Nrf2-ARE Signal Pathway in Adjuvant Arthritis Rats

Sun Y et al. Chinese Herbal Medicines, 2016, 8(2): 143-153

143

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Chinese Herbal Medicines (CHM)  ISSN 1674-6384 Journal homepage: www.tiprpress.com

E-mail: [email protected]

Original article 

Improving Effects of Astragalus Polysaccharides on Cardiac Function via Keap1/Nrf2-ARE Signal Pathway in Adjuvant Arthritis Rats Yue Sun1, Jian Liu2*, Lei Wan2, Fang Wang1, Xiao-jun Zhang1, Ya-jun Qi1 1. Graduate Faculty, Anhui University of Chinese Medicine, Hefei 230031, China 2. Department of Rheumatology, First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei 230038, China

ARTICLE INFO  Article history Received: August 3, 2015 Revised: September 21, 2015 Accepted: December 1, 2015 Available online: April 12, 2016

DOI: 10.1016/S1674-6384(16)60024-2

  ABSTRACT    Objective

To observe the effect of Astragalus polysaccharides (APS) on cardiac function

Methods

Forty-eight rats were randomly divided into normal control (NC) group,

and expression of Keap1/Nrf2-ARE signal pathway in adjuvant arthritis (AA) rats. model control (MC) group, leflunomide (LEF) group, and APS group. The AA model rats were induced with Freund’s complete adjuvant, then administration began from day 19 after modeling for 30 d. The paw swelling and arthritic index (AI), the cardiac function

indexes, the expression of Keap1, maf, Nrf2 mRNAs, and HO-1, r-GCS proteins in

cardiac tissues were observed. Oxidation-related substances (SOD, MDA, ROS, and TAC) and cytokines (IL-10 and TNF-α) were also determined. Results

Compared with NC

group, the paw swelling and AI were increased, and the body weight was decreased in

MC group. HR, HI, LVSP, LVEDP, and levels of MDA, TAC, ROS, RNS, and TNF-α in MC

group were increased, while dp/dtmax and levels of GSH, TRX, and IL-10 were decreased. Compared with MC group, LEF group showed higher HR and ± dp/dtmax, and

lower LVEDP. In APS group, the HR, LVSP, and LVEDP were decreased and ±dp/dtmax was

increased. TNF-α was decreased, TAC, ROS, MDA, and IL-10 were increased in both LEF and APS groups. APS and LEF groups also showed less MDA, TAC, ROS, and SOD, and the differences were statistically significant. Spearman correlation analysis showed that the cardiac function parameters were positively correlated with Keap1, anti-oxidant

indicators, and anti-inflammation cytokines, and negatively correlated with Nrf2, oxidation

Conclusion

indictors,

pro-inflammation

cytokines,

and

inflammation

indictors.

APS can adjust the expression of Keap1/Nrf2-ARE signal pathway in AA

rats, and can also improve their cardiac function. The mechanism may be involved in increasing myocardial anti-oxidant capacity, reducing oxidative stress, and inhibiting inflammation.

Key words

Astragalus polysaccharide; adjuvant arthritis rats; cardiac function; oxidative stress

© 2016 published by TIPR Press. All rights reserved.

*

Correspondence author: Liu J

Tel: +86-13955109537 E-mail: [email protected]

Funds: Key Projects in the National Science and Technology Pillar Program in the Twelfth Five-Year Plan Period (No. 2012BAI26B02); National Key Disciplines of Chinese Medicine Construction Projects (No. [2009]30)

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Sun Y et al. Chinese Herbal Medicines, 2016, 8(2): 143-153

1.    Introduction  Rheumatoid arthritis (RA) is an autoimmune connective tissue disease, which is mainly expressed as a chronic joint disease that may be repeated and protracted, and ultimately leads to joint deformity and loss of function. However, the lesion is not limited to the joints but concurrently with lesions in organs other than joints; it can even cause life-threatening vasculitis in major organs (Ralf and Wagner, 2002). Heart is more easily affected due to its richness in connective tissues and blood vessels. The domestic and international studies showed that the heart disease suffered by RA patients can not be fully explained with the cardiovascular risk factors (Barber et al, 2015), the autoimmune disorders and inflammation of RA may play an important role in the development of heart disease at an early stage (Liu et al, 2012). At present, the RA pathomechanism is still unclear, but some studies have shown that oxidative stress played an important role in the progress. By observation of the change on indicator of cardiac function and oxidative stress in adjuvant arthritis (AA) model rats, this study preliminarily discusses the mechanism of the effect of Astragalus polysaccharides (APS, a traditional Chinese medicine monomer) on improving the cardiac function.

2.    Materials and methods   

2.1    Experimental animal  Forty-eight clean male SD rats, weighing (150 ± 20) g, were provided by Anhui Experimental Animal Center. Animal License: scxk (Wan) 2011-002. All rats were fed at the room temperature of 22−28 oC and at relative humidity of 50%−60%, under natural lighting. All the rats were fed in standard laboratory animal room during the whole process of the experiment.

2.2    Drugs  Freund’s complete adjuvant: 10 mL/vial, produced by Sigma, USA. APS: 100 mg/bottle (purity > 90%), manufactured by Shanghai Yuanye Bio-technology Co., Ltd. Leflunomide Tablet: 10 mg/tablet, manufactured by Fujian Huitian Biological Pharmaceutical Co., Ltd., batch No. 120503.

2.3    Reagents  2.3.1    PCR‐related reagents  Trizol reagent: Invitrogen; RT-qPCR Kit: Fermentas; chloroform: Reagent Factory of Shanghai Jianxin Chemicals Co., Ltd.; anhydrous ethanol: Shanghai Suran Chemical Reagent Co., Ltd.; isopropanol: Nanjing Chemical Reagent Factory; DEPC, Ruibio; primer, and probe synthesis kit: Invitrogen; fluorescent quantitative reagent and laboratory consumables: TaKaRa. 2.3.2    Western blotting‐related reagents  Tissue total protein lysis buffer: Shanghai Beyotime

Bio-Technology Co., Ltd.; electrophoresis (Running), transfer buffer (10 ×), coomassie brilliant blue (CBB) G250: Bio-Rad, USA; nitrocellulose membrane: Bio-Rad, USA; enhanced chemiluminescent (ECL) kit: Thermo, USA; skim milk powder: Fonterra, New Zealand; protein mark (Protein Ladder): Thermo Scientific, USA. SDS-PAGE gel preparation kit, Shanghai Beyotime Bio-Technology Co., Ltd. Primary antibody: Heme oxygenase-1 (HO-1, rabbit antibody): Bioworld Bio-Technology Co., Ltd., USA, batch No. BS1361; γ-glutamylcysteine synthetase (γ-GCS, rabbit antibody): Bioworld Bio-technology Co., Ltd., USA, batch No. BS2398. Secondary antibody: goat anti-rabbit IgG， Bioworld Bio-Technology Co., Ltd., USA, batch No. BS13278; goat anti-mouse IgG: Bioworld Bio-technology Co., Ltd., USA, batch No. BS12478. 2.3.3    SP‐related reagent  Anti-rabbit-IgG monoclonal antibody, reactive oxygen species (ROS), reactive nitrogen species (RNS), glutathione (GSH), and thioredoxin (TRX): Santa Cruz, USA; SP kit and DAB Histostain-Plus Kit: Beijing CoWin Biotech Co., Ltd. 2.3.4    ELISA kits    SOD, MDA, ROS, total antioxidant capacity (T-AOC), interleukin-18 (IL-18), IL-35, interferon-γ (IFN-γ), and tumor necrosis factor (TNF-α) were provided by Shanghai Westang Bio-tech Co., Ltd.

2.4    Apparatus and equipments    2.4.1    Paw Volume Meter and cardiac function test system  Animal Paw Volume Meter was provided by Beijing Gene & I Technology Co., Ltd., type No. XR-YLS-7B; Electronic balance was provided by Shanghai Liheng Instrumentation Co., Ltd., type No. MXX−5001. PowerLab 8/30 Data Acquisition and Analysis System which including Quad Bridge Amplifier, Dual Bio Amplifier and Stimulus Isolator were producted by AD Instruments, Australia.   2.4.2    Tissue paraffin embedding machines  Leica, Germany, type No. A130395; Paraffin slicing machine: LEICA, Germany, type No. RM2245; Tissue Dehydrator: LEICA, Germany, type No. ASP300S; Tissue Bleaching Machine: Bloomington, USA, MP−220; Drying Machine: LEICA, Germany, type No. HI1220; Staining Machine: SLEE, Germany, type No. MAS. Microscope: Olympus, Japan, type No. BX53. 2.4.3    Regular PCR instruments    ABI, USA (type No. ABI2720); Fluorescent quantitative PCR instrument: ABI, USA type No. ABI7500; Trizol: Invitrogen; DYY−6B Steady Current & Voltage ElectroPhoresis Apparatus: Beijing Liuyi Instrument Factory; TGL− 18R Refrigerated Centrifuge: Heima Instrument Co., Ltd.; Adjustable Pipette: Eppendorf; Low Speed Centrifuge:

Sun Y et al. Chinese Herbal Medicines, 2016, 8(2): 143-153 Shanghai Anting Scientific Instrument Factory (type No. TDL−40B), USTC Zonkia (type No. SC−3610) 2.4.4    Ultrapure water system  Sartorius, Germany, type No. Milli Q Biocel; table-top high-speed Refrigerated Centrifuge: Eppendorf, Germany, type No. Centrifμge 5424R; High-Speed Centrifuge: Eppendorf, Germany, type No. Centrifμge 5424; Ultraviolet Spectrophotometer: Hitachi, Japan, type No. U−5100; PCR Amplification Instrument: Biometra, Germany, type No. T1-Thermoblock; Electrophoresis Instrument: Bio-Rad, USA, type No. Power PAC 300; Electrophoresis Tank: Bio-Rad, USA, type No. Mini-Protean® Tetra system; semi-Dry Transfer Unit: Bio-Rad, USA, type No. Trans-B; Gel Image Analysis System: Bio-Rad, USA, type No. Gel Doc XR; Microwave Oven: Stratagene, USA, type No. Stratalinker® 1800; Shaking Table: Labet International, USA, type No. S2025−XLD; Tissue Grinders: Kimble, USA, type No. 749540−0000; Thermostatic Container: VWR, USA, type No. Signature; Water Bath: Lauda, Germany, type No. RM6; Ice Maker: Folett, USA, type No.: MFD400A/2JA1; blending Machine: Scientific Industries, USA, type No. G−560; 8 Micro-tube Rotor Centrifuge: Qualitron, Korea, type No. TZL−1001. 2.4.5    Other instruments  The ELISA was produced by Bio-Jek Co., Ltd., USA, type No. ELX800. Table-top High-speed Refrigerated Centrifuge (5417R): Eppendorf, Germany.

2.5    Methods  2.5.1    Duplication, grouping, and medication of animal model  The rats were randomly divided into four groups: normal control (NC), model control (MC), Astragalus polysaccharide control (APS), and leflunomide (LEF) groups, with 12 rats in each group. Except the NC group, each rat in each group was ip injected in right rear paw with 1 mL/kg of Freund’s complete adjuvant to induce inflammation in order to obtain AA model (Li, 1991). The rats were given the dose from day 19 after inflammation, and the dosage was equivalent to 10 times the clinical dosage. The dosage of each group was as follows: NC and MC groups: 10 mL/kg saline, once daily; APS group: APS grounded into fine powder, added with saline, and then formulated into a suspension (1 mg/mL, 10 mg/kg), performed the gavage at the dosage of 10 mL/kg, once daily; LEF group: LEF Tablets were grounded into fine powder, added with saline, and then formulated as a suspension (LEF 0.5 mg/mL, namely 5 mg/kg), performed the gavage at the dosage of 10 mL/kg, once daily; The rats were continuously administered for 30 d. 2.5.2    Measurement of paw swelling and arthritic index in rats  At the day before modeling, day 18 after inducing inflammation, and the 3-day interval after medication, the rear paw volume was measured and the paw swelling in rats in each group was calculated, respectively (Wei et al, 2010).

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Paw volume was measured by XR−YLS−7B Paw Volume Meter for mice and rats. Paw swelling E = (Vt−Vn)/Vn (Vn and Vt represented the volumes before and after modeling). Calculation of arthritic index (AI): on day 12 after inducing inflammation, the extent of the joint lesion was observed and recorded systemically, once every 3 d. Systemic disease was evaluated according to the 5-grade rating method, and AI was calculated on the basis of cumulative integral of lesion extent in the remaining three rats without injection of adjuvant (Zhang and Du, 2012). 0 point: no swelling; 1 point: redness and swelling little paw joint; 2 points: paw joint and paw swelling; 3 points: paw swelling below the ankle joint; 4 points: all paw swelling includes ankle. The accumulation of point for each joint is the AI of each rat. The highest score is 12 points. 2.5.3    Invasive hemodynamic measurement of cardiac function  After 24 h of the last medication, six rats were randomly taken from each group, weighed, and then performed with ip anesthesia using 300 mg/kg of 10% chloral hydrate (10 mL/kg). The rat was fixed by supine position, and its chest was performed with depilation using 20% sodium sulfide for skin preparation, the skin was vertically cut up for 10 mm along with 5 mm at the edge of the dual forelimb, the fascia and muscle gap were separated using blunt dissection technique with a hemostat clamp. From the inner side of left sternocleidomastoid muscle, the trachea, left carotid artery, left external carotid artery, and left internal carotid artery were successively dissociated and clipped at proximal ends using the artery clamp to block the blood flow, respectively. The external carotid artery was picked up with the ophthalmic tweezers, and the ophthalmic scissors were used to cut a “V”-shaped incision on the arterial wall, the rat PE50 polyethylene catheter, which had been already lubricated by the liquid paraffin, was placed into the incision site and moved forward to the common carotid artery, the ligature was slightly fixed, and the catheter was flushed with heparin saline (40 IU/mL). The artery clamp was removed, and at the moment the waveform observed in the monitor was the waveform of arterial blood pressure. Ligated the left external carotid artery, the catheter slowly inserted through the artery gap into the left ventricle, the waveform of the arterial blood pressure suddenly became the waveform of ventricular internal pressure. Fixed the artery catheter, and then sutured the cervical incision after the hemodynamic measurement. The blood pressure parameter and curve were analyzed by using some professional software including CHART and Scope, and some data such as the heart rate (HR), heart index (HI), left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and the largest increasing/ decreasing rate (dp/dtmax) were obtained, and the left ventricular functions in rats were measured such as LVEDP (Zheng et al, 2013). 2.5.4    Pathological observation of joint synovium  After 24 h of the last medication, the rats were sacrificed. The paw joints were cut open, the specimens were observed

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Sun Y et al. Chinese Herbal Medicines, 2016, 8(2): 143-153

in general to confirm the presence or absence of joint effusion, synovial swelling, and the condition surface, glossiness and smoothness of cartilage. According to the situation, 3−6 paw joint lesion specimens at different lesion sites were taken and prepared to be specimens. All the specimens used were fixed by using 100 mL/L neutral formalin solution for 72 h, decalcificated in 100 mL/L mixture of nitric acid mixed solution (100 mL/L nitrate + 100 mL/L neutral formalin + 600 mL/L double-distilled water) for 48 h, dehydrated conventionally, performed with paraffin embedding with thickness of 5 μm, and then sliced continuously into pieces. HE staining method was used for the grading assessment (Zhang et al, 2012). ① Inflammatory cell infiltration: 0: no inflammatory cell; +: sparsely scattered; + +: fairly intensive; + + +: a large number. ② Fibroplasia: 0: no fibroplasia; +: few hyperplasia; + +: moderate hyperplasia; + + +: a large number. ③ Synovial cell hyperplasia: 0: no synovial cell proliferation; +: dense monolayer cell swelling; + +: dense bi-layer cell swelling; + + +: dense tri-layer cell swelling. ④ Macrophage- like type-A cell hyperplasia: 0: no macrophage-like type-A cell; +: sparsely scattered; + +: fairly dense; + + +: a large number. 2.5.5    Pathological observation of cardiac tissue  When the rats were sacrificed, the chest was cut open and the heart was taken out, the surrounding connective tissues were removed, washed with saline, and the cardiac tissue was fixed in 4% paraformaldehyde for 8 h, then conducted dehydration, transparent processing, waxing and embedding, and then sliced into pieces (6 μm) for HE staining. 2.5.6    Determination of ROS, RNS, GSH, and TRX in cardiac  tissue with immunohistochemical method  The measurement was performed according to the streptavidin-peroxidase (SP) method kit. ROS, RNS, GSH and TRX monoclonal antibodies were diluted with the dilution proportion of l:200, respectively, randomly chose one slice in each group and performed with staining by 0.01 mol/L PBS solution instead of primary antibody, and took it as a negative control. Five fields were randomly chosen in each slice, it was deemed to be a positive control when some yellow or brown granules appeared in the cytoplasm, cytoplast, and nucleus. Semi-quantitative determination of immunohistochemical staining: ① Staining intensity of staining index of protein expression (A): colorless, pale yellow, brown, and tan were counted as 0, l, 2, and 3 points, respectively; staining extent (B): The percentage of the number of positive cells accounted the total number of cells; Table 1

MAF Keap1

IOD = area × mean optical density The larger the positive reaction IOD is, the stronger the protein expression is.

2.5.7    Determination of expression level of Keap1 Design and synthesis of primer were as follows. The primer was designed and analyzed by the Primer Premier 5 according to Kelch-like ECH-associated protein-1 (Keap 1) mRNA sequence (GeneBank No. NM_057152), small Maf proteins (Maf) mRNA sequence (GeneBank No. AF540876) and NF-E2-related factor 2 (Nrf2) mRNA sequence (GeneBank No. NM_031789) provided by GenBank. The PCR primers were designed by Invitrogen, China (Table 1). Maf and Nrf2 mRNA in rat cardiac tissue by real-time quantitative polymerase chain reaction (RT-qPCR) method. Cardiac tissues (50−100 mg) of each group were weighed and homogenized with a homogenizer, and total RNA was extracted using Trizol kit (Trnzol-a +). The reverse transcription was performed according to the method indicated in the product instruction of M−Mulv RT-PCR Kit. The fluorescent quantitative PCR reaction system is 20 μL, including 10 μL of 2 × Goldstar Taqman mixture premixed system, 2 μL of reverse transcription product, 0.4 μL of forward primer and 0.4 μL of reverse primer, 0.4 μL of probe, and then supplemented with water to 20 μL. The reaction conditions were as follows: pre-denaturated at 95 oC for 10 min, and then the reaction was carried out with 40 cycles repeatedly, each cycle was comprised of denaturation at 95 oC for 15 s, annealing for 40 s, extension at 60 oC for 60 s, and terminated at 4 oC. The analytical method used for the experiment was the relative quantification study, and the indicator used for the analysis was Ct values. 2.5.8    Determination  of  expression  levels  of  HO‐1  and  r‐GCS proteins in rat cardiac tissue using Western blotting  The specific procedures were as follows: ① Sample preparation: added with liquid nitrogen with the volume of four times the volume of cardiac tissue, transfer into the

Receptor and ligand fluorescence quantitative PCR primers sequences and predicted amplification length

Genes Nrf-2

The value obtained by multiplying the two values (A and B) was the immunohistochemical staining index, the higher the staining index was, the stronger the protein expression was. ② Integral optical density (IOD) was performed with image analysis by using the image analysis software (professional image analysis software system, image-proplus, Media Cybernetics Inc., USA), the expression of ROS, RNS, GSH, and TRX proteins was indicated by integrated optical density (IOD); The calculation formula was as follows.

Primers forward

5’-ACGGGGCAGTCATGTACCA-3’

reverse

5’-GTTGAAGAACTCCTCCTGCTTG-3’

forward

5’-TTGGTTTATCTGGCTCAT-3’

reverse

5’-AGTCCGTCAGTGTTATTCA-3’

forward

5’-GTGCTGCATGTGATGAACGG-3’

reverse

5’-AAGAACTCCTCCTCCCCGAA-3’

Predicted amplification segments / bp 197 147 197

Sun Y et al. Chinese Herbal Medicines, 2016, 8(2): 143-153 cardiac tissue and grounded, radio immune precipitation assay (RIPA) was used for measuring the total protein of extract tissue in lysis buffer (containing PMSF), performed the colorimetric analysis using biological spectrophotometer, UV absorption method was used for the determination of content of total protein, the protein concentration was adjusted to be 2 μg/μL, took 5 μL of total protein, added with 10 μL of sample buffer and boiled for 5 min, took out and loaded the sample. ② Electrophoresis separation; prepared the separation gel and stacking gel; when the gel was well condensed, loaded the sample for electrophoresis. The voltage used for the upper gel was 40−60 V, when the sample moved to the separating gel, the voltage used was 100−120 V, the time spent was 60−90 min. ③ Membrane transfer: the protein was transferred from gel to membrane by using the semi-dry membrane transfer method. The lower filter papers (3 to 4 layers) were paved on the electronic membrane transfer device; the right corner of membrane was cut off (used for labeling), the membrane was paved on the filter papers attached to the membrane, exhausted the bubbles, poured some transfer buffer on the membrane to make the membrane moisten; removed the stacking gel, stripping out the gel, carefully transferred the gel onto the membrane; covered the upper filter paper (3 to 4 layers) onto the gel; installed the electronic membrane transfer device, the setup of voltage and time was 18 V and 33 min, respectively. ④ Isolation: isolated with 5% skim milk powder for 1−2 h. ⑤ Immunoreactions: incubation of primary antibody: the antibodies HO-1 and r-GCS were diluted with the dilution proportion of 1:1000; the diluted antibodies and membrane were incubated at room temperature for 1−2 h; washing: washed with PBST for three times (5 min, × 5); incubation of secondary antibody: the secondary antibody of anti-Rabbit was used for HO-1 and r-GCS, and were diluted with the dilution proportion of 1 : 5000. Incubated at room temperature for 30−60 min; Washed with PBST (5 min, × 5). ⑥ Color developing: enhanced chemiluminescence (ECL) was used for color developing: the color developing substrates A and B were mixed isometrically, and then covered the mixture uniformly on the membrane surface (1−2 min); wrapped the membrane with the film, put into the clamp plats; covered the X-ray sheet on the membrane in the dark room, exposed for 1−5 min; developed, fixed, and washed. Data analysis and processing: the film was scanned and photographed by using scanner; the strip was analyzed and processed by using the software Band Scan, calculated the gray-scale value strip in each group, and then compared with the gray-scale value of internal reference β-actin in each group, and the ration of the two gray-scale values was taken as the relative expression level of HO-1 and r-GCS proteins in cardiac tissue. 2.5.9    Determination  of  SOD,  MDA,  ROS,  T‐AOC,  IL‐4,  and  TNF‐α using ELISA method  After 24 h of the last medication, the rats were performed with anesthesia by ip injecting 10% chloral hydrate 300 mg/kg (10 mL/kg), the blood was collected with

147

abdominal aortic method, the collected blood was centrifuged at 3000 r/min for 5 min, the serum was taken out, placed into a refrigerator (−80 oC) and waited for testing. Dual-antibody sandwich ELISA method was used for measuring SOD, MDA, ROS, T-AOC, IL-4, and TNF-α, the specific operation procedures were performed according to the kit instructions (The kits were purchased from R&D Company, USA). Determination of results: the OD value of each well was read in an enzyme-labeled instrument at a wavelength of 450 nm; the OD value was taken as the vertical axis, the concentration of standard was taken as the horizontal axis, and then the curve was plotted; the corresponding concentration range was found out according to the OD value of the sample. 2.5.10    Statistics processing  SPSS 17.0 was used for statistics processing, and the continuous variables were expressed as x ± s ; The comparative analysis between groups was compared generally by using variance analysis, when the difference was statistically significant, multiple comparison was performed, pairwise comparisons, the test level (α) was equal to 0.05.

3.    Results   

3.1    Comparison  on  body weight,  paw  swelling,  and  AI in rats in each group  Before inducing inflammation, the body weight, paw swelling and AI were compared in rats in each group, respectively; the differences were not statistically significant (P > 0.05). After medication, when compared with the NC group, in the MC group before medication and each treatment group, the paw swelling and AI in rats were significantly increased (P < 0.01), the body weight was significantly decreased (P < 0. 05). When compared with the MC group, in the LEF group and APS group after medication, the paw swelling and AI in rats were significantly decreased (P < 0.05, 0.01), and the body weight in the APS group was significantly increased (P < 0.05), respectively. As shown in Table 2. 

  3.2    Comparison on each indictor of cardiac function  in rats in each group  When compared with NC group, HR, HI, LVSP, and LVEDP in MC group were significantly increased, and ± dp/dtmax was significantly decreased (P < 0.05, 0.01). HR, HI, LVSP, and LVEDP in rats were decreased in the treatment groups, in addition to APS group, ± dp/dtmax in other groups was increased (P < 0.05, 0.01), respectively. When compared with MC group, in LEF and APS groups after medication, HR, HI, LVSP, and LVEDP in rats were significantly decreased, and ± d p/d tmax was significantly increased (P < 0.05, 0.01), in which ±d p/d tmax in rats of LEF group was significantly increased (P < 0.01), as shown in Table 3. 

148

Sun Y et al. Chinese Herbal Medicines, 2016, 8(2): 143-153 Table 2

Groups

Time

NC MC APS LEF *

P < 0.05

Comparison on body weight, paw swelling, and AI in rats in each group ( x ± s , n = 12 ) Body weight / g

Paw swelling / %

AI

5.34 ± 1.56

0.20 ± 0.05

279.4 ± 37.3

6.54 ± 2.70

0.32 ± 0.07

22.08 ± 8.69**

1.03 ± 0.06**

after medication

169.6 ± 42.5* 225.6 ± 34.9*

18.10 ± 13.00**

6.21 ± 0.75**

pre-administration

173.1 ± 43.0

19.43 ± 7.96**

0.95 ± 0.08**

after medication

282.5 ± 26.1△

12.56 ± 6.40*△

5.03 ± 1.04**△△

pre-administration

176.4 ± 36.1

18.67 ± 9.25**

0.93 ± 0.09**

after medication

232.9 ± 30.2*

11.26 ± 9.20**△

5.21 ± 0.57**△

pre-administration

178.3 ± 31.6

after medication pre-administration

**

△

P < 0.01 vs NC group in same period; P < 0.05

△△

P < 0.01 vs MC group in same period; same as below

Table 3 Comparison on indictors of cardiac function in rats in each group ( x ± s , n = 6 ) Group

HR / (n·min−1)

HI

LVSP / mmHG

LVEDP / mmHG

NC

380.32 ± 15.7

2.34 ± 0.21

112.30 ± 4.12

−1.33 ± 0.52

MC

417.00 ± 15.3*

3.36 ± 0.85**

160.63 ± 6.11**

2.43 ± 0.87

103.19 ± 7.87

3.27 ± 0.72**△

119.99 ± 2.41

*

APS

318.91 ± 29.2

LEF

506.48 ± 21.3*△

3.3    Pathological  characteristics  of  joint  synovium  and cardiac tissue observed by optical microscope    3.3.1    Pathological changes of joint synovium in AA rats No significant synovial hyperplasia was showed in the NC group, the HE staining results showed that, bone layer was clear, the surface was smooth and flat, the chondrocytes were evenly distributed, no cluster was appeared and the tide line was intact. The MC group showed that, the joint surface was rough and irregular, the extent of infiltration of synovial cell, hyperplasia of synovitis cell and fibrous tissue were significantly increased. No hyperplasia of macrophage-like Type-A cell was appeared, as shown in Figure 1.

A

Figure 1

± dp/dtmax / (mmHG·ms−1) 546.82 ± 24.82

6.08 ± 0.76**

291.42 ± 24.27**

−1.10 ± 0.89 1.63 ± 0.75△

618.30 ± 29.82 1147.61 ± 53.71**△△

3.3.2    Pathological changes of heart tissue in AA rats  To observe the HE-staining myocardial slices by optical microscope, the myocardial in the MC group showed that, the myocardial fibers appeared edema and the corresponding separation widened generally. The number of myofibril was increased, and the myocardial fiber in the visible portion of the longitudinal section was deranged, which was of “branching” shape. The myocardial fiber in the visible portion of the cross-section was arranged crowdedly, which was of “swirl” shape. The nuclei were deeply stained and irregular shaped, the transactional diameter of myocardial (TDM/μm) cell was significantly higher than that of the control group. A small part of myocardial tissue was replaced by fibrous tissue,

B

Pathological changes of particular cartilage in AA rats of NC (A) and MC (B) groups

disproportionate connective tissue proliferation and fatty degeneration as well as nuclear shrinking of muscle cell appeared, as shown in Figure 2.

in the MC group expression was increased, respectively, and the expression of GSH and TRX proteins was decreased, respectively, as shown in Figure 3.

3.4    Expression  of  ROS,  RNS,  GSH,  and  TRX  proteins  of cardiac tissue in rats from four groups 

  3.5    Expression  of  Keap1,  maf,  and  Nrf2  mRNA  in  cardiac tissue in rats from four groups 

The immunohistochemistry results showed that, ROS, RNS, GSH, and TRX were mainly located at the cytoplasm of myocardial cells, endothelial cells, and fibroblasts, and also were expressed in the inflammatory cells. When compared with the NC group, the expression of ROS and RNS proteins

As detected by RT-qPCR: When compared with the NC group, the expression of Keap1, maf, and Nrf2 mRNA of cardiac tissue in rats in the MC group, respectively, in which, the expression of Nrf2 mRNA increased was particularly evidently, as shown in Figure 4.

Sun Y et al. Chinese Herbal Medicines, 2016, 8(2): 143-153

A

Figure 2

B

149

C

D

Pathologic changes of heart tissue in rats from NC (A), MC (B), APS (C), and LEF (D) groups

A

a

b

c

d

a

b

c

d

a

b

c

d

aa

b

c

d

B

C

D

A: ROS expression in heart tissue

B: RNS expression in heart tissue C: GSH expression in heart tissue

D: TRX expression in heart tissue

a: NC group b: MC group c: APS group d: LEF group.

Figure 3 8 6

Immunohistochemical staining in different tissues

Keap1 Maf Nrf2

3.6    Expression of HO‐1 and r‐GCS proteins of cardiac  tissue in AA rats 

4 2 0 NC group

MC group

APS group

LEF group

Figure 4 Expression of Keap1, maf, and Nrf2 mRNA in heart tissue of rats in four groups

As detected by Western blotting: Compared with NC group, the expression of HO-1 and r-GCS proteins of cardiac tissue in MC group was increased, respectively. Compared with MC group, the expression of HO-1 and r-GCS proteins of cardiac tissue all decreased in APS and LEF groups, respectively, as shown in Figure 5.

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Sun Y et al. Chinese Herbal Medicines, 2016, 8(2): 143-153

NC

MC

APS

3.7    Comparison  on  oxidative  stress  indexes  and  cytokines of rats in each group 

LEF

β-actin

HO-1

r-GCS

Figure 5

Expression of HO-1 and r-GCS proteins of cardiac

tissue of rats in four groups

Compared with NC group, MDA, ROS, and T-AOC of rats in MC group were increased (P < 0.05). The contents of TAC, MDA, and ROS were increased in rats of treatment groups, the content of SOD was decreased; Compared with MC group, SOD, MDA, TAC, and ROS in APS and LEF groups were decreased (P < 0.05). Compared with NC group, TNF-α in MC group was significantly increased (P < 0.01), and IL-10 was decreased (P < 0.01); Compared with MC group, the content of TNF-α in rats of treatment groups was increased, and IL-10 was decreased (P < 0. 05), as shown in Table 4.

Table 4 Comparison on ROS indexes and cytokines in peripheral blood of rats in each group ( x ± s , n = 12 ) Groups

T-AOC / (U·mL−1)

MDA / (mol·L−1)

SOD / (U·L−1)

NC

1.978 ± 0.072

0.973 ± 0.012

76.05 ± 3.20

91.77 ± 4.82

MC

3.472 ± 0.204**

2.014 ± 0.045**

81.41 ± 4.40

189.78 ± 5.43**

APS LEF

2.510 ± 0.029

*

2.404 ± 0.095

*

1.294 ± 0.067

*

1.255 ± 0.042

*

65.91 ± 3.70

*

56.71 ± 2.23

*

4.    Discussion  AA rat is the classical animal model which is frequently used to study immunological aspects of RA and other arthritic or inflammatory diseases in human. RA is a long lasting disorder that primarily affects joints which is characterized by synovitis, pannus formation, and subsequent joint damage as its main pathological features. It typically result is warm, swollen, and painful joints (Jeffery, 2014). However, RA can affect body parts besides joints. RA is an autoimmune disease, which means the arthritis results from the immune system attacking the body’s own tissues. As discovered in some studies by some foreign researchers (Van Halm et al, 2009), the incidence of cardiovascular disease in RA patients within three years was 9.0%, while only 4.1% in the normal population. RA which now has been seen as an independent cardiovascular risk factor was of equal danger as diabetes. In recent years, many studies had shown that the oxidative stress played an important role in the pathogenesis of RA (Sun et al, 2015). Thus, the study of our team is now focusing on the extra-articular changes of RA, like the decline of cardiac function, and the role of oxidative stress played in the pathogenesis, we designed this experiment which aims to find drugs or therapeutic methods which can not only relief the pain of joints but also improve the cardiac function. LEF, as an immunomodulatory agent, is well recognized to be an effective drug for the treatment of RA (Meier et al, 2013). LEF was neither superior nor inferior to methotrexate (MTX) in several studies. But the two drugs have different dosing methods. To treat RA, a typical dosage of 15–25 mg of MTX once a week is recommended, administered either orally or subcutaneously, and is usually followed by 5 mg of folic or folinic acid the day after to reduce side effects. However, a typical dosage of 10–20 mg of LEF once a day is recommended, which is more similar to our experimental

ROS / (IU·mL−1)

134.32 ± 0.67 112.71 ± 6.41

*

*△

TNF-α / (IU·mL−1)

IL-10 / (IU·mL−1)

49.63 ± 2.07

106.8 ± 21.24

90.79 ± 1.65**

51.8 ± 16.42**

70.15 ± 2.09

*

92.1 ± 29.79*

56.63 ± 3.46

*△

58.8 ± 7.44**△△

drugs. In order to avoid the errors caused by different drug administration, we finally chose LEF as positive control drug. APS is an important natural and active ingredient which is extracted from Astragal Radix. A large number of experiments, in vivo or in vitro, and clinical studies indicated that APS possessed several pharmacological effects on enhancing the body immune function, protecting cardiac and antihypertension, hypoglycemia, antitumor (Zheng et al, 2013), antiviral, anti-radiation, and anti-oxidation (Shao et al, 2004). APS possesses several effects, such as boosting the function of immune organs and the development of antibody effectively, and antibacterial, antiviral, and antitumor effects, and enhancing the phagocytosis effect of reticuloendothelial system on the tumor cell, and promoting the lymphocyte transfer, and activating T and B cells, and promoting the formation of antibodies (Huang et al, 2013). The results of this study indicated that the cardiomyocytes in AA rats showed significant pathological changes. The myocardial fibers appeared in edema and enlargement, the number of myofibril in the cells was increased, some myocardial fibers were deranged, and the myocardial tissue was replaced by the fibrous tissue. The results meet the pathological characteristics of chronic cardiac insufficiency. When compared with NC group, the presence of decreasing cardiac function in AA rats was found, which exhibited that HR, HI, LVSP, and LVEDP were increased and ± d p/d tmax was decreased (P < 0.05, 0.01). It proves that the joint inflammatory reaction in AA rats was occurred simultaneously with the decline of cardiac function. This is consistent with our previous findings (Liu et al, 2012). Compared with the model group, the body weight and ±d p/d tmax in each treatment group were increased, while HR, HI, LVSP, and LVEDP were decreased (P < 0.05, 0.01). However, the improvement in APS group was quite significant. In physiological condition, the production and removal

Sun Y et al. Chinese Herbal Medicines, 2016, 8(2): 143-153 of free radicals in normal body cell were maintained in a dynamic equilibrium state. The effective antioxidant defense system maintained the concentration of free radicals at a physiologically low level (Jalili M et al, 2012). When the equilibrium between oxidation and anti-oxidation in body was broken and tilted to the oxidation side, that the concentration of reactive oxygen cluster was increased, high potent molecules in body such as ROS and RNS were produced extensively, so that oxidative stress arises and the tissue damage were induced (Kundu et al, 2012). On one hand, ROS and RNS can cause oxidative damage to various biological macromolecules such as nucleic acid (DNA and RNA), lipid, protein, and sugar, and organelle such as mitochondria, on the other hand, ROS and RNS as messenger molecule can participate the physiological pathological processes such as regulation of gene expression, thus cause damage to the body or cell tissue (Li et al, 2011). By using immunohistochemistry, this study has observed myocardial cells in AA rats, and found that the expression levels of ROS and RNS in myocardial cells in rats of MC group were significantly increased, respectively, and the levels of ROS and RNS in the other medication groups were higher than these of NC group, respectively, but the level of ROS and RNS was of significantly decreasing trend compared with MC group, and the level of ROS and RNS in LEF and APS groups decreased was of no significant difference (P < 0.05), respectively. Glutathione (GSH), a non-protein anti-oxidant containing hydrosulfide group, is composed of glycine, cysteine, and glutamic acid, the main function of GSH is to protect the enzyme containing hydrosulfide functional group and the protein from oxidation, to protect the integrity of erythrocytes membrane, to prevent the oxidation of ferrous hemoglobin to methemoglobin, and to eliminate the cell damages caused by peroxides and free radicals (Mirshafiey and Mohsenzadegan, 2009). Under the pathological conditions such as oxidative stress, the intracellular GSH would be decreased due to consumption. Thioredoxin (TRX) and thioredoxin reductase (TRxR) are the redox-active system of oxidative stress induction. Some studies have shown that, the local oxidative stress contributed to the up-regulated expression of TRX caused by the RA fibroblast-like synovial cells, which not only possessed the potential ability to stimulate the expression of inflammationinducing cytokines such as TNF-α, IL-1, and IL-6; but also regulated the activities of transcription activator protein 1 (AP-1) and nuclear factor (NF)-κB through the modification and regulation of redox, thus to regulate the transcription of related genes. By using immunohistochemistry, this study had observed the myocardial cells in AA rats, and found that the GSH and TRX expression levels of myocardial cells in rats in MC group were significantly decreased, and the GSH and TRX levels in other medication groups were of no significant difference when compared with NC group (P < 0.05), and the levels of ROS and RNS in LEF and APS groups decreased was of no significant difference (P < 0.05), respectively. Malondialdehyde (MDA) is the main end-product of lipid peroxidation in body produced after the attack of free

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radicals produced, and the level of MDA indirectly reflects the severity extent of the body cells attacked by free radicals. Some studies have shown that, the phenomenon of enhanced peroxidation or decreased antioxidant capacity was present in RA patients, and the MDA content of serum in RA patients was significantly increased, respectively; Some literatures have also reported that, the MDA level of serum in RA patients was significantly higher than that of normal people (Kundu et al, 2012). This study has observed the MDA and ROS levels of serum in AA rats, and found that they were significantly increased, and the ROS level in other medication groups was decreased, respectively, and the MDA levels in other medication groups were higher than these of NC group, but were of significant decreasing trend when compared with these of MC group, and the MDA and ROS levels between LEF and APS groups MDA were of no significant difference (P < 0. 05). The level of serum total anti-oxidant capacity (T-AOC) indirectly reflects the body’s capacity on eliminating reactive oxygen species. SOD can remove the superoxide anion free radicals and protect the cells from damage, so the level of SOD activity is closely related with RA. Some literatures (Vijayakumar et al, 2009) showed that the SOD activity of serum in RA patients was significantly decreased, which indicated that the anti-oxidant defense capacity in body of RA patients was weakened. Some studies found that the T-AOC level of serum in RA patients was significantly decreased compared with that of healthy control group. This study has observed the T-AOC and SOD levels of serum in AA rats, and found that the T-AOC and SOD levels of serum in AA rats were increased at different extents. When compared with the AA rats, the T-AOC and SOD levels in each treatment group were decreased, respectively, and the T-AOC level in LEF and APS groups was significantly decreased (P < 0.05), the SOD level in LEF group was decreased at a more significant extent (P < 0.05). The body in response to the active oxygen damage has formed a complex oxidative stress response system, the coordination response is regulated by antioxidant responsive element (anti-oxidant responsive element, ARE) of these protective genes in upstream regulation region (Itoh et al, 2003). Nrf2 is the activator of ARE. Nrf2, as a critical factor to regulate intracellular expression of many antioxidants, has play a role in keeping the balance between cellular oxidative and antioxidant, apoptosis inhibition, multiple biologically activity of anti-inflammation, its anti-oxidation process has correlated and closely related with various type of cellular factors, and plays multiple protection role in the process (Stewart et al, 2003). Nrf2-ARE is the most important endogenous antioxidant stress pathway ever discovered up to now (Nguyen et al, 2003). Keapl-Nrf2/ARE, as an effective antioxidant regulation pathway, has played an important role for the defense, and reduced the oxidative stress of the human body (Rubiolo et al, 2008). The activated Nrf2 disaggregated from Keap1 has entered the nucleus, and bonded with Maf protein in nuclear to form heterodimer, then combined with the ARE sequences, and then initiated the transcription of

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ARE-regulated genes. Keapl is negative regulation factor for Nrf2, when it is under the stimulation of oxidation, exogenous chemical substances or electrophilic compounds, the cysteine residues are oxidized, thus resulting in the uncoupling of Nrf2 and Keapl, thereby activating the phase Ⅱ detoxifying enzyme gene in downstream elements of antioxidant response sequence and generating the gene transcription of antioxidant enzymes, such as NQO1, HO-l, and GSH, and increasing resistance to the oxidative stress in cells, exogenous chemical substances and electrophilic compounds (Can et al, 2005). Through the observation on the expression of Keapl, maf, and Nrf2 mRNA in myocardial cells in AA rats, when compared with the NC group, it was found that the expression of Keap1, maf, and Nrf2 mRNA in myocardial cell in rats in the MC group were all increased slightly, in which Nrf2 mRNA expression was increased significantly particularly (P < 0.05) (Rajesh et al, 2006). Therefore, it is showed that while AA rats are under the inflammatory immune response, the oxidative stress pathway is also activated. This study has also observed the expression of HO-1 AA myocardial tissue, γ-GCS protein, it was found that after drug intervention, both of the originally increased HO-1 and γ-GCS were becoming normal, it also showed that drug intervention can be regulated by the Keapl-Nrf2/ARE expression pathway, then further to regulate the oxidative stress in body (Cai and Lu, 2010). In our study, the cardiac function is found reduced in AA rats and abnormal pathway activation of Keapl-Nrf2/ARE pathway is also found. Keap l, as a major inhibiting factor for Nrf2 transcription plays an important role in rapid degradation of Nrf2. The results of this study show that massive Keapl proliferation leading to the reduce of activated Nrf2 dissociated from Keap1 as well as the reduction heterodimers generated by binding with Maf protein in nuclear, thereby inhibiting the transcription of ARE-regulated genes, further resulted the function of antioxidant decreased. TNF-α is an important physiological inflammatory mediators and plays a leading role in the generation and the development of RA, it is mainly generated by the synovial membrane and macrophages of cartilage pannus, it is the upstream factor in inflammatory cascade and has the pleiotropy. In joint lesions, it can stimulate the synthesis of synovial cells and chondrocytes, thus resulting in destruction and absorption of bone and cartilage, and finally promotes the proliferation of fibrocyte. TNF-α can stimulate the macrophages to produce IL-6, IL-8, MCP-1, and MCP-1α and oxygen free radicals and allow them involved in the local inflammation. The TNF-α level in synovial fluid has connected with the number of macrophages in synoviale intima and the extent of radiological bone erosion (Sun et al, 2015). IL-10 can inhibit the immune pathological process of RA and is publicly recognized as an anti-inflammatory potential (Liu et al, 2013). The results of this study showed that TNF-α was found significantly increased in rats of MC group, IL-10 was found significantly decreased, and has a significant correlation with the paw swelling and AI by compared with the NC group. Thus, we assume that due to excessive activation of

Keap1-Nrf2/ARE pathway, ROS, RNS, MDA, and other oxidation products were overly produced, the concentration of anti-oxidizing substance such as GSH, TRX, r-GCS, etc. was reduced, the degree of oxidation overwhelmed oxides was removed, the oxidation system and anti-oxidant system became unbalance, which therefore cause the increase of body’s pro-inflammatory factor TNF-α, decrease of suppression of inflammatory factor IL-10, aggravation of inflammatory immune response, thus resulting in long-term chronic inflammation of the body, leading to significant formation of immune complex. The immune complex is deposited in tissue with rich connective tissues and blood vessels such as heart and other regions, which further activate the complementary body and large amount of neutrophile granulocyte to be accumulated in complex sedimentary location; The lysosomal enzymes were released when neutrophil was swallowing the composite, resulting in damage to surrounding tissue. It can also cause diffuse micro-thrombosis, endothelial cells proliferation and macrophages and lymphocytes infiltration directly or indirectly, which further cause the damage to synovial membrane and heart, vascular endothelial tissue, leading to decrease of cardiac function. The modern pharmacological research shows that Astragali Radix can improve myocardial energy metabolism and protect cell structures, mitochondrial structure in particular, increasing the intracellular SOD activity, reducing the damage of oxygen free radicals on myocardial membranes, thereby enhancing myocardial contractility, improving myocardial ischemia (Niu and Ju, 2013). Previous studies have also shown that APS can significantly improve the vitality of free radical scavenger SOD, significantly decrease the assay of end product MDA generated by lipid peroxidation, and can significantly improve the fatigue resistance of swimming mice, cold-resistant ability and atmospheric pressure hypoxia-resistant ability, and significantly improve the exercise capability of mice, and the athletic injury caused by exhaustive exercise-resistant ability, and indicated that APS can eliminate free radicals, protect the cells, and delay the aging (Gao et al, 2010). In conclusion, a decline in cardiac function in AA rats happens, which may be caused by oxidative stress dysfunction in body due to immune inflammation reaction. Traditional Chinese medicine APS can improve the cardiac function, whose action may be related to the mechanism that the APS can inhibit the production of free radicals, and improve the antioxidant effect in body, thus the peroxidation of myocardial cells is inhibited.

  Acknowledgements  We would like to thank Prof. Jian Liu, and Prof. Chuan-bing Huang. at the First Affiliated Hospital of Anhui University of Chinese Medicine for supporting the conduct and monitoring of the study, and PhD. Xiao-jun Zhang, PhD. Lei Wan, and Fang Wang, Ya-jun Qi for assistance with the preparation and review of the manuscript.

Sun Y et al. Chinese Herbal Medicines, 2016, 8(2): 143-153 References    Barber CE, Smith A, Esdaile JM, Barnabe C, Martin LO, Faris P, Hazlewood G, Noormohamed R, Alvarez N, Mancini GB, Lacaille D, Keeling S, Aviña-Zubieta JA, Marshall D, 2015. Best practices for cardiovascular disease prevention in rheumatoid arthritis: A systematic review of guideline recommendations and Quality Indicators. Arthritis Care Res 67(2):169-179. Cai QQ, Lu ZQ, 2010. The effect of Keapl-Nrf2/ARE chemical pathway in the process of antioxidation. Zhejiang Med J 32(7): 1113-1116. Cai WC, Wang H, Li RQ, Gao L, 2005. Determination of oxidation-reduction level in patients with rheumatoid arthritis. J South Med Univ 25(6): 749-750. Gao X, Li LF, Liu BX, 2010. Effects of Astragalus polysaccharides on stress response ability and regulation of free radicals in mice. China Prev Med 11(2): 120-121. Huang JL, Zhang Y, 2013. Mechanism research progress of Astragalus polysaccharides anti-inflammatory effect. Chin J Integr Med (cardio-/cerebrovascul dis) 11(11): 1374-1376. Itoh K, Wakabayashi N, Katoh Y, Ishii T, O'Connor T, Yamamoto M, 2003. Keap1 regulates both cytoplasmic-nuclear shuttling and degradation of Nrf2 in response toelectrophiles. Genes Cells (8): 379-391. Jalili M, Kolahi S, Aref-Hosseini SR, Mamegani ME, Hekmatdoost A, 2014. Beneficial role of antioxidants on clinical outcomes and erythrocyte antioxidant parameters in rheumatoid arthritis patients. Int J Prev Med 5(7): 835-840. Jeffery RC, 2014. Clinical features of rheumatoid arthritis. Med 42(5): 231-236. Kundu S, Ghosh P, Datta S, Ghosh A, Chattopadhyay S, Chatterjee M, 2012. Oxidative stress as a potential biomarker for determining disease activity in patients with rheumatoid arthritis. Free Radic Res 46(12): 1482-1489. Li H, Duan HJ, 2011. Nrf2/ARE pathway and downstream antioxidant genes. Chin Pharmacol Bull 27(3): 300-303. Li YK, 1991. Traditional Chinese Medicine Pharmacology Experiment Methodology. Shanghai Scientific and Technical Publishers, Shanghai. Liu J, Cao YX, Huang CB, Zhang WD, 2012. The changes on cardiac function and MMP-9, TIMP-1 expression in the myocardium in adjuvant arthritis rats. Chin J Immunol 28(3): 251-256, 265. Liu J, Cao YX, Zhu Y, 2012. Effects of Xinfeng capsule on the cardiac function and the myocardial ultrastructure in rats with adjuvant arthritis. Chin J Integr Tradit West Med 32(11): 1543-1548. Liu J, Sun Y, 2013. How does Chinese medicine target cytokine imbalance in rheumatoid arthritis. Chin J Integr Med 19(11): 874-880. Meier FM, Frerix M, Hermann W, Müller-Ladner U, 2013. Current immunotherapy in rheumatoid arthritis. Immunotherapy 2013; 5(9): 955-974. Mirshafiey A, Mohsenzadegan M, 2009. The role of reactive oxygen species in immunopathogenesis of rheumatoid arthritis. Iran J Allergy Asthma Immunol 7(4): 195-202. Nguyen T, Sherratt P J, Huang HC, Yang CS, Pickett CB, 2003.

153

Increased protein stability as a mechanism that enhances Nrf2-mediated transcriptional activation of the antioxidant response element: Degradation of Nrf2 by the 26S proteasome. J Biol Chem 278(7): 4536-4541. Niu LL, Ju BJ, 2012. Pharmacology and application research on astragalus in cardiovascular diseases. Clin J Chin Med 4(7): 46-47. Rajesh K, Thimmulappa R, Hannah L, Reddy SP, Yamamoto M, Kensler TW, Biswal S, 2006. Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis. J Clin Invest 1(16): 984-995. Ralf W, Wagner U, 2002. Prognostic use of human leukocyte antigen genotyping for rheumatoid arthritis susceptibility, disease course, and clinical stratification. Rheum Dis Clin North Am 28(1): 13-37. Rubiolo JA, Mithieux G, Vega FV, 2008. Resveratrol protects primary rat hepatocytes against oxidative stress damage: Activation of the Nrf2 transcription factor and augmented activities of antioxidant enzymes. Eur J Pharmacol 591: 66-72. Shao BM, Xu W, Dai H, 2004. A study on the immune receptors for polysaccharides from the roots of Astragalus membranaceus, a Chinese medicinal herb. Biochem Biophys Res Commun 20(4): 1103-1111. Stewart D, Killeen E, Naquin R, Alam S, Alam J, 2003. Degradation of transcription factor Nrf2 via the ubiquitin-proteasome pathway and stabilizationby cadmium. J Biol Chem (278): 396-402. Sun Y, Liu J, Wan L, Wang F, Qi YJ, 2015. Xinfeng Capsule increases peripheral blood BTLA expression of CD19(+) and CD24(+) B cells and relieves oxidative stress damage to improve cardiac function of patients with rheumatoid arthritis. Chin J Cellular Mol Immunol 31(1): 93-96. Sun Y, Liu J, Wan L, Qi YJ, 2015. Changes of cardio-pulmonary function in rheumatoid arthritis patients on the basis of BTLA and oxidative stress. Immunological J 31(3): 234-239. Van Halm VP, Peters MJ, Voskuyl AE, Boers M, Lems WF, Visser M, Stehouwer CD, Spijkerman AM, Dekker JM, Nijpels G, Heine RJ, Bouter LM, Smulders YM, Dijkmans BA, Nurmohamed MT., 2009. Rheumatoid arthritis versus diabetes as a risk factor for cardiovascular disease: a cross-sectional study, the CARRE Investigation. Ann Rheum Dis 68(9): 1395-1400. Vijayakumar D, Suresh K, Manoharan S, 2006. Lipid peroxidation and antioxidant status in blood of rheumatoid arthritis patients. Indian J Clin Biochem 21(1): 104-108. Wei W, Wu XM, Li YJ, 2010. Experimental Methodology of Pharmacology. 4th Ed. Beijing: People’s Medical Publishing House 919-921. Zhang JT, Du GH, 2012. Modern Pharmacological Experiment Method. Peking Union Medical College Press, Beijing 1383. Zheng YQ, Xu BX, Wang Y, Wang XY, Wang CJ, Zhan Q, Hou ZZ, Yang F, Ni FY, Chen HD, 2013. Improved method of left ventricular cannulation and measurement of cardiac function parameters in model rats with the heart-Qi deficiency Syndrome. J Shanxi Coll Tradit Chin Med 14(2): 19-21. Zheng ZP, Yang WB, Li N, Bo YJ, Xing SY, Shao Q, Huang LB, 2013. Clinical observation on injection of astragalus polysaccharides protecting marrow suppression of non-small-cell lung cancer after chemotherapy. Chin Tradit Herb Drugs 44(2): 208-209.