAT activities. The protein expression of Bcl-2, PDX-1, GLUT-2, and GCK but not caspase 3 was significantly enhanced in the oil-treated rats compared with diabetic rats. However, Bax content was not significantly
L. An C. Wang X. Han G. Xu G. Yuan H. Li Y. Sheng M. Wang J. Sun J. Zhan H. Sun N. Li P. Du (&) College of Pharmacy, Beihua University, Jilin 132013, Jilin, People’s Republic of China e-mail: [email protected] Y. Wang Institute of Special Economic Plants and Wildlives Utilization, Chinese Academy of Agricultural Sciences, Jilin 132019, People’s Republic of China M. Fan F. Ding (&) Affiliated Hospital, Beihua University, Jilin 132013, Jilin, People’s Republic of China e-mail: [email protected]
different between the control and DM groups. Schisandra chinensis oil improves pancreatic b-cell function by enhancing antioxidant potential of the pancreas, upregulating the expression of anti-apoptotic genes, increasing expression of glucose metabolism, and delaying islet cell apoptosis. Keywords Diabetes Schisandra chinensis oil Apoptosis Pancreatic b-Cells
Introduction Type 2 diabetes is characterized by impaired insulin secretion due to beta cell dysfunction and reduced beta cell mass. Beta cell mass is regulated by a balance of cell growth, including replication and neogenesis, and cell loss through apoptosis, necrosis, and senescence [1, 2]. The regulation of beta cell mass plays a pivotal role in the pathogenesis of type 2 diabetes [3]. Chronic hyperglycemia in diabetes leads to progressive loss of beta cell mass with a prolonged increase in the rate of beta cell apoptosis, despite new islet formation and normal beta cell replication. Therefore, increased beta cell apoptosis is regarded as one of the major factors in the pathogenesis of diabetes [4]. Therapeutic approaches designed to arrest apoptosis are a significant advance in the management of type 2 diabetes, to actually reverse the disease rather than just lower glycemia. Traditional Chinese medicine (TCM) has been used extensively to prevent and treat DM for thousands of years. The dried ripe fruit of Schisandra chinensis (Turcz.) Baill. is officially listed in the Chinese Pharmacopoeia as Wuweizi in Chinese and mainly used as a tonic and sedative [5]. Pharmacological studies have indicated that
123
Endocrine
Schisandra chinensis is associated with potent antiinflammatory and antioxidant function, with a protective role in central nervous system [6]. A new variety of Schisandra chinensis has been named as Optimal Red 01 by Chinese Academy of Agricultural Sciences. The Schisandrae chinensis oil is a low-temperature concentrate of the seed extract with ethyl acetate. Schisandra lignans is the main component of Schisandrae chinensis oil, which can lower the blood sugar, increase the serum insulin secretion, and decrease the secretion of glucagon [7]. However, the anti-diabetic mechanism of action is still unclear. In the present study, we observed the protective effects of Schisandrae chinensis oil on pancreatic b-cells in a type 2 diabetes mellitus rat model induced by high-fat diet plus multiple low dosages of streptozotocin.
Materials and methods Reagents Schisandrae chinensis oil was kindly provided by Chinese Academy of Agricultural Sciences (Changchun, China). Streptozotocin was purchased from Sigma Aldrich (USA). Kits to measure blood glucose, total cholesterol, and triglyceride were obtained from Beijing BHKT Clinical Reagent Co., Ltd (Beijing, China). Iodine [125I] insulin radioimmunoassay kit was purchased from Tianjing Nine Tripods Medical & Bioengineering Co., Ltd (Tianjing, China). Kits to measure SOD, MDA, GSH, and GSH-Px were provided by Nanjing Jiancheng Chemical Factory (Nanjing, China). Chemical agents for Western blot were obtained from Sigma Aldrich. All other chemical reagents were from a commercial source. Animals Six-week-old male Wistar rats were housed in an environmentally controlled breeding room (temperature: 20 ± 2 °C, humidity: 60 ± 5 %, 12 h light/dark cycle). All rats were provided with free access to tap water. All procedures were approved by the Ethics Committee for the Use of Experimental Animals of Jilin University. Animal model and experimental groups The diabetic rat model was developed using a high-fat diet plus multiple low doses of streptozotocin, as reported previously by Wang et al. [8]. The high-fat diet consisted of 22 % fat, 48 % carbohydrate, and 20 % protein (the total calorific value was 44.3 kJ/kg). The animals were housed at the Stoyer Center of Experimental Animal Holding, Changchun, China. The control rats were given
123
regular chow consisting of 5 % fat, 53 % carbohydrate, and 23 % protein (the total calorific value was 25 kJ/kg). Following 4 weeks of dietary intervention, the diabetic group was injected intraperitoneally (i.p.) with low doses of streptozotocin (30 mg/kg, dissolved in 0.1 M sodium citrate buffer, pH 4.4). One week later, blood samples were collected by tail incision for fasting blood glucose measurements by glucose oxidase peroxidase. Rats with fasting blood glucose of \7.8 mmol/L were injected with streptozotocin again (30 mg/kg). Control rats were administered vehicle citrate buffer (pH 4.4) in a matched volume (0.25 ml/kg) via intraperitoneal injections. The fasting blood glucose was measured again 4 weeks after the streptozotocin injections, and rats with fasting blood glucose of C7.8 mmol/L were considered diabetic. The diabetic rats were fed high-fat diet for another 4 weeks. The control and diabetic rats were then randomly divided into four groups: (1) control group (CON, rats treated with matched saline), (2) control Schisandrae chinensis oiltreated group (CON ? SCO, control rats treated with Schisandrae chinensis oil 1 mg/kg), (3) diabetic model group (DM, diabetic rats treated with saline in a matched volume), (4) diabetic Schisandrae chinensis oil-treated group (DM ? SCO, diabetic rats treated with 1 mg/kg of Schisandrae chinensis oil). Schisandrae chinensis oil was administered via oral gavage daily for 8 weeks. All the rats were allowed to feed on their respective diets until the end of the study. Measurement of glucose, insulin, and lipid metabolic parameters After a 12-h fast, rats were anesthetized with 20 % urethane (100 mg/kg). Blood samples were obtained from abdominal aorta, allowed to clot for 30 min at 4 °C, centrifuged (3,5009g, 10 min, 4 °C), and the supernatant was used for the measurement of glucose, insulin, and lipid metabolic parameters. Blood glucose was estimated by a commercially available glucose kit based on the glucose oxidase method. Insulin was measured by radioimmunoassay method. Total cholesterol and triglyceride levels were measured according to the manufacturer’s instructions, respectively. Determination of superoxide dismutase (SOD) and catalase (CAT) activities and lipid peroxidation levels (MDA) Pancreatic tissues were removed from each rat. The tail segment of pancreas was washed with saline and the homogenate was prepared using phosphate-buffered solution (pH 7.2) to obtain a 1:5 (w/v) homogenate. The homogenate was stored at -80 °C, and later thawed, to
Endocrine
determine the superoxide dismutase (SOD) and catalase (CAT) activities and lipid peroxidation levels (MDA). Immunohistochemistry Pancreata were rapidly fixed in 4 % paraformaldehyde in 0.1 M PBS at 4 °C for 24 h and embedded in paraffin. Paraffin-embedded implants were completely sectioned at 7 lm section thickness. Sections were stained using antiinsulin antibodies (1: 1000; Abcam). Secondary antibodies were biotin-linked anti-rabbit IgG (Fuzhou Maixin Biotechnology Development Co., Ltd.;1:200, CHN). The peroxidase reaction was developed with a DAB development kit (Fuzhou Maixin Biotechnology Development Co., Ltd., CHN). Western blot analysis Pancreatic islets were isolated by static digestion and discontinuous gradient centrifugation according to the method described by Zywert A [9]. Hanks’ solution (containing in mM: NaCl 137, KCl 5.36, MgSO4 0.81, Na2HPO4 0.34, KH2PO4. 0.44, CaCl2 1.26, NaHCO3 4.17) saturated was used during the isolation. Frozen pancreata were cut down with scissors and were incubated in Hanks’ solution with collagenase for 15 min. After the digestion with collagenase, islets were washed with Hanks’ solution without the enzyme. Islets homogenate was prepared at 4 °C. Bax, Bcl2, caspase 3, pancreatic duodenal homeobox-1(PDX-1), glucose transporter 2(GLUT-2), and glucokinase (GCK) protein levels were assessed by Western blot. Briefly, protein samples 80 lg were subjected to sodium dodecyl sulfate–PAGE using 10 % gels, followed by electrotransfer to polyvinylidene difluoride (PVDF) membranes (BioRad). Blots were probed with rabbit polyclonal antibodies (Bax, Bcl-2, caspase 3, PDX-1, GLUT-2, 1: 800; GCK, 1: 1,000; Cell Signaling Technology) and actin (1: 2,000; Santa Cruz Biotechnology) followed by incubation with horseradish peroxidase–conjugated secondary antibodies (goat Anti-rabbit IgG Horseradish Peroxidase Conjugate, 1: 2,000; Jackson ImmunoResearch). Protein bands were then radiographically visualized with enhanced chemiluminescence. An Imaging Densitometer was used to scan the protein bands and quantified using the Image Analysis Software.
from each rat. The same slice was taken from at least three islets and at least three different sections were taken in each group. After the rats were sacrificed, paraffin slices were obtained from 1-cm-thick pancreas tissues. The pancreas sections were deparaffinized by xylene, hydrated by graded ethanol, and stained using TUNEL assay kit (Roche, Applied Science, Basel, Switzerland), according to the manufacturer’s instructions. The Image-Plus medical image statistical analysis system was applied to measure the positive rate of apoptotic cells. Positive apoptotic cells were characterized by a brown nucleus. The percentage of apoptotic cells was determined with the Soft Imaging System (Image Pro Plus 5.02, Media Cybernetics, Inc., USA). Statistical analysis All data were expressed as mean ± S.E.M. ‘‘n’’ denotes the sample size in each group. Statistical analysis was performed using one-way analysis of variance (ANOVA) with post hoc test for multiple comparisons. SPSS software (version 13.0 for Windows) was used for statistical analysis. P \ 0.05 was considered statistically significant.
Results Body weight and biochemical parameters Mean values of the body weight and biochemical parameters from normal and diabetic rats were summarized in Table 1. Body weight was not significantly different among the four groups. Diabetic rats showed higher fasting blood glucose, total cholesterol, and triglyceride levels compared with those of control (P \ 0.05). The serum insulin levels were reduced slightly in diabetic group. The treatment with Schisandrae chinensis oil significantly decreased fasting blood glucose, triglyceride, and total cholesterol levels compared with the untreated diabetic group. Schisandrae chinensis oil did not affect these parameters in control rats. The data indicate that the oil improved the blood glucose and lipid metabolism in diabetic rats induced by a high-fat diet along with multiple low doses of streptozotocin.
TUNEL staining
Effect of Schisandrae chinensis oil on MDA, SOD, and CAT levels in the pancreatic tissues
The level of apoptosis in pancreatic tissues was assessed by TUNEL staining. The experimental procedures were carried out according to the manufacturer’s protocol. There were 8 rats in each group and three sections were taken
Schisandrae chinensis oil treatment significantly lowered MDA contents and upregulated SOD and CAT levels (P \ 0.05) in the pancreatic tissues compared with the untreated diabetic group, as shown in Table 2.
123
Endocrine Table 1 Body weight and biochemical parameters
Weight (g)
CON
CON ? SCO
DM
DM ? SCO
386.78 ± 15.12
388.61 ± 15.62
396.92 ± 13.52
392.15 ± 16.67
4.52 ± 0.42
4.97 ± 0.53
17.06 ± 1.57a
8.34 ± 0.46b
a
14.48 ± 1.23b
3.52 ± 0.43a
2.72 ± 0.56b
a
1.25 ± 0.34b
Glucose (mmol/L) Insulin (mIU/L)
17.58 ± 1.339
17.63 ± 1.102
2.53 ± 0.08
2.63 ± 0.16
Total Cholesterol (mmol/L) Triglyceride (mmol/L)
1.26 ± 0.09
1.14 ± 0.1
11.09 ± 0.93 1.56 ± 0.25
Data are expressed as mean ± S.E.M (n = 8) CON control group, CON ? SCO control rats treated with Schisandrae chinensis oil 1 mg/kg, DM diabetic group, DM ? SCO diabetic rats treated with Schisandrae chinensis oil 1 mg/kg a
Different from CON and CON ? SCO groups
b
Different from DM group (P \ 0.05)
Table 2 Pancreatic MDA levels and SOD and CAT activity
MDA (nmol/mgprot) SOD (U/mgprot) CAT (U/mgprot)
CON
CON ? SCO
DM
12.72 ± 0.59
12.42 ± 0.72
24.831 ± 1.67a
18.76 ± 0.71b
27.04 ± 0.27
12.87 ± 0.69
a
18.92 ± 0.54b
17.71 ± 0.38
a
22.25 ± 0.54b
27.24 ± 0.47 25.58 ± 0.46
24.67 ± 0.66
DM ? SCO
Data are expressed as mean ± S.E.M (n = 8) CON control group, CON ? SCO control rats treated with Schisandrae chinensis oil 1 mg/kg, DM diabetic group, DM ? SCO diabetic rats treated with Schisandrae chinensis oil 1 mg/kg, MDA malondialdehyde, SOD superoxide dismutase, CAT catalase a
Different from CON and CON ? SCO groups
b
Different from DM group (P \ 0.05)
Fig. 1 Insulin expression of rats pancreatic islet cells in all groups (9200). a CON: control group; b CON ? SCO: control rats treated with Schisandrae chinensis oil 1 mg/kg; c DM: diabetic group; d DM ? SCO: diabetic rats treated with Schisandrae chinensis oil 1 mg/kg
123
Endocrine
Fig. 2 TUNEL staining of rat pancreatic islet cells (9400). a CON: control group; b CON ? SCO: control rats treated with Schisandrae chinensis oil 1 mg/kg; c DM: diabetic group; d DM ? SCO: diabetic rats treated with Schisandrae chinensis oil 1 mg/kg
Immunohistochemistry Under a light microscope, the structure of rat islets in the control group and the control rats treated with Schisandrae chinensis oil group was integrated, the size of them was larger and the boundary was clear; insulin-positive granules after the staining showed a brown color and were widely distributed within the islets. Islets of rats in the diabetic model group presented various sizes, most of them were narrow and their boundaries were not clear, and insulin-positive granules after the staining showed a shallow color and there were a few of them. Compared with those in the model group, positively stained insulin granules significantly increased and the volume of islets was enlarged in the Schisandra chinensis oil-treated group (Figs. 1, 2).
Pancreatic cell apoptosis by TUNEL assay TUNEL assay revealed an increased number of apoptotic pancreatic cells in DM groups, whereas the treatment of Schisandrae chinensis oil dramatically decreased the cellular apoptosis (P \ 0.05). Schisandrae chinensis oil did not alter the rate of apoptosis in the control pancreatic tissues (Figs. 2, 3).
Effect of Schisandrae chinensis oil on protein expressions of Bax, Bcl-2, Caspase3, PDX-1, GLUT-2, and GCK by western blotting analysis Effect of Schisandrae chinensis oil treatment on Bax, Bcl-2, Caspase3, PDX-1, GLUT-2, and GCK protein expressions was observed and analyzed by Western immunoblotting (Fig. 3). Western blotting analysis showed an increase of Bcl-2 protein levels (P \ 0.05) in rats treated with the oil in contrast to those in diabetic rats, while Bax content was not significantly different between the control and DM groups (Fig. 4a). Caspase 3 expression was slightly decreased in the oil-treated rats compared with diabetic rats (Fig. 4b). The protein expression of PDX-1, GLUT-2, and GCK was significantly enhanced in treated rats compared with diabetic rats (P \ 0.05) (Fig. 4c, d). Further, the oil did not change the expression of Bax, Bcl-2, Caspase 3, PDX-1, GLUT-2, and GCK in the pancreatic tissues from normal rats.
Discussion The active ingredients in natural medicine extraction have a better hypoglycemic effect. Schisandra contains lignans, polysaccharides, volatile oils, fatty acids, vitamins, and
123
Endocrine
Fig. 3 TUNEL assay: Apoptosis rates. CON: control group; CON ? SCO: control rats treated with Schisandrae chinenesis oil 1 mg/kg; DM: diabetic group; DM ? SCO: diabetic rats treated with Schisandrae chinensis oil 1 mg/kg; Data are expressed as mean ± S.E. *P \ 0.05 versus CON and CON ? SCO groups; # P \ 0.05 versus DM group
amino acids etc. Schisandra lignans is one of the most important ingredients with pharmacological activities. Schisandra lignans from Schisandra can improve the sensitivity of cells to insulin at the molecular level, which has been demonstrated by some experiments. Schisandra lignans is also one of the main Schisandra Chinensis Oil [7, 10]. Schisandra chinensis oil reduced blood glucose levels in animal models, increased the serum insulin level and lowered levels of glucagon, and therefore, therapeutically effective in diabetes [11, 12]. The anti-diabetic effect of the oil was also confirmed, with markedly reduced blood glucose and lipid levels and improved pathological changes in pancreatic tissues of diabetic rats [13]. Type 2 diabetes mellitus is characterized by insulin resistance and pancreatic b-cell dysfunction [14]. In decompensated b-Cells, fasting blood glucose (FBG) was increased, further aggravating the pancreatic b-cell dysfunction. Uncontrolled pancreatic b-cell dysfunction leads to b-cell failure and the cessation of insulin secretion results in low hyperinsulinemia due to insulin resistance [15, 16]. In this study, serum FBG and FINS contents in diabetic model group were significantly decreased, indicating a severely impaired function of pancreatic b-cells. The impaired b-cells function might be closely associated with genetic and environmental factors, and was probably induced by oxidative stress. The declined antioxidant mechanisms elevated the oxidative stress and generated reactive oxygen species (ROS) [17, 18]. The results revealed that MDA level was significantly increased but the SOD and CAT activity was decreased in pancreatic tissues of the diabetic rats, suggesting that the increased ROS was due to the enhanced oxidative stress. The diminished activity of expression associated with antioxidant enzymes, such as SOD and GSH-Px in pancreatic
123
b-Cells, increases the damage by oxidative stress. High levels of ROS may cause large-scale non-specific oxidative damage including disruption of membrane integrity in pancreatic b-Cells, leading to apoptosis [17]. It also causes disorders in apoptosis-related gene expression, downregulates anti-apoptotic gene Bc1-2 expression, and upregulates pro-apoptotic genes Bad, Bid, and Bik [19, 20]. The TUNEL assay also demonstrated the increase in the rate of islet cell apoptosis and Bax/bcl-2 ratio, decrease in Bcl-2 expression of pancreatic tissue, no pronounced changes in Bax expression, and increase in the expression of apoptosis-related factor caspase 3 in the diabetic rats. Six weeks later, the blood glucose of rats in the oil-treated group returned to normal, the serum insulin levels were significantly increased, the MDA content was decreased, and the SOD and CAT activities were enhanced. The increase of apoptosis may be associated with the decreased expression of PDX-1-related protein. PDX-1 is a transcription factor expressed specifically in pancreatic b-Cells, and inhibits the apoptosis of pancreatic cells [21]. It is also a key transcription factor of insulin gene expression, and induces the differentiation of pancreatic b-Cells, promotes pancreatic the b-cell proliferation, and inhibits the apoptosis, playing a key role in the growth and function of the pancreatic b-Cells [22]. Studies demonstrated that in diabetic rats, the sensitivity of pancreatic b-Cells to apoptotic factors was increased. The activity of caspase 3 and pancreatic b-Cells apoptosis was increased. The anti-apoptotic Bcl-2 was abnormal. However, the number and volume of islets and the released insulin were decreased [21]. Sustained high blood sugar inhibited PDX1 gene expression. The transient spikes in blood sugar level promoted binding of PDX-1 and insulin gene in the pancreatic b-Cells, increased insulin mRNA levels, while the chronically elevated blood sugar level decreased both PDX-1 and insulin expressions. Alquobaili et al. [23] reported that sustained high blood sugar suppressed PDX-1 and insulin gene expressions, and inhibited the binding of PDX-1 protein and insulin gene promoters, thereby decreasing the synthesis and secretion of insulin. PDX-1 is also known to be a transcription factor for insulin releaserelated genes such as GLUT-2 and GCK. GLUT-2 is the major membrane transport protein that transports glucose into cells, and regulates synthesis and secretion of insulin in the glucose-stimulated islet cells. It is also one of the rate-limiting steps of glycolysis in pancreatic b-Cells. GCK is regulated by blood glucose, and is also the first ratelimiting enzyme in glucose metabolism. It stimulates the secretion of insulin and its activity determines the changes in glucose metabolism and insulin secretion, and regulates blood sugar levels. Thus, GCK plays an important role in maintaining glucose homeostasis [24]. GLUT-2 and GCK are important in glucose-stimulated GSIS, mediated by
Endocrine
Fig. 4 Western blotting analysis of the effects of Schisandrae chinensis oil on Bax, Bcl-2, Caspase3, PDX-1, GLUT-2, and GCK protein expression in rat pancreatic tissue. Expression and quantification of Bax, Bcl-2, Caspase3, PDX-1, GLUT-2, and GCK protein expression relative to the amount of actin; CON, control rat;
CON ? SCO, control rats treated with the oil 1 mg/kg; DM, diabetic rat; DM ? SCO, diabetic rats treated with Schisandrae chinensis oil 1 mg/kg. Representative bands are shown. Data are expressed as mean ± S.E.M. (n = 8). *P \ 0.05 versus CON, CON ? SC, DM ? SCO; #P \ 0.05 versus DM
PDX-1 binding to insulin gene promoter. The decrease in PDX-1 gene expression was associated with significantly downregulated GLUT-2 and GCK gene expression in our study. It reduced gene synthesis, resulting in a reduction of glucose-induced GSIS [25]. Chronically high blood sugar levels inhibit PDX-1 gene expression. Similar decreases in GLUT-2 expression, followed by downregulation of the PDX-1 and GCK expressions in pancreatic tissue of diabetic rats, were reported previously [26, 27]. The results showed that Schisandra chinensis oil increased the expression of PDX-1, GLUT-2, and GCK in diabetic rats to regulate glucose metabolism and promote insulin secretion. It is believed that Schisandra chinensis oil can enhance the antioxidant capacity of pancreas, upregulate the expression of anti-apoptotic gene, increase the expression
of glucose metabolism factors such as PDX-1, GLUT-2, and GCK, delay the apoptosis of islet cells, and further improve B cell functions. Acknowledgments This work was funded by project ‘‘2013-199’’ supported by the Education Department of Jilin Province, project ‘‘201262503’’ supported by Sci-tech Department of Jilin City and project ‘‘20122082’’ the Healthe Department of Jilin Province. Conflict of interest of interest.
The authors declare that they have no conflict
References 1. H. Sone, Y. Kagawa, Pancreatic beta cell senescence contributes to the pathogenesis of type 2 diabetes in high-fat diet-induced
123
Endocrine
2.
3.
4. 5. 6.
7.
8.
9.
10.
11.
12.
13.
14. 15.
diabetic mice. Diabetologia 48(1), 58–67 (2005). doi:10.1007/ s00125-004-1605-2 P. Marchetti, M. Bugliani, U. Boggi, M. Masini, L. Marselli, The pancreatic beta cells in human type 2 diabetes. Adv. Exp. Med. Biol. 771, 288–309 (2012) B. Thorens, The required beta cell research for improving treatment of type 2 diabetes. J. Intern. Med. 274(3), 203–214 (2013). doi:10.1111/joim.12096 M.O. Larsen, Beta-cell function and mass in type 2 diabetes. Dan. Med. Bull. 56(3), 153–164 (2009) C.P. Commission, China Pharmacopoeia. In vol. 1. (Chemical Industry Press, Beijing 2010), pp. 61–62 A. Panossian, G. Wikman, Pharmacology of Schisandra chinensis Bail.: an overview of Russian research and uses in medicine. J. Ethnopharmacol. 118(2), 183–212 (2008). doi:10.1016/j.jep. 2008.04.020 D.Y. Kwon, S. da Kim, H.J. Yang, S. Park, The lignan-rich fractions of Fructus Schisandrae improve insulin sensitivity via the PPAR-gamma pathways in in vitro and in vivo studies. J. Ethnopharmacol. 135(2), 455–462 (2011). doi:10.1016/j.jep. 2011.03.037 C. Wang, J. Li, X. Lv, M. Zhang, Y. Song, L. Chen, Y. Liu, Ameliorative effect of berberine on endothelial dysfunction in diabetic rats induced by high-fat diet and streptozotocin. Eur. J. Pharmacol. 620(1–3), 131–137 (2009). doi:10.1016/j.ejphar. 2009.07.027 A. Zywert, K. Szkudelska, T. Szkudelski, Inhibition of phosphodiesterase 3B in insulin-secreting cells of normal and streptozocin-nicotinamide-induced diabetic rats implications for insulin secretion. J. Physiol. Pharmacol. 65(3), 425–433 (2014) W. Xie, L. Du, Diabetes is an inflammatory disease: evidence from traditional Chinese medicines. Diabetes Obes. Metab. 13(4), 289–301 (2011). doi:10.1111/j.1463-1326.2010.01336.x K.F. Cai, Z.C. Qin, Y.L. Wang, Effects of Fructus Schisandrae oil on islet cell morphology and function of diabetic mice. Chin. J. Tradit. Med. Sci. Technol. 14(3), 177–178 (2007) K.F. Cai, Z.C. Qin, Y.L. Wang, Effects of Fructus Schisandrae oil on antioxidant and the expression of glucose transporter protein 4 mRNA of diabetic mice. Chin. Arch. Tradit. Chin. Med. 24(7), 1199–1200 (2006) L.P. An, Y.P. Wang, X.M. Liu, Effect of Fructus Schisandrae oil on blood glucose of type 2 diabetic rats induced by Streptozotocin. Chin. Tradit. Herb. Drugs 43(3), 52–556 (2012) J.H. Yki, Pathogenesis of noninsulin dependent diabetes mellitus. Lancet 343(8889), 91–95 (1994) W.H. Kim, J.W. Lee, Y.H. Suh, S.H. Hong, J.S. Choi, J.H. Lim, J.H. Song, B. Gao, M.H. Jung, Exposure to chronic high glucose
123
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
induces beta-cell apoptosis through decreased interaction of glucokinase with mitochondria: downregulation of glucokinase in pancreatic beta-cells. Diabetes 54(9), 2602–2611 (2005) M. Bensellam, D.R. Laybutt, J.C. Jonas, The molecular mechanisms of pancreatic beta-cell glucotoxicity: recent findings and future research directions. Mol. Cell. Endocrinol. 364(1–2), 1–27 (2012). doi:10.1016/j.mce.2012.08.003 T. van de Weijer, V.B. Schrauwen-Hinderling, P. Schrauwen, Lipotoxicity in type 2 diabetic cardiomyopathy. Cardiovasc. Res. 92(1), 10–18 (2011). doi:10.1093/cvr/cvr212 J.R. Zierath, A. Krook, H. Wallberg-Henriksson, Insulin action and insulin resistance in human skeletal muscle. Diabetologia 43(7), 821–835 (2000). doi:10.1007/s001250051457 U.K. Prospective Diabetes Study Group, U.K. prospective diabetes study 16. Overview of 6 years’ therapy of type II diabetes: a progressive disease. Diabetes 44(11), 1249–1258 (1995) G.C. Weir, D.R. Laybutt, H. Kaneto, S. Bonner-Weir, A. Sharma, Beta-cell adaptation and decompensation during the progression of diabetes. Diabetes 50(Suppl 1), S154–S159 (2001) J.D. Johnson, N.T. Ahmed, D.S. Luciani, Z. Han, H. Tran, J. Fujita, S. Misler, H. Edlund, K.S. Polonsky, Increased islet apoptosis in Pdx1 ± mice. J. Clin. Investig. 111(8), 1147–1160 (2003). doi:10.1172/JCI16537 W. Kim, J.M. Egan, The role of incretins in glucose homeostasis and diabetes treatment. Pharmacol. Rev. 60(4), 470–512 (2008). doi:10.1124/pr.108.000604 F. Al-Quobaili, M. Montenarh, Pancreatic duodenal homeobox factor-1 and diabetes mellitus type 2 (review). Int. J. Mol. Med. 21(4), 399–404 (2008) R. Singh, E.R. Pearson, P.M. Clark, A.T. Hattersley, The longterm impact on offspring of exposure to hyperglycaemia in utero due to maternal glucokinase gene mutations. Diabetologia 50(3), 620–624 (2007). doi:10.1007/s00125-006-0541-8 R.N. Kulkarni, U.S. Jhala, J.N. Winnay, S. Krajewski, M. Montminy, C.R. Kahn, PDX-1 haploinsufficiency limits the compensatory islet hyperplasia that occurs in response to insulin resistance. J. Clin. Investig. 114(6), 828–836 (2004). doi:10.1172/ JCI21845 H. Kaneto, T. Miyatsuka, D. Kawamori, T.A. Matsuoka, Pleiotropic roles of PDX-1 in the pancreas. Rev. Diabet. Stud. 4(4), 209–225 (2007). doi:10.1900/RDS.2007.4.209 H. Kaneto, T. Miyatsuka, T. Shiraiwa, K. Yamamoto, K. Kato, Y. Fujitani, T.A. Matsuoka, Crucial role of PDX-1 in pancreas development, beta-cell differentiation, and induction of surrogate beta-cells. Curr. Med. Chem. 14(16), 1745–1752 (2007)
