A problem in using natural herbal material is the difficulty in standardization of efficacy, which is partially due to factors such as differences in region of origin, harvest period, and cultivation time. Also, for the discovery and development of new treatments, further understanding of the processes leading to disease progression and in particular, the pathways leading to the expression of the MMPs, aggrecanases, and cartilage destruction are of pivotal importance. Therefore, we measured the major components and examined their efficacy to set a standard for use of WIN-34B in practice and in medicine development. In this study, we demonstrated the cartilage-protective effects of WIN-34B compared to standard compounds, CA and MF, in IL-1β-stimulated cartilage explants culture. WIN-34B more effectively improved the cartilage protection without cytotoxicity by modulating MMPs, ADADMTs, TIMPs, and inflammatory mediators, and possibly by inhibiting MAPK pathways.
WIN-34B did not exert cytotoxic effects in the absence of IL-1β in human OA cartilage explants culture and did not affect cell viability in chondrocytes. However, CA was cytotoxic in human cartilage explants culture and chondrocytes.
Previously, we found that even high doses of WIN-34B did not cause toxicity or gastric injury when orally administered to rats . Both single and multiple doses of WIN-34B had no effect on mortality, body weight changes, gross findings, or clinical signs in patients of either sex . This finding was in contrast to those for diclofenac and celecoxib, which cause inflammation and hemorrhage [19, 20]. Furthermore, clinical study on patients with OA revealed that WIN-34B not only had a good analgesic efficacy and safety profile, but also showed functional improvements on the time taken to go up and down a standard flight of stairs, duration of morning stiffness, and softening of the affected knee joint. These results support the safety and therapeutic usefulness of WIN-34B for development as an OA treatment.
WIN-34B markedly prevented the release of GAG and type II collagen, which are associated with the down-regulation of matrix proteinases (MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5), inflammatory cytokines (PGE2, NO, IL-1β, and TNF-α), and up-regulation of TIMP-1 and TIMP-3 levels in IL-1β-stimulated human OA cartilage culture. However, CA and MF significantly inhibited only collagen release, which is associated with the inhibition of MMP-1, MMP-13, and inflammatory cytokines in IL-1β-stimulated human OA cartilage culture. The inhibition of GAG release and recovery of aggrecan expression by CA and MF was not evident in IL-1β-stimulated human OA cartilage culture. Therefore, we suggest that WIN-34B could be a potential candidate for effective anti-osteoarthritic therapy with cartilage protective properties better than CA or MF.
Protecting ECM components is critical to modifying OA progression and protecting joint functions [21, 22]. A number of studies have documented the fact that aggrecan not only resists mechanical loading by enabling the cartilage matrix to attract and imbibe water molecules, but also plays a partial role in preventing collagen degradation in OA pathogenesis [21, 23, 24]. For this reason, many researchers have investigated the OA-modifying effects of drugs designed to inhibit ADAMTS-4 and ADAMTS-5 . Several studies have reported that glucosamine down-regulates ADAMTS and MMPs including MMP-3, MMP-9, MMP-10, and MMP-12 [25, 26]. SKI306X, a commercially-available herbal mixture for OA treatment, inhibits cartilage degradation through the production of MMPs and inflammatory mediators . Inflammatory mediators, such as PGE2, NO, IL-1, and TNF-α, play key roles in the progression of cartilage destruction in OA [28, 29]. Particularly, IL-1β produces PGE2 and NO, and stimulates the expression of other inflammatory cytokines and MMPs . PGE2 is a pathologic mediator responsible for the remodeling of cartilage and bone . NO is a pleiotropic mediator involved in the catabolic process of OA, which inhibits the synthesis of proteoglycan and collagen, resulting in the promotion of cartilage destruction . Presently, WIN-34B decreased the level of inflammatory mediators including PGE2 and NO, as well as the proinflammatory cytokines, IL-1β, and TNF-α, which are all recognized as inducers of MMPs and aggrecanases. The inhibition of PGE2 release, NO production, and TNF-α secretion by WIN-34B was superior to CA or MF. These results suggest that WIN-34B inhibits the pathologic inflammatory molecules in the cartilage destruction of OA.
MAPKs regulate pro-inflammatory cytokine production and downstream signaling cascades leading to catabolic joint destruction [8, 30]. Studies in human cells have suggested that MAPK signaling is important for the MMP-derived catabolic response of chondrocytes. Liacini et al. showed that TNF-stimulated human OA chondrocytes up-regulated expression of MMP-13, through MAPK 44/42 and Janus-NH2-terminal kinase JNK . Similar results in human chondrosarcoma cells supported these findings . Taken together, these findings suggest that MAPK signaling is important for the MMP-derived catabolic response of chondrocytes. Recently reported that intra-articular injections of the p38 MAPK inhibitor SB203580 in the anterior cruciate ligament transection (ACLT) rat model of OA inhibited the expression of MMP-3 and MMP-13 and protected against cartilage damage . Other research showed WIN-34B dose-dependently diminished phosphorylation of ERK, JNK, and p38 MAPK, as well as MMPs and aggrecanases in IL-1β-stimulated cartilage explants culture. However, CA and MF increased phosphorylation of p38 and suppressed phosphorylation of JNK, but did not affect the phosphorylation of ERK. Inhibition of the MAPK P44/42 pathway by either U0126 or PD98059 leads to abrogation of the expression and activity of MMPs and aggrecanases and ADAMTS. Inhibitors of the p38 MAPK and JNK pathways were also investigated by SB203580 or SB202190 and PP1, respectively. However, inhibition of these pathways resulted in inhibition of MMP expression and activity, but did not influence aggrecanases activity . The current line of investigations suggests that p38 MAPK and JNK activity could be associated with MMP-mediated irreversible cartilage damage, whereas the processes needed for normal repair mechanism and aggrecanase activity may in part be controlled by MAPK p44/42-activities [33, 34]. From these results we can suggest that WIN-34B may be critical role on cartilage protection and anti-inflammatory effect by the downregulation of pERK, p38 MAPK and pJNK signaling pathways.