Carnosic acid (CA) attenuates collagen-induced arthritis in db/db mice via inflammation suppression by regulating ROS-dependent p38 pathway

Highlights

• Carnosic acid inhibits insulin resistance and lipid accumulation in db/db mice.

• Carnosic acid inhibits CIA-promoted oxidative stress and arthritis.

• Carnosic acid inhibits M-CSF- and RANKL-induced osteoclast differentiation.

• Carnosic acid suppressed ROS-dependent p38 pathway in osteoclastogenesis.

Abstract

Rheumatoid arthritis (RA) is a multifactorial autoimmune disease, characterized by inflammation of synovial joints. Carnosic acid (CA) is a phenolic diterpene isolated from Rosmarinus officinailis, playing a central role in cytoprotective responses to oxidative stress and inflammation response. Our study aimed to investigate the effects of CA on RA progression in diabetic animals. Carnosic acid (CA) was used to treat collagen-induced arthritis (CIA)-induced db/db mice. Blood glucose, oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were investigated to explore insulin resistance. CA significantly down-regulated fasting blood glucose, glucose level in OGTT and ITT, ameliorated CIA-induced bone loss, and reduced pro-inflammatory cytokines and reactive oxygen species (ROS) in db/db mice with arthritis induced by CIA. In vitro, CA suppressed Receptor Activator for Nuclear Factor-κ B Ligand (RANKL)- and Macrophage colony-stimulating factor (M-CSF)-induced osteoclastogenesis. The osteoclastic specific markers were inhibited by CA. Signal transduction studies showed that CA significantly decreased the expression of molecules contributing to ROS and increased anti-oxidants. Additionally, CA inactivated the RANKL- and M-CSF-induced p38 mitogen activated protein kinases (MAPK), inhibited NF-κB phosphorylation, causing pro-inflammatory cytokines down-regulation. Together, CA ameliorated osteoclast formation and CIA-induced bone loss in db/db mice through inflammation suppression by regulating ROS-dependent p38 pathway.

Introduction

Rheumatoid arthritis (RA), characterized by progressive inflammation of the synovial joints, leads to the breakdown of cartilage and bone, and eventually results in malformation of hands and feet, reducing the quality of life for the patients [1], [2]. Inflammation likely enhances development of several cardiovascular risk factors such as diabetes [3], [4]. Also, accumulating evidences report that insulin resistance, oxidative stress and inflammatory response influence diabetic patients with RA [5], [6]. Previous studies have indicated improvements in glucose and insulin metabolism among RA users of various immunosuppressive agents [7], [8]. Thus systemic immunosuppression may also reduce the risk for diabetes. For example, several investigations have illustrated that tumor necrosis factor (TNF) inhibitors improve insulin resistance [9], [10]. In spite of extensive efforts before, the molecular pathogenesis and aetiological factors contributing to diabetic patients with RA are not understood clearly, and effective therapeutic strategies with limited side-effects to patients remain lacking.

Carnosic acid (CA), extracted from rosemary, displays multiple pharmacological activities [11]. CA regulates ER stress to ameliorate disease progression [12]. CA can also cross the blood-brain barrier readily and exert its protective effects via its potent anti-oxidative role [13]. In addition, CA ameliorates acute promyelocytic leukemia cells through activating nuclear transcription factor E2-related factor 2 (Nrf2), which plays an essential role in cytoprotective responses to oxidative stress [14]. Being different with other antioxidants, CA does not remove the endogenous antioxidant glutathione [14]. Increasing evidence from animal models of RA suggests that oxidative stress may be the critical molecular mechanisms underlying the breakdown of cartilage and bone [15], [16], [17]. An increase in the production of ROS has been implicated in inflammatory arthritis [18], [19]. ROS oxidation can result in the inflammatory response, apoptosis, aging, and cancer [20], [21], [22], [23]. RANKL induced intracellular ROS production, influencing RANKL signaling pathways activation, which is related to osteoclast differentiation. RANKL has been known as an upstream component of the signaling pathway, which could cause bone breakdown via promoting osteoclast activation and survival [24], [25]. However, the exact role of CA in osteoclasts differentiation has not to be elucidated.

To our best knowledge, it was the first time that the effect of CA was investigated on db/db mice with RA induced by CIA in vivo, and on osteoclast differentiation in vitro. We found that CA protects db/db mice with insulin resistance and lipid accumulation and acts as an inflammatory inhibitor via NF-κB suppression. The inhibitory role might be due to blockage of ROS-regulated p38 signaling pathway, which was involved in osteoclast differentiation. Our study illustrated that CA suppressed collagen-induced arthritis in db/db mice by inflammation suppression through regulating ROS-dependent p38 pathway.