Succinate/NLRP3 Inflammasome Induces Synovial Fibroblast Activation: Therapeutical Effects of Clematichinenoside AR on Arthritis.

Clematichinenoside AR (C-AR) is a triterpene saponin isolated from the root of Clematis manshurica Rupr., which is a herbal medicine used in traditional Chinese medicine for the treatment of arthritis. C-AR exerts anti-inflammatory and immunosuppressive properties, but little is known about its action in the suppression of fibroblast activation. Low oxygen tension and transforming growth factor-ß (TGF-ß1) induction in the synovium contribute to fibrosis in arthritis. This study was designed to investigate the effect of C-AR on synovial fibrosis from the aspects of hypoxic TGF-ß1 and hypoxia-inducible transcription factor-1α (HIF-1α) induction. In the synovium of rheumatoid arthritis (RA) rats, hypoxic TGF-ß1 induction increased succinate accumulation due to the reversal of succinate dehydrogenase (SDH) activation and induced NLRP3 inflammasome activation in a manner dependent on HIF-1α induction. In response to NLRP3 inflammasome activation, the released IL-1ß further increased TGF-ß1 induction, suggesting the forward cycle between inflammation and fibrosis in myofibroblast activation. In the synovium of RA rats, C-AR inhibited hypoxic TGF-ß1 induction and suppressed succinate-associated NLRP3 inflammasome activation by inhibiting SDH activity, and thereby prevented myofibroblast activation by blocking the cross-talk between inflammation and fibrosis. Taken together, these results showed that succinate worked as a metabolic signaling, linking inflammation with fibrosis through NLRP3 inflammasome activation. These findings suggested that synovial succinate accumulation and HIF-1α induction might be therapeutical targets for the prevention of fibrosis in arthritis.

Mouse myosin-19 is a plus-end-directed, high-duty ratio molecular motor.

Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament.

Studies on the stability of salvianolic acid B as potential drug material.

INTRODUCTION: Salvianolic acid B (Sal B) is one of the major water-soluble compounds isolated from the roots of Salvia miltiorrhiza, which is widely used as a traditional Chinese medicine. Although much research on the general stability of Sal B has been undertaken and reported, there is still a need for further study of the stability required as a potential drug material. OBJECTIVE: To study the stability of Sal B in the solid state and in normal saline (NS) solution during storage, as required in the ICH guidelines (2003) and Chinese Pharmacopoeia (2005). METHODOLOGY: Sal B stability was analysed using the high-performance liquid chromatography (HPLC) method described in the Chinese Pharmacopoeia. HPLC coupled with time-of-flight mass spectrometry (HPLC-TOFMS) was applied for the separation and identification of the degradation products of Sal B. RESULTS: In the solid state, Sal B packaged in aluminium foil bags was stable for 6 months under 'accelerated conditions' (40°C, 75% relative humidity, RH). However, solid Sal B degradation was observed under open exposure to stress conditions of high temperature (60°C) or high humidity (92.5 or 75% RH). In NS solution, Sal B underwent severe degradation under accelerated conditions. Through HPLC-TOFMS, nine degradation products were identified and the possible degradation pathway was deduced. CONCLUSION: The results demonstrate that the potential drug material Sal B could be used in a solid formulation, but is not suitable for use as a liquid formulation.