Dentin Matrix Metalloprotease Inhibition by Berberine

Dentin organic matrix degradation in caries progression and in hybrid layers is related to endogenous proteolytic activity. Matrix metalloproteases (MMPs) are the main enzymes responsible for collagen breakdown. Hypothesis: Berberine, a plant-derived quaternary ammonium compound will inhibit MMP activity and provide a protective effect against collagen degradation. Objective: The aim of this study is to determine if berberine will have an inhibitory effect on selective recombinant and endogenous MMPs. Methods: Gelatin zymography assays were conducted to assess the inhibitory effect of various concentrations of berberine (AnaSpec) against selective recombinant MMPs. Recombinant MMP-2 and MMP-9 were electrophoresed under non-reducing conditions in 10% SDS-PAGE co-polymerized with 0.2% of fluorescent MDPF-labeled gelatin. Following, gels were incubated with: plain MMPs buffer, MMPs buffer containing 0.02%, 0.002% or 0.0002% berberine, or MMPs buffer containing 2 mM phenantroline. After a 48 hour incubation at 37 °C, images were captured under UV light using Quantity One analysis software (Bio-Rad). Endogenous MMP activity was assessed through quantification of hydroxyproline release. Demineralized human dentin powder samples were treated with one of five different solutions: water, 0.02% berberine, 0.2% berberine, 0.002% chlorhexidine; 0.02% chlorhexidine. Powder specimens were treated with the various solutions for 1 hour at 37° C under agitation. Afterwards, the specimens were washed and then incubated in artificial saliva at 37° C under agitation for a total of 7 days. The amount of hydroxyproline released in supernatant was evaluated at 24 hours, 48 hours, and 7 days. Statistical analysis was performed using two-way and one-way ANOVA and Games Howell post hoc tests (p < 0.05). Results: All berberine concentrations were able to decrease both rMMP-2 and rMMP-9 activities. The higher concentration (0.02%) showed nearly complete inhibition of both recombinant enzymes. No statistical differences in the amount of collagen degradation were observed among the groups treated with berberine as compared to the control group treated with water.