Attenuation of diabetic nephropathy by Chaihuang ...

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a Department of Pharmacology, Institute of Clinical Medical Sciences, China–Japan Friendship Hospital, Beijing 100029, China b Faculty of Pharmacy, ...
Journal of Ethnopharmacology 151 (2014) 556–564

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Attenuation of diabetic nephropathy by Chaihuang-Yishen granule through anti-inflammatory mechanism in streptozotocin-induced rat model of diabetics Haojun Zhang a, Tingting Zhao a, Yuewen Gong b, Xi Dong a, Weiku Zhang a, Sifan Sun a, Hua Wang a, Yanting Gu a, Xiaoguang Lu c, Meihua Yan a, Ping Li a,n a b c

Department of Pharmacology, Institute of Clinical Medical Sciences, China–Japan Friendship Hospital, Beijing 100029, China Faculty of Pharmacy, University of Manitoba, Winnipeg, MB, Canada Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China

art ic l e i nf o

a b s t r a c t

Article history: Received 8 August 2013 Received in revised form 30 September 2013 Accepted 10 November 2013 Available online 22 November 2013

Ethnopharmacological relevance: Traditional Chinese medical herbs have been used in China for a long time to treat different diseases. Based on traditional Chinese medicine (TCM) principle, ChaihuangYishen granule (CHYS) was developed and has been employed clinically to treat chronic kidney disease including diabetic nephropathy (DN). The present study was designed to investigate its mechanism of action in treatment of DN. Materials and methods: Diabetic rats were established by having a right uninephrectomy plus a single intraperitoneal injection of STZ. Rats were divided into four groups of sham, diabetes, diabetes with CHYS and diabetes with fosinopril. CHYS and fosinopril were given to rats by gavage for 20 weeks. Samples from blood, urine and kidney were collected for biochemical, histological, immunohistochemical and molecular analyses. Results: Rats treated with CHYS showed reduced 24 h urinary protein excretion, decreased serum TC and TG levels, but CHYS treatment did not affect blood glucose level. Glomerular mesangial expansion and tubulointerstitial fibrosis in diabetic rats were significantly alleviated by CHYS treatment. Moreover, CHYS administration markedly reduced mRNA levels of NF-κB p65 and TGF-β1, as well as decreased protein levels of NF-κB p65, MCP-1, TNF-α and TGF-β1 in the kidney of diabetic rats. Conclusions: CHYS ameliorates renal injury in diabetic rats through reduction of inflammatory cytokines and their intracellular signaling. & 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Diabetic nephropathy Chinese herbs Anti-inflammatory NF-κB

1. Introduction Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus, which is the leading cause of end-stage renal disease (ESRD) in many developed countries and a major cause of morbidity and mortality in patients with kidney diseases worldwide. DN is characterized by a series of

Abbreviations: DN, diabetic nephropathy; CHYS, Chaihuang-Yishen granule; STZ, streptozotocin; NF-κB, nuclear factor kappaB; IκB, inhibitory proteins of nuclear factor kappaB; MCP-1, monocyte chemotactic peptide-1; TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1beta; TGF-β1, transforming growth factor-beta1; EMT, epithelial-mesenchymal transition; TCM, traditional Chinese medicine; ACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker; BMPRII, bone morphogenetic protein receptor type II; PAN, puromycin aminonucleoside; TC, total cholesterol; TG, triglycerides; BUN, urea nitrogen; Cre, creatinine; HPLC, high performance liquid chromatography. n Corresponding author. Tel.: þ 861064227163; fax: þ 861084206084. E-mail address: [email protected] (P. Li). 0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.11.020

renal structure abnormality including basement membrane thickening, mesangial expansion, glomerulosclerosis and tubulointerstitial fibrosis (Abe et al., 2011). However, the pathogenesis of DN is still not fully understood. Although great care has been taken to patients with diabetes such as strict control of glycemia and blood pressure, many patients still develop progressive renal failure (Cooper, 2012). Accumulating evidences suggest that inflammation plays an important role in the development and progression of DN (Lim and Tesch, 2012). Several clinical and animal studies revealed the increased macrophage infiltration and leukocyte adhesion molecules in diabetic kidney. Moreover, circulating inflammatory markers and pro-inflammatory cytokines are closely related with the risk of DN (Lim and Tesch, 2012). Furthermore, some antiinflammatory agents such as mycophenolate mofetil and retinoic acid could prevent development of glomerular injury in streptozotocin (STZ)-induced diabetic rats (Luis-Rodriguez et al., 2012). In addition, macrophage accumulation and activation in the kidney

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contributed to albuminuria and renal fibrosis (Elmarakby et al., 2010). Traditional Chinese medicine (TCM) has been in practice for thousands of years in China and is becoming more popular worldwide especially in the prevention and treatment of diabetes and its complications. However, more extensive investigation of their mechanisms and evidence-based knowledge is still required (Li et al., 2004; Xie et al., 2011b). Chaihuang-Yishen (CHYS) granule, also named as Qilong-Lishui granule (Li et al., 2007), is a TCM preparation developed from famous and experienced TCM doctors, which is composed of seven herbs (Radix Bupleuri, Radix Astragali, Radix Angelicae sinensis, Rhizoma Dioscoreae nipponicae, Polyporus, Folium Pyrrosiae and Hirudo). Clinically, CHYS possessed the actions of supplementing qi and activating blood circulation, promoting urination. It has been used to treat chronic kidney diseases and has shown significant effect on decreasing proteinuria. Our previous study found that CHYS could improve tubulointerstitial injury and down-regulate the renal expression of bone morphogenetic protein receptor type II (BMPRII) and smad1mRNA in a puromycin aminonucleoside (PAN)-induced rat model of nephrotic syndrome (Li et al., 2007). It has not yet been evaluated whether this traditional herbal medicine is also beneficial in renal complications associated with diabetes. A DN model established by STZ-induction in uninephrectomized rats, which caused progressive renal injuries by increasing glomerular capillary pressure (Tesch and Allen, 2007), is thought to be a good model for pharmacological study of DN. Therefore, in the current investigation, we employed this model to examine the effects of CHYS on renal function and renal pathological changes.

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Pyrrosiae (Pyrrosia petiolosa (Christ) Ching, Polypodiaceae, voucher specimen no. DP-C006) and Hirudo (Hirudo nipponica Whitman, Hirudinidae, voucher specimen no. DP-C007) were purchased from Beijing Tong Ren Tang Group Co. Ltd (Beijing, China) and identified by the botanist Prof. Zongwan Xie of Institute of Chinese Materia Medica at China Academy of Chinese Medical Sciences. These seven herbs that constitute CHYS were prepared as previously described (Li et al., 2007). 2.2. Chromatographic analysis of CHYS CHYS was dissolved in 50% methonal and filtered through a 0.45 μm filter (Microgen, Laguna Hills, CA, USA) before high performance liquid chromatography (HPLC) analyses. The HPLC system consisted of Agilent G1311A quat pump, G1313A autosampler and Agilent G1315B diode array detector were used for all analyses. HPLC analyses were performed using a Phenomenex Luna C18 (2)(4.6  250 mm, particle size 5 μm) with acetonitrile (as Solvent A): 0.5% phosphoric acid (as Solvent B) as mobile phase at a flow rate of 1.0 ml/min at the column temperature of 30 1C. A linear gradient elution was applied from 5% of Solvent A starting from 0 to 10 min, 5–30% of Solvent A starting from 10 to 80 min, 30–100% of Solvent A starting from 80 to 120 min. Pure standards of protocatechuic acid (PA), chlorogenic acid (CA), calycosin 7-O-βD-glucoside (CG), formononetin and dioscin purchased from National Institutes for Food and Drug Control, were used as external standards in the HPLC analyses. CHYS mainly contained a constant volume of PA (0.429 mg/g), CA (0.160 mg/g), CG (1.720 mg/g), formononetin (0.005 mg/g) and dioscin (2.092 mg/g) for quality control (Fig. 1).

2. Materials and methods

2.3. Animals and diabetic model

2.1. Preparation of CHYS

Male Wistar rats weighing 200 720 g, purchased from Beijing HFK Bio-Technology Co. Ltd. (Beijing, Certificate No. SCXK 2002– 0010) were used in the study. All animals were divided into diabetic rats and non-diabetic rats. Diabetic rats were anesthetized by intraperitoneal injection of chloral hydrate at a dosage of 330 mg/kg. A right uninephrectomy was performed to accelerate development of DN. One week after surgical recovery, diabetes was induced by a single intraperitoneal injection of STZ (Sigma, USA) at a dose of 40 mg/kg diluted in citrate buffer (0.1 mol/L, pH 4.5). About 72 h after STZ injection, rats with blood glucose over

Radix Bupleuri (Bupleurum chinense DC., Umbelliferae, voucher specimen no. DP-C001), Radix Astragali (Astragalus membranaceus (Fisch.) Bge. Var. mongholicus (Bge.) Hsiao, Leguminosae, voucher specimen no. DP-C002), Radix Angelicae sinensis (Angelica sinensis (Oliv.) Diels, Umbelliferae, voucher specimen no. DP-C003), Rhizoma Dioscoreae nipponicae (Dioscorea nipponica Makino, Dioscoreaceae, voucher specimen no. DP-C004), Polyporus (Polyporus umbellatus (Pers.) Fries, Polyporaceae, voucher specimen no. DP-C005), Folium

Fig. 1. Results of HPLC analyses of CHYS granule (above) and five compounds including protocatechuic acid (PA), chlorogenic acid (CA), calycosin 7-O-β-D-glucoside (CG), formononetin and dioscin (below) in 210 nm. Column: Phenomenex Luna C18 (2)(4.6  250 mm, 5 μm); mobile phase: acetonitrile-0.5% H3PO4 (acetonitrile: 5% in 10 min, 5– 30% in 10–80 min, 30–100% in 80–120 min); flow rate: 1.0 mL/min; detection wavelength: 210 nm.

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16.7 mmol/L were confirmed to be in diabetic state. Non-diabetic rats were received sham operation, one week later, the rats were given an intraperitoneal injection of citrate buffer (0.1 mol/L, pH 4.5). The following groups were generated for the current study: (1) Non-diabetic rats with treatment of distilled water (n ¼10 and presents as Sham), (2) DN rats with treatment of distilled water (n ¼14 and presents as DN), (3) DN rats with treatment of Chaihuang-Yishen granule (0.56 g/kg body weight/day, n ¼14 and presents as CHYS), and (4) DN rats with treatment of fosinopril (1.67 mg/kg body weight/day, n ¼14 and presents as fosinopril). All drugs were dissolved in distilled water and administered once daily by gastric gavage. At the end of study, venous blood was collected from both eye sockets of each rat and kidney was removed after in situ cardio-perfusion. Renal cortex was isolated immediately and stored in liquid nitrogen for pathological and molecular studies. During experimental period, all rats were kept in cages at a temperature of 20–25 1C, humidity of 65–69%, and were submitted to a 12-h light/dark cycle with free access to food and water. This study was approved by the Ethics Committee of China–Japan Friendship Institute of Clinical Medical science and performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. 2.4. Measurement of body weight, blood glucose and urinary protein Body weight of rats was measured at 4-week intervals. Blood was sampled from the tail vein at 4-week intervals, and blood glucose levels measured by One Touch Ultra II blood glucose monitoring system (LifeScan, USA). Rats were housed individually in metabolic cages (Fengshi, China) for 24-h urinary collection at 4-week intervals and urinary protein excretion was determined by the Bradford method. 2.5. Measurement of biochemical parameters At the end of study, blood samples were centrifuged at 3000 g/ min for 15 min. Serum was separated for detection of total cholesterol (TC), triglycerides (TG), urea nitrogen (BUN), creatinine (Cre), total protein, and albumin. Examination of the above biochemical indicators was performed by an automatic biochemistry analyzer (CD-1600CS, Abbott Labs, USA). 2.6. Histological examination of the kidney Sections of kidney tissue were removed and immediately fixed in 10% phosphate buffered formalin solution and embedded in paraffin. Sections (3-μm) from each sample were cut and stained with periodic acid Schiff's stain for determination of glomerulosclerosis. The degree of glomerulosclerosis, defined as percentage mesangial matrix, was evaluated as described previously (Yuan et al., 2008). In brief, the prepared kidney sections were observed under an Olympus BX51 light microscope (Olympus, Japan) at a magnification of  400 with an Olympus DP70 digital imaging system (Olympus, Japan). Twenty glomeruli were randomly selected from each kidney, and the extent of mesangial extracellular matrix was identified by PAS-positive material in the mesangium. Glomeruli from the outer and middle thirds of the renal cortex were selected for area measurements with the aid of Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD). Care was taken to exclude juxtamedullary glomeruli. Then, percentage of mesangial matrix occupying the selected glomerular tuft was calculated. Interstitial fibrosis score (%) was given to each microscopic 10 fields viewed at  200 magnification on green with Masson's trichrome stain. The lesions were quantified by a color image analyzer (Image Pro-Plus for Windows). The fibrotic area was

digitized and subjected to color threshold analysis. Scores from 10 non-overlapping fields per kidney were averaged as final percentage positive stain (Yang et al., 2008). 2.7. Western blot analyses of nuclear factor kappaB (NF-κB) p65 and transforming growth factor-beta1 (TGF-β1) Renal cortexes were frozen under liquid nitrogen, ground into powder and added into protein lysate (Sanbio, Beijing, China). After centrifugation at 12,000 rpm for 15 min at 4 1C, supernatants were transferred into new tubes and protein content was determined by the Bradford assay. Samples containing 30 μg of protein were resolved on a 12% SDS-polyacrylamide gel and electrotransfered onto PVDF membranes, which were then incubated with 5% nonfat skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2hs following overnight incubation with monoclonal antibodies against NF-κB p65, and TGF-β1 (Abcam, Cambridge, MA) at 1:1000 dilutions respectively. After rinsing in TBST, membranes were incubated with horseradish peroxidase–conjugated secondary antibodies against rabbit or mouse IgG (Abmart, Shanghai, China) in 1:3000 dilutions respectively. After rinsing with TBST, the membrane was exposed to an x-ray film (Eastman Kodak Co., USA) using ECL Western blot detection reagents (Amersham Pharmacia Biotech, USA), density of the bands was analyzed using Quantity One Image software (Bio-rad, USA). 2.8. Immunohistochemistry staining of renal tumor necrosis factoralpha (TNF-α) and monocyte chemotactic peptide-1 (MCP-1) Renal slides (4 μm) were dewaxed by two 10-min washes in xylene and dehydrated in graded ethanol. For antigen retrieval, the slides were immersed in 10 mM citrate buffer, pH 6.0, heated in a microwave oven for 10 min and cooled for 30 min at room temperature (RT). After this, the slides were blocked with 3% H2O2 for 15 min and incubated with primary antibodies against MCP-1 at dilution of 1:75 and TNF-α at dilution of 1:75 (Santa Cruz, USA) overnight at 4 1C. The slides were then incubated with peroxidase-conjugated goat immunoglobulin G against mouse or rabbit IgG respectively (Dako, Denmark) for 30 min at RT. After washing, the slides were incubated in diaminobenzidine for color development (Dako, Denmark). The slides were counterstained with hematoxylin and eosin before being examined under a light microscope and the integrated optical density was measured by computer analysis with Image-Pro Plus 6.0 (Media Cybernetics, USA). 2.9. Reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA from kidney was isolated by using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. Two μg RNA was employed for the first strand cDNA synthesis by using moloney murine leukemia virus reverse transcriptase (Thermo

Table 1 Primer and conditions of PCR. Gene

Primer

Tm Size (1C) (bp)

NF-κB p65

Forward 5′-GTGTGCACCAACTGCCCCCA-3′ Reverse 5′-GTTGGCAGGGCAGGAGCTCC-3′ Forward 5′-CGAGGTGACCTGGGCACCATCCATGAC-3′ Reverse 5′-CTGCTCCACCTTGGGCTTGCGACCCAC-3′ Forward 5′-GATGGCACAGGAGGAAAGAG-3′ Reverse 5′-CTTGTGACTGGCTGCTTTCAC-3′

54

408

56

405

56

367

TGF-β1 Cyclophilin B

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Scentific, Lithuania). PCR amplification was performed using a thermocycler (ABI system, USA) by the following steps: denaturing at 94 1C for 2 min followed by 32 cycles of denaturing at 94 1C for 30 s, annealing for 30 s at temperature listed in Table 1 and extending at 72 1C for 1 min. PCR was ended after elongation at 72 1C for 10 min. The sequences of primers for NF-κB p65, TGF-β1 and cyclophilin-B were displayed in Table 1. Density of the bands was quantified using Quantity One software (Bio-rad, USA), and results were represented as ratio of target genes to cyclophilin-B.

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in DN group had significant elevation of 24h urinary protein level after 8 weeks of STZ injection. 3.2. Effects of CHYS on renal structure

Data are presented as mean 7SEM unless stated otherwise. Significant differences between groups were compared using ANOVA with p o0.05 being considered statistically significant.

Glomerular and tubular structures were examined by PAS and Masson's stains respectively. As shown in Fig. 3, there were no obvious abnormalities in glomerular and tubular structures of the kidneys in rats of sham group. However, there was a significant mesangial expansion, thickened basement membranes and occluded capillaries in rats of DN group. Moreover, there was a significant collagen deposition and fibrosis in the tubulo-interstitium of rats in DN group. However, both CHYS and fosinopril treatment significantly reduced the changes of both glomerular and tubular structure in rats from CHYS or fosinopril respectively.

3. Results

3.3. Effects of CHYS on renal cortical NF-κB p65 expression in rats

2.10. Statistics

3.1. Effects of CHYS on body weight, blood glucose and 24 h urinary protein By the end of study, one rat died in the group treated with fosinopril. In the other groups all rats were alive. As shown in Fig. 2A, rats in sham group displayed progressive gain of body weight while rats in DN group showed less gain of body weight. However, treatments of either CHYS or fosinopril did not prevent the reduction of body weight in CHYS and fosinopril groups. Moreover, blood glucose of DN rats was significantly higher than that of sham control and the treatments of either CHYS or fosinopril did not reverse the elevation of blood glucose in these rats (Fig. 2B). However, CHYS and fosinopril did reduce 24 h urinary protein level of rats in CHYS and fosinopril groups (Fig. 2C) and rats

In order to understand why CHYS could improve glomerular structure and reduce tubulointerstitial fibrosis, we examined the expression of NF-κB p65 in the kidney cortex of these rats. As shown in Fig. 4, both NF-κB p65mRNA (Fig. 4A) and protein (Fig. 4B) were significantly elevated in the kidney cortex of rats from DN group. However, treatment of CHYS or fosinopril restored the mRNA and protein levels of NF-κB p65 back to the levels of sham control. 3.4. Effects of CHYS on renal MCP-1 and TNF-α expression in rats Inflammatory factors (MCP-1 and TNF-α) were further investigated in the kidney. Both MCP-1 and TNF-α were dramatically increased in the kidney cortex of rats in DN group (Fig. 5). However,

Fig. 2. Effect of CHYS on body weight (A), blood glucose (B), and 24 h urinary protein(C) in rats. Body weight, blood glucose and 24 h urinary protein were determined according to description in the materials and methods. Data were presented as mean7 SEM, * indicates Po 0.05 between DN group and CHYS or fosinopril groups, respectively.

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Fig. 3. Effects of CHYS on renal pathology of experimental rats. Representative pictures of PAS staining (Original magnification 400  ) and Masson's trichrome staining (Original magnification 200  ) are presented as A to D and E to H respectively. Pictures A and E represent Sham group. Pictures B and F are from DN group. Pictures C and G are from CHYS group and pictures D and H are from fosinopril group. The bottom panels represent quantitative assessments of glomerular mesangial expansion (I) and tubulo-interstitial collagen area (J) respectively. Data were presented as mean 7 SEM, n indicates P o 0.05 between DN group and other treated groups.

Fig. 4. Effects of CHYS on renal NF-κB p65 mRNA and protein abundance. Panel A displays mRNA abundance of NF-κB p65 while panel B shows protein abundance of NF-κB p65. Top panels indicate typical pictures of RT-PCR and Western blot. Bottom panels displays histogram of the band densities. Data are presented as mean 7 SEM, and n indicates Po 0.05 between DN group and other treated groups.

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Fig. 5. Effects of CHYS on renal MCP-1 and TNF-α expression. Immunohistochemical staining of MCP-1 and TNF-α are shown in the top panels with Sham (A and E), DN (B and F), CHYS (C and G) and fosinopril (D and H) respectively (original magnification 200  ). Panel I represents quantitative analysis of MCP-1 in tubulo-interstitium and panel J displays quantitative analysis of TNF-α in tubulo-interstitium. Data are presented as mean 7SEM and n indicates Po 0.05 between DN group and other treated groups.

treatments of CHYS or fosinopril significantly reduced the MCP-1 and TNF-α immune-reactivity in the tubulo-interstitium. 3.5. Effects of CHYS on renal cortical TGF-β1 expression in rats Since TGF-β1 is an important factor mediating fibrogenesis, we examined the renal cortical TGF-β1 gene expression. As shown in Fig. 6, there was about 2.1-fold induction of TGF-β1 in DN rats compared with sham control rats. This increase in renal TGF-β1 transcripts was markedly suppressed by CHYS or fosinopril treatment in the DN rats respectively. Western blot analysis also revealed that treatments of CHYS or fosinopril significantly reduced TGF-β1 protein level in the diabetic kidneys.

3.6. Regulation of kidney function by CHYS in rats As shown above, CHYS improved kidney structure and reduced the expressions of inflammatory and fibrotic factors. We also found that CHYS could improve kidney function as indicated in Table 2. Although treatment of CHYS did not affect serum total protein and Cre levels, CHYS or fosinopril did reverse the levels of serum albumin, TC, TG and BUN in CHYS or fosinopril group as compared to DN group respectively.

4. Discussion DN affects approximately one third of patients with type 1 or type 2 diabetes mellitus. Treatment of patients with DN only limited to angiotensin converting enzyme inhibitor (ACEI) and angiotensin receptor blocker (ARB) due to their approved efficacy in clinical and animal studies (Abdel-Rahman et al., 2012). However, with recent advancement in understanding of DN, antiinflammatory therapy may emerge as another standard treatment for patients with DN (Luis-Rodriguez et al., 2012). The current study investigates the effect of a Chinese herbal formulation on inflammatory reaction in the kidney of diabetic rats and an ACEI was used as control for the treatment of DN. In recent years, several clinical and animal studies indicated that inflammatory cytokines play an important role in the development and progression of DN (Lim and Tesch, 2012; Luis-Rodriguez et al., 2012). One of the key intracellular molecules regulating inflammatory cytokines is NF-κB, which interacts closely with inhibitory proteins (IκB). Inactive NF-κB is sequestered in the cytosol by IκB to prevent its nuclear translocation and activity. Many physiological and non-physiological stimuli including interleukin-1beta (IL-1β), TNF-α activate NF-κB by dissociating it from the inhibitory unit IκB by phosphorylating p65 subunit (Sanz et al., 2010), After translocation into the nucleus, active NF-κB promotes the transcription of pro-inflammatory cytokines, such as TNF-α and IL-1β.

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Fig. 6. Effects of CHYS on renal TGF-β1 mRNA and protein expression. Panel A shows TGF-β1mRNA level while panel B represents the protein level of TGF-β1. The top panel displays typical RT-PCR and Western blot and the bottom panel shows histogram of the band densities. Data are presented as mean 7 SEM and n indicates P o 0.05 between DN group and CHYS or fosinopril group.

Table 2 Biochemical and metabolic parameters for all animals at week 20.

n Total protein (g/L) Albumin (g/L) TC (mmol/L) TG (mmol/L) BUN (mmol/L) Cre (μmol/L) n

Sham

DN

CHYS

Fosinopril

10 63.727 1.53 34.78 7 0.63* 2.007 0.11* 0.89 7 0.10* 6.55 7 0.31* 59.11 71.90

14 60.767 1.85 30.3370.91 4.02 7 0.57 3.80 7 0.65 10.69 70.89 61.007 2.03

14 62.12 71.19 31.337 0.68 2.62 7 0.18* 2.12 70.31* 12.077 0.79 62.08 7 1.31

13 61.88 7 1.47 32.377 0.53 2.65 7 0.18* 1.73 70.15* 12.50 7 0.68 62.177 1.82

Indicates Po 0.05 between other groups versus DN group.

Several studies of renal diseases showed that NF-κB was activated in tubules and glomeruli (Sanz et al., 2010) and there was a correlation between NF-κB activation and DN progression (Iwamoto et al., 2005). Moreover, clinical studies of patients with both type 1 and type 2 diabetes demonstrated that increased NF-κB binding activity in peripheral blood mononuclear cells correlates with severity of DN (Hofmann et al., 1999). Schmid et al. (2006) reported that up-regulation of NF-κB plays a role in inflammatory response in the kidney of the patients with progressive DN. Furthermore, progression of DN could be associated with NF-κB stimulated overexpression of chemokines, such as MCP-1 and regulated on activation normal T-cell expressed and secreted chemokine (Mezzano et al., 2004). Our findings in the current study are consistent with previous studies. Moreover, we observed Chinese herbs could alleviate NF-κB activation and inflammatory cytokines production. This finding supports our clinical application of these herbs in patients with DN, which is also easy to be understood for the suppression of NF-κB by Radix astragli, Radix Angelicae sinensis, Polyporus polysaccharide in CHYS (Chao et al., 2010; Qin et al., 2012; Wei et al., 2011). MCP-1 is a key chemokine mediating progression of diabetic renal injury. High glucose or advanced glycation end products could also induce MCP-1 in mesangial cells, podocytes, and renal tubular epithelial cells (Nam et al., 2012; Park et al., 2008; Tesch, 2008). Elevated expression of MCP-1 was found in glomeruli and tubulo-interstitium in animal models of DN and diabetic patients (Chow et al., 2007, 2006). Moreover, increased level of renal MCP-1 in diabetic patients is associated with macrophage recruitment and progression of DN. MCP-1-mediated macrophage infiltration in the kidney contributed to progression of DN (Chow et al., 2007; Tam et al., 2009; Wada et al., 2000), because inhibition of MCP-1 activity could ameliorate development of DN in diabetic mice (Kanamori et al., 2007). Furthermore, urinary level of MCP-1 correlated with severities of macroalbuminuria and deterioration

of kidney function (Tam et al., 2009; Tesch, 2008; Titan et al., 2012). Our results showed that renal MCP-1 protein expression was significantly increased in diabetic rats. CHYS treatment obviously decreased renal MCP-1 expression, which was supported by the reports about activity of inhibiting MCP-1 of Radix Astragali and Bupleurum polysaccharides from CHYS (Hoo et al., 2010; Jiang et al., 2012), and this improvement might be obtained via the inhibition of NF-κB. The role of TNF-α in DN is supported by the findings that elevated levels of TNF-α in serum, kidney and urine were observed in STZ-induced diabetic rats and patients with DN (Elmarakby et al., 2010; Navarro-Gonzalez et al., 2009). Moreover, increased level of TNF-α correlated with urinary albumin excretion and progression of DN. Furthermore, TNF-α contributes to sodium retention and renal hypertrophy in STZ-induced DN (DiPetrillo et al., 2003). Several studies indicated that TNF-α induced direct damage to glomerular, mesangial and epithelial cells. TNF-α also promoted local generation of superoxide, which affected barrier function of glomerular capillary wall resulting in enhanced albumin permeability. In addition, inhibition of TNF-α resulted in decreased urinary albumin excretion in experimental diabetic rats (Moriwaki et al., 2007). Therefore, TNF-α plays an important role in the development and progression of DN. The current findings suggested that therapeutic activity of CHYS in DN was partly mediated by inhibition of TNF-α expression under diabetic condition, which was consistent with that Radix Astragali, Radix Angelicae sinensis, Rhizoma Dioscoreae nipponicae, Radix Bupleuri, Polyporus polysaccharide and Hirudo presented in CHYS could reduced TNF-α expression in vitro and in vivo (Chao et al., 2010; Kim et al., 2004; Qin et al., 2012; Wei et al., 2011; Xie et al., 2011a). Numerous studies have demonstrated that TGF-β1 plays a pivotal role in experimental diabetic kidney disease and human DN (Lan, 2012). TGF-β1 is a key profibrotic cytokine that acts by stimulating synthesis of extracellular matrix components, decreasing matrix degradation, and promoting cell–matrix interactions. Moreover, TGF-β1 is important for the epithelial–mesenchymal transition (EMT) through which tubular cells become fibroblasts in DN (Hills and Squires, 2011). TGF-β1 is also involved in tubuleglomerular sclerosis and podocyte apoptosis in diabetes. Treatment with neutralizing antibodies against TGF-β prevented glomerular hypertrophy and mesangial matrix expansion in several experimental model of DN (McGowan et al., 2004). Furthermore, activation of NF-κB upregulates the TGF-β1 expression due to a NF-κB binding site in the promoter region of TGF-β1 (Wada and Makino, 2013). MCP-1 and TNF-α can also promote TGF-β1 production in renal cells under high glucose condition (Phillips et al., 1996; Tesch, 2008). These inflammatory factors collectively

H. Zhang et al. / Journal of Ethnopharmacology 151 (2014) 556–564

Fig. 7. A diagram demonstrates the possible mechanism of CHYS in improving diabetic nephropathy through anti-inflammatory and anti-fibrotic effects. CHYS alleviated massive proteinuria, mesangial expansion and tubulointerstital fibrosis by inhibition of NF-κB pathway and TGF-β1.

led to increased TGF-β1 level in DN. As major constituents of CHYS, Radix Astragali and Radix Angelicae sinensis inhibited TGF-β1 in different models (Gao et al., 2011; Xie et al., 2006). In the present study, similar to fosinopril, CHYS treatment reversed the increase in renal TGF-β1 expression in diabetic rat kidney, which was dependent of its anti-inflammation action to some extent. We also determined the contents of PA, CA, CG, formononetin and dioscin in CHYS by HPLC, and these ingredients were reported to inhibit inflammation. PA reduced the phosphorylation of IκB-α, the nuclear translocation of NF-κB in LPS-induced RAW 264.7 cells and inhibited the leukocyte number and the levels of TNF-α, PGE2 in the exudates of the air pouch in carrageenan-treated mice, as well as COX-2 expression and NF-κB activation (Min et al., 2010). CA suppressed LPS-induced COX-2 expression via attenuating the activation of NF-κB and JNK/AP-1 signaling pathways in RAW 264.7 cells (Shan et al., 2009). CG ameliorated upregulation of inflammatory proteins such as MCP-1, interleukin-6, ICAM-1 and TGF-β1 through supression of activated ERK1/2 and NF-κB phosphorylation in AGEs treated human umbilical vein endothelial cells (Xu et al., 2011). Formononetin significantly inhibited IL-1βinduced activation of NF-κB in INS-1 cell (Wang et al., 2012). Dioscin decreased hepatic expressions of inflammatory cytokines including TNF-α, IL-6 and ICAM-1 in CCl4 treated mice (Lu et al., 2012). These findings supported that these ingredients were the active substances of CHYS for the suppression of inflammation in DN. In summary, CHYS treatment attenuated glomerularsclerosis and tubulointerstitial fibrosis in diabetic rats. An increase in renal expression of MCP-1 and TNF-α was greatly reduced by CHYS in the model. Moreover, the anti-inflammatory and anti-fibrotic effects of CHYS were accompanied by a suppression of NF-κB pathway and TGF-β1 in the diabetic kidneys, as shown in Fig. 7 (Elmarakby et al., 2010; Wada and Makino, 2013). Therefore, our research results proved that the administration of CHYS may slow the progression of DN by inhibiting the inflammatory and consequently fibrotic processes.

Acknowledgements This work was supported by the International Science and Technology Cooperation Program of China (Grant no. 2011DFA31860) and the National Natural Science Foundation of China (Grant no. 81173422, 30973911). References Abdel-Rahman, E.M., Saadulla, L., Reeves, W.B., Awad, A.S., 2012. Therapeutic modalities in diabetic nephropathy: standard and emerging approaches. J. Gen. Internal Med. 27, 458–468.

563

Abe, H., Matsubara, T., Arai, H., Doi, T., 2011. Role of Smad1 in diabetic nephropathy: molecular mechanisms and implications as a diagnostic marker. Histol. Histopathol. 26, 531–541. Chao, W.W., Hong, Y.H., Chen, M.L., Lin, B.F., 2010. Inhibitory effects of Angelica sinensis ethyl acetate extract and major compounds on NF-kappaB transactivation activity and LPS-induced inflammation. J. Ethnopharmacol. 129, 244–249. Chow, F.Y., Nikolic-Paterson, D.J., Ma, F.Y., Ozols, E., Rollins, B.J., Tesch, G.H., 2007. Monocyte chemoattractant protein-1-induced tissue inflammation is critical for the development of renal injury but not type 2 diabetes in obese db/db mice. Diabetologia 50, 471–480. Chow, F.Y., Nikolic-Paterson, D.J., Ozols, E., Atkins, R.C., Rollin, B.J., Tesch, G.H., 2006. Monocyte chemoattractant protein-1 promotes the development of diabetic renal injury in streptozotocin-treated mice. Kidney Int. 69, 73–80. Cooper, M.E., 2012. Diabetes: treating diabetic nephropathy-still an unresolved issue. Nat. Rev. Endocrinol. 8, 515–516. DiPetrillo, K., Coutermarsh, B., Gesek, F.A., 2003. Urinary tumor necrosis factor contributes to sodium retention and renal hypertrophy during diabetes. Am. J. Physiol. Renal Physiol. 284, F113–121. Elmarakby, A.A., Abdelsayed, R., Yao Liu, J., Mozaffari, M.S., 2010. Inflammatory cytokines as predictive markers for early detection and progression of diabetic nephropathy. EPMA J. 1, 117–129. Gao, M., Zhang, J.H., Zhou, F.X., Xie, C.H., Han, G., Fang, S.Q., Zhou, Y.F., 2011. Angelica sinensis suppresses human lung adenocarcinoma A549 cell metastasis by regulating MMPs/TIMPs and TGF-beta1. Oncol. Rep. 27, 585–593. Hills, C.E., Squires, P.E., 2011. The role of TGF-beta and epithelial-to mesenchymal transition in diabetic nephropathy. Cytokine Growth Factor Rev. 22, 131–139. Hofmann, M.A., Schiekofer, S., Isermann, B., Kanitz, M., Henkels, M., Joswig, M., Treusch, A., Morcos, M., Weiss, T., Borcea, V., Abdel Khalek, A.K., Amiral, J., Tritschler, H., Ritz, E., Wahl, P., Ziegler, R., Bierhaus, A., Nawroth, P.P., 1999. Peripheral blood mononuclear cells isolated from patients with diabetic nephropathy show increased activation of the oxidative-stress sensitive transcription factor NF-kappaB. Diabetologia 42, 222–232. Hoo, R.L., Wong, J.Y., Qiao, C., Xu, A., Xu, H., Lam, K.S., 2010. The effective fraction isolated from Radix Astragali alleviates glucose intolerance, insulin resistance and hypertriglyceridemia in db/db diabetic mice through its anti-inflammatory activity. Nutr. Metab. (London) 7, 67. Iwamoto, M., Mizuiri, S., Arita, M., Hemmi, H., 2005. Nuclear factor-kappaB activation in diabetic rat kidney: evidence for involvement of P-selectin in diabetic nephropathy. Tohoku J. Exp. Med. 206, 163–171. Jiang, Y.W., Li, H., Zhang, Y.Y., Li, W., Jiang, Y.F., Ou, Y.Y., Chen, D.F., 2012. Beneficial effect of Bupleurum polysaccharides on autoimmune-prone MRL-lpr mice. Clin. Dev. Immunol. 2012, 842928. Kanamori, H., Matsubara, T., Mima, A., Sumi, E., Nagai, K., Takahashi, T., Abe, H., Iehara, N., Fukatsu, A., Okamoto, H., Kita, T., Doi, T., Arai, H., 2007. Inhibition of MCP-1/CCR2 pathway ameliorates the development of diabetic nephropathy. Biochem. Biophys. Res. Commun. 360, 772–777. Kim, M.J., Kim, H.N., Kang, K.S., Baek, N.I., Kim, D.K., Kim, Y.S., Jeon, B.H., Kim, S.H., 2004. Methanol extract of Dioscoreae Rhizoma inhibits pro-inflammatory cytokines and mediators in the synoviocytes of rheumatoid arthritis. Int. Immunopharmacol. 4, 1489–1497. Lan, H.Y., 2012. Transforming growth factor-beta/Smad signalling in diabetic nephropathy. Clin. Exp. Pharmacol. Physiol. 39, 731–738. Li, P., Yan, J., Sun, Y., Burczynski, F.J., Gong, Y., 2007. Chinese herbal formula QilongLishui granule improves puromycin aminonucleoside-induced renal injury through regulation of bone morphogenetic proteins. Nephrology (Carlton) 12, 466–473. Li, W.L., Zheng, H.C., Bukuru, J., De Kimpe, N., 2004. Natural medicines used in the traditional Chinese medical system for therapy of diabetes mellitus. J. Ethnopharmacol. 92, 1–21. Lim, A.K., Tesch, G.H., 2012. Inflammation in diabetic nephropathy. Mediators Inflamm. 2012, 146154. Lu, B., Xu, Y., Xu, L., Cong, X., Yin, L., Li, H., Peng, J., 2012. Mechanism investigation of dioscin against CCl4-induced acute liver damage in mice. Environ. Toxicol. Pharmacol. 34, 127–135. Luis-Rodriguez, D., Martinez-Castelao, A., Gorriz, J.L., De-Alvaro, F., NavarroGonzalez, J.F., 2012. Pathophysiological role and therapeutic implications of inflammation in diabetic nephropathy. World J. Diabetes 3, 7–18. McGowan, T.A., Zhu, Y., Sharma, K., 2004. Transforming growth factor-beta: a clinical target for the treatment of diabetic nephropathy. Curr. Diabetes Rep. 4, 447–454. Mezzano, S., Aros, C., Droguett, A., Burgos, M.E., Ardiles, L., Flores, C., Schneider, H., Ruiz-Ortega, M., Egido, J., 2004. NF-kappaB activation and overexpression of regulated genes in human diabetic nephropathy. Nephrol. Dialysis, Transplantation 19, 2505–2512. Min, S.W., Ryu, S.N., Kim, D.H., 2010. Anti-inflammatory effects of black rice, cyanidin-3-O-beta-D-glycoside, and its metabolites, cyanidin and protocatechuic acid. Int. Immunopharmacol. 10, 959–966. Moriwaki, Y., Inokuchi, T., Yamamoto, A., Ka, T., Tsutsumi, Z., Takahashi, S., Yamamoto, T., 2007. Effect of TNF-alpha inhibition on urinary albumin excretion in experimental diabetic rats. Acta Diabetol. 44, 215–218. Nam, B.Y., Paeng, J., Kim, S.H., Lee, S.H., do, Kim, Kang, H., Li, H.Y., Kwak, J.J., Park, S.J., Yoo, J.T., Han, T.H., Kim, S.H., Kang, S.W., D.K., 2012. The MCP-1/CCR2 axis in podocytes is involved in apoptosis induced by diabetic conditions. Apoptosis 17, 1–13.

564

H. Zhang et al. / Journal of Ethnopharmacology 151 (2014) 556–564

Navarro-Gonzalez, J.F., Jarque, A., Muros, M., Mora, C., Garcia, J., 2009. Tumor necrosis factor-alpha as a therapeutic target for diabetic nephropathy. Cytokine Growth Factor Rev. 20, 165–173. Park, J., Ryu, D.R., Li, J.J., Jung, D.S., Kwak, S.J., Lee, S.H., Yoo, T.H., Han, S.H., Lee, J.E., Kim, D.K., Moon, S.J., Kim, K., Han, D.S., Kang, S.W., 2008. MCP-1/CCR2 system is involved in high glucose-induced fibronectin and type IV collagen expression in cultured mesangial cells. Am. J. Physiol. Renal Physiol. 295, F749–757. Phillips, A.O., Topley, N., Steadman, R., Morrisey, K., Williams, J.D., 1996. Induction of TGF-beta 1 synthesis in D-glucose primed human proximal tubular cells by IL-1 beta and TNF alpha. Kidney Int. 50, 1546–1554. Qin, Q., Niu, J., Wang, Z., Xu, W., Qiao, Z., Gu, Y., 2012. Astragalus membranaceus inhibits inflammation via phospho-P38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB pathways in advanced glycation end product-stimulated macrophages. Int. J. Mol. Sci. 13, 8379–8387. Sanz, A.B., Sanchez-Nino, M.D., Ramos, A.M., Moreno, J.A., Santamaria, B., RuizOrtega, M., Egido, J., Ortiz, A., 2010. NF-kappaB in renal inflammation. J. Am. Soc. Nephrol. 21, 1254–1262. Schmid, H., Boucherot, A., Yasuda, Y., Henger, A., Brunner, B., Eichinger, F., Nitsche, A., Kiss, E., Bleich, M., Grone, H.J., Nelson, P.J., Schlondorff, D., Cohen, C.D., Kretzler, M., 2006. Modular activation of nuclear factor-kappaB transcriptional programs in human diabetic nephropathy. Diabetes 55, 2993–3003. Shan, J., Fu, J., Zhao, Z., Kong, X., Huang, H., Luo, L., Yin, Z., 2009. Chlorogenic acid inhibits lipopolysaccharide-induced cyclooxygenase-2 expression in RAW264.7 cells through suppressing NF-kappaB and JNK/AP-1 activation. Int. Immunopharmacol. 9, 1042–1048. Tam, F.W., Riser, B.L., Meeran, K., Rambow, J., Pusey, C.D., Frankel, A.H., 2009. Urinary monocyte chemoattractant protein-1 (MCP-1) and connective tissue growth factor (CCN2) as prognostic markers for progression of diabetic nephropathy. Cytokine 47, 37–42. Tesch, G.H., 2008. MCP-1/CCL2: a new diagnostic marker and therapeutic target for progressive renal injury in diabetic nephropathy. Am. J. Physiol. Renal Physiol. 294, F697–701. Tesch, G.H., Allen, T.J., 2007. Rodent models of streptozotocin-induced diabetic nephropathy. Nephrology (Carlton) 12, 261–266. Titan, S.M., Vieira Jr., J.M., Dominguez, W.V., Moreira, S.R., Pereira, A.B., Barros, R.T., Zatz, R., 2012. Urinary MCP-1 and RBP: independent predictors of renal

outcome in macroalbuminuric diabetic nephropathy. J. Diabetes Complications 26, 546–553. Wada, J., Makino, H., 2013. Inflammation and the pathogenesis of diabetic nephropathy. Clin. Sci. (London) 124, 139–152. Wada, T., Furuichi, K., Sakai, N., Iwata, Y., Yoshimoto, K., Shimizu, M., Takeda, S.I., Takasawa, K., Yoshimura, M., Kida, H., Kobayashi, K.I., Mukaida, N., Naito, T., Matsushima, K., Yokoyama, H., 2000. Up-regulation of monocyte chemoattractant protein-1 in tubulointerstitial lesions of human diabetic nephropathy. Kidney Int. 58, 1492–1499. Wang, Y., Zhu, Y., Gao, L., Yin, H., Xie, Z., Wang, D., Zhu, Z., Han, X., 2012. Formononetin attenuates IL-1beta-induced apoptosis and NF-kappaB activation in INS-1 cells. Molecules 17, 10052–10064. Wei, J.A., Zeng, X., Han, L., Huang, Y., 2011. The regulatory effects of polyporus polysaccharide on the nuclear factor kappa B signal pathway of bladder cancer cells stimulated by Bacillus Calmette-Guerin. Chin. J. Integr. Med. 17, 531–536. Xie, C.H., Zhang, M.S., Zhou, Y.F., Han, G., Cao, Z., Zhou, F.X., Zhang, G., Luo, Z.G., Wu, J.P., Liu, H., Chen, J., Zhang, W.J., 2006. Chinese medicine Angelica sinensis suppresses radiation-induced expression of TNF-alpha and TGF-beta1 in mice. Oncol. Rep. 15, 1429–1436. Xie, J.Y., Di, H.Y., Li, H., Cheng, X.Q., Zhang, Y.Y., Chen, D.F., 2011a. Bupleurum chinense DC polysaccharides attenuates lipopolysaccharide-induced acute lung injury in mice. Phytomedicine 19, 130–137. Xie, W., Zhao, Y., Zhang, Y., 2011b. Traditional Chinese medicines in treatment of patients with type 2 diabetes mellitus. Evid. Based Complementary Altern. Med. 2011, 726723. Xu, Y., Feng, L., Wang, S., Zhu, Q., Lin, J., Lou, C., Xiang, P., He, B., Zheng, Z., Tang, D., Zuo, G., 2011. Phytoestrogen calycosin-7-O-beta-D-glucopyranoside ameliorates advanced glycation end products-induced HUVEC damage. J. Cell. Biochem. 112, 2953–2965. Yang, S.H., Shin, S.J., Oh, J.E., Jin, J.Z., Chung, N.H., Lim, C.S., Kim, S., Kim, Y.S., 2008. The protective role of uteroglobin through the modulation of tissue transglutaminase in the experimental crescentic glomerulonephritis. Nephrol Dialysis, Transplantation 23, 3437–3445. Yuan, H., Lanting, L., Xu, Z.G., Li, S.L., Swiderski, P., Putta, S., Jonnalagadda, M., Kato, M., Natarajan, R., 2008. Effects of cholesterol-tagged small interfering RNAs targeting 12/15-lipoxygenase on parameters of diabetic nephropathy in a mouse model of type 1 diabetes. Am. J. Physiol. Renal Physiol. 295, F605–617.