Effects And Mechanisms Of Uncoupling Protein 2 In Lipopolysaccharide-induced Acute Lung Injury

1. Backgroud and Purposes:Acute lung injury(ALI), acute respiratory distress syndrome(ARDS) are common clinical critically ills. Despite long period of research on ALI/ARDS, there is still lack of effective treatment and mortality reachs to 30 to 40%. Th development of ALI / ARDS is the result of uncontrolled systemic inflammatory response. ALI/ARDS development is the result of uncontrolled inflammatory responses in the lungs, which involve neutrophil accumulation, diffuse endothelium and epithelial damage, air-blood barrier disruption, and the subsequent infiltration of peripheral inflammatory cells into lung tissues. This leads to the up-regulation of inflammatory cytokines that induce lung edema, which ultimately results in tissue injury and severe immunopathology. Mitochondria are considered an important factor in alveolar epithelial damage. The mitochondria are involved in numerous apoptosis signaling pathways, such as regulating reactive oxygen species(ROS) production, adenosine triphosphate(ATP) balance, stabilizing mitochondrial membrane potential, or controlling calcium homeostasis. Moreover, prior studies suggested that the intersection of mitochondrial biogenesis and inflammatory responses are important in disease. However, the interaction of mitochondrial dysfunction and inflammatory response and their roles in the pathogenesis of ALI is not clear.Uncoupling proteins(UCPs), members of the anionic proton transporter family, are located in the mitochondrial inner membrane, pumping protons from the inner membrane into the matrix to uncouple electron transport from ATP synthesis. There are 5 isoforms of UCPs, which named UCP1 ~ 5. Uncoupling protein 2(UCP2) contributes to the inflammation of heart, cerebrum and liver. Prior studies found that expression of UCP2 was increased in patients with systemic inflammation and infection, while the functional role of UCP2 in LPS-induced lung injury is still unclear.. In the present study, we explored the association between pulmonary inflammation and UCP2 expression.We constructed adenoviral vectors to overexpress UCP2 in mice lungs, while genipin was used to down-regulate the expression of UCP2 in mice lungs. In LPS-induced acute lung injury model in mice, Western Blot(WB), Immunohistochemistry were used to detected the expression of UCP2 in lungs. We assessed the pathological changes of the lung tissue, alveolar capillary permeability, levels of inflammatory factors, cell apoptosis, and the activation of MAPK and NF-κB pathways after ALI. This study provided experimental evidence for the pathogenesis of ALI/ARDS.2. Methods:(1) Construct UCP2 overexpression lentiviral vectorsWe isolated peripheral blood mononuclear cells to extract total RNA, UCP2 c DNA was amplification by RT-PCR. The DNA fragment was subcloned into the viral shuttle vector PYr-adshuttle-1 which were identified by sequencing DNA. The vector was viral packaging in HEK293 cells to obtain recombinant adenovirus UCP2-Ad. UCP2-Ad infected C57 BL / 6J mice through a nasal drip. The expression of UCP2 in mice lungs was measured by immunohistochemistry and Western Blot. Empty virus vector UCP2-NC was used as a negative control.(2) The role of UCP2 in inflammation after ALILipopolysaccharide(LPS;15 mg/kg) was intraperitoneal injected to induce ALI after adenoviral transfection. Genipin was pretreated before LPS treatment to inhibit UCP2 expression in lung tissue.The pathological changes in the mice lung tissue, pulmonary edema, inflammatory cytokines in bronchoalveolar lavage fluid(BALF) were assessed in 24 h after LPS treatment. The expression of cleaved caspase 3, cytochrome C and Bcl-2 family protein in lung tissue in mice were assessed by Western Blot. TUNEL was used to detect the cell apoptosis in lungs.(3) The role of UCP2 in MAPK and NF-κB pathway after ALITo observate the role of UCP2 in mitogen-activated protein kinase(MAPKs) pathway after acute lung injury, activation of Jun N-terminal kinase(JNK), extracellular signal-regulated kinase(ERK), p38 MAPK were assessed by Western Blot. P38 MAPK inhibitor SB203580, JNK inhibitor SP600125 or ERK inhibitor PD98059 were used before LPS treatment to comfirm UCP2 via which MAPKs pathway regulate ALI. Additionally, the the effect of UCP2-Ad and genipin in NF-κB pathway was assessed.To identify the mechanism of UCP2 in NF-κB regulation, the level of ATP and the activation of 5’-AMP-activated protein kinase(AMPK) were measured.3. Results(1) The recombinant adenovirus containing the mouse UCP2 gene(UCP2-Ad) was constructed and adenovirus expressing no transgene was used as negative control(UCP2-NC). The results of immunohistochemistry and Western Blot showed that UCP2 was overexpressed in in alveolar cells. UCP2-Ad(5 x 108 pfu/mouse) was used to overxpressed UCP2 and avoided lung injury. The increase of UCP2 expression was highest at 3 day.(2) UCP2 increased LPS-induced pathological changes, lung permeability(wet/dry weight ratio and protein concentration in bronchoalveolar lavage fluid(BALF), lung inflammation(neutrophils infiltration, tumor necrosis factor alpha and interleukin-1 beta in BALF), whereas survival rates were lowered. Furthermore, ATP levels and mitochondrial membrane potential were decreased, while reactive oxygen species production was increased. On the other hand, LPS-induced alveolar epithelial cell death and inflammation were attenuated by genipin.(3) In UCP2-overexpressed mice, MAPKs activity was elevated, which increased the sensitivity to LPS-induced apoptosis and inflammation. LPS-induced apoptosis and release of inflammatory factors was alleviated by pretreatment of the JNK inhibitor SP600125 or the p38 MAPK inhibitor SB203580, but not with the ERK inhibitor PD98059 in UCP2-overexpressing mice. Additionally, UCP2 increased the expression of NF-κB via ATP depletion induced activation of AMPK. Genipin attenuated activity of JNK, p38 MAPK and NF-κB, cell death and inflammation factors after LPS treatment.4. Conclusion(1) UCP2 up-regulation aggravated inflammation damage of ALI, while UCP2 down-regulation protected ALI;(2) UCP2 aggravated mitochondrial dysfunction in lung tissue after ALI, and promoted alveolar epithelial cell apoptosis;(3) UCP2 may regulated ALI via JNK, p38 MAPK and NF-κB signaling pathway.