Oral administration of berberine limits post-traumatic osteoarthritis development and associated pain via AMP-activated protein kinase (AMPK) in mice.

OBJECTIVE: We investigated the effect of berberine, a natural plant product that can activate AMP-activated protein kinase (AMPK), on Osteoarthritis (OA) development and associated pain in mice. DESIGN: Human primary knee chondrocytes were utilized to investigate how AMPK is activated by berberine. Both global knockout (KO) of AMPKα1 and congenic wild type (WT) mice were subjected to the post-traumatic OA through destabilization of medial meniscus (DMM) surgery. Two weeks after surgery, the mice were randomly divided into two groups with one group receiving berberine chloride daily via drinking water and were sacrificed at 6 and 12 weeks after surgery. OA severity was assessed by histological and histomorphometric analyses of cartilage degradation, synovitis, and osteophyte formation. OA-associated pain behavior was also determined. Immunohistochemistry (IHC) analyses were carried out to examine changes in AMPK signaling. RESULTS: Berberine induced phosphorylation of AMPKα (Thr172) via liver kinase B1 (LKB1), the major upstream kinase of AMPK, in chondrocytes in vitro. Both WT and AMPKα1KO developed OA and associated pain post DMM surgery. However, treatment with berberine significantly reduced severity of OA and associated pain in WT but not AMPKα1KO mice. IHC analysis of WT DMM knee cartilage further revealed that berberine inhibited concomitant loss of expression and phosphorylation of AMPKα and expression of SIRT1 and SIRT3, suggesting an important role of activation of AMPK signaling in mediating beneficial effect of berberine. CONCLUSIONS: Berberine acts through AMPK to reduce joint structural damage and pain associated with post-traumatic OA in mice in vivo.

Role of TLR2 and TLR4 in regulation of articular chondrocyte homeostasis.

OBJECTIVE: Toll-like receptor (TLR)-mediated catabolic responses are implicated to contribute to osteoarthritis (OA). However, deficiency of TLRs has little chondroprotection in mice in vivo. Here, we studied the effect of deficiency of TLR2 and TLR4 in articular chondrocytes on cellular stress responses in vitro. DESIGN: Chondrocytes isolated from TLR2 and TLR4 double knockout (TLR2/4dKO) and wild type (WT) mice and recombinant HMGB1 (rHMGB1) and LPS were used. Expression of anti-oxidant and DNA repair enzymes including SOD1, SOD2 and OGG1, and phosphorylation of H2AX (a marker for DNA damage) were examined by Western blotting. MitoSOX Red staining was used for assessing mitochondrial superoxide generation. Autophagic activity was monitored by flow cytometry analysis of mean fluorescence intensity (MFI) of GFP and RFP in chondrocytes transfected with a tandem GFP-mRFP-LC3 plasmid, and by Western blot analysis of expression of LC3 and p62, a selective autophagy adaptor. RESULTS: Basal expression of SOD2 but not SOD1 was largely reduced in TLR2/4dKO compared to WT chondrocytes, correlated with significantly enhanced menadione-induced mitochondrial superoxide generation (2.85-3.92 and 3.39 to 8.97 with mean difference 3.39 and 6.18 for 25 and 50µM menadione, respectively) and phosphorylation of H2AX. LPS and rHMGB1 induced expression of SOD2, OGG1 and p62 in WT but not TLR2/4dKO chondrocytes. Autophagy flux was impaired in TLR2/4dKO chondrocytes after acute nutrient stress and by LPS and rHMGB1. CONCLUSIONS: TLR2 and TLR4 deficiency appears to reduce chondrocyte anti-oxidative stress and autophagy flux capacity, which may compromise cartilage homeostasis as a result of chondrocyte dysfunction.

Activation of AMPK-SIRT3 signaling is chondroprotective by preserving mitochondrial DNA integrity and function.

OBJECTIVE: In osteoarthritis (OA), articular chondrocytes manifest mitochondrial damage, including mitochondrial DNA 4977-bp (mtDNA4977) deletion that impairs mitochondrial function. OA chondrocytes have decreased activity of AMPK, an energy biosensor that promotes mitochondrial biogenesis. Here, we tested if pharmacologic AMPK activation, via downstream activation of predominately mitochondrially localized sirtuin 3 (SIRT3), reverses existing decreases in mitochondrial DNA (mtDNA) integrity and function in human OA chondrocytes and limits mouse knee OA development. DESIGN: We assessed mtDNA integrity and function including the common mtDNA4977 deletion and mtDNA content, mitochondrial reactive oxygen species (mtROS) generation, oxygen consumption and intracellular ATP levels. Phosphorylation of AMPKα, expression and activity of SIRT3, acetylation and expression of the mitochondrial antioxidant enzyme SOD2 and DNA repair enzyme 8-oxoguanine glycosylase (OGG1), and expression of subunits of mitochondrial respiratory complexes were examined. We assessed effect of pharmacologic activation of AMPK on age-related spontaneous mouse knee OA. RESULTS: The mtDNA4977 deletion was detected in both OA chondrocytes and menadione-treated normal chondrocytes, associated with increased mtROS, decreased SIRT3, and increased acetylation of SOD2 and OGG1. AMPKα1 deficient chondrocytes exhibited significantly reduced SIRT3 activity. AMPK pharmacologic activation attenuated existing mtDNA4977 deletion and improved mitochondrial functions in OA chondrocytes via SIRT3 by reducing acetylation and increasing expression of SOD2 and OGG1, and limited aging-associated mouse knee OA development and progression. CONCLUSIONS: AMPK activation, via SIRT3, limits oxidative stress and improves mtDNA integrity and function in OA chondrocytes. These effects likely contribute to chondroprotective effects of AMPK activity.

Inflammation and intracellular metabolism: new targets in OA.

Articular cartilage degeneration is hallmark of osteoarthritis (OA). Low-grade chronic inflammation in the joint can promote OA progression. Emerging evidence indicates that bioenergy sensors couple metabolism with inflammation to switch physiological and clinical phenotypes. Changes in cellular bioenergy metabolism can reprogram inflammatory responses, and inflammation can disturb cellular energy balance and increase cell stress. AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) are two critical bioenergy sensors that regulate energy balance at both cellular and whole-body levels. Dysregulation of AMPK and SIRT1 has been implicated in diverse human diseases and aging. This review reveals recent findings on the role of AMPK and SIRT1 in joint tissue homeostasis and OA, with a focus on how AMPK and SIRT1 in articular chondrocytes modulate intracellular energy metabolism during stress responses (e.g., inflammatory responses) and how these changes dictate specific effector functions, and discusses translational significance of AMPK and SIRT1 as new therapeutic targets for OA.

Hyperalgesia, synovitis and multiple biomarkers of inflammation are suppressed by interleukin 1 inhibition in a novel animal model of gouty arthritis.

BACKGROUND: Monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystal-induced interleukin 1 beta (IL1beta) release contributes to inflammation in subcutaneous air pouch and peritoneal models of acute gout and pseudogout. However, consequences of IL1 inhibition have not been explored in more clinically relevant models of crystal-induced arthritis. OBJECTIVE: To develop a novel mouse model of acute gouty ankle arthritis and use it to assess the effects of genetic deletion of IL1 receptor type (IL1R1) and of exogenous mIL1 Trap (a high-affinity blocker of mouse IL1alpha and IL1beta) on pain, synovitis and systemic inflammatory biomarkers. METHODS: MSU crystals were injected into the mouse ankle joint and pain and ankle swelling were measured over 4 days. The effects of IL1 inhibition were determined in this model, and in the comparator models of crystal-induced peritonitis and subcutaneous air pouch inflammation. RESULTS: Both IL1R1-null mice and mice pretreated with mIL1 Trap showed reduced neutrophil influx in MSU and CPPD crystal-induced peritonitis and air pouch models (p<0.05). In the ankle joint model, both IL1R1 knockout mice and pretreatment with mIL1 Trap were associated with significant reductions in MSU crystal-induced elevations in hyperalgesia, inflammation, serum amyloid A and the levels of multiple inflammatory cytokines and chemokines (p<0.05). Additionally, it was found that administration of mIL1 Trap after MSU crystal injection reduced established hyperalgesia and ankle swelling. CONCLUSIONS: IL1 inhibition both prevented and relieved pain and ankle joint inflammation in response to intra-articular MSU crystals in mice. Results suggested that IL1 Trap has the potential to both prevent and treat gouty arthritis.

Overview of hyperuricaemia and gout.

In most mammals purine degradation ultimately leads to the formation of allantoin. Humans lack the enzyme uricase, which catalyzes the conversion of uric acid to allantoin. The resulting higher level of uric acid has been hypothesized to play a role as an antioxidant. Hyperuricaemia is usually an asymptomatic condition which is hypothesized to play a role in cardiovascular disease and hypertension. Some hyperuricaemic individuals develop gout, an inflammatory arthritis caused by the deposition of monosodium urate crystals in joints. Over time, acute intermittent gouty arthritis can develop into a chronic condition with deposits of monosodium urate (MSU) crystals in joints and as tophi. The mechanisms by which MSU crystals lead to an acute inflammatory arthritis are under investigation and current knowledge is reviewed here. Treatment of gout includes management of acute flares with anti-inflammatory medications such as non-steroidal anti-inflammatory drugs or corticosteroids and long term management with urate-lowering therapy when indicated. Future directions in the treatment of gout, in part guided by a better understanding of pathophysiology, are discussed.