Studies On The Role Of Angiopoietin-like Protein 4 (ANGPTL4)in The LPS-induced ALI In Mice

1. Background and objectiveAcute lung injury(ALI) is a critical illness caused by excessive inflammation, with the manifestations of acute respiratory distress, non-cardiogenic pulmonary edema and hypoxemia. Despite airway management and protective ventilation strategies have been advanced, the mortality rate among ALI patients remains high due to a lack of understanding the mechanisms underlying ALI pathogenesis.Oxidative stress caused by neutrophils activation in the lungs is a critical event in the development of ALI pulmonary inflammation. Researches have demonstrated that lipopolysaccharide(LPS) not only oxidizes membrane lipid, but also produces oxygen free radicals through the activation of NADPH oxidase, therefore, clarify the mechanisms that LPS-induced acute lung injury is essential for the treatment of acute lung injury. Angiopoietin-like protein 4(ANGPTL4), which was also called as fasting-induced adipose factor(FIAF), participates in the liver and adipose tissue inflammation. Moreover, our unpublished data showed that LPS increased angptl4 expression in lung tissues. However, the role of Angptl4 in LPS-induced ALI is not clear. Based on the previous studies, the present study was to knockdown of Angptl4 expression in lung tissues with lentiviral vector Angsi RNA, and further clarified the role of Angptl4 in LPS-induced ALI. Another purpose was to knockdown of Angptl4 expression in A549 cells with plasmid, and further explored the underlying mechanisms for Angptl4 regulation of LPS-induced ALI and a new target for treatment of ALI/ARDS.2. Methods(1) Construction of ANGPTL4 RNAi lentiviral vector(Angsi RNA)The Angsi RNA lentiviral vector contains Angptl4 short interfering RNA fragments. It was generated by three steps. First, design si RNA targetment according to the mouse ANGPTL4 gene, and synthesis single stranded DNA oligo, and further anneal to produce double-stranded DNA. The second step was to transformation, that is, the GV118-lentiviral vector was transformation into Escherichia coli cells. The third step was to identify RNAi lentiviral vector whether contain Angptl4 short interfering RNA via polymerase chain reaction amplification and sequencing verification. Lentivirus was produced using 293 T packaging cells co-transfected with p Helper1.0, p Helper2.0 plasmid and Angsi RNA lentiviral vector. The titer of Angsi RNA was identified by means of 50% tissue culture infections dose. And then, Angsi RNA was administered into lung tissues to establish mouse model. The effects of Angsi RNA on interference efficiency and pathological changes in the lungs were determined.(2) Angptl4 knockdown alleviates LPS-induced ALI in miceC57/BL6 mice were anesthetized via ether inhalation and inoculated intranasally with 4x107 PFUs of Angsi RNA or NCsi RNA. Forty eight hours after the virus administration, a 15mg/kg dose of LPS was injected into the mice peritoneal cavities to establish ALI mouse model. The transfection efficiency of Angsi RNA in lung tissues was determined with the methods of quantitative real time PCR, Western blot and immunohistochemistry. Subsequently, the condition of lung inflammation was assessed by indicators that neutrophil infiltration and expression of inflammatory cytokines in lung tissues. Besides, the extent of vascular leakage was determined by wet to dry ratio in lung tissues and protein content in bronchoalveolar lavage fluid. Also, the extent of tissue damage was determined by the expression of cleaved-caspase3 in lung tissues. In addition, 7-days survival rate was used to determine the protective effects of Angsi RNA on LPS-induced ALI. Furthermore, the underlying mechanisms for Angptl4 regulation of LPS-induced ALI were investigated by measurement expression of SIRT1 and NF-k B, and content of MDA in lung tissues with methods of quantitative real time PCR, Western blot and MDA detection kits respectively.(3) The underlying mechanisms for Angptl4 regulation of LPS-induced ALI were investigated with the model of A549 cells in vitroThe expression pattern of Angptl4 in A549 cells after LPS challenged was investigated. Subsequently, the effects of Angptl4 on LPS-induced A549 inflammatory injury and SIRT1 expression were determined after Angptl4 expression was downregulated by Angsi RNA plasmid. Furthermore, to further determine the role of SIRT1 signaling pathway in Angptl4 regulation of LPS-induced ALI, we decreased both Angptl4 and SIRT1 expression in A549 cells with Angsi RNA plasmid and SIRT1 antagonist nicotinamide(NAM) to observe the inflammatory cytokines IL6 and NF-k B expression.3. Results(1) The lentivirus vector Angsi RNA that target ANGPTL4 gene in mice and the negative control virus(NCsi RNA) were constructed successfully. The titer of Angsi RNA and NCsi RNA were 1x109 PFU/m L and 2x109PFU/m L, respectively, which met the requirement of subsequent experiments. The quantitative real time PCR, Western blot and immunohistochemistry results have demonstrated that Angsi RNA administration can significantly decrease Angptl4 expression in lung tissues(P<0.05), and did not cause obviously pathological changes in lung tissues.(2) Angptl4 was expressed in alveolar epithelial cells in lung tissues. LPS significantly increased Angptl4 expression in lung tissues(P<0.05). Compared with LPS and NCsi RNA+LPS group, the extent of neutrophils infiltration, inflammatory cytokines TNFα and IL6 expression, lung edema, protein content in bronchoalveolar lavage fluid, MPO expression and cleaved-caspase3 expression in lung tissues were significantly decreased in Angsi RNA+LPS group(P<0.05). Furthermore, Angsi RNA can significantly increase the survival rate of LPS-induced ALI mice(P<0.05).(3) Compared with LPS and NCsi RNA+LPS treated mice, SIRT1 expression was significantly increased in Angsi RNA+LPS group(P<0.05), whereas, NF-k B expression and MDA content was significantly decreased in Angsi RNA+LPS treated mice(P<0.05).(4) LPS significantly increased Angptl4 expression in A549 cells, knockdown of Angptl4 can significantly decrease LPS-induced inflammatory cytokine TNFα and IL6 secretion and apoptosis rate in A549 cells(P<0.05). Compared with LPS and NCsi RNA+LPS group, the SIRT1 expression was significantly increased in the Angsi RNA+LPS treated A549 cells. To further demonstrate the role of SIRT1 signaling pathway in Angptl4’s modulation of LPS-induced ALI, we knockdown of Angptl4 and SIRT1 expression at the same time, the IL6 and NF-k B expression in Angsi RNA+LPS+NAM treated group was significantly increased compared to Angsi RNA+LPS-treated group(P<0.05).4. Conclusion(1) LPS increased Angptl4 expression in mouse lung tissues, knockdown of Angptl4 expression protects aganist LPS-induced ALI pulmonary inflammatory injury.(2) SIRT1 signaling pathway and lipid oxidation may be invovled in Angptl4’s modulation of LPS-induced ALI.