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Osteolysis around the prosthesis and subsequent aseptic loosening are the main causes of prosthesis failure. Inflammation due to wear particles and osteoclast activation are the key factors in osteolysis and are also potential targets for the treatment of osteolysis. However, it is not clear whether puerarin can inhibit chronic inflammation and alleviate osteolysis. In this study, we investigated the effect of puerarin on Ti particle‐induced inflammatory osteolysis in vivo in rat femoral models and in vitro in receptor activator of nuclear factor kappa‐B ligand (RANKL)‐induced osteoclast activation models. Our in vivo results showed that puerarin significantly inhibited Ti particle‐induced osteolysis and the expression of matrix metallopeptidase 9 (MMP‐9), nuclear factor of activated T cells 1 (NFATc1), tumour necrosis factor (TNF)‐α and interleukin (IL)‐6. In vitro, puerarin prevented RANKL‐induced osteoclast differentiation, bone resorption and F‐actin ring formation in a concentration‐dependent manner. Furthermore, puerarin decreased the phosphorylation of p65 and prevented p65 moving from the cytoplasm to the nucleus. Puerarin also reduced the expression of osteoclast‐specific factors and inhibited the inflammatory response. In conclusion, our study proves that puerarin can block the NF‐κB signalling pathway to inhibit osteoclast activation and inflammatory processes, which provides a new direction for the treatment of osteolysis‐related diseases.

For a linear response in a competitive bioaffinity assay of a ligand, an optimized system requires CRT greater than 3-fold of CPT, CPT greater than 50-fold of KdR, and KdR greater than 260-fold of KdX, based on chemometrics for bioaffinity interactions (CRT and CPT are the concentrations of the probe and the biomacromolecule and KdX and KdR are the dissociation constants of complexes with the ligand and the probe, respectively). These criteria were tested for a competitive bioaffinity assay of biotin. The probe was a conjugate of monomethyl-poly-(ethylene glycol)-5000, 1-naphthyl-ethylenediamine and biotin. The complex of the probe with streptavidin was quantified by the fluorescence at 430 nm based on Forster resonance energy transfer with tryptophan residues as the intrinsic donors. By fluorometric titration, KdR of the probe was 5.4 ± 1.4 nM (n = 4). At 1.5 μM probe plus 0.50 μM streptavidin, there was a linear decrease of fluorescence at 430 nm for biotin concentrations ranging from ∼36 to ∼500 nM; the linear response slope was consistent with that for the fluorescence at 430 nm to the concentration of the complex of streptavidin and the probe. Biotin at 81 and 414 nM was estimated with variation coefficients below 7%. These proposed criteria may be universally applicable for linear responses in competitive assays of ligands.

To characterize streptavidin immobilization on magnetic submicron particles (MSPs), residual streptavidin after magnetic removal of immobilized streptavidin was quantified with N-biotinyl- N′-(1-naphthyl)-ethylenediamine (BNEDA) based on Forster resonance energy transfer. Residual BNEDA after magnetic removal of bound BNEDA was measured by its own fluorescence. Streptavidin was immobilized at about 12 mg per gram of MSPs and easily retained over 50% of its original activity. These assays facilitated optimized streptavidin immobilization on MSPs.

To determine inhibition constant (K i) of tight-binding inhibitor, the putative method estimated an apparent K i from the response of initial rates to total concentrations of the inhibitor considering its depletion during binding for conversion into the true K i, but was impractical with glutathione S-transferase of sophisticated kinetics. A fluorometric titration assay of dissociation constant (K d) was thus proposed. Schistosoma japonicum glutathione S-transferase (SjGST) action on a nonfluorescent divalent pro-inhibitor and glutathione yielded a divalent product in active site to act as a tight-binding inhibitor, whose binding quenched fluorescence of SjGST at 340 nm under the excitation at 280 nm. K d was estimated from the response of fluorescence of SjGST at 340 nm to total concentrations of the divalent product considering its depletion during binding. By fluorometric titration assay, K d of two tested nonfluorescent divalent products varied from subnanomolar to nanomolar, but both were resistant to change of SjGST levels and consistent with their apparent K i estimated via the putative method. Hence, fluorometric titration assay of K d of nonfluorescent tight-binding inhibitors/ligands was effective to GST and may be universally applicable to common enzymes/proteins; affinities of tight-binding inhibitors of GST can be approximated by their apparent K i estimated via the putative method.