HMGB1 Effects On Changes In Apoptosis Of PMN In LPS-induced Acute Lung Injury

The pathogenesis of endotoxin-induced acute lung injury (ALI) remains obscure and hasn't been fully elucidated hitherto. It is generally accepted that ALI in essence is an excessive, uncontrolled inflammatory response within the lung. Polymorphonuclear granulocytes (PMN) are important inflammatory cells implicated in the excessive inflammatory response. Studies in recent years show that there exists a delay in apoptosis of PMN in lung tissue during acute lung injury. Delay in apoptosis may lead to prolonged release of PMN products and direct tissue injury, therefore participate in the development of ALI.High mobility group box protein 1(HMGB1) was previously described as a DNA-binding protein involved in DNA replication, repair, and transcription; and as a membrane-associated protein that mediates neurite outgrowth. It is newly discovered that HMGBl may act as a late mediator of endotoxemia. When released by activated monocytes, it participates in the development of lethality and activates downstream cytokine release. As a proinflammatory cytokine, HMGB1 is lethal to otherwise healthy animals. Elevated levels of HMGBl were observed in the serum of patients with sepsis, and the highest levels were found in those patients that died. The delayed kinetics of HMGB1 release indicates that it may be useful to target this toxic cytokine in the development of future therapies. Much evidence demonstrates that HMGB1 has been linked to delayed endotoxin lethality. However, the role of HMGB1 in lipopolysaccharide (LPS)-induced delay and inhibition of PMN apoptosis continues to evolve. It is poorly described that whether HMGB1 contributes to apoptotic changes during ALI. Furthermore, the interaction between HMGB1 expression and PMN apoptosis, and HMGB1's ability to regulate apoptosis remains largely undefined. Thus, we developed the LPS-induced acute lung injury model in rats to investigate the following: (1) the apoptotic changes of PMN in peripheral blood and bronchoalveolar lavage fluid (BALF); (2) lung tissue HMGB1 mRNA expression level at different time point was examinated by reverse transcription-polymerase chain reaction (RT-PCR); (3)phogocytosis changes of alveolar macrophage (AM) after LPS challenge; (4) the relationship between lung tissue HMGBl mRNA expression and percentage of PMN apoptosis in peripheral blood and in BALF; (5) the relationship between AM phocytosis changes and lung tissue HMGBl mRNA expression; (6) effects of Sodium butyrate(SB), a HMGBl inhibitor, on apoptotic changes in PMN, phocytosis changes in AM, and lung tissue HMGBl mRNA expression. After that, we conducted a special cell culture system to clarify effects of HMGBl on PMN apoptosis. Human circulating PMN freshly isolated from peripheral blood of human healthy donors were coincubated with different doses of HMGB 1 (10, 100, 1000 ng/ml, respectively) at 37°C in 5% CO2. LPS (lOug/ml) and RMPI-1640 were used as positive and negative control respectively. After 24h of culture, PMN were collected and treated appropriately. PMN apoptosis was examined by FCM and TUNEL. The aim of this study is to provide some laboratory evidence and a potential theoretical basis at the point of HMGBl regulation on PMN apoptosis in order to further elucidate the molecular mechanism for excessive, uncontrolled inflammatory response. Results:1. Results from morphologic examination indicated that the edema severity and pathological changes of lung tissues were excessively aggravated in rats after LPS administration. By comparison, SB treatment diminished the severity of lung damage. Combined with lung HMGBl expression level, the above changes indicate that the pathological changes of lung tissue were related to the injuried lung HMGBl expression, as well as apoptotic changes in PMN.2. The percentage of apoptosis of PMN in rat model of LPS-induced ALI was gradually decreasing as compared with that of normal control (43.67%+/-3.82% at 2h time point vs 64.82%+/-9.15% in peripheral blood; 53.24%+/-2.67% at 2h time point and 15.65+/-9.42% at 6h time point vs 64.82+/-9.15% in BALF) within 6h after LPS infusion. The PMN apoptosis-initiation time and non-survival time in rat BALF prolonged as compared with that of normal control [(18.4+/-3.5) h vs (11.5+/-2.9) h, and (63.3+/-2.1) h vs (47.2+/-8.2) h, P<0.0\]. Meanwhile, compared with that of normal control, the injuried rat lung tissue HMGBl mRNA expression was upregulated after LPS exposure (0.521+/-0.107 at 6h time point and 0.648+/-0.120 at 24h time point vs 0.227+/-0.045, P<0.05 and <0.01, respectively).3. At 6h, 12h, 24h time point, as compared with that of normal control (86.5+/-9.69)%, the injuried rat AM phagocytosis ratio decreased to (18.94+/-2.01)%, (12.64+/-2.0l)% and (12.52+/-2.41)%, respectively; phagocytosis index after LPS challenge was 1.39+/-0.15, 1.34+/-0.02 and 1.30+/-0.41 vs con 2.45+0.39, respectively. The relative analysis showed a significant negative correlative relationship between AM phagocytosis changes and HMGB1 expression in rat lung tissue.4. Human circulating PMN were coincubated for 24 hours with different doses of HMGB1 (0, 10, 100,1000 ng/ml, respectively) at 37°C in 5%CO2. By appling FCM, PMN apoptosis ratio was (40.53+/- 4.12)%, (40.52+/- 2.73)%, (34.89+/- 1.15)%, and (18.77+/-3.02)%, respectively. Meanwhile, PMN TUNEL positive rate was (31.42+/- 4.40)%, (31.39+/- 3.80)%, (25.62+/- 2.46)%, and(l7.98+/- 3.20)%, respectively.Conclusions:1. We successfully conducted a LPS-induced acute lung injury model in rats.2. The study provides a new evidence that there exists apoptosis inhibition and delay of PMN in rats during LPS-induced acute lung injury. After LPS challenge, rats lung HMGB1 mRNA high expression occurs at a later phase, but keeps for a long time. Sodium butyrate (SB) treatment attenuated LPS-induced PMN apoptosis delay and inhibition, and downreguiated injuried lung HMGB1 mRNA expression. The results demonstrate that HMGB1 may contribute to the development of PMN apoptotic changes during LPS-induced acute lung injury.3. LPS challenge weakens the injuried rat AM phagocytosis significantly. SB treatment ameliorates LPS-induced rat AM phagocytosis changes. The relative analysis showed a significant negative correlative relationship between AM phagocytosis changes and HMGB1 expression in rat lung tissue. The result shows high expression of HMGB1 may be implied in AM phagocytosis changes.4. HMGB1 in vitro inhibited human circulating PMN apoptosis markedly. The inhibitory effect was HMGB1 dose-depended.In conclusion, the present research demonstrates that HMGB1 may play an important role as a modulator in apoptotic changes in PMN during LPS-induced ALL The contribution of HMGB 1 to apoptotic changes may be associated with LPS-induced AM phagocytosis changes. The modulatotion of HMGB1 may be one of the importantmechanisms for lipopolysaccharide-induced acute lung injury. HMGB1 targeted treatment may bring new light to the prevention and treatment of LPS-induced acute lung injury.