Protective Effects And Mechanism Of Ethyl Pyruvate On Acute Lung Injury Induced By Sepsis

PartⅠProtective Effects of Ethyl Pyruvate on Acute Lung Injury Rats induced by SepsisObjective To investigate the effects of ethyl pyruvate on TNF-α,IL-1βand inducible NO synthase (iNOS) expression , and the mechanism of ethyl pyruvate protect against acute lung injury induced by sepsis. Methods In a model of cecal ligation and puncture (CLP), 40 Wistar rats were randomly divided into normal control,cham operation ,acute lung injury ,and ethyl pyruvate treatment (40 mg/kg intra-peritoneally every 6 hrs) groups.2h later serum were collected to determine TNF-αand IL-1βby enzyme-linked immunosorbent assay (ELISA).At the time points 24 hours animals in each group were sacrificed, the serum and lung tissues were taken. The iNOS mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and serum NO production was quantified by spectrophotometric. PaO2,PaCO2 and pH were measured. Histological examination of the lungs was also performed. Results Ethyl pyruvate significantly inhibited TNF-α,IL-1βand iNOS mRNA expression(P<0.01) . Ethyl pyruvate significantly decreased Serum NO level(P<0.01). PaO2 and pH were significantly higher,PaCO2 was significantly lower compared with acute lung injury group (P<0.01).The histological changes of lung injury were ameliorated. Conclusions This study showed that ethyl pyruvate administered inhibit of TNF-α,IL-1βand NO derived from iNOS,and provided protective effects on the lungs against acute injury induced by sepsis. PartⅡEffects of Ethyl Pyruvate on High Mobility Group Box1 in acute lung injury induced by sepsisObjective To investigate the effects of ethyl pyruvate on High Mobility Group Box1(HMGB1) expression and the possible mechanism of ethyl pyruvate protect against acute lung injury induced by sepsis. Methods In a sepsis model by cecal ligation and puncture, 40 Wistar rats were randomly divided into normal controls,cham operation ,acute lung injury ,and ethyl pyruvate treatment (40 mg/kg intra-peritoneally every 6 hrs) groups.At the time points 24 hours animals in each group were sacrificed, the serum and lung tissues were harvested. The expression of HMGB1 mRNA was detected by RT-PCR. HMGB1 in lungs were measured by immunocytochemical staining and Western blot analysis. Wet/dry lung weight ratio, the protein in the bronchoalveolar lavage fluid,and pulmonary permeability index(PPI) were determined.The histological appearance of lung was observed under microscope. Results Ethyl pyruvate significantly inhibited the HMGB1 mRNA expression(P<0.01)and HMGB1 protein level(P<0.01). Ethyl pyruvate treatment decreased wet/dry lung weight ratio, the protein in the bronchoalveolar lavage fluid,and PPI(P<0.01). The histological appearance of lung injury was ameliorated. HMGB1 mRNA expression and HMGB1 protein level in lungs positively correlation with wet/dry lung weight ratio, the protein in the bronchoalveolar lavage fluid,and PPI respectively(P<0.01).Conclusions These observations suggest ethyl pyruvate administered inhibit of HMGB1,and provided protective effects on the lungs against acute injury induced by sepsis.PartⅢEthyl pyruvate attenuates Janus kinase2-signal transducer and activator of transcription3 pathway in lungs of septic ratsObjective To investigate the hypothesis that ethyl pyruvate is effective in mediating JAK2-STAT3 signaling pathways and protecting against acute lung injury in a rat model of sepsis.Methods In a model of cecal ligation and puncture (CLP), 48 Wistar rats were randomly divided into normal controls,cham operation ,acute lung injury , ethyl Pyruvate treatment (40 mg/kg intra-peritoneally every 6 hrs), AG490 treatment, and rapamycin treatment groups (n=8 in each group). All animals were killed at 24h, pulmonary tissues and bronchoalveolar lavage fluid(BALF) were harvested. Lung tissues were examined by immunohistochemical to identify phospho-STAT3 alterations in intracellular signaling pathways. Specific protein expression in JAK2 , phospho-JAK2, STAT3,and phospho-STAT3 were examined by Western blot analyses. White blood cell count in BALF, Wet/dry lung weight ratio,and pulmonary myeloperoxidase(MPO) activity were determined. Histopathological assessment of lungs was performed. Results Immunohistochemical examination demonstrated a marked increase in immunoreactivity for phospho-STAT3 in the lungs of septic rats. In contrast, treatment with ethyl pyruvate, AG490,and rapamycin markedly reduced the immunoreactivity for phospho-STAT3 . Septic rats lung tissue phospho-JAK2 and phospho-STAT3 protein was upregulated compared to normal controls(P<0.05). The treatment of rats with ethyl pyruvate decreased phospho-JAK2 protein expression(P<0.05). The treatment of rats with ethyl pyruvate ,AG490,and rapamycin attenuated the sepsis-induced phospho-STAT3 protein expression(P<0.05). Ethyl pyruvate treatment decreased white blood cell count in BALF, Wet/dry lung weight ratio,and pulmonary MPO activity. Ethyl pyruvate,AG490,and rapamycin treatment reduced the degree of histopathological injury compared with the sepsis group .Conclusions These findings suggest that activation of the JAK2-STAT3 signaling pathway is a significant contributing factor to the pathogenesis of acute lung injury induced by sepsis and that treatment with ethyl pyruvate interference in activation of the pathway potentiates recovery. Ethyl pyruvate is beneficial in the treatment of acute lung injury induced by sepsis like specific inhibitor.PartⅣThe Role of ethyl pyruvate in the Endotoxin-induced Alveolar MacrophagesObjective To investigate the Role of ethyl pyruvate in the Alveolar Macrophages induced by lipopolysaccharide (LPS), and the cytological mechanism of ethyl pyruvate protect against acute lung injury. Methods Alveolar Macrophages harvested from rats were exposured to ethyl pyruvate(5mmol/L) and LPS(100 ng/ml), the expression of TNF-αmRNA was detected by RT-PCR 2h later, levels of HMGB1 in culture medium were examined by Western blot analyses 24h later, Specific protein expression in JAK2 , phospho-JAK2, STAT3,and phospho-STAT3 were examined by Western blot analyses, protein levels of phospho-STAT3 were evaluated by immunocytochemistry. Results Compared with those of the control group, LPS alone significantly increased the expression of TNF-αmRNA (P<0.01) ,HMGB1 (P<0.01) ,phospho-JAK2 (P<0.05), and phospho-STAT3 (P<0.05).The treatment of ethyl pyruvate significantly reduced LPS-induced positive cell numbers of phospho-STAT3. Ethyl pyruvate significantly inhibited the expression of TNF-αmRNA (P<0.01), HMGB1(P<0.01) ,phospho-JAK2(P<0.05),and phospho-STAT3(P<0.05). Conclusions In Alveolar Macrophages ,ethyl pyruvate could suppress TNF-αand HMGB1 expression. Ethyl pyruvate specifically inhibited the activation of JAK2-STAT3 signaling pathways,and the signaling pathways are relation to HMGB1 release. This results describes the cytological mechanism of ethyl pyruvate protect against acute lung injury.