Effects of Alcohol Extract of Giantleaf Ardisia Rhizome on Levels of Inflammator

Affiliated Zhongshan Hospital of Guangzhou University of Chinese Medicine, Zhongshan 528401, China

Abstract [Objectives] The purpose of this study was to investigate the effects of ethanol extract of Giantleaf Ardisia Rhizome on the serum levels of interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in adjuvant arthritis (AA) rats induced by Freund’s complete adjuvant (CFA), so as to investigate its role in the treatment of rheumatoid arthritis. [Methods] Rat models of AA were induced by CFA, and they were given with different doses of ethanol extract of Giantleaf Ardisia Rhizome, followed by scoring of systemic and joint inflammation and swelling degree and determination of serum IL-6, TNF-α and IL-1β levels. [Results] Compared with the model group, the ethanol extract of Giantleaf Ardisia Rhizome significantly reduced the scores of systemic and joint inflammation and swelling degree and the serum levels of IL-6, TNF-α and IL-1β in AA rats (P<0.05, P<0.01). [Conclusions] The ethanol extract of Giantleaf Ardisia Rhizome can suppress the development of AA in rats and the mechanism may be related to the down-regulation of the expression of serum inflammatory factors (IL-6, TNF-α, IL-1β).

Key words Giantleaf Ardisia Rhizome, Adjuvant arthritis, IL-6, TNF-α, IL-1β

1 Introduction

Rheumatoid arthritis (RA) is a multifactorial chronic inflammatory disease involving the surrounding joints, mainly manifested as persistent joint pain, redness, deformation and dysfunction of activity. There are currently no specific drugs for clinical treatment of RA. Clinically, non-steroidal anti-inflammatory drugs and immunosuppressive drugs are used to relieve clinical symptoms and control the disease. However, these drugs have toxic and side effects such as gastrointestinal and liver and kidney function damage[1]. Therefore, the treatment of RA with relatively low toxic and side effects has gradually attracted attention. Giantleaf Ardisia Rhizome is the dry root ofArdisiagigantifoliastapf., with effects of dispelling wind, eliminating dampness, promoting blood circulation and removing blood stasis. It is used for the treatment of rheumatism, bruises, postpartum blood stasis, postpartum abdominal pain and sore ulcer[2], and is one of the few Chinese folks commonly used in the treatment of RA[3]. A number of clinical reports have also shown that single[4]and compound[5-7]preparations of Giantleaf Ardisia Rhizome have a good therapeutic effect on RA, and the side effects are small, which is worthy of research and development. However, there has been no experimental research on the effect of Giantleaf Ardisia Rhizome on RA. In this paper, CFA-induced adjuvant arthritis (AA) rat models were established to explore the anti-RA effect of ethanol extract of Giantleaf Ardisia Rhizome and the mechanism, in order to provide a scientific experimental basis for its application in clinical treatment of RA.

2 Materials

2.1 Experimental animalsMale SD rats of SPF grade, weighing (170±20) g, were provided by Guangdong Experimental Animal Center[animal certificate, SCXK (Guangdong) 2013-0034].

2.2 Main drugs and reagentsGiantleaf Ardisia Rhizome (Guangzhou Zhixin Traditional Chinese Medicine Decoction Pieces Co., Ltd., batch No. 20131201, identified as the dry root ofArdisiagigantifoliastapf. by Associate Professor Tian Suying from Zhongshan Campus of Guangdong Pharmaceutical University); BCG vaccine (Beijing Institute of Biological Products, batch No. 201506DG); methotrexate (Shanghai Xinyi Pharmaceutical Co., Ltd., batch No. 036140901); liquid paraffin (Guangdong Hengjian Pharmaceutical Co., Ltd., batch No. 140102); 0.9% NaCl injection (Sichuan Kelun Pharmaceutical Co., Ltd., batch No. B14012712); IL-1β Elisa kit (Cusabio Biotech Co., Ltd., batch No.O290150033); IL-6Elisa kit (Cusabio Biotech Co., Ltd., batch No. R13015031); TNF-α Elisa kit (Cusabio Biotech Co., Ltd., batch No. Q23015032).

2.3 Main apparatus and instrumentsRE-2000B rotary evaporator (Shanghai Yalong Biochemical Instrument Factory); SHZ-DCIII circulating water vacuum pump (Shanghai Yuhua Instrument Equipment Co., Ltd.); DK-8AS electric heating constant-temperature water bath (Shanghai Huitai Instrument Manufacturing Co., Ltd.); KQ5200E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Muitiskan mLB3 microplate reader [Thermo Electron (Shanghai) Instrument Co., Ltd.]; GL-20G-II high-speed refrigerated centrifuge (Shanghai Anting Scientific Instrument Factory).

3 Methods

3.1 Preparation of test drug solutionsThe medicinal material of Giantleaf Ardisia Rhizome was pulverized. A certain amount (400 g) of the powder of Giantleaf Ardisia Rhizome was added with five times the amount of 70% ethanol and refluxed twice with 1 h each time. The mixed extract was filtered, rotary-evaporated (65 ℃) to an ethanol-free taste, and added with distilled water to prepare into solution with Giantleaf Ardisia Rhizome content of 0.6 and 0.3 g/mL, respectively. The prepared test drug solutions were prepared at 4 ℃ for use.

3.2 Preparation of CFA[8]The liquid paraffin was placed in a 100-mL jar with a blue lid and sterilized according to the procedure for liquids. A mortar was wrapped in newspaper and autoclaved. The BCG vaccine was placed in a constant-temperature water bath at 80 ℃ for 1 h for inactivation. Using the sterilized liquid paraffin and mortar and inactivated BCG, Freund’s complete adjuvant was prepared on a clean bench. A certain volume (10 mL) of the sterilized liquid paraffin was transferred to the autoclaved mortar with a sterile syringe, added with 100 mg of BCG, and ground clockwise to an emulsified state until a droplet (10 μL) of CFA would not scatter in clean water (i.e., the emulsion drop floated on the water surface and did not spread). The content of BCG in the prepared CFA was 10 mg/mL. The total grinding time was 3.5 h.

3.3 Establishment of AA models[9]After one week of adaptive feeding, 10 rats were randomly selected from the test SD rats and taken as a normal control group, and the remaining 52 rats were subjected to CFA modeling. The left hind plantar of each rat in the model group was disinfected with ethanol, and injected with 100 μL of CFA intradermally into the left footpad. After 15 d, if the contralateral joint showed inflammatory response, the modeling was considered to be successful. The rats in the control group were injected with 100 μL/rat of sodium chloride injection in the same manner.

3.4 Grouping and administrationThe 10 rats in the normal control group were continuously taken as the normal control group. A total of 40 rats were selected randomly from the 44 rat models, and they were randomly and evenly divided into four groups: model group, methotrexate positive group (0.5 mg/kg), high-dose Giantleaf Ardisia Rhizome group (6 g/kg) and low-dose Giantleaf Ardisia Rhizome group (3 g/kg). The rats in the Giantleaf Ardisia Rhizome groups were administered once a day; the rats in the methotrexate positive group were administered once every 2 d; and the rats in the normal group and model group were administered with 1 mL/100 g of sodium chloride injection once a day. The administration lasted for 20 d.

3.5 Examined indices

3.5.1Systemic inflammatory status. After the onset of inflammation in AA rats, their whole body performance was scored once every 5 d. The scoring criteria are shown in Table 1. Each rat scored a maximum of 8 points[10].

Table 1 Criteria for scoring of whole body performance of AA rats

PositionScore∥point012NoseNo swelling in connective tissueObvious swelling in connective tissue/EarsNo nodule or rednessNodule and redness in one earNodule and redness in both earsTailNo noduleNodule/Front pawsNo swellingSwelling in one pawSwelling in both pawsHind pawsNo swellingSwelling in one pawSwelling in both paws

3.5.2Joint inflammation and swelling degree. Since the 15th d after the beginning of the modeling, every 5 d (D15, D20, D25, D30 and D35), the joint inflammation and swelling degree of each AA rat was scored, and the secondary lesions of the limbs of each group were observed. The specific scoring criteria are as follows: 0 point, normal; 1 point, red spots and slight swelling in individual areas of the paws and footpads; 2 points, red spots and slight swelling in any two (or all) of paw, footpad and ankle joint; 3 points, red spots and slight swelling from ankle joint to metatarsal joint or metacarpal joint; 4 points, stiffness, deformity, dysfunction and severe swelling in ankle joint. The scores of the four limbs of each rat were added together, and the total score was up to 16 points[11].

3.5.3IL-1β, IL-6 and TNF-α levels. After 1 h of the last administration, the rats were anesthetized with 10% chloral hydrate solution through intraperitoneal injection according to a dose of 300 mg/kg,i.e., 0.3 mL/100 g. Then, the blood of each rat was collected through abdominal aorta. The blood samples were centrifuged at 3 500 r/min, and the serum obtained as preserved at -20 ℃ for analysis. According to the instructions of the ELISA kits, the serum IL-1β, TNF-α and IL-6 levels were determined.

4 Results

4.1 Evaluation on AA rat modelsAfter the injection of CFA, the first 3 days were the acute inflammatory phase of the rats. On the second day of the injection, the swelling of the ankle site at the CFA injection side reached a peak (Fig.1). The swelling lasted for 3-5 d. During the 5th to 8th d, the inflammation at the injection side of most rats gradually decreased. During the 11th to 15th d, the inflammation spread to the contralateral limbs and even throughout the body of some rats (Fig.2). On day 15, inflammation was observed in the four limbs of some AA rats. In individual rats, the joints were stiff, red spots appeared in the tail and ears, or inflammation occurred in the genital (Fig.3). The overall coat color of the AA rats was dull, their movement was disordered (e.g. claudication), and their body weight was reduced compared with the normal group. The success rate of the modeling was 84.62%.

Fig.1 Inflammation occurred at the injection side on day 2 of modelingFig.2 Contralateral joints became red and swollen from day 11 to day 15 of modeling Fig.3 Nodules and inflammation were observed in ear, tail and genitalia of in-dividual rats

4.2 Effects on score of systemic inflammatory statusOn day 15 of the modeling (i.e., on day 1 of the administration), no significant difference was observed in the systemic inflammatory status between the groups (P>0.05), indicating that the overall inflammatory status of the rats in each group was the same before administration. On days 20, 25, 30 and 35, the score of systemic inflammatory status differed significantly between the methotrexate and high and low-dose Giantleaf Ardisia Rhizome groups (P<0.05,P<0.01), indicating that the ethanol extract of Giantleaf Ardisia Rhizome had a better improvement effect on systemic inflammation in the AA rats. There was no significant difference between the high and low-dose Giantleaf Ardisia Rhizome groups (P>0.05), and the dose-dependent relationship was not obvious. The results are shown in Table 2.

GroupScore of systemic inflammatory status∥pointD15D20D25D30D35Model3.80±1.405.90±1.295.10±1.295.20±0.794.10±0.99Methotrexate3.50±1.274.10±1.29∗∗3.40±1.58∗∗2.50±1.27∗∗2.50±1.08∗High-dose ethanol extract of Giantleaf Ardisia Rhizome3.40±1.844.20±1.40∗∗2.70±1.16∗∗2.80±1.40∗∗1.60±1.43∗∗Low-dose ethanol extract of Giantleaf Ardisia Rhizome3.60±1.434.00±1.25∗∗3.10±1.37∗∗2.80±1.62∗∗2.20±1.81∗∗

Note: Compared with model group,*P<0.05,**P<0.01, the same in Table 3 and Table 4.

4.3 Effects on score of joint inflammation and swelling degreeOn day 15 of the modeling (i.e., on day 1 of the administration), there was no significant difference in the score of joint inflammation and swelling degree between the groups (P>0.05), indicating that the joint inflammation and swelling degree of the rats in each group was the same before administration. From day 20 to day 35, the joint inflammation and swelling in the administration groups was milder than that in the model group. Compared with the model group, on day 20, the score of joint inflammation and swelling degree in the methotrexate group was significantly reduced; on day 25, the score of joint inflammation and swelling degree in the high-dose Giantleaf Ardisia Rhizome group was significantly reduced; and on day 35, the score of joint inflammation and swelling degree in the methotrexate and high and low-dose Giantleaf Ardisia Rhizome groups reduced significantly (P<0.05,P<0.01), indicating that ethanol extract of Giantleaf Ardisia Rhizome and methotrexate had better improvement effects on joint inflammation and swelling in rat models, especially after 35 d, the improvement effect was more obvious (Table 3).

GroupScore of joint inflammation and swelling∥pointD15D20D25D30D35Model6.70±3.5310.9±3.519.20±4.268.00±3.977.10±3.67Methotrexate7.60±3.507.40±3.10∗6.60±3.605.10±3.143.80±2.82∗High-dose ethanol extract of Giantleaf Ardisia Rhizome6.70±3.508.70±4.525.70±3.23∗5.60±4.402.50±2.55∗∗Low-dose ethanol extract of Giantleaf Ardisia Rhizome6.30±3.278.00±3.656.10±3.765.40±4.123.60±4.17∗

4.4 Effects on serum levels of IL-1β, IL-6 and TNF-α in AA ratsAs shown in Table 4, the serum IL-1β, IL-6 and TNF-α levels in the model group were higher than those in the normal control group (P<0.01); and compared with the model group, methotrexate and high and low-dose Giantleaf Ardisia Rhizome reduced the serum IL-1β, IL-6 and TNF-α levels in the AA rats (P<0.05,P<0.01), indicating that both high and low doses of Giantleaf Ardisia Rhizome could significantly reduce the levels of IL-1β, IL-6 and TNF-α cytokines in the serum of AA rat models.

5 Discussions

Modern medicine believes that the man pathogenesis of RA is immune disorders. It is a chronic, inflammatory autoimmune disease that damages the synovium, cartilage and bone. It mainly invades the joints throughout the body, with early manifestations of joint pain, obvious swelling and unfavorable flexion and extension and late manifestations of cartilage necrosis and shedding, and joint destruction, deformity and loss of function[11-12]. In this study, CFA was used to induce AA rat model, which has many similar characteristics to human RA in terms of clinical manifesta-

GroupIL-1βIL-6TNF-αModel105.27±85.251.377±0.36524.27±0.79Control28.59±22.39∗∗0.521±0.126∗∗2.48±0.58∗∗Methotrexate37.44±21.41∗∗1.044±0.217∗10.27±2.31∗∗High-dose ethanol extract of Giantleaf Ardisia Rhizome50.86±28.51∗0.777±0.359∗∗12.56±2.20∗∗Low-dose ethanol extract of Giantleaf Ardisia Rhizome46.61±7.92∗0.850±0.263∗∗13.24±4.19∗∗

tions, pathology, serology, immunological changes and pathological mechanisms, and is an experimental animal model for studying the pathogenesis of RA and evaluating the efficacy of RA drugs[13-14]. A study[11]believes that AA rat is a kind of RA model with localized inflammation and systemic symptoms, and systemic inflammatory status, arthritis index and inflammation-related cytokines are the main indices for evaluating the clinical manifestations of the AA model[11].

Chinese medicine believes that RA belongs to the category of arthralgia, which inflicts the bones and joints of the human body by wind, cold and dampness and causes local blood stagnation, vein obstruction, joint pain, physical heaviness, joint stiffness and swelling, and unfavorable flexion and extension. Damp evil invades into joints and causes swelling of joints, thus forming arthritis[15]. Giantleaf Ardisia Rhizome has effects of dispelling wind, eliminating dampness, promoting blood circulation and removing blood stasis, and is mainly used for the treatment of rheumatism, bruises, postpartum blood stasis, postpartum abdominal pain and sore ulcer[2]. Clinical research data show that in the treatment of RA, the efficacy of single preparation of Giantleaf Ardisia Rhizome is similar to that of tripterygium wilfordii polyglycosidium tablets, with small side effects[4]. Giantleaf Ardisia Rhizome is an ideal therapeutic agent for RA.

The results of this study show that compared with the normal control group, the lesions were more severe, and varying degrees of joint inflammation and swelling and systemic inflammation occurred in the model group. Different doses of ethanol extract of Giantleaf Ardisia Rhizome could reduce the score of joint inflammation and swelling degree and systemic inflammatory status in AA rats (P<0.05,P<0.01), indicating that the daily administration of the ethanol extract of Giantleaf Ardisia Rhizome can effectively relieve the symptoms of arthritis in the AA rat models.

Currently, the pathogenesis of RA is still unclear. It is generally believed to be associated with abnormalities in the immune system and dysregulation of cytokine levels in the body. The abnormal expression and imbalance of IL-1β, IL-6 and TNF-α are one of the typical characteristics of RA, and they are important cytokines directly related to the pathogenesis of RA and play a central role in the cytokine network[16]. IL-1β is an osteoclast activating factor. It can induce synovial cells and chondrocytes synthesize and release prostaglandin E (PGE2) and collagenase, causing joint damage. TNF-α and IL-1β have similar effects and target cell ranges. They often synthesize and secrete at the same time, jointly destroying articular cartilage and bone. IL-6 can be produced by synovial cells induced by TNF-α. It can induce osteoclast precursor differentiation into true osteoclasts in bone metabolism, further destroying bone and cartilage[17-18]. In this study, compared with those of the model group, the serum levels of IL-1β, IL-6 and TNF-α in the rats of the Giantleaf Ardisia Rhizome groups changed significantly (P<0.05,P<0.01), suggesting that the ethanol extract of Giantleaf Ardisia Rhizome can suppress inflammation through down-regulating the levels of IL-1β, IL-6 and TNF-α in serum, thus alleviating the symptoms of RA such as joint swelling. The related mechanism remains to be further explored.

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