HMGB1 neutralizing antibody attenuates hemorrhagic shock-induced cardiac injury and modulates apoptotic gene expression in murine heart

Abstract

To explore the possible protective effects and mechanisms of neutralizing antibody of HMGB1 on hemorrhagic shock-induced cardiac injury in mice. The KM mice of hemorrhagic shock model were divided into sham (sham), control+IgG, hemorrhagic shock (HS) and HMGB1 neutralizing antibody treatment (HS+α-HMGB1) groups (n = 8 each). After modeling, the animals were anesthetized and blood samples collected. The concentrations of creatine kinase (CK) and lactate dehydrogenase (LDH) were measured. And the serum levels of such inflammatory factors as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and HMGB1 were analyzed by enzyme-linked immunosorbent assay (ELISA). The cardiac tissues were harvested, fixed; hemotoxylin-eosin stained and observed under transmission electron microscopy. The histopathological cardiac changes were examined after hemorrhagic shock and resuscitation (HS/R). Immunohistochemical staining was performed to detect the cardiac expressions of HMGB1 and Fas/FasL after HS/R. Reverse transcription polymerase chain reaction (PCR) was performed to analyze the RNA levels of Bax, Bcl-2 and Caspase-3 in cardiac tissues. And the protein levels of HMGB1, Bax, Bcl-2 and Caspase-3 in cardiac tissues were tested by Western blot. Hemorrhagic shock and resuscitation resulted in significantly cardiac cell damages, enhanced inflammatory factors in sera and up-regulated the expressions of pro-apoptotic genes and proteins in murine hearts. The lowered protein level of Bcl-2 induced by HS/R was reversed by neutralizing HMGB1 antibody treatment. Neutralizing HMGB1 antibody administration obviously attenuated HS/R-induced cardiac damages and apoptosis (HS +α-HMGB1 group vs HS group, 4.5 ± 1.3 vs 8.9 ± 1.9), inhibited inflammatory responses (HS+α-HMGB1 vs HS, IL-1β: 127.7 ± 21.2 vs 164.3 ± 30.2; IL-6: 226.7 ± 33.4 vs 402.5 ± 59.5; TNF-α:100.6 ± 10.7 vs 146.5 ± 15.4), and modulated apoptosis-associated genes (HS+α-HMGB1 group vs HS group, Bcl-2: 0.25 ± 0.02 vs 0.19 ± 0.02; Bax: 0.38 ± 0.04 vs 0.50 ± 0.03; Caspase-3: 0.38 ± 0.04 vs 0.56 ± 0.04) and proteins expression (HS+α-HMGB1 group vs HS group, Bcl-2: 0.47 ± 0.04 vs 0.3 ± 0.03; Bax: 0.11 ± 0.02 vs 0.17 ± 0.02; Caspase-3: 0.62 ± 0.04 vs 0.8 ± 0.04) in murine hearts after HS/R. Neutralizing HMGB1 antibody may protect mice from HS/R-induced cardiac damages and apoptosis. Such an effect is probably due to its anti-inflammatory responses and anti-apoptosis related gene expression.