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Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats
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Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats Young Sook Kim, Dong Ho Jung, Eunjin Sohn, Yun Mi Lee, Chan-Sik Kim, Jin Sook Kim ∗ Korean Medicine-Based Herbal Drug Development Group, Herbal Medicine Research Division, Korea Institute of Oriental Medicine (KIOM), Daejeon 305-811, Republic of Korea
a r t i c l e
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Article history: Received 30 August 2013 Received in revised form 11 October 2013 Accepted 28 November 2013 Keywords: Cassiae semen Advanced glycation end products Diabetic nephropathy
a b s t r a c t Chronic hyperglycemia leads to the formation of advanced glycation end products (AGEs), which accelerates the development of diabetic complications. Previous studies have shown that extract of Cassiae semen (CS), the seed of Cassia tora, has inhibitory activity on AGEs formation in vitro and reduces transforming growth factor-beta1 (TGF-1) and extracellular matrix protein expression via inhibition of AGEs-mediated signaling in glomerular mesangial cells. In this study, to examine the preventive effects of CS extract on the development of diabetic nephropathy in vivo, streptozotocin (STZ)-injected diabetic rats were orally administered CS extract (200 mg/kg body weight/day) for 12 weeks. Serum glucose, triglycerides, and total cholesterol in diabetic rats were significantly higher compared to control rats. CS or aminoguanidine (AG) treatment significantly reduced these factors. Proteinuria and creatinine clearance were also significantly decreased in the CS-treated group compared with the untreated diabetic group. The CS-treated group had significantly inhibited COX-2 mRNA and protein, which mediates the symptoms of inflammation in the renal cortex of diabetic rats. Furthermore, histopathological studies of kidney tissue showed that in diabetic rats, AGEs, the receptor for AGEs, TGF-1, and collagen IV were suppressed by CS treatment. Our data suggest that oral treatment of CS can inhibit the development of diabetic nephropathy via inhibition of AGEs accumulation in STZ-induced diabetic rats. © 2013 Elsevier GmbH. All rights reserved.
Introduction Chronic hyperglycemia leads to macrovascular and microvascular diabetic complications, including accelerated atherosclerosis, diabetic retinopathy, neuropathy, and nephropathy (Vlassara et al., 1994; Yang et al., 1994). Advanced glycation end products (AGEs) have been implicated in the pathogenesis of diabetic complications, and the irreversible formation of AGEs promotes deposition of the extracellular matrix (ECM) such as collagen in the glomeruli via receptor for AGEs (RAGE) and abnormal glomerular remodeling in the kidney (Brownlee, 2005; Raptis and Viberti, 2001). Inhibitors of AGEs formation and cross-linking prevent the progression of diabetic nephropathy (Kim et al., 2011; Waanders et al., 2008). Inflammation plays a key role in the development and progression of diabetic nephropathy. AGEs increase inflammation and accelerate the formation of reactive oxygen species during normal aging, as well as in diabetic patients, and its restriction reduces insulin resistance and improves inflammation in type 2 diabetic
∗ Corresponding author. Tel.: +82 42 868 9465; fax: +82 42 868 9471. E-mail address: [email protected] (J.S. Kim).
patients (Uribarri et al., 2011). Cyclooxygenase-2 (COX-2), which shows anti-inflammatory properties via inhibition of its enzymatic activity, is increased in diabetic nephropathy. Its inhibition reduces proteinuria and glomerular injury in animal models of diabetes (Cheng et al., 2011). Transforming growth factor-beta1 (TGF-1), a multifunctional cytokine, is a key mediator of diabetic nephropathy and increases glomeruli expression of ECM proteins, such as collagen I, collagen IV, laminin, and fibronectin (Grande et al., 1997). These effects are blocked by TGF-1 neutralizing antibodies or antisense strategies (Sharma et al., 1996; Ziyadeh et al., 2000). AGEs induce the expression of TGF-1 and oxidative stress, inducing transcription factors such as nuclear transcription factor-kappa B (NF-B), which transcribe genes involved in glomerulosclerosis (Ohga et al., 2007). Natural products have been reportedly used for the treatment of diabetes and diabetic complications (Marles and Farnsworth, 1995). In traditional Korean herbal medicine, CS (Cassia tora seed, Cassia semen) is widely used to reduce inflammation in the liver, to improve eyesight, and to relax the bowels, and it is drunk as a roasted tea in Korea and China. CS also exhibits an antidiabetic effect related to the reduced levels of serum triglycerides and low-density lipoprotein cholesterol in diabetes patients (Cho
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Please cite this article in press as: Kim, Y.S., et al., Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.11.002
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et al., 2005; Guan and Zhao, 1995). CS has a neuroprotective effect on transient cerebral global ischemia in mice based on its antiinflammatory action (Kim et al., 2009). Recently, our laboratory reported that naphthopyrone glucosides, rubrofusarin-6-O--dgentiobioside, toralactone-9-O--d-gentiobioside, and cassiaside, can be isolated from butanol soluble extracts of CS, and that these compounds show more potent in vitro inhibitory activity against AGEs formation than the aminoguanidine (AG) positive control (Lee et al., 2006; Jung et al., 2010). Furthermore, CS extract and its major compound (rubrofusarin-6-O-beta-d-gentiobioside) inhibit TGF-1 and ECM protein expression in glomerular mesangial cells cultured under diabetic conditions (Jung et al., 2010). In this study, we examined whether CS could prevent the development of diabetic nephropathy in streptozotocin-induced diabetic rats. AGEs, RAGE, TGF-1, and ECM expression were examined. Furthermore, inflammation mediated factors were tested. Material and methods Plant materials and chemicals CS was purchased from a commercial supplier (Gyeongbuk, Korea, in January 2005) and identified by Professor J.H. Kim, Department of Life Science, Gachon University, Republic of Korea. A herbarium voucher specimen (no. KIOM-H021) has been deposited at the herbarium of the Diabetic Complications Research Team. CS extracts were prepared as previously described (Jung et al., 2010; Lee et al., 2006). Antibodies were purchased from Cell Signaling (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents were from Sigma–Aldrich (St. Louis, MO, USA). Reagents used for cell culture were from GIBCO-BRL (Grand Island, NY, USA). Animals and induction of diabetes All experiments were performed according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Korea Institute of Oriental Medicine Institutional Animal Care and Use Committee. Diabetes was induced by a single injection of streptozotocin (STZ, 60 mg/kg, intraperitoneally) in rats. Age-matched control rats (aged 6 weeks) received an equal volume of vehicle (0.01 M citrate buffer, pH 4.5). Two days after injection, blood glucose levels were measured from the tail vein. Rats with a blood glucose levels over 300 mg/dl were considered diabetes-induced rats. To investigate the effects of CS, treatment was begun one week after the onset of diabetes and the compound was orally administered to the rats once a day for 12 weeks. Animals were divided into five groups: (1) normal SD rats (N, n = 9), (2) normal SD rats treated with CS (N + CS, n = 9), (3) STZ-induced diabetic rats (DM, n = 9), (4) STZ-induced diabetic rats treated with CS (DM + CS, 100 mg/kg body weight, n = 9), and (5) STZ-induced diabetic rats treated with aminoguanidine, a positive control for AGEs inhibitor (DM + AG, 100 mg/kg body weight, n = 9).
RNA extraction and semi-quantitative RT-PCR analyses Total cellular RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA), quantified by measuring absorbance at 260 nm, and stored at −80 ◦ C until assayed. The expression of TGF-1 and fibronectin mRNAs was detected using RT-PCR analyses. The extracted RNA (1 g) was subjected to reverse transcriptase reactions with the Maxime RT premix (Intron, Daejeon, Korea) at 42 ◦ C for 60 min and 72 ◦ C for 10 min. Subsequently, semiquantitative PCR was performed with Accupower® PCR premix (iNtRON, Daejeon, Korea). The primer sequences were: rat COX1 (sense) 5 -ATG AGT CGA AGG CTC TC-3 , (anti-sense) 5 -TCC AGT ATC CGT GTG TCA GC-3 ; COX-2 (sense) 5 -ATG CTC TTC CGA GCT GTG C-3 , (anti-sense) 5 -ATC TCA TGG ATT GAA TTC GAA G-3 ; -actin (sense) 5 -GGA AAG ACA ACG GAC AAA TC-3 and (antisense) 5 -GCT GCT GGT TTC CAA GTT CA-3 . Aliquots of the PCR products were electrophoresed on 1.2% agarose gels and visualized after ethidium bromide staining.
Western blot analyses Aliquots of protein (100 g) were treated with Laemmli sample buffer (Bio-Rad, Hercules, CA), heated at 100 ◦ C for 5 min, and electrophoresed with 20 g/lane on a denaturing SDS–polyacrylamide gel. Proteins were transferred to nitrocellulose membranes (Whatman, GmbH, Hahnestr, Germany) using a Bio-Rad tank blotting apparatus (Bio-Rad). Membranes were probed with polyclonal antibodies against COX-1, COX-2, and -actin (1:1000 dilution; Santa Cruz Biotechnology). Membranes were washed and incubated with horseradish peroxidase-linked goat anti-rabbit IgG (Santa Cruz Biotechnology). After washing membranes three times, signals were detected using WEST-one ECL solution (iNtRON) and Fugifilm LAS-3000 (LAS-3000, Fuji Photo, Tokyo, Japan). The intensity of the bands was determined using multi-gauge v3.0 software.
Immunohistochemical staining Renal cortices were fixed in 10% formaldehyde and embedded in paraffin, and 4-m thick sections were prepared. Staining was performed as previously described (Kim et al., 2012). Antibodies used were rabbit anti-collagen IV (1:250, Santa Cruz, CA, USA), mouse anti-AGEs (Transgenic Inc. Kobe, Japan), rabbit antiRAGE (1:250, Santa Cruz), and rabbit anti-TGF-1 (1:200, Santa Cruz). For detection of collagen IV, AGEs, RAGE, and TGF-1, sections were incubated with the Envision kit (DAKO, Carpinteria, CA, USA) and visualized by 3,3 -diaminobenzidine tetrahydrochloride. Negative controls for immunohistochemistry were performed by incubating sections with nonimmune serum instead of primary antibodies. For morphometric analyses, positive cell numbers or positive signal areas per one glomerulus in a total of 40 glomeruli was determined using Image J software (NIH, Bethesda, MD, USA).
Metabolic and morphological analyses When the rats reached 18 weeks of age, blood glucose and HbA1c (A1C) were measured using an automated analyzer (Wako, Japan). Blood samples were collected from the tail vein after a 16-h fast. Individual rats were placed in metabolic cages to obtain 24-h urine collections, and urinary protein and albumin excretion levels were measured. Renal cortices were fixed in 10% formaldehyde and embedded in paraffin, and 4-m thick sections were prepared. Sections were stained with periodic acid-Schiff (PAS) reagent and hematoxylin as a counterstain.
Statistical analyses All experiments were repeated three times, and representative data are shown. Data are expressed as the mean ± S.E.M. Differences between groups were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests or unpaired Student’s t-tests using GraphPad Prism 5.0 software (San Diego, CA, USA). p < 0.05 was considered statistically significant.
Please cite this article in press as: Kim, Y.S., et al., Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.11.002
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∗∗
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proteinuria and creatinine clearance in the DM rats (Fig. 2A and B; p < 0.01). Administration of AG did not alter proteinuria. These results suggest that CS can effectively improve renal function in DM rats.
Blood glucose (mg/dl)
500 400
##
∗∗
Effect of CS on COX-1 and COX-2 expression in the renal cortex
#
300 200
N N+CS DM DM+CS DM+AG
100 0 6
3
8
10
12
14
16
To investigate the effect of CS on inflammation, we examined the mRNA and protein expression of COX-2 and COX-1 in the renal cortex. COX-1 mRNA and protein did not significantly change in any of the groups. COX-2 mRNA and protein were elevated in the renal cortex of diabetic rats (Fig. 3). When comparing the diabetic group and treated groups (CS or AG), there were significant differences in COX-2 mRNA or protein levels (Fig. 3C and D; # p < 0.01, ## p < 0.05).
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CS ameliorates renal histopathologic changes in glomeruli of STZ-induced diabetic rats
Weeks Fig. 1. Inhibitory effect of CS on blood glucose in STZ-diabetic rats. Groups: N: normal group; N + CS: normal group treated with CS (200 mg/kg); DM: STZinduced diabetic rat group; D + CS: STZ-induced diabetic rat group treated with CS (200 mg/kg); DM + AG: STZ-induced diabetic rat group treated with AG (50 mg/kg). Data are expressed as the mean ± S.E.M. **p < 0.01 compared with normal group, # p < 0.01, ## p < 0.05, compared with DM (n = 9, each group).
As shown in Fig. 1, the increased fasting blood glucose levels were significantly reduced in diabetic rats treated with CS (p < 0.01) or AG (p < 0.05) for 12 weeks. The effect of CS on HbA1c, total cholesterol, and triglyceride levels in control and experimental rats is shown in Table 1. Diabetic rats showed significant increases (p < 0.05) in the level of HbA1c, total cholesterol, and triglycerides when compared to normal control rats. Oral administration of CS to diabetic rats extensively reversed total cholesterol and triglyceride levels. However, the level of HbA1c was unchanged in diabetic rats treated with CS or AG compared to the diabetic group of rats. Table 1 shows the effect of CS on the activities of serum ALP, AST, and ALT in control and diabetic group of rats. ALP, AST, and ALT activities were increased significantly (*p < 0.05, **p < 0.01) in the serum from diabetic rats when compared to control rats. Oral treatment with CS or AG showed the activities of these enzymes were close to that of diabetic rats.
Consistent with the renal functional findings, diabetic kidneys showed significantly increased glomerulus size, with mesangial matrix expansion, mesangial cell proliferation, and capillary wall thickness. Renal histopathological examination using PAS staining demonstrated that compared with the control group, CS-treated diabetic rats displayed normal sized glomerulus without obvious mesangial matrix expansion, capillary collapse, and mesangial cell proliferation (Fig. 4Aa–e). Previous studies suggested CS possesses stronger inhibitory activity against AGEs formation in vitro than AG (Guan and Zhao, 1995; Jung et al., 2010; Lee et al., 2006). Therefore, to examine whether CS could inhibit AGEs and RAGE expression in vivo, we performed immunostaining with specific AGEs or RAGE antibodies in renal tissue. As shown in Fig. 4A, immunohistochemical staining of AGEs and RAGE in the glomeruli was significantly increased in diabetic rats compared with normal control rats. CS or AG treatment significantly decreased the expression of AGEs and RAGE (Fig. 4A, C, and D). To investigate the effect of CS on expression of ECM, we measured collagen IV and TGF-1 in renal tissue. Diabetic rats are associated with increased expression of collagen protein and TGF1. TGF-1, a key regulator of ECM expression, has been implicated in the pathogenesis of diabetic nephropathy. Collagen IV and TGF1 expression was decreased in diabetic rats treated with CS or AG (Fig. 4A, B and E).
CS improves renal function in STZ-induced diabetic rats
Discussion
In our experiments, proteinuria and creatinine clearance in the DM group rats was significantly higher than in the N group (p < 0.05), which suggested that the DM group had kidney damage. When compared to the DM group, CS significantly reduced
Diabetic nephropathy is one of the major microvascular complications of diabetes and is a leading cause end-stage renal disease. During the course of diabetes, hyperglycemia induced-the formation AGEs and RAGE, pro-inflammatory cytokine overexpression,
Results Effect of CS on functional parameters in blood
Table 1 Effect of CS on plasma function parameters. N (n = 9) HbA1c (%) HDL (mg/dl) LDL (md/dl) Total cholesterol (md/dl) Triglycerides (md/dl) ALP (U/L) AST (U/L) ALT (U/L)
3.89 17.7 7.42 65.6 114.4 441.1 214.2 87.7
± ± ± ± ± ± ± ±
N + CS (n = 9) 0.06 1.13 0.29 3.43 10.6 35.0 14.8 6.9
3.82 13.7 5.15 55.9 63.88 200.9 188.7 78.4
± ± ± ± ± ± ± ±
0.03 0.60 0.58 5.05 14.05 11.5 13.7 9.5
DM (n = 9) 6.89 24.5 6.30 87.5 475.3 1650 357.6 207.2
± ± ± ± ± ± ± ±
0.12* 1.95 1.81 14.3* 152.9* 158.8** 58.0* 21.4**
DM + CS (n = 9) 6.75 19.1 3.42 34.3 124.1 1204 389.8 233.5
± ± ± ± ± ± ± ±
0.11 1.80 0.48 5.0# 27.7# 215.0 46.9 33.5
DM + AG (n = 9) 6.84 21.7 5.43 54.6 81.71 1317 305.6 157.8
± ± ± ± ± ± ± ±
0.14 2.76 1.0 7.53# 21.43# 184.6 28.6 15.7
Groups: N: normal group; N + CS: normal group treated with CS (200 mg/kg); DM: STZ-induced diabetic rat group; D + CS: STZ-induced diabetic rat group treated with CS (200 mg/kg); DM + AG: STZ-induced diabetic rat group treated with AG (50 mg/kg). Values are the mean ± S.E.M. * p < 0.05 compared with the control group. ** p < 0.01 compared with the control group. # p < 0.05 compared with DM.
Please cite this article in press as: Kim, Y.S., et al., Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.11.002
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(B)
3000
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1500 1000 500 0
Creatinine clearance (ml/min/kg)
Proteinuria (mg/kg/day)
(A)
∗
10 8
∗∗
6
∗∗
4 2 0
N
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DM
DM+CS DM+AG
N
N+CS
DM
DM+CS DM+AG
Fig. 2. Effect of CS on proteinuria and creatinine clearance in STZ-diabetic rats. CS (200 mg/kg) and AG (50 mg/kg) were provided 12 weeks after STZ treatment. Data are expressed as the mean ± S.E.M. (A) Proteinuria *p < 0.001 vs. N; **p < 0.05 vs. DM. (B) Creatinine clearance *p < 0.001 vs. N; **p < 0.05 vs. DM.
and oxidative stress activation play major role in kidney damage (Brownlee, 2005). Inhibition of AGEs formation, down-regulation of RAGE expression, or blockade of RAGE downstream signaling are potentially therapeutic avenues in diabetic nephropathy (Sakurai et al., 2003; Yamamoto et al., 2007). CS inhibits AGEs formation. CS and its major compound (rubrofusarin-6-O-beta-d-gentiobioside) decrease TGF-1 and fibronectin expression, as well as NF-B DNA binding activity, in mouse mesangial cells (Lee et al., 2006; Sharma et al., 1996). In the present study, we first investigated the
protective effects of CS on diabetic nephropathy in a type 1 diabetic animal model. CS-treated diabetic rats exhibited significantly ameliorated levels of blood glucose, proteinuria, and creatinine clearance. Kidney tissue expression of COX-2, AGEs, RAGE, TGF-1, and collagen IV was significantly reduced upon CS treatment. STZ-induced diabetic rats have been described as a useful experimental model to study the preventive effect of diabetic nephropathy because the selective destruction of pancreatic -cells by STZ leads to the poor sensitivity of insulin for glucose uptake
Fig. 3. Effect of CS on COX-1 and COX-2 expression in the renal cortex of STZ-diabetic rats. Expression of COX-1 and COX-2 mRNA (A, C) was quantified using RT-PCR (n = 5) and COX-2 protein expression (B, D) was evaluated using western blot analyses (n = 3). Data are expressed as the mean ± S.E.M.
Please cite this article in press as: Kim, Y.S., et al., Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.11.002
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Fig. 4. Effect of CS on histopathological changes in the kidney. (A) PAS staining (a–e) and immunohistochemical staining of collagen IV (f–j), AGEs (k–o), RAGE (p–t), and TGF-1 (u–y) in the kidney. Glomeruli of normal rats (a, f, k, p, and u), normal treated with CS (200 mg/kg; b, g, l, q, and v), diabetic rats (c, h, m, r, and w), diabetic rats treated with CS (200 mg/kg; d, i, n, s, and x), and diabetic rats treated with AG (50 mg/kg; e, j, o, t, y). Hematoxylin counterstain. Original magnification, 400×. The average score of 60 randomly selected glomeruli was calculated. Data are expressed as the mean ± S.E.M. (B) Quantitative analyses of collagen IV in glomeruli. *p < 0.001 vs. N; **p < 0.001 vs. DM; # p < 0.01 vs. DM. (C) AGEs, *p < 0.001 vs. N; **p < 0.001 vs. DM. (D) RAGE, *p < 0.001 vs. N; **p < 0.001 vs. DM. (E) TGF-1, *p < 0.001 vs. N; **p < 0.001 vs. DM.
(Burns and Gold, 2007). Increased blood glucose levels and AGEs formation are involved in the development of diabetic nephropathy (Brownlee, 2005). Although the CS- or AG-treated diabetic groups did not have altered blood glucose levels at 10 weeks, these levels were significantly decreased at 12 weeks (Fig. 1). The increased fasting blood glucose levels in at least 12 h fasted rats were significantly reduced in diabetic rats treated with CS or AG for 12 weeks (Fig. 1). This effect on blood glucose control is reflected in the HbA1c levels that did not decrease in CS- or AG-treated groups compared with the untreated diabetic group (Table 1). Diabetic rats had significantly increased plasma levels of plasma glucose, total cholesterol, triglycerides, low density lipoprotein (LDL)-cholesterol, very lowdensity lipoprotein (VLDL)-cholesterol, and HbA1c, but decreased plasma high-density lipoprotein (HDL)-cholesterol and insulin levels (Saravanan and Ramachandran, 2013). Although Hb1Ac was not decreased in CS- or AG-treated rats, total cholesterol and triglyceride levels were significantly decreased in these animals. Liver ALP, AST, and ALT enzymes are used as indices of liver damage. Increased AST and ALT activities indicates that diabetes likely induces hepatic dysfunction. Therefore, increased AST and ALT activities in the liver tissue are predominantly indicative of defective glucose utilization (Murali et al., 2013). In the present study, significantly higher levels
of ALP, AST, and ALT were observed in STZ-induced diabetic rats whereas the CS-treated diabetic group were unchanged (Table 1). COX-2 expression is dramatically upregulated during inflammation, but COX-1 displays the characteristics of a housekeeping gene that is constitutively expressed in almost all tissues (Crofford, 1997). Overexpression of COX-2 mRNA and protein was significantly reduced in CS- or AG-treated diabetic groups compared with the untreated diabetic group (Fig. 3C and D). A previous study suggests that overexpression of COX-2 in podocytes exacerbates diabetic nephropathy and treatment with a COX-2 inhibitor can reduce albuminuria and mesangial matrix expansion (Cheng et al., 2011). Because CS has been shown to have inhibitory effects on the formation of AGEs, we tested whether CS can inhibit AGEs accumulation in the glomeruli of diabetic rats. As shown as Fig. 4A, CSor AG-treated diabetic rats had significantly decreased AGEs and RAGE expression in the kidney. A number of AGEs inhibitors such as pyridoxamine, aminoguanidine, and LR-90 prevent the development of complications (Degenhardt et al., 2002; Figarola et al., 2003; Kim et al., 2007). KIOM-79, an inhibitor of AGEs protein crosslinking, prevents progression of nephropathy in type 1 and type 2 diabetes animal models (Kim et al., 2007; Lee et al., 2006). New
Please cite this article in press as: Kim, Y.S., et al., Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.11.002
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therapies, including those targeting the accumulation of AGEs, ROS generation, glycosaminoglycan sulodexide, and a protein kinase C inhibitor, are likely to be involved in the future treatment of diabetic nephropathy (Goh et al., 2008). In conclusion, our work demonstrates the preventive effect of CS was achieved via inhibition of AGEs accumulation and RAGE expression in experimental models of diabetic nephropathy. Furthermore, CS treatment inhibited COX-2 expression in the renal cortex of STZ-diabetic rats. Conflict of interest statement The authors declare no conflicts of interest. Acknowledgements This research was supported by grants [K11040 and K12040] from the Korea Institute of Oriental Medicine (KIOM). References Brownlee, M., 2005. The pathobiology of diabetic complications: a unifying mechanism. Diabetes 54, 1615–1625. Burns, N., Gold, B., 2007. The effect of 3-methyladenine DNA glycosylase-mediated DNA repair on the induction of toxicity and diabetes by the beta-cell toxicant streptozotocin. Toxicological Sciences: An Official Journal of the Society of Toxicology 95, 391–400. Cheng, H., Fan, X., Moeckel, G.W., Harris, R.C., 2011. Podocyte COX-2 exacerbates diabetic nephropathy by increasing podocyte (pro)renin receptor expression. Journal of the American Society of Nephrology: JASN 22, 1240–1251. Cho, S.H., Kim, T.H., Lee, N.H., Son, H.S., Cho, I.J., Ha, T.Y., 2005. Effects of Cassia tora fiber supplement on serum lipids in Korean diabetic patients. Journal of Medicinal Food 8, 311–318. Crofford, L.J., 1997. COX-1 and COX-2 tissue expression: implications and predictions. Journal of Rheumatology Suppl. 49, 15–19. Degenhardt, T.P., Alderson, N.L., Arrington, D.D., Beattie, R.J., Basgen, J.M., Steffes, M.W., Thorpe, S.R., Baynes, J.W., 2002. Pyridoxamine inhibits early renal disease and dyslipidemia in the streptozotocin-diabetic rat. Kidney International 61, 939–950. Figarola, J.L., Scott, S., Loera, S., Tessler, C., Chu, P., Weiss, L., Hardy, J., Rahbar, S., 2003. LR-90 a new advanced glycation endproduct inhibitor prevents progression of diabetic nephropathy in streptozotocin-diabetic rats. Diabetologia 46, 1140–1152. Goh, S.Y., Jasik, M., Cooper, M.E., 2008. Agents in development for the treatment of diabetic nephropathy. Expert Opinion on Emerging Drugs 13, 447–463. Grande, J.P., Melder, D.C., Zinsmeister, A.R., 1997. Modulation of collagen gene expression by cytokines: stimulatory effect of transforming growth factorbeta1, with divergent effects of epidermal growth factor and tumor necrosis factor-alpha on collagen type I and collagen type IV. Journal of Laboratory and Clinical Medicine 130, 476–486. Guan, Y., Zhao, S., 1995. Yishou jiangzhi (de-blood-lipid) tablets in the treatment of hyperlipemia. Journal of Traditional Chinese Medicine 15, 178–179. Jung, D.H., Kim, Y.S., Kim, N.H., Lee, J., Jang, D.S., Kim, J.S., 2010. Extract of Cassiae semen and its major compound inhibit S100b-induced TGF-beta1 and fibronectin expression in mouse glomerular mesangial cells. European Journal of Pharmacology 641, 7–14. Kim, C.S., Sohn, E.J., Kim, Y.S., Jung, D.H., Jang, D.S., Lee, Y.M., Kim, D.H., Kim, J.S., 2007. Effects of KIOM-79 on hyperglycemia and diabetic nephropathy in type 2 diabetic Goto-Kakizaki rats. Journal of Ethnopharmacology 111, 240–247.
Kim, D.H., Kim, S., Jung, W.Y., Park, S.J., Park, D.H., Kim, J.M., Cheong, J.H., Ryu, J.H., 2009. The neuroprotective effects of the seeds of Cassia obtusifolia on transient cerebral global ischemia in mice. Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association 47, 1473–1479. Kim, Y.S., Jung, D.H., Sohn, E., Kim, J., Kim, J.S., 2012. Glycoxidised LDL induced the upregulation of Axl receptor tyrosine kinase and its ligand in mouse mesangial cells. PloS ONE 7, e50297. Kim, Y.S., Kim, J., Kim, C.S., Sohn, E.J., Lee, Y.M., Jeong, I.H., Kim, H., Jang, D.S., Kim, J.S., 2011. KIOM-79, an inhibitor of AGEs-protein cross-linking, prevents progression of nephropathy in Zucker diabetic fatty rats. Evidence-based Complementary and Alternative Medicine: eCAM 2011, 761859. Lee, G.Y., Jang, D.S., Lee, Y.M., Kim, J.M., Kim, J.S., 2006. Naphthopyrone glucosides from the seeds of Cassia tora with inhibitory activity on advanced glycation end products (AGEs) formation. Archives of Pharmacal Research 29, 587–590. Marles, R.J., Farnsworth, N.R., 1995. Antidiabetic plants and their active constituents. Phytomedicine: International Journal of Phytotherapy and Phytopharmacology 2, 137–189. Murali, R., Srinivasan, S., Ashokkumar, N., 2013. Antihyperglycemic effect of fraxetin on hepatic key enzymes of carbohydrate metabolism in streptozotocin-induced diabetic rats. Biochimie. Ohga, S., Shikata, K., Yozai, K., Okada, S., Ogawa, D., Usui, H., Wada, J., Shikata, Y., Makino, H., 2007. Thiazolidinedione ameliorates renal injury in experimental diabetic rats through anti-inflammatory effects mediated by inhibition of NF-kappaB activation. American Journal of Physiology. Renal Physiology 292, F1141–F1150. Raptis, A.E., Viberti, G., 2001. Pathogenesis of diabetic nephropathy. Experimental and Clinical Endocrinology & Diabetes: Official Journal, German Society of Endocrinology [and] German Diabetes Association 109 (Suppl. 2), S424–S437. Sakurai, S., Yonekura, H., Yamamoto, Y., Watanabe, T., Tanaka, N., Li, H., Rahman, A.K., Myint, K.M., Kim, C.H., Yamamoto, H., 2003. The AGE-RAGE system and diabetic nephropathy. Journal of the American Society of Nephrology: JASN 14, S259–S263. Saravanan, R., Ramachandran, V., 2013. Modulating efficacy of rebaudioside A, a diterpenoid on antioxidant and circulatory lipids in experimental diabetic rats. Environmental toxicology and pharmacology 36, 472–483. Sharma, K., Jin, Y., Guo, J., Ziyadeh, F.N., 1996. Neutralization of TGF-beta by anti-TGF-beta antibody attenuates kidney hypertrophy and the enhanced extracellular matrix gene expression in STZ-induced diabetic mice. Diabetes 45, 522–530. Uribarri, J., Cai, W., Ramdas, M., Goodman, S., Pyzik, R., Chen, X., Zhu, L., Striker, G.E., Vlassara, H., 2011. Restriction of advanced glycation end products improves insulin resistance in human type 2 diabetes: potential role of AGER1 and SIRT1. Diabetes Care 34, 1610–1616. Vlassara, H., Bucala, R., Striker, L., 1994. Pathogenic effects of advanced glycosylation: biochemical, biologic, and clinical implications for diabetes and aging. Laboratory Investigation: A Journal of Technical Methods and Pathology 70, 138–151. Waanders, F., van Goor, H., Navis, G., 2008. Adverse renal effects of the AGE inhibitor pyridoxamine in combination with ACEi in non-diabetic adriamycin-induced renal damage in rats. Kidney & Blood Pressure Research 31, 350–359. Yamamoto, H., Watanabe, T., Yamamoto, Y., Yonekura, H., Munesue, S., Harashima, A., Ooe, K., Hossain, S., Saito, H., Murakami, N., 2007. RAGE in diabetic nephropathy. Current Molecular Medicine 7, 752–757. Yang, C.W., Vlassara, H., Peten, E.P., He, C.J., Striker, G.E., Striker, L.J., 1994. Advanced glycation end products up-regulate gene expression found in diabetic glomerular disease. Proceedings of the National Academy of Sciences of the United States of America 91, 9436–9440. Ziyadeh, F.N., Hoffman, B.B., Han, D.C., Iglesias-De La Cruz, M.C., Hong, S.W., Isono, M., Chen, S., McGowan, T.A., Sharma, K., 2000. Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice. Proceedings of the National Academy of Sciences of the United States of America 97, 8015–8020.
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Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats Young Sook Kim, Dong Ho Jung, Eunjin Sohn, Yun Mi Lee, Chan-Sik Kim, Jin Sook Kim ∗ Korean Medicine-Based Herbal Drug Development Group, Herbal Medicine Research Division, Korea Institute of Oriental Medicine (KIOM), Daejeon 305-811, Republic of Korea
a r t i c l e
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Article history: Received 30 August 2013 Received in revised form 11 October 2013 Accepted 28 November 2013 Keywords: Cassiae semen Advanced glycation end products Diabetic nephropathy
a b s t r a c t Chronic hyperglycemia leads to the formation of advanced glycation end products (AGEs), which accelerates the development of diabetic complications. Previous studies have shown that extract of Cassiae semen (CS), the seed of Cassia tora, has inhibitory activity on AGEs formation in vitro and reduces transforming growth factor-beta1 (TGF-1) and extracellular matrix protein expression via inhibition of AGEs-mediated signaling in glomerular mesangial cells. In this study, to examine the preventive effects of CS extract on the development of diabetic nephropathy in vivo, streptozotocin (STZ)-injected diabetic rats were orally administered CS extract (200 mg/kg body weight/day) for 12 weeks. Serum glucose, triglycerides, and total cholesterol in diabetic rats were significantly higher compared to control rats. CS or aminoguanidine (AG) treatment significantly reduced these factors. Proteinuria and creatinine clearance were also significantly decreased in the CS-treated group compared with the untreated diabetic group. The CS-treated group had significantly inhibited COX-2 mRNA and protein, which mediates the symptoms of inflammation in the renal cortex of diabetic rats. Furthermore, histopathological studies of kidney tissue showed that in diabetic rats, AGEs, the receptor for AGEs, TGF-1, and collagen IV were suppressed by CS treatment. Our data suggest that oral treatment of CS can inhibit the development of diabetic nephropathy via inhibition of AGEs accumulation in STZ-induced diabetic rats. © 2013 Elsevier GmbH. All rights reserved.
Introduction Chronic hyperglycemia leads to macrovascular and microvascular diabetic complications, including accelerated atherosclerosis, diabetic retinopathy, neuropathy, and nephropathy (Vlassara et al., 1994; Yang et al., 1994). Advanced glycation end products (AGEs) have been implicated in the pathogenesis of diabetic complications, and the irreversible formation of AGEs promotes deposition of the extracellular matrix (ECM) such as collagen in the glomeruli via receptor for AGEs (RAGE) and abnormal glomerular remodeling in the kidney (Brownlee, 2005; Raptis and Viberti, 2001). Inhibitors of AGEs formation and cross-linking prevent the progression of diabetic nephropathy (Kim et al., 2011; Waanders et al., 2008). Inflammation plays a key role in the development and progression of diabetic nephropathy. AGEs increase inflammation and accelerate the formation of reactive oxygen species during normal aging, as well as in diabetic patients, and its restriction reduces insulin resistance and improves inflammation in type 2 diabetic
∗ Corresponding author. Tel.: +82 42 868 9465; fax: +82 42 868 9471. E-mail address: [email protected] (J.S. Kim).
patients (Uribarri et al., 2011). Cyclooxygenase-2 (COX-2), which shows anti-inflammatory properties via inhibition of its enzymatic activity, is increased in diabetic nephropathy. Its inhibition reduces proteinuria and glomerular injury in animal models of diabetes (Cheng et al., 2011). Transforming growth factor-beta1 (TGF-1), a multifunctional cytokine, is a key mediator of diabetic nephropathy and increases glomeruli expression of ECM proteins, such as collagen I, collagen IV, laminin, and fibronectin (Grande et al., 1997). These effects are blocked by TGF-1 neutralizing antibodies or antisense strategies (Sharma et al., 1996; Ziyadeh et al., 2000). AGEs induce the expression of TGF-1 and oxidative stress, inducing transcription factors such as nuclear transcription factor-kappa B (NF-B), which transcribe genes involved in glomerulosclerosis (Ohga et al., 2007). Natural products have been reportedly used for the treatment of diabetes and diabetic complications (Marles and Farnsworth, 1995). In traditional Korean herbal medicine, CS (Cassia tora seed, Cassia semen) is widely used to reduce inflammation in the liver, to improve eyesight, and to relax the bowels, and it is drunk as a roasted tea in Korea and China. CS also exhibits an antidiabetic effect related to the reduced levels of serum triglycerides and low-density lipoprotein cholesterol in diabetes patients (Cho
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Please cite this article in press as: Kim, Y.S., et al., Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.11.002
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et al., 2005; Guan and Zhao, 1995). CS has a neuroprotective effect on transient cerebral global ischemia in mice based on its antiinflammatory action (Kim et al., 2009). Recently, our laboratory reported that naphthopyrone glucosides, rubrofusarin-6-O--dgentiobioside, toralactone-9-O--d-gentiobioside, and cassiaside, can be isolated from butanol soluble extracts of CS, and that these compounds show more potent in vitro inhibitory activity against AGEs formation than the aminoguanidine (AG) positive control (Lee et al., 2006; Jung et al., 2010). Furthermore, CS extract and its major compound (rubrofusarin-6-O-beta-d-gentiobioside) inhibit TGF-1 and ECM protein expression in glomerular mesangial cells cultured under diabetic conditions (Jung et al., 2010). In this study, we examined whether CS could prevent the development of diabetic nephropathy in streptozotocin-induced diabetic rats. AGEs, RAGE, TGF-1, and ECM expression were examined. Furthermore, inflammation mediated factors were tested. Material and methods Plant materials and chemicals CS was purchased from a commercial supplier (Gyeongbuk, Korea, in January 2005) and identified by Professor J.H. Kim, Department of Life Science, Gachon University, Republic of Korea. A herbarium voucher specimen (no. KIOM-H021) has been deposited at the herbarium of the Diabetic Complications Research Team. CS extracts were prepared as previously described (Jung et al., 2010; Lee et al., 2006). Antibodies were purchased from Cell Signaling (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents were from Sigma–Aldrich (St. Louis, MO, USA). Reagents used for cell culture were from GIBCO-BRL (Grand Island, NY, USA). Animals and induction of diabetes All experiments were performed according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Korea Institute of Oriental Medicine Institutional Animal Care and Use Committee. Diabetes was induced by a single injection of streptozotocin (STZ, 60 mg/kg, intraperitoneally) in rats. Age-matched control rats (aged 6 weeks) received an equal volume of vehicle (0.01 M citrate buffer, pH 4.5). Two days after injection, blood glucose levels were measured from the tail vein. Rats with a blood glucose levels over 300 mg/dl were considered diabetes-induced rats. To investigate the effects of CS, treatment was begun one week after the onset of diabetes and the compound was orally administered to the rats once a day for 12 weeks. Animals were divided into five groups: (1) normal SD rats (N, n = 9), (2) normal SD rats treated with CS (N + CS, n = 9), (3) STZ-induced diabetic rats (DM, n = 9), (4) STZ-induced diabetic rats treated with CS (DM + CS, 100 mg/kg body weight, n = 9), and (5) STZ-induced diabetic rats treated with aminoguanidine, a positive control for AGEs inhibitor (DM + AG, 100 mg/kg body weight, n = 9).
RNA extraction and semi-quantitative RT-PCR analyses Total cellular RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA), quantified by measuring absorbance at 260 nm, and stored at −80 ◦ C until assayed. The expression of TGF-1 and fibronectin mRNAs was detected using RT-PCR analyses. The extracted RNA (1 g) was subjected to reverse transcriptase reactions with the Maxime RT premix (Intron, Daejeon, Korea) at 42 ◦ C for 60 min and 72 ◦ C for 10 min. Subsequently, semiquantitative PCR was performed with Accupower® PCR premix (iNtRON, Daejeon, Korea). The primer sequences were: rat COX1 (sense) 5 -ATG AGT CGA AGG CTC TC-3 , (anti-sense) 5 -TCC AGT ATC CGT GTG TCA GC-3 ; COX-2 (sense) 5 -ATG CTC TTC CGA GCT GTG C-3 , (anti-sense) 5 -ATC TCA TGG ATT GAA TTC GAA G-3 ; -actin (sense) 5 -GGA AAG ACA ACG GAC AAA TC-3 and (antisense) 5 -GCT GCT GGT TTC CAA GTT CA-3 . Aliquots of the PCR products were electrophoresed on 1.2% agarose gels and visualized after ethidium bromide staining.
Western blot analyses Aliquots of protein (100 g) were treated with Laemmli sample buffer (Bio-Rad, Hercules, CA), heated at 100 ◦ C for 5 min, and electrophoresed with 20 g/lane on a denaturing SDS–polyacrylamide gel. Proteins were transferred to nitrocellulose membranes (Whatman, GmbH, Hahnestr, Germany) using a Bio-Rad tank blotting apparatus (Bio-Rad). Membranes were probed with polyclonal antibodies against COX-1, COX-2, and -actin (1:1000 dilution; Santa Cruz Biotechnology). Membranes were washed and incubated with horseradish peroxidase-linked goat anti-rabbit IgG (Santa Cruz Biotechnology). After washing membranes three times, signals were detected using WEST-one ECL solution (iNtRON) and Fugifilm LAS-3000 (LAS-3000, Fuji Photo, Tokyo, Japan). The intensity of the bands was determined using multi-gauge v3.0 software.
Immunohistochemical staining Renal cortices were fixed in 10% formaldehyde and embedded in paraffin, and 4-m thick sections were prepared. Staining was performed as previously described (Kim et al., 2012). Antibodies used were rabbit anti-collagen IV (1:250, Santa Cruz, CA, USA), mouse anti-AGEs (Transgenic Inc. Kobe, Japan), rabbit antiRAGE (1:250, Santa Cruz), and rabbit anti-TGF-1 (1:200, Santa Cruz). For detection of collagen IV, AGEs, RAGE, and TGF-1, sections were incubated with the Envision kit (DAKO, Carpinteria, CA, USA) and visualized by 3,3 -diaminobenzidine tetrahydrochloride. Negative controls for immunohistochemistry were performed by incubating sections with nonimmune serum instead of primary antibodies. For morphometric analyses, positive cell numbers or positive signal areas per one glomerulus in a total of 40 glomeruli was determined using Image J software (NIH, Bethesda, MD, USA).
Metabolic and morphological analyses When the rats reached 18 weeks of age, blood glucose and HbA1c (A1C) were measured using an automated analyzer (Wako, Japan). Blood samples were collected from the tail vein after a 16-h fast. Individual rats were placed in metabolic cages to obtain 24-h urine collections, and urinary protein and albumin excretion levels were measured. Renal cortices were fixed in 10% formaldehyde and embedded in paraffin, and 4-m thick sections were prepared. Sections were stained with periodic acid-Schiff (PAS) reagent and hematoxylin as a counterstain.
Statistical analyses All experiments were repeated three times, and representative data are shown. Data are expressed as the mean ± S.E.M. Differences between groups were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests or unpaired Student’s t-tests using GraphPad Prism 5.0 software (San Diego, CA, USA). p < 0.05 was considered statistically significant.
Please cite this article in press as: Kim, Y.S., et al., Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.11.002
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proteinuria and creatinine clearance in the DM rats (Fig. 2A and B; p < 0.01). Administration of AG did not alter proteinuria. These results suggest that CS can effectively improve renal function in DM rats.
Blood glucose (mg/dl)
500 400
##
∗∗
Effect of CS on COX-1 and COX-2 expression in the renal cortex
#
300 200
N N+CS DM DM+CS DM+AG
100 0 6
3
8
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12
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To investigate the effect of CS on inflammation, we examined the mRNA and protein expression of COX-2 and COX-1 in the renal cortex. COX-1 mRNA and protein did not significantly change in any of the groups. COX-2 mRNA and protein were elevated in the renal cortex of diabetic rats (Fig. 3). When comparing the diabetic group and treated groups (CS or AG), there were significant differences in COX-2 mRNA or protein levels (Fig. 3C and D; # p < 0.01, ## p < 0.05).
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CS ameliorates renal histopathologic changes in glomeruli of STZ-induced diabetic rats
Weeks Fig. 1. Inhibitory effect of CS on blood glucose in STZ-diabetic rats. Groups: N: normal group; N + CS: normal group treated with CS (200 mg/kg); DM: STZinduced diabetic rat group; D + CS: STZ-induced diabetic rat group treated with CS (200 mg/kg); DM + AG: STZ-induced diabetic rat group treated with AG (50 mg/kg). Data are expressed as the mean ± S.E.M. **p < 0.01 compared with normal group, # p < 0.01, ## p < 0.05, compared with DM (n = 9, each group).
As shown in Fig. 1, the increased fasting blood glucose levels were significantly reduced in diabetic rats treated with CS (p < 0.01) or AG (p < 0.05) for 12 weeks. The effect of CS on HbA1c, total cholesterol, and triglyceride levels in control and experimental rats is shown in Table 1. Diabetic rats showed significant increases (p < 0.05) in the level of HbA1c, total cholesterol, and triglycerides when compared to normal control rats. Oral administration of CS to diabetic rats extensively reversed total cholesterol and triglyceride levels. However, the level of HbA1c was unchanged in diabetic rats treated with CS or AG compared to the diabetic group of rats. Table 1 shows the effect of CS on the activities of serum ALP, AST, and ALT in control and diabetic group of rats. ALP, AST, and ALT activities were increased significantly (*p < 0.05, **p < 0.01) in the serum from diabetic rats when compared to control rats. Oral treatment with CS or AG showed the activities of these enzymes were close to that of diabetic rats.
Consistent with the renal functional findings, diabetic kidneys showed significantly increased glomerulus size, with mesangial matrix expansion, mesangial cell proliferation, and capillary wall thickness. Renal histopathological examination using PAS staining demonstrated that compared with the control group, CS-treated diabetic rats displayed normal sized glomerulus without obvious mesangial matrix expansion, capillary collapse, and mesangial cell proliferation (Fig. 4Aa–e). Previous studies suggested CS possesses stronger inhibitory activity against AGEs formation in vitro than AG (Guan and Zhao, 1995; Jung et al., 2010; Lee et al., 2006). Therefore, to examine whether CS could inhibit AGEs and RAGE expression in vivo, we performed immunostaining with specific AGEs or RAGE antibodies in renal tissue. As shown in Fig. 4A, immunohistochemical staining of AGEs and RAGE in the glomeruli was significantly increased in diabetic rats compared with normal control rats. CS or AG treatment significantly decreased the expression of AGEs and RAGE (Fig. 4A, C, and D). To investigate the effect of CS on expression of ECM, we measured collagen IV and TGF-1 in renal tissue. Diabetic rats are associated with increased expression of collagen protein and TGF1. TGF-1, a key regulator of ECM expression, has been implicated in the pathogenesis of diabetic nephropathy. Collagen IV and TGF1 expression was decreased in diabetic rats treated with CS or AG (Fig. 4A, B and E).
CS improves renal function in STZ-induced diabetic rats
Discussion
In our experiments, proteinuria and creatinine clearance in the DM group rats was significantly higher than in the N group (p < 0.05), which suggested that the DM group had kidney damage. When compared to the DM group, CS significantly reduced
Diabetic nephropathy is one of the major microvascular complications of diabetes and is a leading cause end-stage renal disease. During the course of diabetes, hyperglycemia induced-the formation AGEs and RAGE, pro-inflammatory cytokine overexpression,
Results Effect of CS on functional parameters in blood
Table 1 Effect of CS on plasma function parameters. N (n = 9) HbA1c (%) HDL (mg/dl) LDL (md/dl) Total cholesterol (md/dl) Triglycerides (md/dl) ALP (U/L) AST (U/L) ALT (U/L)
3.89 17.7 7.42 65.6 114.4 441.1 214.2 87.7
± ± ± ± ± ± ± ±
N + CS (n = 9) 0.06 1.13 0.29 3.43 10.6 35.0 14.8 6.9
3.82 13.7 5.15 55.9 63.88 200.9 188.7 78.4
± ± ± ± ± ± ± ±
0.03 0.60 0.58 5.05 14.05 11.5 13.7 9.5
DM (n = 9) 6.89 24.5 6.30 87.5 475.3 1650 357.6 207.2
± ± ± ± ± ± ± ±
0.12* 1.95 1.81 14.3* 152.9* 158.8** 58.0* 21.4**
DM + CS (n = 9) 6.75 19.1 3.42 34.3 124.1 1204 389.8 233.5
± ± ± ± ± ± ± ±
0.11 1.80 0.48 5.0# 27.7# 215.0 46.9 33.5
DM + AG (n = 9) 6.84 21.7 5.43 54.6 81.71 1317 305.6 157.8
± ± ± ± ± ± ± ±
0.14 2.76 1.0 7.53# 21.43# 184.6 28.6 15.7
Groups: N: normal group; N + CS: normal group treated with CS (200 mg/kg); DM: STZ-induced diabetic rat group; D + CS: STZ-induced diabetic rat group treated with CS (200 mg/kg); DM + AG: STZ-induced diabetic rat group treated with AG (50 mg/kg). Values are the mean ± S.E.M. * p < 0.05 compared with the control group. ** p < 0.01 compared with the control group. # p < 0.05 compared with DM.
Please cite this article in press as: Kim, Y.S., et al., Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.11.002
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(B)
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Creatinine clearance (ml/min/kg)
Proteinuria (mg/kg/day)
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Fig. 2. Effect of CS on proteinuria and creatinine clearance in STZ-diabetic rats. CS (200 mg/kg) and AG (50 mg/kg) were provided 12 weeks after STZ treatment. Data are expressed as the mean ± S.E.M. (A) Proteinuria *p < 0.001 vs. N; **p < 0.05 vs. DM. (B) Creatinine clearance *p < 0.001 vs. N; **p < 0.05 vs. DM.
and oxidative stress activation play major role in kidney damage (Brownlee, 2005). Inhibition of AGEs formation, down-regulation of RAGE expression, or blockade of RAGE downstream signaling are potentially therapeutic avenues in diabetic nephropathy (Sakurai et al., 2003; Yamamoto et al., 2007). CS inhibits AGEs formation. CS and its major compound (rubrofusarin-6-O-beta-d-gentiobioside) decrease TGF-1 and fibronectin expression, as well as NF-B DNA binding activity, in mouse mesangial cells (Lee et al., 2006; Sharma et al., 1996). In the present study, we first investigated the
protective effects of CS on diabetic nephropathy in a type 1 diabetic animal model. CS-treated diabetic rats exhibited significantly ameliorated levels of blood glucose, proteinuria, and creatinine clearance. Kidney tissue expression of COX-2, AGEs, RAGE, TGF-1, and collagen IV was significantly reduced upon CS treatment. STZ-induced diabetic rats have been described as a useful experimental model to study the preventive effect of diabetic nephropathy because the selective destruction of pancreatic -cells by STZ leads to the poor sensitivity of insulin for glucose uptake
Fig. 3. Effect of CS on COX-1 and COX-2 expression in the renal cortex of STZ-diabetic rats. Expression of COX-1 and COX-2 mRNA (A, C) was quantified using RT-PCR (n = 5) and COX-2 protein expression (B, D) was evaluated using western blot analyses (n = 3). Data are expressed as the mean ± S.E.M.
Please cite this article in press as: Kim, Y.S., et al., Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.11.002
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Fig. 4. Effect of CS on histopathological changes in the kidney. (A) PAS staining (a–e) and immunohistochemical staining of collagen IV (f–j), AGEs (k–o), RAGE (p–t), and TGF-1 (u–y) in the kidney. Glomeruli of normal rats (a, f, k, p, and u), normal treated with CS (200 mg/kg; b, g, l, q, and v), diabetic rats (c, h, m, r, and w), diabetic rats treated with CS (200 mg/kg; d, i, n, s, and x), and diabetic rats treated with AG (50 mg/kg; e, j, o, t, y). Hematoxylin counterstain. Original magnification, 400×. The average score of 60 randomly selected glomeruli was calculated. Data are expressed as the mean ± S.E.M. (B) Quantitative analyses of collagen IV in glomeruli. *p < 0.001 vs. N; **p < 0.001 vs. DM; # p < 0.01 vs. DM. (C) AGEs, *p < 0.001 vs. N; **p < 0.001 vs. DM. (D) RAGE, *p < 0.001 vs. N; **p < 0.001 vs. DM. (E) TGF-1, *p < 0.001 vs. N; **p < 0.001 vs. DM.
(Burns and Gold, 2007). Increased blood glucose levels and AGEs formation are involved in the development of diabetic nephropathy (Brownlee, 2005). Although the CS- or AG-treated diabetic groups did not have altered blood glucose levels at 10 weeks, these levels were significantly decreased at 12 weeks (Fig. 1). The increased fasting blood glucose levels in at least 12 h fasted rats were significantly reduced in diabetic rats treated with CS or AG for 12 weeks (Fig. 1). This effect on blood glucose control is reflected in the HbA1c levels that did not decrease in CS- or AG-treated groups compared with the untreated diabetic group (Table 1). Diabetic rats had significantly increased plasma levels of plasma glucose, total cholesterol, triglycerides, low density lipoprotein (LDL)-cholesterol, very lowdensity lipoprotein (VLDL)-cholesterol, and HbA1c, but decreased plasma high-density lipoprotein (HDL)-cholesterol and insulin levels (Saravanan and Ramachandran, 2013). Although Hb1Ac was not decreased in CS- or AG-treated rats, total cholesterol and triglyceride levels were significantly decreased in these animals. Liver ALP, AST, and ALT enzymes are used as indices of liver damage. Increased AST and ALT activities indicates that diabetes likely induces hepatic dysfunction. Therefore, increased AST and ALT activities in the liver tissue are predominantly indicative of defective glucose utilization (Murali et al., 2013). In the present study, significantly higher levels
of ALP, AST, and ALT were observed in STZ-induced diabetic rats whereas the CS-treated diabetic group were unchanged (Table 1). COX-2 expression is dramatically upregulated during inflammation, but COX-1 displays the characteristics of a housekeeping gene that is constitutively expressed in almost all tissues (Crofford, 1997). Overexpression of COX-2 mRNA and protein was significantly reduced in CS- or AG-treated diabetic groups compared with the untreated diabetic group (Fig. 3C and D). A previous study suggests that overexpression of COX-2 in podocytes exacerbates diabetic nephropathy and treatment with a COX-2 inhibitor can reduce albuminuria and mesangial matrix expansion (Cheng et al., 2011). Because CS has been shown to have inhibitory effects on the formation of AGEs, we tested whether CS can inhibit AGEs accumulation in the glomeruli of diabetic rats. As shown as Fig. 4A, CSor AG-treated diabetic rats had significantly decreased AGEs and RAGE expression in the kidney. A number of AGEs inhibitors such as pyridoxamine, aminoguanidine, and LR-90 prevent the development of complications (Degenhardt et al., 2002; Figarola et al., 2003; Kim et al., 2007). KIOM-79, an inhibitor of AGEs protein crosslinking, prevents progression of nephropathy in type 1 and type 2 diabetes animal models (Kim et al., 2007; Lee et al., 2006). New
Please cite this article in press as: Kim, Y.S., et al., Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.11.002
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therapies, including those targeting the accumulation of AGEs, ROS generation, glycosaminoglycan sulodexide, and a protein kinase C inhibitor, are likely to be involved in the future treatment of diabetic nephropathy (Goh et al., 2008). In conclusion, our work demonstrates the preventive effect of CS was achieved via inhibition of AGEs accumulation and RAGE expression in experimental models of diabetic nephropathy. Furthermore, CS treatment inhibited COX-2 expression in the renal cortex of STZ-diabetic rats. Conflict of interest statement The authors declare no conflicts of interest. Acknowledgements This research was supported by grants [K11040 and K12040] from the Korea Institute of Oriental Medicine (KIOM). References Brownlee, M., 2005. The pathobiology of diabetic complications: a unifying mechanism. Diabetes 54, 1615–1625. Burns, N., Gold, B., 2007. The effect of 3-methyladenine DNA glycosylase-mediated DNA repair on the induction of toxicity and diabetes by the beta-cell toxicant streptozotocin. Toxicological Sciences: An Official Journal of the Society of Toxicology 95, 391–400. Cheng, H., Fan, X., Moeckel, G.W., Harris, R.C., 2011. Podocyte COX-2 exacerbates diabetic nephropathy by increasing podocyte (pro)renin receptor expression. Journal of the American Society of Nephrology: JASN 22, 1240–1251. Cho, S.H., Kim, T.H., Lee, N.H., Son, H.S., Cho, I.J., Ha, T.Y., 2005. Effects of Cassia tora fiber supplement on serum lipids in Korean diabetic patients. Journal of Medicinal Food 8, 311–318. Crofford, L.J., 1997. COX-1 and COX-2 tissue expression: implications and predictions. Journal of Rheumatology Suppl. 49, 15–19. Degenhardt, T.P., Alderson, N.L., Arrington, D.D., Beattie, R.J., Basgen, J.M., Steffes, M.W., Thorpe, S.R., Baynes, J.W., 2002. Pyridoxamine inhibits early renal disease and dyslipidemia in the streptozotocin-diabetic rat. Kidney International 61, 939–950. Figarola, J.L., Scott, S., Loera, S., Tessler, C., Chu, P., Weiss, L., Hardy, J., Rahbar, S., 2003. LR-90 a new advanced glycation endproduct inhibitor prevents progression of diabetic nephropathy in streptozotocin-diabetic rats. Diabetologia 46, 1140–1152. Goh, S.Y., Jasik, M., Cooper, M.E., 2008. Agents in development for the treatment of diabetic nephropathy. Expert Opinion on Emerging Drugs 13, 447–463. Grande, J.P., Melder, D.C., Zinsmeister, A.R., 1997. Modulation of collagen gene expression by cytokines: stimulatory effect of transforming growth factorbeta1, with divergent effects of epidermal growth factor and tumor necrosis factor-alpha on collagen type I and collagen type IV. Journal of Laboratory and Clinical Medicine 130, 476–486. Guan, Y., Zhao, S., 1995. Yishou jiangzhi (de-blood-lipid) tablets in the treatment of hyperlipemia. Journal of Traditional Chinese Medicine 15, 178–179. Jung, D.H., Kim, Y.S., Kim, N.H., Lee, J., Jang, D.S., Kim, J.S., 2010. Extract of Cassiae semen and its major compound inhibit S100b-induced TGF-beta1 and fibronectin expression in mouse glomerular mesangial cells. European Journal of Pharmacology 641, 7–14. Kim, C.S., Sohn, E.J., Kim, Y.S., Jung, D.H., Jang, D.S., Lee, Y.M., Kim, D.H., Kim, J.S., 2007. Effects of KIOM-79 on hyperglycemia and diabetic nephropathy in type 2 diabetic Goto-Kakizaki rats. Journal of Ethnopharmacology 111, 240–247.
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Please cite this article in press as: Kim, Y.S., et al., Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.11.002