Change In High Mobility Group-1 Protein Gene Expression And Its Potential Regulation Mechanism In Septic Rats

Objecttive: The changes and the distribution of HMG-1 mRNA in differenttissues were observed in a CLP (cecal ligation puncture) model, (a) to investigate thepotential role of HMG-1 in the development of systemic inflammatory response andsubsequent multiple organ dysfunction syndrome, (b) to identify the molecularregulation mechanism(s) underlying HMG-1 induction in sepsis, (c) to evaluate theeffect of treatment with HMG-1 inhibitor on survival rate in rats secondary toCLP-induced sepsis.Material and methods: l20 male Wistar rats were randomly divided into fourgroups as follows: (1) normal control group (n=10); (2) sham operation group (n=10);(3) CLP group (n=60): being further divided respectively into 2h, 6h, l2h, 24h, 48hand 72h subgroups post-CLP; (4) bactericidal/permeability increasing protein (BPI)treatment group (n=20): intravenous injection of rBPI₂1 at a dose of 1mg/kg at 0.5hand 4h after CLP, being further divided respectively into 12h, 24h post-CLPsubgroups; (5) sodium butyate (NaB) treatment group(n=20): intravenous injectionof sodium butytate at a dose of 500mg/kg at 0.5h and 4h after CLP, being furtherdivided respectively into l2h, 24h subgroups post-CLP At serial time points in eachgroup, animals were sacrificed, and blood and tissue samples from liver, lungs,kidneys and small intestine were harvested. Blood and tissue endotoxinconcentrations were measured by the chromogenic Limulus Amebocyte Lysate(LAL), which was modified by perchloric acid (PCA) pretreatment for samples, andboth blood and tissue TNF-α Levels were determined by enzyme-linkedimmunosorbent assay (ELISA). HMG-1,LBP, CD14 and TNF-α mRNAs weresemi-quanitated by the reverse transcription polymerase chain reaction (RT-PCR)taking GAPDH as an internal standard. The major organ functional indices, lungtissue myeloperoxidase (MPO) and small intestine diamine oxidase (DAO) were alsomeasured.In addition, additional exPeriments were pefformed to observe the effect oftreatment with sodiurn butyrate on survival rate. (l): intravenous injection of sodiumbW at a dose of 500mg/kg at 0.5h and 4h after CLP (2). Both sodium butyratetreatInent grouP (n=22) and CLP grouP (n=35) received fluid resuscitation andanibiotic treatIneni followng puncture. The mortality of rats in each group wasrecorded up to 7 days.Results: 1. HMG-l mRNA levels significamly increased in various tissuesduring 6-l2 h after CLP peakng at 24-72 h post-in jmp. HMG-l mRNA leve1s weresignificanly decreased at l2h and 24h in tissues by sodium butyrate (NaB) treatmentgrouP comPared to CLP colltTols. 2. Plasma endotoxin levels increased significanlyfollowing CLP with double peaks at 2-12h and 48-72h. MeanWhile, endotoxin levelsin the liver, lungs and kidneys markedly increased at 2h fOllowing CLP. and kepthigh values uP to 24h. Treatment with BPI could significanly reduced endotoxinlevels in liver, lungs and kidneys except plasma at l2h after injUry. Moreover,tfetheeni with NaB could decreased endotoxin levels in liver and kidneys, withoutmarked influence on pulonmary and plasma levels at l2h after injury. 3. LBP mRNAwas significanly expressed in liver, lungs and kidney as early as 2h post-CLP whichwere not recovered unil 72h. LBP mRNA levels in small intestine elevated only at2h and 72h after CLP Concomitantly, CDl4 mRNA levels in various tissuessignificanly increased with two peaks at 2-l2h and 48-72h. Treatment with rBPI2lcould inhibit LBP mRNA expression in the liver and lungs at both l2h and 24h, andinhibit CDl4 mRNA expression in all tissues at l2h and in intestine at 24hpost-injury TreatInent with NaB also couId significanly inhibit LBP mANAexpression in the liver and lungs at both l2h and 24h, as well as CDl4 mRNAexPression in all tissues at 12h bllt not at 24h. 4. TNF-a mRNA and proteinexpression was significanly augmented at 2h following CLP and kePt such trend tol2h. Eary treatInent with rBPI2l could inhibit tissue TNF-...