Calpain and lipopolysaccharide mediated hepatitis
Rose, Robert Edward
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This study tested the role of the calcium dependent cytosolic protein calpain in neutrophilic hepatitis. We hypothesized that inhibition of calpain would protect against LPS-induced neutrophilic liver damage. To test our hypothesis, a reliable LPS-mediated hepatitis model to investigate the mechanisms of hepatic neutrophil infiltration following LPS administration was developed by repeat intravenous injection of LPS at a dose of 10 mg/kg to rats. Blood was collected for hematologic and biochemical analysis and multiple organs including liver were collected for evaluation of histopathologic changes. Flow cytometry was employed to investigate L-selectin (CD 62L) and MAC-1 (CD11b/18) expression on neutrophils both in vivo and in vitro. Significant hematologic changes included neutrophilia, elevation in neutrophil to lymphocyte ratio with toxic changes and left shift. Biochemical changes were observed in several liver (AST, GGT) and kidney (BUN) parameters generally at the earliest time points. Histopathology revealed a time-dependent neutrophil and mononuclear infiltration around the periportal areas in the single dose study and mid-zonal inflammation with multifocal coagulative necrosis in the repeated dose study. CD 11b was up-regulated both in vitro and in vivo. After development of a suitable model, the first goal was to investigate the role of the intracellular enzyme calpain in the development of neutrophilic hepatitis and midzonal necrosis. A second goal was to compare the observed protective effects of calpain inhibition with a relatively selective inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine and an inhibitor of coagulation, heparin. When compared to rats administered LPS alone, administration of calpain 1 inhibitor prior to LPS significantly reduced hepatic iNOS expression, hepatic neutrophil infiltration and attenuated midzonal hepatic necrosis. Administration of heparin and aminoguanidine prior to LPS also decreased liver iNOS expression, hepatic neutrophil infiltration and liver pathology comparable to calpain inhibition. Blood neutrophil activation, as measured by the neutrophil adhesion molecule CD11b integrin, was upregulated in all the LPS treated groups regardless of inhibitor administration. We conclude that amelioration of liver pathology via calpain inhibition is likely dependent on the down-regulation of iNOS expression in the rat model of LPS mediated hepatitis.