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Therapeutic effect of 7, 3′-dimethoxy hesperetin on adjuvant arthritis in rats through inhibiting JAK2-STAT3 signal pathway
International Immunopharmacology 14 (2012) 157–163
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Therapeutic effect of 7, 3′-dimethoxy hesperetin on adjuvant arthritis in rats through inhibiting JAK2-STAT3 signal pathway Rong Li a, b, Li Cai c, Dan-yang Ren a, b, Xue-feng Xie a, b, Cheng-mu Hu a, b, Jun Li a, b,⁎ a b c
School of Pharmacy, Anhui Medical University, 81 Meishan Road, Hefei, China Key Laboratory for Bioactivity of Natural Medicine of Anhui Province, Hefei, China Department of Pathology, School of Basic Medicine, Anhui Medical University, 81 Meishan Road, Hefei, China
a r t i c l e
i n f o
Article history: Received 4 May 2012 Received in revised form 22 June 2012 Accepted 1 July 2012 Available online 15 July 2012 Keywords: 7, 3′-dimethoxy hesperetin Adjuvant arthritis IL-6 JAK2-STAT3 signal pathway
a b s t r a c t In our previous study, we have demonstrated that 7, 3′-dimethoxy hesperetin (DMHP), an active derivative of hesperidin, showed pro-apoptotic effect on synoviocytes in vitro. The present study was to investigate the potential therapeutic effect of DMHP on adjuvant arthritis (AA) in rat and its possible mechanisms. Freund's complete adjuvant was used to induce AA in rats. DMHP were administered intragastrically once a day from days 12 to 21 after AA induction. Secondary paw swelling, arthritis index, and pathological assessments were observed. IL-6 production in serum and IL-6 mRNA expression in synovium was detected by ELISA and real-time RT-PCR respectively. The expression of mRNA (JAK2, STAT3) and protein (JAK2, p-JAK2, STAT3, p-STAT3) in synovium were determined. We found that DMHP significantly inhibited hind paw swelling and arthritis index, and ameliorated pathological changes of ankle joint in AA rats. DMHP suppressed the level of IL-6 in serum and the expression of IL-6 mRNA in synovium of AA rats in a dose-dependent manner. DMHP apparently decreased mRNA expression of JAK2 and STAT3 as well as protein expression of p-JAK2 and p-STAT3 in the synovium of the AA rats. Correlation analysis indicated that p-JAK2 or p-STAT3 protein expression was highly correlated with joint damage severity. In conclusion, DMHP has a powerful therapeutic effect on AA in rats and its mechanisms might be partly related to inhibiting excessive activation of JAK2-STAT3 pathway. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by joint swelling, synovium hyperplasia, inflammatory cell infiltrates, and cartilage or bone destruction [1]. The janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is the signalling target of a proinflammatory cytokine such as IL-6, which plays an important role in rheumatoid synovial inflammation [2,3]. So far, four mammalian JAKs (JAK1, 2, 3 and Tyk2) and seven mammalian STATs (STAT1, 2, 3, 4, 5a, 5b and 6) have been identified [4]. In recent years, many studies have confirmed that JAK/STAT signal pathway is hyperactivated in synovium of RA patient or arthritis animal model, suggesting an important role of this signaling pathway in regulating the inflammatory response [5–7]. RA synoviocytes exhibit abnormal phenotypes characterized by increased proliferation, resistance to apoptosis and invasiveness of adjacent tissues [8]. It had been verified that synoviocyte survival or overproliferation was directly dependent on STAT3 activation in RA [7,9]. Thus, modulating JAK-STAT signal pathway,
⁎ Corresponding author at: School of Pharmacy, Anhui Medical University, 81 Meishan Road, Hefei 230032, China. Tel./fax: +86 551 5161001. E-mail address: [email protected] (J. Li). 1567-5769/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2012.07.001
especially STAT3, may provide an effective therapeutic strategy in treatment of inflammatory arthritis including RA [6,10,11]. Adjuvant arthritis in rats is a classical animal model of experimental arthritis that can be induced by intradermal injection of Freund's complete adjuvant (FCA). The AA in rats shares many pathologic characteristics with RA in human, such as extremities swelling, synovium hyperplasia and cartilage or bone degradation. The similarities in joint pathology between AA and RA make it possible to screen new drugs for RA treatment and explore the potential pathogenesis of RA [12–14]. Natural products are desirable in treating RA due to lower toxicity and fewer side effects than currently approved therapeutic drugs, such as glucocorticosteroid, non-steroidal anti-inflammatory drug, immunosuppressant [15,16]. Hesperidin, a natural flavanone glycoside composed of hesperetin (aglycone) and rutinose, can be obtained from a plentiful and inexpensive byproduct of citrus cultivation [17]. Although hesperidin showed antiarthritic effects on adjuvant or collagen induced arthritis [18,19], low bioavailability (b25%) of oral administration and low water-solubility (20 mg/L) limits its further prospect in pharmaceutical application [20]. Thus, we have synthesized a series of hesperidin derivatives and screened their anti-inflammatory activity in vivo and ex vivo. Among them, 7, 3′-dimethoxy hesperetin (DMHP, Fig. 1) was found to have a higher anti-inflammatory activity than its parent
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2.3. AA induction and drug administration Freund's complete adjuvant (FCA), containing 10 mg heat-inactivated BCG in 1 mL paraffin oil, was given by intradermal injection into the left hind paw in 0.1 mL for each rat [13]. The same volume of paraffin oil alone was given to rat in normal group. DMHP or TPT in 0.5% carboxymethylcellulose sodium (CMC-Na) solution at concentrations of 2, 4, 8 g/L or 4 g/L, were administered intragastrically in a volume of 10 mL/kg, once a day from days 12 to 21 after AA induction. Each rat in normal and AA model group was given an equivalent volume of 0.5% CMC-Na solution. 2.4. Evaluation of AA in rat Fig. 1. Structure of 7, 3′-dimethoxy hesperetin (DMHP).
compound hesperidin [21] and can effectively induce apoptosis of fibroblast-like synoviocytes in rats with adjuvant arthritis (AA) in vitro [22]. However, the therapeutic effects of DMHP on AA and its mechanism have not been studied. In the present study, we therefore evaluated the effects of oral administration of DMHP on treating AA in rats. Inhibitory effect of DMHP on both IL-6 production in serum and IL-6 mRNA expression in synovium was also observed. Then we investigated the inhibitory effect of DMHP on JAK2-STAT3 signal pathway by assaying mRNA and protein levels of several related molecules in synovial tissues. Tripterygium glycosides (TPT), extracted from root of the traditional medicinal plant Tripterygium wilfordii Hook F, are isolated by Institute of Dermatology of Chinese Academy of Medical Sciences for the first time in the 1970s. In China, TPT was approved by State Food and Drug Administration (SFDA) for marketing as a prevalent drug in the treatment of several rheumatic diseases including RA and systemic lupus erythematosus (SLE) [23,24]. Therefore, TPT was used as a suitable positive control in our study, which is consistent with previous studies [19,25,26].
The volume of right hind paw was assayed by a digiti pedis volume meter before AA induction (basic value, day 0) and days 12, 16, 20, 24, and 28. The paw swelling at each time point was defined as the increase in right hind paw volume since immunization, given in milliter. Arthritis severity was graded on a scale of 0–4: 0, no swelling; 1, isolated phalanx joint involvement; 2, involvement of the phalanx joint and digits; 3, involvement of the entire region down to the ankle; 4, involvement of the entire paw, including the ankle [27]. The arthritis index was the total score of the three secondary paws and had a maximum score of 12. 2.5. Tissue preparation Animals were killed on day 28 after AA induction. The ankle joints of right hind paws were promptly removed, trimmed, fixed in 4% buffered paraformaldehyde and decalcified in 10% EDTA. The tissue was paraffin embedded and sliced at 5 μm for histopathological analysis. Fresh synovial tissues were obtained from knee joints of rats for the subsequent molecular biology experiments. Synovial tissues were quickly frozen in liquid nitrogen and preserved at − 80 °C. 2.6. Histological examination and assessment of joint damage
2. Materials and methods 2.1. Materials and reagents 7, 3′-dimethoxy hesperetin (purity >99%) was synthesized and provided by Prof. Tian-pa You (School of Chemistry, University of Science and Technology of China). Tripterygium glycosides (TPT) used as a positive control was obtained from Fudan Fuhua Pharmaceutical Factory (Shanghai, China). Bacillus Calmette–Guerin (BCG) was obtained from Beijing Biological Products Factory (Beijing, China). IL-6 ELISA kit was purchased from Senxiong Technology Industry Co, Ltd (Shanghai, China). All PCR primers were produced by Sangon Biotech Company (Shanghai, China). STAT3 and p-STAT3 antibody were obtained from Santa Cruz Biotechnology (California, USA). JAK2 and p-JAK2 antibodies were purchased from Cell Signaling Technology (California, USA).
Paraffin sections of ankle joints were prepared as above and stained with hematoxylin and eosin (HE). Sections were evaluated under a light microscope (Nikon 80i, Japan) and representative photos were taken. Histological analysis was carried out on the basis of synovial hyperplasia, bone or cartilage destruction, inflammatory cells infiltrate and vascular proliferation. Severity of lesions was classified into four grades [28]: 0, no detectable changes; 1, mild; 2, moderate; and 3, severe. Total histological score of joint damage was showed as the sum of the above every pathological index score. The pathological evaluation was performed by a trained pathologist blind to the groups of specimens. 2.7. Measurement of IL-6 level in serum Serum samples were collected and stored in − 20 °C until analysis. The concentration of IL-6 in serum was measured by ELISA according to the manufacturer's instructions. The samples and standards were all run in duplicates and the data were then averaged.
2.2. Animals Male Sprague–Dawley rats obtained from Animal Department of Anhui Medical University were housed in plastic cages with free access to food and water. Rats were maintained in a room controlled at 21–23 °C with a relative humidity of 55–60% and a 12 h light– dark cycle. After a 7-day acclimatization, rats were randomly divided into six groups (10 rats per group): normal, AA, DMHP (20, 40, 80 mg/kg) and TPT (40 mg/kg). All experiments and animal care procedures were approved by the Animal Resource Center of Anhui Medical University in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
2.8. RNA isolation and real-time quantitative polymerase chain reaction (Q-PCR) Total RNA was extracted from synovium using Trizol method. cDNA was synthesized using reverse transcriptase (Promega, Wisconsin, USA). Q-PCR was performed by SYBR Green PCR Kit (Applied Biosystems, USA) and an ABI Prism 7000 Sequence Detector system in 25 μl volume for 40 cycles: 15 s at 95 °C; 60 s at 65 °C (IL-6) or 60 °C (JAK2, STAT3). The following primers were used: IL-6, 5′-TCC TACCCCAACTTCCAATGCTC-3′ and 5′-TTGGATGGTCTTGGTCCTTAGCC -3′; JAK2, 5′-GTGGAGATGTGCCGCTATG-3′ and 5′-CCTTGTACTTCACG
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ATGTTGTC-3′; STAT3, 5′-CACCCATAGTGAGCCCTTGGA-3′ and 5′-TT TGAGTGCAGTGACCAGGACAG-3′; β-actin, 5′-TTGCTGACAGGATGCAG AA-3′ and 5′-ACCAATCCACACAGAGTACTT-3′. The relative amount of mRNA was calculated by the 2-ΔΔCt method [29]. The relative amplification efficiencies of the primers were tested and shown to be similar.
2.9. Western blot analysis Aliquots (100 mg) of synovial tissues were treated with RIPA lysis buffer. The protein concentrations were determined by Bradford assay. An equal amount (30 μg) of protein was separated by 10% SDS-PAGE and then electrophoretic transferred onto a nitrocellulose membrane. After blocking with 5% skim milk, the membranes were incubated respectively with antibodies against JAK2, p-JAK2, STAT3 and p-STAT3, followed with appropriate horseradish peroxidaseconjugated secondary antibodies. The protein bands were detected by ECL reagent according to a commercial protocol (Pierce, Rockford, USA). The optic density of each band was measured using the software LabWorks 4.5. The ratio of optical density value of each protein band over its corresponding β-actin band was used for representing the relative protein level.
2.10. Statistical analysis Statistical analysis was performed by SPSS 11.5 software. Data of arthritis index and histopathological scores were analyzed by non-parametric Kruskal–Wallis test. Other data were analyzed by one-way analysis of variance followed by Students–Newman–Keuls test. Pearson's correlation test was used to analyze the correlation between p-JAK2 or p-STAT3 protein expression in synovium and total histological scores of joint damage. p b 0.05 was considered statistically significant.
3. Results
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Table 2 Effects of DMHP on arthritis index in rats with AA. Group
Dose (mg/kg)
Normal – AA – DMHP 20 40 80 TPT 40
Arthritis index Day 12
Day 16
Day 20
Day 24
Day 28
0.6 ± 0.7 4.9 ± 0.7†† 4.7 ± 0.7 4.4 ± 0.8 4.2 ± 0.9 4.2 ± 0.9
0.8 ± 0.8 6.5 ± 1.1†† 6.0 ± 0.7 5.5 ± 0.8⁎ 5.4 ± 0.5⁎
0.9 ± 0.6 7.9 ± 0.7†† 7.5 ± 0.9 6.9 ± 0.6⁎⁎ 6.5 ± 0.5⁎⁎ 7.0 ± 0.9⁎
0.8 ± 0.4 9.3 ± 0.9†† 8.6 ± 0.8 7.8 ± 0.9⁎⁎ 7.3 ± 1.1⁎⁎ 7.5 ± 1.1⁎⁎
0.7 ± 0.7 7.5 ± 0.9†† 7.1 ± 0.6 6.6 ± 1.0⁎ 5.7 ± 1.3⁎⁎ 5.9 ± 1.2⁎⁎
5.8 ± 1.2
Data are mean±SD (n=10). AA, adjuvant arthritis; DMHP, 7, 3′-dimethoxy hesperetin; TPT, Tripterygium glycosides, was the positive control. †† pb 0.01 compared with normal group. ⁎ pb 0.05. ⁎⁎ pb 0.01 compared with AA group.
3.2. Histopathological evaluation Photomicrographs of sections stained with HE illustrated the disease severity of AA and the effect of DMHP on joint histology (Fig. 2). No inflammation or joint destruction was seen in normal rats (Fig. 2A). In contrast, AA rats exhibited extensive inflammation, synovial hyperplaisia, cartilage destruction and vascular proliferation (Fig. 2B and C). AA rats treated with DMHP (40 mg/kg) exhibited moderate synovial hyperplasia and inflammatory cells infiltration, as well as mild cartilage damage (Fig. 2D). DMHP (80 mg/kg) and TPT (40 mg/kg) ameliorated inflammatory cells infiltration and joint destruction, although slight synovial hyperplasia remained (Fig. 2E and F). Histological analysis suggested that DMHP inhibited synovial hyperplasia, bone or cartilage destruction and inflammatory cells infiltration in a dose-dependent manner (Fig. 3A-C). Moreover, DMHP obviously attenuated vascular proliferation at a dose of 80 mg/kg (Fig. 3D).
3.3. Effects of DMHP on IL-6 production in serum and IL-6 mRNA expression in synovial tissues of AA rats
3.1. Effect of DMHP on secondary inflammation in AA rats DMHP was administered intragastrically from days 12 to 21 after AA immunization. Paw swelling and arthritis index were measured every 4 days from days 12 to 28. As shown in Tables 1 and 2, the swelling peak of AA rats appeared on day 24. Administration of DMHP (20 mg/kg) showed a significant inhibitory effect on hind paw swelling of AA rats on day 28; while DMHP at 40, 80 mg/kg significantly suppressed secondary hind paw swelling and reduced arthritis index of AA rats on days 16, 20, 24 and 28, which was similar to the TPT-treated positive control group.
As shown in Fig. 4A, the concentration of cytokine IL-6 in serum significantly elevated in AA group compared with that in normal group. AA rats receiving the treatment of DMHP showed a dose dependent decrease in serum IL-6 production which was significantly different from the AA group. In addition, IL-6 mRNA expression in synovium was also remarkably increased in the AA group. DMHP (40, 80 mg/kg) treatment significantly inhibited IL-6 mRNA expression in the AA rats (Fig. 4B). TPT showed similar inhibitory effects on IL-6 production in serum and IL-6 mRNA expression in synovial tissues.
Table 1 Effects of DMHP on hind paw swelling in rats with AA. Group
Dose (mg/kg)
Normal AA DMHP
– – 20 40 80 40
TPT
Hind paw swelling (ΔmL) Day 12
Day 16
Day 20
Day 24
Day 28
0.058 ± 0.011 0.338 ± 0.124†† 0.290 ± 0.082 0.285 ± 0.080 0.309 ± 0.061 0.272 ± 0.099
0.076 ± 0.010 0.529 ± 0.131†† 0.456 ± 0.095 0.427 ± 0.143⁎ 0.387 ± 0.083⁎⁎ 0.412 ± 0.133⁎⁎
0.097 ± 0.013 0.729 ± 0.212†† 0.633 ± 0.098 0.514 ± 0.106⁎⁎ 0.529 ± 0.090⁎⁎ 0.497 ± 0.145⁎⁎
0.111 ± 0.015 0.896 ± 0.246†† 0.780 ± 0.102 0.629 ± 0.122⁎⁎ 0.578 ± 0.095⁎⁎ 0.613 ± 0.203⁎⁎
0.119 ± 0.013 0.724 ± 0.261†† 0.601 ± 0.085⁎ 0.419 ± 0.089⁎⁎ 0.361 ± 0.078⁎⁎ 0.335 ± 0.067⁎⁎
Data are mean ± SD (n = 10). AA, adjuvant arthritis; DMHP, 7, 3′-dimethoxy hesperetin; TPT, Tripterygium glycosides, was the positive control. †† pb 0.01 compared with normal group. ⁎ p b 0.05. ⁎⁎ p b 0.01 compared with AA group.
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Fig. 2. Histopathologic examinations under a light microscope on ankle joints by HE staining (magnification×100). (A) Normal; (B, C) AA; (D) DMHP 40 mg/kg; (E) DMHP 80 mg/kg; (F) TPT 40 mg/kg. AA, adjuvant arthritis; DMHP, 7, 3′-dimethoxy hesperetin; TPT, Tripterygium glycosides, was the positive control.
3.4. Effects of DMHP on JAK2 and STAT3 mRNA expressions in synovial tissues The mRNA expressions of JAK2 and STAT3 in synovial tissues from different groups were detected by real time Q-PCR. As shown in Fig. 5, mRNA expression of JAK2 and STAT3 in synovium from the AA group was significantly higher than the normal group (p b 0.01). DMHP (40, 80 mg/kg) treatment significantly decreased JAK2 and STAT3 mRNA expressions in the AA rats. As a positive control, TPT remarkably inhibited STAT3 mRNA expression (p b 0.05), and an approximate significant decrease was detected in JAK2 mRNA expression (p = 0.059). 3.5. Effect of DMHP on JAK2, p-JAK2, STAT3 and p-STAT3 protein expressions in synovial tissues The JAK2, p-JAK2, STAT3 and p-STAT3 protein expressions were evaluated by western blot analysis. Representative examples of interest protein expressions from various groups were shown in Fig. 6A. The relative expressions of interest proteins were quantified densitometrically using software LabWorks 4.5 and β-actin was used as a control. There were no significant changes in the amounts of non-phosphorylated JAK2 and STAT3 proteins in various groups
(Fig. 6A). Statistical analysis indicated that p-JAK2 and p-STAT3 in synovium were significantly elevated in the AA group as compared to the normal group and that DMHP treatments significantly suppressed phosphorylation of JAK2 and STAT3 in the AA rats (Fig. 6B). 3.6. Relationships between p-JAK2 or p-STAT3 protein expression in synovium and total histological scores on joint damage Total histological scores on joint damage were showed as the sum of the score of following pathological index: synovial hyperplasia, bone or cartilage destruction, inflammatory cells infiltrate and vascular proliferation. The total histological scores were used to represent the severity of joint damage. Correlation analysis (Fig. 7) indicated a positive correlation between protein levels of p-JAK2 (a: r = 0.828 with p b 0.01) and p-STAT3 (b: r = 0.909 with p b 0.01) in synovium and total histological scores on joint damage. These data imply that the increase of p-JAK2 or p-STAT3 protein expression in synovial tissues is significantly correlated with the increase of total histological scores on joint damage, indicating that inhibition of JAK-STAT pathway by DMHP is highly associated with its therapeutic effect on rats with AA.
Fig. 3. Inhibitory effects of DMHP on histological scores in ankle joints of rats with AA: (A) synovial hyperplasia, (B) bone or cartilage destruction, (C) inflammatory cells infiltration, (D) vascular proliferation. Data are mean ± SEM (n = 5). ††p b 0.01 compared with normal group. *pb 0.05, **p b 0.01 compared with AA group. AA, adjuvant arthritis; DMHP, 7, 3′-dimethoxy hesperetin; TPT, Tripterygium glycosides, was the positive control.
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Fig. 4. Effects of DMHP on IL-6 production in serum and IL-6 mRNA expression in synovial tissues of AA rats. Data are mean ± SEM (n = 5). ††p b 0.01 compared with normal group. *p b 0.05, **p b 0.01 compared with AA group. AA, adjuvant arthritis; DMHP, 7, 3′-dimethoxy hesperetin; TPT, Tripterygium glycosides, was the positive control.
4. Discussion
Fig. 5. Effects of DMHP on the mRNA expression of JAK2 and STAT3 in synovial tissues, detected by Q-PCR. Data are mean ± SEM (n = 5). ††p b 0.01 compared with normal group. *p b 0.05, **p b 0.01 compared with AA group. AA, adjuvant arthritis; DMHP, 7, 3′-dimethoxy hesperetin; TPT, Tripterygium glycosides, was the positive control.
Rat AA induced by FCA has many similar characteristics to RA in the aspects of histology and immunology. In the AA rat model, clinical signs of arthritis developed within 2 weeks and reached its peak about on day 24 after induction. The main pathological features in ankle joint of AA rats could be observed, including synovial hyperplaisia, cartilage or bone damage, inflammatory cell infiltration and pannus formation. Here, our results indicated that DMHP remarkably inhibited hind paw swelling and reduced arthritis index of the AA rats. Histological examination also revealed that DMHP significantly ameliorated the above pathological changes in ankle joint of the AA rats. These results clearly suggest that DMHP can reduce secondary inflammation of AA rats and may be effective in treating RA. Proinflammatory cytokines, particularly IL-1β, TNF-α and IL-6, play a vital pathogenic role in RA development [30]. Many previous studies demonstrated that IL-6 overproduction was found in both synovial fluid and serum of RA patients [31,32]. The serum level of IL-6 correlated positively with RA disease activity, and IL-6 level in serum remarkably decreased with medical treatment [33,34]. The binding of IL-6 to its receptors (gp130 and sIL-6R) initiates cellular events including activation of JAK kinases. Activated JAK kinases phosphorylate and activate STAT transcription factors, particularly STAT3. Phosphorylated STAT3 then forms a dimer and translocates into the nucleus to activate
Fig. 6. Western blot analysis for JAK2, p-JAK2, STAT3 and p-STAT3 in synovial tissues. (A) Representative examples of JAK2, p-JAK2, STAT3, p-STAT3 protein expressions in synovium from various groups. (B) Semi-quantitative statistical graph of p-JAK2, p-STAT3 protein levels in various groups, β-actin serves as the house-keeping protein. Data are mean ± SEM (n = 5). ††p b 0.01 compared with normal group. *p b 0.05, **p b 0.01 compared with AA group. AA, adjuvant arthritis; DMHP, 7, 3′-dimethoxy hesperetin; TPT, Tripterygium glycosides, was the positive control.
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Fig. 7. Correlations between p-JAK2 (a) or p-STAT3 (b) protein level in synovium and total histological scores on joint damage. The correlation analysis was performed by the Pearson's correlation test (n = 30). Asterisks (*) show the significance of the correlation (two tails). **p b 0.01.
transcription of genes [35,36]. Thus, the blockade of IL-6 may be beneficial in treating RA [37]. Indeed, a humanized mAb to IL-6R (tocilizumab) has proven to be effective against RA [38]. In our study, consistent with the previous study [13], IL-6 concentration in serum and IL-6 mRNA expression in synovial tissues were both significantly elevated in the AA group compared with the normal group. Our results indicated that DMHP treatment reversed the high levels of IL-6 in serum and IL-6 mRNA in synovium of the AA rats. It is well known that JAKs associate with cytokine receptors and translate signals triggered by cytokine binding into intracellular responses [39]. Recent evidence suggested that inhibition of JAKs in the signaling complex would effectively inhibit cytokine signaling and thus treat RA [40]. As latent cytoplasmic transcription factors, STATs have critical roles in mediating cellular transcriptional responses to cytokines. Among 7 mammalian STATs, STAT3 has been verified to play a significant role in RA and animal arthritis model. It was reported that STAT3 was highly tyrosine phosphorylated in synovium of RA patients and periarticular injection of SOCS3 adenovirus (a negative regulator of JAK-STAT3 pathway) drastically reduced arthritis severity in collagen induced arthritis [10]. Similarly, a previous study also indicated that STAT3 was over activated in inflamed synovium of mice with zymosan-induced arthritis throughout the course of disease [5]. In addition, STAT3 contributed to RA development by promoting synovial fibroblast survival and abnormal proliferation. Ablation of STAT3 function using a dominant-negative STAT3 mutant (STAT3-YF) apparently induced RA synoviocytes apoptosis in vitro [9]. These findings suggested that modulating STAT3 expression was a potential therapeutic target in RA. In order to elucidate whether the antiarthritic effect of DMHP is related to inhibiting JAK2-STAT3 signal pathway, we analyzed the mRNA levels of JAK2 and STAT3 as well as protein expressions of several key molecules in JAK2-STAT3 pathway, such as JAK2, p-JAK2, STAT3 and p-STAT3. Our data showed that DMHP significantly inhibited JAK2 and STAT3 mRNA expressions which remarkably elevated in AA rats. Although no significant changes of non-phosphorylated JAK2 and STAT3 proteins in synovium were found among various groups, significant increased p-JAK2 and p-STAT3 were observed in the AA group. Treatment with DMHP remarkably decreased the levels of p-JAK2 and p-STAT3 in the AA rats. Moreover, correlation analysis indicated that the protein levels of p-JAK2 and p-STAT3 in synovium were highly correlated with joint damage severity. These results suggested that DMHP suppressed hyper activated JAK2-STAT3 pathway in synovium of AA rats, which might be one possible molecular mechanism underlying the therapeutic effect of DMHP on AA. In conclusion, DMHP has a powerful protective action against FCA-induced AA in rats, as assessed by paw swelling, arthritis index
and histological evaluation. The possible antiarthritic mechanisms of DMHP on AA in rats might be partly associated with inhibition of excessive activation of JAK2-STAT3 signal pathway. Our findings suggest the possible utility of DMHP against human RA. Acknowledgments This study was supported by the National Natural Science Foundation of China (81102273), Specialized Research Fund for the Doctoral Program of Higher Education (20113420120005), Anhui Provincial Natural Science Foundation (11040606Q15) and Grants for Scientific Research of BSKY (XJ201020) from the Anhui Medical University. References [1] Sindhu G, Ratheesh M, Shyni GL, Nambisan B, Helen A. Anti-inflammatory and antioxidative effects of mucilage of Trigonella foenum graecum (Fenugreek) on adjuvant induced arthritic rats. Int Immunopharmacol 2012;12:205–11. [2] Gadina M, Hilton D, Johnston JA, Morinobu A, Lighvani A, Zhou YJ, et al. Signaling by type I and II cytokine receptors: ten years after. Curr Opin Immunol 2001;13: 363–73. [3] Walker JG, Smith MD. The Jak-STAT pathway in rheumatoid arthritis. J Rheumatol 2005;32:1650–3. [4] Kisseleva T, Bhattacharya S, Braunstein J, Schindler CW. Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene 2002;285:1–24. [5] de Hooge AS, van de Loo FA, Koenders MI, Bennink MB, Arntz OJ, Kolbe T, et al. Local activation of STAT-1 and STAT-3 in the inflamed synovium during zymosan-induced arthritis: exacerbation of joint inflammation in STAT-1 gene-knockout mice. Arthritis Rheum 2004;50:2014–23. [6] Mori T, Miyamoto T, Yoshida H, Asakawa M, Kawasumi M, Kobayashi T, et al. IL-1beta and TNFalpha-initiated IL-6-STAT3 pathway is critical in mediating inflammatory cytokines and RANKL expression in inflammatory arthritis. Int Immunol 2011;23:701–12. [7] Walker JG, Ahern MJ, Coleman M, Weedon H, Papangelis V, Beroukas D, et al. Expression of Jak3, STAT1, STAT4, and STAT6 in inflammatory arthritis: unique Jak3 and STAT4 expression in dendritic cells in seropositive rheumatoid arthritis. Ann Rheum Dis 2006;65:149–56. [8] Karouzakis E, Neidhart M, Gay RE, Gay S. Molecular and cellular basis of rheumatoid joint destruction. Immunol Lett 2006;106:8–13. [9] Krause A, Scaletta N, Ji JD, Ivashkiv LB. Rheumatoid arthritis synoviocyte survival is dependent on Stat3. J Immunol 2002;169:6610–6. [10] Shouda T, Yoshida T, Hanada T, Wakioka T, Oishi M, Miyoshi K, et al. Induction of the cytokine signal regulator SOCS3/CIS3 as a therapeutic strategy for treating inflammatory arthritis. J Clin Invest 2001;108:1781–8. [11] Walker JG, Ahern MJ, Coleman M, Weedon H, Papangelis V, Beroukas D, et al. Changes in synovial tissue Jak-STAT expression in rheumatoid arthritis in response to successful DMARD treatment. Ann Rheum Dis 2006;65:1558–64. [12] Jawed H, Shah SU, Jamall S, Simjee SU. N-(2-hydroxy phenyl) acetamide inhibits inflammation-related cytokines and ROS in adjuvant-induced arthritic (AIA) rats. Int Immunopharmacol 2010;10:900–5. [13] Liu M, Mao W, Guan H, Li L, Wei B, Li P. Effects of taurochenodeoxycholic acid on adjuvant arthritis in rats. Int Immunopharmacol 2011;11:2150–8. [14] Wang D, Chang Y, Wu Y, Zhang L, Yan S, Xie G, et al. Therapeutic effects of TACI-Ig on rat with adjuvant arthritis. Clin Exp Immunol 2011;163:225–34.
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Contents lists available at SciVerse ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Therapeutic effect of 7, 3′-dimethoxy hesperetin on adjuvant arthritis in rats through inhibiting JAK2-STAT3 signal pathway Rong Li a, b, Li Cai c, Dan-yang Ren a, b, Xue-feng Xie a, b, Cheng-mu Hu a, b, Jun Li a, b,⁎ a b c
School of Pharmacy, Anhui Medical University, 81 Meishan Road, Hefei, China Key Laboratory for Bioactivity of Natural Medicine of Anhui Province, Hefei, China Department of Pathology, School of Basic Medicine, Anhui Medical University, 81 Meishan Road, Hefei, China
a r t i c l e
i n f o
Article history: Received 4 May 2012 Received in revised form 22 June 2012 Accepted 1 July 2012 Available online 15 July 2012 Keywords: 7, 3′-dimethoxy hesperetin Adjuvant arthritis IL-6 JAK2-STAT3 signal pathway
a b s t r a c t In our previous study, we have demonstrated that 7, 3′-dimethoxy hesperetin (DMHP), an active derivative of hesperidin, showed pro-apoptotic effect on synoviocytes in vitro. The present study was to investigate the potential therapeutic effect of DMHP on adjuvant arthritis (AA) in rat and its possible mechanisms. Freund's complete adjuvant was used to induce AA in rats. DMHP were administered intragastrically once a day from days 12 to 21 after AA induction. Secondary paw swelling, arthritis index, and pathological assessments were observed. IL-6 production in serum and IL-6 mRNA expression in synovium was detected by ELISA and real-time RT-PCR respectively. The expression of mRNA (JAK2, STAT3) and protein (JAK2, p-JAK2, STAT3, p-STAT3) in synovium were determined. We found that DMHP significantly inhibited hind paw swelling and arthritis index, and ameliorated pathological changes of ankle joint in AA rats. DMHP suppressed the level of IL-6 in serum and the expression of IL-6 mRNA in synovium of AA rats in a dose-dependent manner. DMHP apparently decreased mRNA expression of JAK2 and STAT3 as well as protein expression of p-JAK2 and p-STAT3 in the synovium of the AA rats. Correlation analysis indicated that p-JAK2 or p-STAT3 protein expression was highly correlated with joint damage severity. In conclusion, DMHP has a powerful therapeutic effect on AA in rats and its mechanisms might be partly related to inhibiting excessive activation of JAK2-STAT3 pathway. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by joint swelling, synovium hyperplasia, inflammatory cell infiltrates, and cartilage or bone destruction [1]. The janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is the signalling target of a proinflammatory cytokine such as IL-6, which plays an important role in rheumatoid synovial inflammation [2,3]. So far, four mammalian JAKs (JAK1, 2, 3 and Tyk2) and seven mammalian STATs (STAT1, 2, 3, 4, 5a, 5b and 6) have been identified [4]. In recent years, many studies have confirmed that JAK/STAT signal pathway is hyperactivated in synovium of RA patient or arthritis animal model, suggesting an important role of this signaling pathway in regulating the inflammatory response [5–7]. RA synoviocytes exhibit abnormal phenotypes characterized by increased proliferation, resistance to apoptosis and invasiveness of adjacent tissues [8]. It had been verified that synoviocyte survival or overproliferation was directly dependent on STAT3 activation in RA [7,9]. Thus, modulating JAK-STAT signal pathway,
⁎ Corresponding author at: School of Pharmacy, Anhui Medical University, 81 Meishan Road, Hefei 230032, China. Tel./fax: +86 551 5161001. E-mail address: [email protected] (J. Li). 1567-5769/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2012.07.001
especially STAT3, may provide an effective therapeutic strategy in treatment of inflammatory arthritis including RA [6,10,11]. Adjuvant arthritis in rats is a classical animal model of experimental arthritis that can be induced by intradermal injection of Freund's complete adjuvant (FCA). The AA in rats shares many pathologic characteristics with RA in human, such as extremities swelling, synovium hyperplasia and cartilage or bone degradation. The similarities in joint pathology between AA and RA make it possible to screen new drugs for RA treatment and explore the potential pathogenesis of RA [12–14]. Natural products are desirable in treating RA due to lower toxicity and fewer side effects than currently approved therapeutic drugs, such as glucocorticosteroid, non-steroidal anti-inflammatory drug, immunosuppressant [15,16]. Hesperidin, a natural flavanone glycoside composed of hesperetin (aglycone) and rutinose, can be obtained from a plentiful and inexpensive byproduct of citrus cultivation [17]. Although hesperidin showed antiarthritic effects on adjuvant or collagen induced arthritis [18,19], low bioavailability (b25%) of oral administration and low water-solubility (20 mg/L) limits its further prospect in pharmaceutical application [20]. Thus, we have synthesized a series of hesperidin derivatives and screened their anti-inflammatory activity in vivo and ex vivo. Among them, 7, 3′-dimethoxy hesperetin (DMHP, Fig. 1) was found to have a higher anti-inflammatory activity than its parent
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2.3. AA induction and drug administration Freund's complete adjuvant (FCA), containing 10 mg heat-inactivated BCG in 1 mL paraffin oil, was given by intradermal injection into the left hind paw in 0.1 mL for each rat [13]. The same volume of paraffin oil alone was given to rat in normal group. DMHP or TPT in 0.5% carboxymethylcellulose sodium (CMC-Na) solution at concentrations of 2, 4, 8 g/L or 4 g/L, were administered intragastrically in a volume of 10 mL/kg, once a day from days 12 to 21 after AA induction. Each rat in normal and AA model group was given an equivalent volume of 0.5% CMC-Na solution. 2.4. Evaluation of AA in rat Fig. 1. Structure of 7, 3′-dimethoxy hesperetin (DMHP).
compound hesperidin [21] and can effectively induce apoptosis of fibroblast-like synoviocytes in rats with adjuvant arthritis (AA) in vitro [22]. However, the therapeutic effects of DMHP on AA and its mechanism have not been studied. In the present study, we therefore evaluated the effects of oral administration of DMHP on treating AA in rats. Inhibitory effect of DMHP on both IL-6 production in serum and IL-6 mRNA expression in synovium was also observed. Then we investigated the inhibitory effect of DMHP on JAK2-STAT3 signal pathway by assaying mRNA and protein levels of several related molecules in synovial tissues. Tripterygium glycosides (TPT), extracted from root of the traditional medicinal plant Tripterygium wilfordii Hook F, are isolated by Institute of Dermatology of Chinese Academy of Medical Sciences for the first time in the 1970s. In China, TPT was approved by State Food and Drug Administration (SFDA) for marketing as a prevalent drug in the treatment of several rheumatic diseases including RA and systemic lupus erythematosus (SLE) [23,24]. Therefore, TPT was used as a suitable positive control in our study, which is consistent with previous studies [19,25,26].
The volume of right hind paw was assayed by a digiti pedis volume meter before AA induction (basic value, day 0) and days 12, 16, 20, 24, and 28. The paw swelling at each time point was defined as the increase in right hind paw volume since immunization, given in milliter. Arthritis severity was graded on a scale of 0–4: 0, no swelling; 1, isolated phalanx joint involvement; 2, involvement of the phalanx joint and digits; 3, involvement of the entire region down to the ankle; 4, involvement of the entire paw, including the ankle [27]. The arthritis index was the total score of the three secondary paws and had a maximum score of 12. 2.5. Tissue preparation Animals were killed on day 28 after AA induction. The ankle joints of right hind paws were promptly removed, trimmed, fixed in 4% buffered paraformaldehyde and decalcified in 10% EDTA. The tissue was paraffin embedded and sliced at 5 μm for histopathological analysis. Fresh synovial tissues were obtained from knee joints of rats for the subsequent molecular biology experiments. Synovial tissues were quickly frozen in liquid nitrogen and preserved at − 80 °C. 2.6. Histological examination and assessment of joint damage
2. Materials and methods 2.1. Materials and reagents 7, 3′-dimethoxy hesperetin (purity >99%) was synthesized and provided by Prof. Tian-pa You (School of Chemistry, University of Science and Technology of China). Tripterygium glycosides (TPT) used as a positive control was obtained from Fudan Fuhua Pharmaceutical Factory (Shanghai, China). Bacillus Calmette–Guerin (BCG) was obtained from Beijing Biological Products Factory (Beijing, China). IL-6 ELISA kit was purchased from Senxiong Technology Industry Co, Ltd (Shanghai, China). All PCR primers were produced by Sangon Biotech Company (Shanghai, China). STAT3 and p-STAT3 antibody were obtained from Santa Cruz Biotechnology (California, USA). JAK2 and p-JAK2 antibodies were purchased from Cell Signaling Technology (California, USA).
Paraffin sections of ankle joints were prepared as above and stained with hematoxylin and eosin (HE). Sections were evaluated under a light microscope (Nikon 80i, Japan) and representative photos were taken. Histological analysis was carried out on the basis of synovial hyperplasia, bone or cartilage destruction, inflammatory cells infiltrate and vascular proliferation. Severity of lesions was classified into four grades [28]: 0, no detectable changes; 1, mild; 2, moderate; and 3, severe. Total histological score of joint damage was showed as the sum of the above every pathological index score. The pathological evaluation was performed by a trained pathologist blind to the groups of specimens. 2.7. Measurement of IL-6 level in serum Serum samples were collected and stored in − 20 °C until analysis. The concentration of IL-6 in serum was measured by ELISA according to the manufacturer's instructions. The samples and standards were all run in duplicates and the data were then averaged.
2.2. Animals Male Sprague–Dawley rats obtained from Animal Department of Anhui Medical University were housed in plastic cages with free access to food and water. Rats were maintained in a room controlled at 21–23 °C with a relative humidity of 55–60% and a 12 h light– dark cycle. After a 7-day acclimatization, rats were randomly divided into six groups (10 rats per group): normal, AA, DMHP (20, 40, 80 mg/kg) and TPT (40 mg/kg). All experiments and animal care procedures were approved by the Animal Resource Center of Anhui Medical University in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
2.8. RNA isolation and real-time quantitative polymerase chain reaction (Q-PCR) Total RNA was extracted from synovium using Trizol method. cDNA was synthesized using reverse transcriptase (Promega, Wisconsin, USA). Q-PCR was performed by SYBR Green PCR Kit (Applied Biosystems, USA) and an ABI Prism 7000 Sequence Detector system in 25 μl volume for 40 cycles: 15 s at 95 °C; 60 s at 65 °C (IL-6) or 60 °C (JAK2, STAT3). The following primers were used: IL-6, 5′-TCC TACCCCAACTTCCAATGCTC-3′ and 5′-TTGGATGGTCTTGGTCCTTAGCC -3′; JAK2, 5′-GTGGAGATGTGCCGCTATG-3′ and 5′-CCTTGTACTTCACG
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ATGTTGTC-3′; STAT3, 5′-CACCCATAGTGAGCCCTTGGA-3′ and 5′-TT TGAGTGCAGTGACCAGGACAG-3′; β-actin, 5′-TTGCTGACAGGATGCAG AA-3′ and 5′-ACCAATCCACACAGAGTACTT-3′. The relative amount of mRNA was calculated by the 2-ΔΔCt method [29]. The relative amplification efficiencies of the primers were tested and shown to be similar.
2.9. Western blot analysis Aliquots (100 mg) of synovial tissues were treated with RIPA lysis buffer. The protein concentrations were determined by Bradford assay. An equal amount (30 μg) of protein was separated by 10% SDS-PAGE and then electrophoretic transferred onto a nitrocellulose membrane. After blocking with 5% skim milk, the membranes were incubated respectively with antibodies against JAK2, p-JAK2, STAT3 and p-STAT3, followed with appropriate horseradish peroxidaseconjugated secondary antibodies. The protein bands were detected by ECL reagent according to a commercial protocol (Pierce, Rockford, USA). The optic density of each band was measured using the software LabWorks 4.5. The ratio of optical density value of each protein band over its corresponding β-actin band was used for representing the relative protein level.
2.10. Statistical analysis Statistical analysis was performed by SPSS 11.5 software. Data of arthritis index and histopathological scores were analyzed by non-parametric Kruskal–Wallis test. Other data were analyzed by one-way analysis of variance followed by Students–Newman–Keuls test. Pearson's correlation test was used to analyze the correlation between p-JAK2 or p-STAT3 protein expression in synovium and total histological scores of joint damage. p b 0.05 was considered statistically significant.
3. Results
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Table 2 Effects of DMHP on arthritis index in rats with AA. Group
Dose (mg/kg)
Normal – AA – DMHP 20 40 80 TPT 40
Arthritis index Day 12
Day 16
Day 20
Day 24
Day 28
0.6 ± 0.7 4.9 ± 0.7†† 4.7 ± 0.7 4.4 ± 0.8 4.2 ± 0.9 4.2 ± 0.9
0.8 ± 0.8 6.5 ± 1.1†† 6.0 ± 0.7 5.5 ± 0.8⁎ 5.4 ± 0.5⁎
0.9 ± 0.6 7.9 ± 0.7†† 7.5 ± 0.9 6.9 ± 0.6⁎⁎ 6.5 ± 0.5⁎⁎ 7.0 ± 0.9⁎
0.8 ± 0.4 9.3 ± 0.9†† 8.6 ± 0.8 7.8 ± 0.9⁎⁎ 7.3 ± 1.1⁎⁎ 7.5 ± 1.1⁎⁎
0.7 ± 0.7 7.5 ± 0.9†† 7.1 ± 0.6 6.6 ± 1.0⁎ 5.7 ± 1.3⁎⁎ 5.9 ± 1.2⁎⁎
5.8 ± 1.2
Data are mean±SD (n=10). AA, adjuvant arthritis; DMHP, 7, 3′-dimethoxy hesperetin; TPT, Tripterygium glycosides, was the positive control. †† pb 0.01 compared with normal group. ⁎ pb 0.05. ⁎⁎ pb 0.01 compared with AA group.
3.2. Histopathological evaluation Photomicrographs of sections stained with HE illustrated the disease severity of AA and the effect of DMHP on joint histology (Fig. 2). No inflammation or joint destruction was seen in normal rats (Fig. 2A). In contrast, AA rats exhibited extensive inflammation, synovial hyperplaisia, cartilage destruction and vascular proliferation (Fig. 2B and C). AA rats treated with DMHP (40 mg/kg) exhibited moderate synovial hyperplasia and inflammatory cells infiltration, as well as mild cartilage damage (Fig. 2D). DMHP (80 mg/kg) and TPT (40 mg/kg) ameliorated inflammatory cells infiltration and joint destruction, although slight synovial hyperplasia remained (Fig. 2E and F). Histological analysis suggested that DMHP inhibited synovial hyperplasia, bone or cartilage destruction and inflammatory cells infiltration in a dose-dependent manner (Fig. 3A-C). Moreover, DMHP obviously attenuated vascular proliferation at a dose of 80 mg/kg (Fig. 3D).
3.3. Effects of DMHP on IL-6 production in serum and IL-6 mRNA expression in synovial tissues of AA rats
3.1. Effect of DMHP on secondary inflammation in AA rats DMHP was administered intragastrically from days 12 to 21 after AA immunization. Paw swelling and arthritis index were measured every 4 days from days 12 to 28. As shown in Tables 1 and 2, the swelling peak of AA rats appeared on day 24. Administration of DMHP (20 mg/kg) showed a significant inhibitory effect on hind paw swelling of AA rats on day 28; while DMHP at 40, 80 mg/kg significantly suppressed secondary hind paw swelling and reduced arthritis index of AA rats on days 16, 20, 24 and 28, which was similar to the TPT-treated positive control group.
As shown in Fig. 4A, the concentration of cytokine IL-6 in serum significantly elevated in AA group compared with that in normal group. AA rats receiving the treatment of DMHP showed a dose dependent decrease in serum IL-6 production which was significantly different from the AA group. In addition, IL-6 mRNA expression in synovium was also remarkably increased in the AA group. DMHP (40, 80 mg/kg) treatment significantly inhibited IL-6 mRNA expression in the AA rats (Fig. 4B). TPT showed similar inhibitory effects on IL-6 production in serum and IL-6 mRNA expression in synovial tissues.
Table 1 Effects of DMHP on hind paw swelling in rats with AA. Group
Dose (mg/kg)
Normal AA DMHP
– – 20 40 80 40
TPT
Hind paw swelling (ΔmL) Day 12
Day 16
Day 20
Day 24
Day 28
0.058 ± 0.011 0.338 ± 0.124†† 0.290 ± 0.082 0.285 ± 0.080 0.309 ± 0.061 0.272 ± 0.099
0.076 ± 0.010 0.529 ± 0.131†† 0.456 ± 0.095 0.427 ± 0.143⁎ 0.387 ± 0.083⁎⁎ 0.412 ± 0.133⁎⁎
0.097 ± 0.013 0.729 ± 0.212†† 0.633 ± 0.098 0.514 ± 0.106⁎⁎ 0.529 ± 0.090⁎⁎ 0.497 ± 0.145⁎⁎
0.111 ± 0.015 0.896 ± 0.246†† 0.780 ± 0.102 0.629 ± 0.122⁎⁎ 0.578 ± 0.095⁎⁎ 0.613 ± 0.203⁎⁎
0.119 ± 0.013 0.724 ± 0.261†† 0.601 ± 0.085⁎ 0.419 ± 0.089⁎⁎ 0.361 ± 0.078⁎⁎ 0.335 ± 0.067⁎⁎
Data are mean ± SD (n = 10). AA, adjuvant arthritis; DMHP, 7, 3′-dimethoxy hesperetin; TPT, Tripterygium glycosides, was the positive control. †† pb 0.01 compared with normal group. ⁎ p b 0.05. ⁎⁎ p b 0.01 compared with AA group.
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Fig. 2. Histopathologic examinations under a light microscope on ankle joints by HE staining (magnification×100). (A) Normal; (B, C) AA; (D) DMHP 40 mg/kg; (E) DMHP 80 mg/kg; (F) TPT 40 mg/kg. AA, adjuvant arthritis; DMHP, 7, 3′-dimethoxy hesperetin; TPT, Tripterygium glycosides, was the positive control.
3.4. Effects of DMHP on JAK2 and STAT3 mRNA expressions in synovial tissues The mRNA expressions of JAK2 and STAT3 in synovial tissues from different groups were detected by real time Q-PCR. As shown in Fig. 5, mRNA expression of JAK2 and STAT3 in synovium from the AA group was significantly higher than the normal group (p b 0.01). DMHP (40, 80 mg/kg) treatment significantly decreased JAK2 and STAT3 mRNA expressions in the AA rats. As a positive control, TPT remarkably inhibited STAT3 mRNA expression (p b 0.05), and an approximate significant decrease was detected in JAK2 mRNA expression (p = 0.059). 3.5. Effect of DMHP on JAK2, p-JAK2, STAT3 and p-STAT3 protein expressions in synovial tissues The JAK2, p-JAK2, STAT3 and p-STAT3 protein expressions were evaluated by western blot analysis. Representative examples of interest protein expressions from various groups were shown in Fig. 6A. The relative expressions of interest proteins were quantified densitometrically using software LabWorks 4.5 and β-actin was used as a control. There were no significant changes in the amounts of non-phosphorylated JAK2 and STAT3 proteins in various groups
(Fig. 6A). Statistical analysis indicated that p-JAK2 and p-STAT3 in synovium were significantly elevated in the AA group as compared to the normal group and that DMHP treatments significantly suppressed phosphorylation of JAK2 and STAT3 in the AA rats (Fig. 6B). 3.6. Relationships between p-JAK2 or p-STAT3 protein expression in synovium and total histological scores on joint damage Total histological scores on joint damage were showed as the sum of the score of following pathological index: synovial hyperplasia, bone or cartilage destruction, inflammatory cells infiltrate and vascular proliferation. The total histological scores were used to represent the severity of joint damage. Correlation analysis (Fig. 7) indicated a positive correlation between protein levels of p-JAK2 (a: r = 0.828 with p b 0.01) and p-STAT3 (b: r = 0.909 with p b 0.01) in synovium and total histological scores on joint damage. These data imply that the increase of p-JAK2 or p-STAT3 protein expression in synovial tissues is significantly correlated with the increase of total histological scores on joint damage, indicating that inhibition of JAK-STAT pathway by DMHP is highly associated with its therapeutic effect on rats with AA.
Fig. 3. Inhibitory effects of DMHP on histological scores in ankle joints of rats with AA: (A) synovial hyperplasia, (B) bone or cartilage destruction, (C) inflammatory cells infiltration, (D) vascular proliferation. Data are mean ± SEM (n = 5). ††p b 0.01 compared with normal group. *pb 0.05, **p b 0.01 compared with AA group. AA, adjuvant arthritis; DMHP, 7, 3′-dimethoxy hesperetin; TPT, Tripterygium glycosides, was the positive control.
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Fig. 4. Effects of DMHP on IL-6 production in serum and IL-6 mRNA expression in synovial tissues of AA rats. Data are mean ± SEM (n = 5). ††p b 0.01 compared with normal group. *p b 0.05, **p b 0.01 compared with AA group. AA, adjuvant arthritis; DMHP, 7, 3′-dimethoxy hesperetin; TPT, Tripterygium glycosides, was the positive control.
4. Discussion
Fig. 5. Effects of DMHP on the mRNA expression of JAK2 and STAT3 in synovial tissues, detected by Q-PCR. Data are mean ± SEM (n = 5). ††p b 0.01 compared with normal group. *p b 0.05, **p b 0.01 compared with AA group. AA, adjuvant arthritis; DMHP, 7, 3′-dimethoxy hesperetin; TPT, Tripterygium glycosides, was the positive control.
Rat AA induced by FCA has many similar characteristics to RA in the aspects of histology and immunology. In the AA rat model, clinical signs of arthritis developed within 2 weeks and reached its peak about on day 24 after induction. The main pathological features in ankle joint of AA rats could be observed, including synovial hyperplaisia, cartilage or bone damage, inflammatory cell infiltration and pannus formation. Here, our results indicated that DMHP remarkably inhibited hind paw swelling and reduced arthritis index of the AA rats. Histological examination also revealed that DMHP significantly ameliorated the above pathological changes in ankle joint of the AA rats. These results clearly suggest that DMHP can reduce secondary inflammation of AA rats and may be effective in treating RA. Proinflammatory cytokines, particularly IL-1β, TNF-α and IL-6, play a vital pathogenic role in RA development [30]. Many previous studies demonstrated that IL-6 overproduction was found in both synovial fluid and serum of RA patients [31,32]. The serum level of IL-6 correlated positively with RA disease activity, and IL-6 level in serum remarkably decreased with medical treatment [33,34]. The binding of IL-6 to its receptors (gp130 and sIL-6R) initiates cellular events including activation of JAK kinases. Activated JAK kinases phosphorylate and activate STAT transcription factors, particularly STAT3. Phosphorylated STAT3 then forms a dimer and translocates into the nucleus to activate
Fig. 6. Western blot analysis for JAK2, p-JAK2, STAT3 and p-STAT3 in synovial tissues. (A) Representative examples of JAK2, p-JAK2, STAT3, p-STAT3 protein expressions in synovium from various groups. (B) Semi-quantitative statistical graph of p-JAK2, p-STAT3 protein levels in various groups, β-actin serves as the house-keeping protein. Data are mean ± SEM (n = 5). ††p b 0.01 compared with normal group. *p b 0.05, **p b 0.01 compared with AA group. AA, adjuvant arthritis; DMHP, 7, 3′-dimethoxy hesperetin; TPT, Tripterygium glycosides, was the positive control.
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Fig. 7. Correlations between p-JAK2 (a) or p-STAT3 (b) protein level in synovium and total histological scores on joint damage. The correlation analysis was performed by the Pearson's correlation test (n = 30). Asterisks (*) show the significance of the correlation (two tails). **p b 0.01.
transcription of genes [35,36]. Thus, the blockade of IL-6 may be beneficial in treating RA [37]. Indeed, a humanized mAb to IL-6R (tocilizumab) has proven to be effective against RA [38]. In our study, consistent with the previous study [13], IL-6 concentration in serum and IL-6 mRNA expression in synovial tissues were both significantly elevated in the AA group compared with the normal group. Our results indicated that DMHP treatment reversed the high levels of IL-6 in serum and IL-6 mRNA in synovium of the AA rats. It is well known that JAKs associate with cytokine receptors and translate signals triggered by cytokine binding into intracellular responses [39]. Recent evidence suggested that inhibition of JAKs in the signaling complex would effectively inhibit cytokine signaling and thus treat RA [40]. As latent cytoplasmic transcription factors, STATs have critical roles in mediating cellular transcriptional responses to cytokines. Among 7 mammalian STATs, STAT3 has been verified to play a significant role in RA and animal arthritis model. It was reported that STAT3 was highly tyrosine phosphorylated in synovium of RA patients and periarticular injection of SOCS3 adenovirus (a negative regulator of JAK-STAT3 pathway) drastically reduced arthritis severity in collagen induced arthritis [10]. Similarly, a previous study also indicated that STAT3 was over activated in inflamed synovium of mice with zymosan-induced arthritis throughout the course of disease [5]. In addition, STAT3 contributed to RA development by promoting synovial fibroblast survival and abnormal proliferation. Ablation of STAT3 function using a dominant-negative STAT3 mutant (STAT3-YF) apparently induced RA synoviocytes apoptosis in vitro [9]. These findings suggested that modulating STAT3 expression was a potential therapeutic target in RA. In order to elucidate whether the antiarthritic effect of DMHP is related to inhibiting JAK2-STAT3 signal pathway, we analyzed the mRNA levels of JAK2 and STAT3 as well as protein expressions of several key molecules in JAK2-STAT3 pathway, such as JAK2, p-JAK2, STAT3 and p-STAT3. Our data showed that DMHP significantly inhibited JAK2 and STAT3 mRNA expressions which remarkably elevated in AA rats. Although no significant changes of non-phosphorylated JAK2 and STAT3 proteins in synovium were found among various groups, significant increased p-JAK2 and p-STAT3 were observed in the AA group. Treatment with DMHP remarkably decreased the levels of p-JAK2 and p-STAT3 in the AA rats. Moreover, correlation analysis indicated that the protein levels of p-JAK2 and p-STAT3 in synovium were highly correlated with joint damage severity. These results suggested that DMHP suppressed hyper activated JAK2-STAT3 pathway in synovium of AA rats, which might be one possible molecular mechanism underlying the therapeutic effect of DMHP on AA. In conclusion, DMHP has a powerful protective action against FCA-induced AA in rats, as assessed by paw swelling, arthritis index
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