Gallotannin Causes Differentiation and Inflammation Via ERK‑1/‑2 and P38 Kinase Pathways in Rabbit Articular Chondrocytes
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MOLECULAR MEDICINE REPORTS 7: 701-707, 2013 Gallotannin causes differentiation and inflammation via ERK‑1/‑2 and p38 kinase pathways in rabbit articular chondrocytes WON-KIL LEE, KI-WHA CHUNG, GWANG-HOON KIM and SONG-JA KIM Department of Biological Sciences, College of Natural Sciences, Kongju National University, Gongju, Chungnam 314‑701, Republic of Korea Received September 29, 2012; Accepted November 26, 2012 DOI: 10.3892/mmr.2012.1204 Abstract. Gallotannin (GT) is a type of tannic acid, derived osteoarthritis (OA) and rheumatoid arthritis (RA) (3). The from plant polyphenols, that is an agonist of plant defense biochemical and structural changes in chondrocytes and carti- mechanisms. Tannins have two types of structure; condensed lage that characterize arthritis include the degradation of the tannins are a polymer of flavonoid units, while hydrolysable cartilage matrix and insufficient ECM synthesis due to a loss of tannins are carbohydrates. GT is used in medical agents for chondrocyte phenotype. OA is the most common form of joint its anti‑viral, anti‑bacterial and anti‑parasitic effects. The disease that evolves from a local inflammatory disease into a present study investigated the effects of GT on differentiation chronic process with a variable degree of degeneration of the and inflammation in rabbit articular chondrocytes. GT caused articular cartilage and inflammation. This ultimately exposes differentiation and inflammatory responses in the rabbit the underlying bone and results in pain and disability (4). The articular chondrocytes. GT treatment induced the expression second type of arthritis, RA, is a destructive and inflammatory, of type Ⅱ collagen and sulfated proteoglycan, as determined polyarticular joint disease, with an etiology that remains to be by western blot analysis and alcian blue staining, respectively, clarified. RA is characterized massive synovial proliferation in a dose- and time-dependent manner. Additionally, treatment and subintimal infiltration of the inflammatory cells followed with GT increased the expression of cyclooxygenase-2 (COX-2) by the destruction of cartilage and bone (5). and the production of prostaglandin E2 (PGE2), as determined Tannins are water‑soluble polyphenols that are widely by western blot analysis and PGE2 assay. GT was confirmed to distributed in the plant kingdom, including in food grains and cause phosphorylation of ERK‑1/‑2 and p38 kinase. Inhibition fruits (6). Based on their structural characteristics, tannins of pERK with PD98059 promoted GT‑induced type Ⅱ collagen may be separated into four major groups: gallotannins (GTs), expression. However, the inhibition of p38 with SB203580 ellagitannins, complex tannins and condensed tannins. These suppressed GT-induced COX-2 expression and PGE2 produc- tannins are thought to have notable biological and pharmaco- tion. In summary, the results demonstrated that GT-induced logical activities (7,8). The hydrolysable tannin, gallotannin, is ERK-1/-2 and p38 kinase have opposite effects on differentia- a compound of the polygalloyl esters of glucose. Gallotannin is tion and inflammation in rabbit articular chondrocytes. a type of tannic acid derived from plant polyphenols. Tannins have two types of structure; condensed tannins are a polymer Introduction of flavonoid units, while hydrolysable tannins are carbohy- drates. The hydroxyl groups of the carbohydrate are partially Chondrocytes in cartilage are differentiated from mesenchymal or completely esterified with phenolic groups, including gallic cells during embryonic development (1,2). They are the only acid. Hydrolysable tannins are hydrolyzed by weak acids or cell type located in normal mature cartilage and they function weak bases to produce carbohydrates and phenolic acids. to maintain extracellular matrix (ECM) integrity by synthe- These hydrolysable tannins are used in medical agents for sizing cartilage‑specific ECM in sufficient quantities. This their useful properties, including their anti‑viral, anti‑bacterial homeostasis is destroyed in degenerative diseases, including and anti‑parasitic effects. GTs have been shown to exhibit diverse biological abilities, ranging from anti‑inflammation to anti-oxidant effects (9,10). Cyclooxygenases (COXs) are known to exist in two Correspondence to: Professor Song-Ja Kim, Department of isoforms, COX-1 and COX-2. In addition, the two COX Biological Sciences, College of Natural Sciences, Kongju National isoforms were identified with a similar sequence (11). COX‑1 University, 182 Shinkwan-Dong, Gongju, Chungnam 314‑701, is a constitutive enzyme located in the majority of mammalian Republic of Korea cells (12). COX‑2, however, is undetectable in the majority E-mail: [email protected] of normal tissues (13). COX‑2 is an inducible enzyme that becomes abundant in activated macrophages and other cells at Key words: gallotannin, differentiation, inflammation sites of inflammation as a result of various stimuli, including cytokines. Expression of COX-2 was demonstrated to increase prostaglandin E2 (PGE2) production (14,15) and induce various 702 LEE et al: EFFECTS OF GALLOTANNIN IN RABBIT ARTICULAR CHONDROCYTES inflammatory reactions (16). Mice that lack COX‑2, but electrophoresis and transferred to a nitrocellulose membrane. possess COX‑1 expression, exhibit reduced bone resorption The nitrocellulose sheet was then blocked with 3% skimmed in response to parathyroid hormone (PTH) or 1, 25-hydroxyl dry milk in Tris‑buffered saline. The following antibodies vitamin D3 (17). COX‑2 may also have a role in bone forma- were used: anti‑COX‑2 (Cayman Chemical, Ann Arbor, MI, tion as local or systemic injections of PGE2 stimulate bone USA), pp38 (Cell Signaling, Beverly, MA, USA), pERK, formation (18,19). COX-2 mediates the increase in lamellar ERK‑2 and p38 (Santa Cruz Biotechnology Inc., Santa Cruz, bone formation that occurs as a response to mechanical CA, USA) and type II collagen (Chemicon, Temecula, CA, strain (20,21). USA). The western blot samples were developed using a The mitogen-activated protein kinase (MAPK) cascades peroxidase‑conjugated secondary antibody on a chemilumi- make up one of the major signaling systems by which cells nescence system. transduce and integrate diverse intracellular signals. MAPKs are serine/threonine kinases that regulate a variety of Alcian blue staining assay. The cells were fixed with 95% processes, including cell growth, proliferation, apoptosis and methanol at -20˚C for 2 min and stained with 0.1% alcian blue extracellular matrix accumulation. The three MAPK subfami- in 0.1 M HCl overnight. The chondrocytes were washed three lies consist of extracellular signal-regulated kinases (ERKs), times with PBS buffer and 6 M guanidine HCl was added p38 kinases and c-Jun NH2-terminal kinases (JNKs) (22). for 6 h. The production level of sulfated proteoglycan was Previous studies in articular chondrocytes indicated that NO measured at 620 nm by enzyme‑linked immunosorbent assay caused apoptosis and dedifferentiation, which were mediated (ELISA). by the MAPK subtypes, ERK and p38 kinase (23). These MAP kinases play opposing roles; activated ERK-1/-2 induces dedif- PGE2 assay. PGE2 production was determined by measuring ferentiation, COX-2 expression and NO-induced apoptosis the levels of cellular and secreted PGE2 using an assay kit inhibition, whereas p38 kinase signaling triggers apoptosis, (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Briefly, COX‑2 expression and maintains the differentiated states (24). chondrocytes were seeded in standard 96-well microtiter plates In the present study, ERK-1/-2 and p38 kinase are at 2x104 cells/well. Following addition of the indicated phar- demonstrated to regulate GT-induced differentiation and macological reagents, the supernatant was used to quantify the inflammation of chondrocytes, respectively. amount of PGE2, according to the manufacturer's protocol. The PGE2 levels were calculated using a PGE2 standard curve. Materials and methods Immunohistochemistry. Rabbit joint cartilage explants were Cell culture. Rabbit articular chondrocytes were isolated from fixed in 4% paraformaldehyde in PBS for 24 h at 4˚C, washed cartilage slices obtained from 2‑week‑old New Zealand white with PBS, dehydrated in ethanol, embedded in paraffin and rabbits using enzymatic digestion, as described previously (25). sliced into 4‑µm sections, as described previously (26). The The cartilage slices were dissociated enzymatically for 6 h sections were stained by standard procedures, using antibodies in 0.2% collagenase type II (381 U/mg solid; Sigma Aldrich, against type II collagen and COX‑2, then alcian blue staining Louis, MO, USA), in Dulbecco's modified Eagle's medium followed by visualization by development with a kit purchased (DMEM; Gibco‑BRL, Gaithersburg, MD, USA). Individual from Dako (Carpinteria, CA, USA), following the manufac- cells were suspended in DMEM supplemented with 10% (v/v) turer's instructions. fetal bovine‑calf serum, 50 g/ml streptomycin and 50 U/ml penicillin, after which they were plated on culture dishes at a Immunofluorescence staining. Expression and distribution of density of 5x104 cells/cm2. The medium was changed every type II collagen and COX‑2 in rabbit articular chondrocytes 2 days following seeding and the cells reached confluence in were determined by indirect immunofluorescence microscopy, ~5 days. At 3.5 days the cell cultures were treated with GT as described previously (26). Briefly, chondrocytes were fixed purchased from Sigma Aldrich