Synthesis and biological evaluations of oleanolic acid indole derivatives as hyaluronidase inhibitors with enhanced skin permeability

Oleanolic acid (OA) is a natural cosmeceutical compound with various skin beneficial activities including inhibitory effect on hyaluronidase but the anti-hyaluronidase activity and mechanisms of action of its synthetic analogues remain unclear. Herein, a series of OA derivatives were synthesised and evaluated for their inhibitory effects on hyaluronidase. Compared to OA, an induction of fluorinated (6c) and chlorinated (6g) indole moieties led to enhanced anti-hyaluronidase activity (IC₅₀ = 80.3 vs. 9.97 and 9.57 µg/mL, respectively). Furthermore, spectroscopic and computational studies revealed that 6c and 6g can bind to hyaluronidase protein and alter its secondary structure leading to reduced enzyme activity. In addition, OA indole derivatives showed feasible skin permeability in a slightly acidic environment (pH = 6.5) and 6c exerted skin protective effect by reducing cellular reactive oxygen species in human skin keratinocytes. Findings from the current study support that OA indole derivatives are potential cosmeceuticals with anti-hyaluronidase activity.     

Dynamic compression influences interleukin-1beta-induced nitric oxide and prostaglandin E2 release by articular chondrocytes via alterations in iNOS and COX-2 expression

Interleukin-1beta (IL-1beta) induces the release of nitric oxide (.NO) and prostaglandin E2 (PGE2) by chondrocytes and this effect can be reversed with the application of dynamic compression. Previous studies have indicated that integrins may play a role. In addition, IL-1beta upregulates the expression of iNOS and COX-2 mRNA via upstream activation of p38 MAPK. The current study examines the involvement of these pathways in mediating .NO and PGE2 release in IL-1beta stimulated bovine chondrocytes subjected to dynamic compression. Bovine chondrocytes were seeded in agarose constructs and cultured with 0 or 10 ng.ml(-1) IL-1beta with or without the application of 15% dynamic compressive strain at 1 Hz. Selected inhibitors were used to interrogate the role of alpha5beta1 integrin signalling and p38 MAPK activation in mediating the release of .NO and PGE2 in response to both IL-1beta and dynamic compression. The relative expression levels of iNOS and COX-2 were assessed using real-time quantitative PCR. Nitrite, a stable end product of .NO, was measured using the Griess assay and PGE2 release was measured using an enzyme immunoassay. IL-1beta enhanced .NO and PGE2 release and this effect was reversed by the application of dynamic compression. Co-incubation with an integrin binding peptide (GRGDSP) abolished the compression-induced effect. Real-time quantitative PCR analysis revealed that IL-1beta enhanced iNOS and COX-2 mRNA levels, with the maximum expression at 6 or 12 hours. Dynamic compression reduced this effect via a p38 MAPK sensitive pathway. These results suggest that dynamic compression acts to abrogate of .NO and PGE2 release by directly influencing the expression levels of iNOS and COX-2.     

The effects of direct current stimulation on isolated chondrocytes seeded in 3D agarose constructs

Endogenous electrical activity has been detected in articular cartilage. It has previously been suggested that the associated electrical currents and potentials are important to the mechanotransduction processes in cartilage. The present study investigates the effects of direct current on cell proliferation and matrix synthesis, using the well established 3D chondrocyte--agarose model system. Bovine chondrocytes isolated from metacarpalphalangeal joints were seeded in agarose constructs and exposed to a current density of 4 mA/cm2 for 6 h, a magnitude and period which was shown to maintain cell viability. The influence of the optimized electric stimulus was assessed by protein incorporation and mRNA measurements, using radiolabels and real-time QPCR, respectively. Results indicated no systematic influences of electrical current on protein synthesis, cell proliferation and mRNA expression levels. These data suggest that both the mode of stimulation and the model system are critical for the in vitro modulation of chondrocyte metabolism.     

Dynamic compression counteracts IL-1β induced inducible nitric oxide synthase and cyclo-oxygenase-2 expression in chondrocyte/agarose constructs

Background  

Nitric oxide and prostaglandin E₂ (PGE₂play pivotal roles in both the pathogenesis of osteoarthritis and catabolic processes in articular cartilage. These mediators are influenced by both IL-1β and mechanical loading, and involve alterations in the inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 enzymes. To identify the specific interactions that are activated by both types of stimuli, we examined the effects of dynamic compression on levels of expression of iNOS and COX-2 and involvement of the p38 mitogen-activated protein kinase (MAPK) pathway.     

Effect of chloroquine on the stability of rat kidney lysosomes in vivo and in vitro

1. Chronic administration of chloroquine to rats results in increased urinary excretion of lysosomal acid phosphatase, muramidase and cathepsin D. 2. Various concentrations of chloroquine caused lysosomal membrane swelling as shown by decrease of light absorbance in lysosomal suspensions. 3. Incubating lysosomal suspensions in the presence of chloroquine resulted in a marked lysosomal acid phosphatase release. 4. Addition of acetylsalicylic acid, a lysosomal membrane stabilizer, into a lysosomal suspension containing chloroquine, reduced the degree of lysosomal membrane swelling and acid phosphatase release. 5. The results suggest a labilizing effect of chloroquine on rat kidney lysosomes.