Anti-inflammatory and Anti-apoptotic Effect of Valproic Acid and Doxycycline Independent from MMP Inhibition in Early Radiation Damage

Abstract

Background: Matrix metalloproteinase (MMP) inhibitors decrease inflammation in normal tissues and suppress cancer progress in normal tissues. Valproic acid (VA) and doxycycline (DX) are MMP inhibitors that have radio-protective effects. Their ability to inhibit MMPs in irradiated tissue is unknown and the role of MMPs in radio-protective effects has not been tested to date. Aims: The purpose of this study was to examine whether administration of VA and DX to rats before irradiation affects tissue inflammation and apoptosis in the early phase of radiation, and whether the effect of these drugs is mediated by MMP inhibition. Study Design: Animal experimentation. Methods: Twenty-six Wistar rats were randomized into four groups: control (CTRL), radiation (RT), VA plus radiation (VA+RT), and DX plus radiation (DX+RT). Three study groups were exposed to a single dose of abdominal 10 Gy gamma radiation; the CTRL group received no radiation. Single doses of VA 300 mg/kg and DX 100 mg/kg were administered to each rat before radiation and all rats were sacrificed 8 hours after irradiation, at which point small intestine tissue samples were taken for analyses. Levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and matrix metalloproteinases (MMP-2 and MMP 9) were measured by ELISA, MMP activities were measured by gelatin and casein zymography and apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Results: VA decreased the levels of TNF-α and IL-1β proteins insignificantly and decreased apoptosis significantly in the irradiated tissue, but did not inhibit MMPs. In contrast, VA protected the basal MMP activities, which decreased in response to irradiation. No effect of DX was observed on the levels of inflammatory cytokines or activities of MMPs in the early phases of radiation apoptosis. Conclusion: Our findings indicated that VA protects against inflammation and apoptosis, and DX exhibits anti-apoptotic effects in early radiation and these effects are independent from MMP inhibition.

An Improved Collagen Zymography Approach for Evaluating the Collagenases MMP-1, MMP-8, and MMP-13

“…We detected 2 distinct MMP-13 bands of 59 and 46 kDa (Figure 2E), which correspond to proMMP-13 (60 kDa) and active MMP-13 (48 kDa) (19). Previously, we reported that despite using rh-proMMP-2 and rh-proMMP-7 for gelatin and casein zymography, active forms of the recombinant proenzymes could be also observed (20). These results indicate that the commercial rh-proMMPs probably contain low amounts of other forms of MMPs that could be detected by highly sensitive zymographic techniques.…”