Inhibition of matrix metalloproteinases by chemically modified tetracyclines in sepsis.

Sepsis precipitates a systemic inflammatory stimulus that causes systemic release of cytokines and sequestration of polymorphonuclear neutrophils, resulting in degranulation of matrix metalloproteinases (MMPs), which causes extracellular matrix basement membrane degradation. One of the important anti-inflammatory properties of tetracyclines is their ability to inhibit MMPs. In this study, we focused on the regulation of MMPs in sepsis and their reduction by treatment with nonantimicrobial chemically modified tetracyclines (CMTs), which retain their anti-inflammatory activity. Sepsis was induced by cecal ligation and puncture (CLP) method. At 24 h and 1 h before CLP, some rats received CMT-3 (25 mg/kg), another group of rats received hydroxamate (H; an inhibitor of MMP; 25 mg/kg), and untreated rats received saline by gavage. At 0 h, 0.5 h, 1.5 h, and 24 h after CLP, blood and liver samples were collected. Plasma and liver MMP-9 by zymography and Western immunoblotting, plasma nitric oxide by measuring nitrate level, plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) by enzymatic method, and liver gelatinase by radiolabeled gelatin lysis assay and 24 h mortality were determined. Plasma MMP-9 (92 kDa), nitrate, and GOT and GPT levels were elevated compared with the time 0 level and reached peak at 1.5 h CLP and remained high for 24 h. Both CMT-3 and H treatment reduced GOT,GPT, 92-kDa gelatinase, and nitrate levels throughout the 24 h. CMT-3 and H are equally effective in sepsis treatment. The 24-h mortality for CLP rats was 30%, whereas pretreatment with CMT-3 and H resulted in 0% mortality. Hepatic MMP-9 and gelatinase activity increased significantly after CLP, and pretreatment with CMT-3 and H inhibited these expressions. These results indicate the beneficial effect of CMT-3 in preventing the increase in GOT, GPT, NO, MMP-9, gelatinase activity, and the ensuing septic shock.

Modulations of signal transduction pathways during sepsis and the effects of insulin and mifepristone.

OBJECTIVES: The phosphorylation states and the inferred activation of specific members of the mitogen-activated protein kinase (MAPK) pathways (p42/44, p38, and SAPK/JNK) were quantitated in the livers of rats by Western blot analysis during the progression of sepsis. In addition, the authors examined the effects of insulin and mifepristone (RU-486) administration on these signal transduction pathways during sepsis. METHODS: Sepsis was brought about by the cecal ligation and puncture (CLP) method. The control group underwent sham operation. One experimental group was injected with insulin (0.5 U/kg, IV); prior to the CLP or sham procedure, the second group was injected with RU-486, a glucocorticoid antagonist (mifepristone, 20 mg/kg, IP), and the third group was injected with saline (control). RESULTS: The activities of SAPK/JNK were unaltered throughout the course of sepsis, while those of p42/44 MAPK increased four-fold after 24 hours of sepsis. The activation of p38 MAPK was also increased after 0.5 and 1.5 hours of sepsis, but returned to normal during late sepsis (24 hours). Rats that had received RU-486 before CLP showed no change in p42/44 MAPK compared with the untreated group, but the drug appeared to suppress the activation after 24 hours post-CLP. SAPK/JNK activation, on the other hand, was slightly enhanced by RU-486 pretreatment after 1.5 and 24 hours of CLP compared with the untreated CLP and the treated sham-operated rats. Insulin, given preoperatively, had no effect on the activation of SAPK/JNK, although p42/44 MAPK pathways were activated by the hormone at 0.5 hours and 1.5 hours post-CLP only, but not in the sham-operated controls or at 24 hours post-CLP. The p38 MAPK activation seen after 0.5 and 1.5 hours of CLP was suppressed by RU-486. Insulin pretreatment appears to suppress the phosphorylation of p38 in both the CLP and sham-operated groups. CONCLUSIONS: The data suggest that sepsis induces two phases of MAPK activation, an early phase in which p38 is active, followed by a second, more pronounced phase of p42/44 MAPK activation that occurs only late in sepsis. Hormonal administration results suggest that RU-486 suppresses the activation of p38 seen in early sepsis, and the activation of p42/44 MAPK seen in late sepsis.