Study On Molecular Mechanism Of DsRNA Production And Signal Pathway Regulation In The Development And Progression Of Acute Lung Injury

Objective: Alu RNA is the main member of human SINE family. B type RNA is homologous to Alu in mouse SINE. Recently, more researches indicate that non coding RNA(nc RNA)is related to gene regulation and disease. However, Alu sequence accounts for about 10% of human genome. It has been thought as the “junk” sequence. Therefore, the production and function of Alu RNA has not been revealed in development and progression of diseases. Acute lung injury(ALI) is characterized as high morbidity and mortality. Recent researches show that pathogenic factors(such as lipopolysaccharide, LPS) would lead to “respiratory burst” followed oxidative stress(OS), which plays a critical role in the pathological development of ALI. Some researches indicate that OS inducing the production of Alu RNA. But the production and mechanism is not clear. Here, we established the ALI mouse model to explore the function and mechanism to provide new theory for the research of pathogenesis in ALI.Methods: In this study, we investigate the production of B type RNA in the inflammation response in ALI by establishing the LPS induced ALI mouse model by using molecular cloning technology, primary cell culture technique, immunofluorescence, flow cytometry, immunohistochemistry, Real-Time PCR, Western blot, ELISA, cell transfection and MTS in vivo and in vitro, to reveal the mechanism underlying B1 RNA inducing NLRP3 activation and inflammatory factors expression.Results:Part I: LPS induced the production of endogenous ds RNA, which including B type RNA, in mouse primary type II alveolar epithelial cells(AECs).Firstly, we established an OS model with H2O2 in A549 cells(human lung adenocarcinoma cell line). Dot-blot assay was applied to detect ds RNA in total RNA extracted from A549 cells, which was induced by H2O2. J2 antibody was used to recognize ds RNA specially. The result indicated that ds RNA level was significantly increased in H2O2 stimulated A549 cells. After sequencing and blasting, we found that the ds RNA was derived from different subtypes of Alu RNA. Then we detected the production of Alu RNA in A549 cells induced by LPS, which has been identified as an OS factor. On this basis, we isolated primary type II ACEs from BALB/c mice by magnetic beads and identified the purity of isolated cells. LPS was challenged in primary type II AECs to establish the cell model. After 24 hours, the cells were collected to acquire ds RNA by RNA Binding Protein Immunoprecipitation(RIP) assay. We constructed c DNA library of endocellular ds RNA by T-A-cloning technology. Gene sequencing was performed where B1 RNA as the dominant sequence was identified in the isolated ds RNA.Part II: B type RNA expression and related signal pathway identification in LPS induced mouse primary type II AECs.First, we detected B1, B2 and B4 RNA expression level in LPS-induced primary type II AECs by Real-Time PCR. The results indicated that B1 and B2 RNA were increased in LPS-induced primary type II AECs.Then, NLRP3 related inflammatory signal pathway activation was identified in LPS-induced primary type II AECs. We detected increased transcription of NLRP3, IL-1β, IL-18 and TNF-α using q RT-PCR. We also detected IL-1β, IL-18 and TNF-α secretion using ELISA assay in LPS-induced Type II AECs.After then, we revealed that B1 RNA initiated the NLRP3 pathway activation through NF-κB phosphorylation. Western blot and Real-Time PCR showed that after LPS challenge, not only NLRP3 and downstream cytokines were upregulated, NF-κB was also be activated by phosphorylation in LPS-induced primary type II AECs. To identify the relationship between NF-κB phosphorylation and ds RNA, confocal assay was used and result exhibited that ds RNA was elevated and mainly accumulated around and inside the nucleus, which was partly collocated with phosphorylated NF-κB. It suggested strongly that NF-κB was involved in the NLRP3 inflammatory pathway activation. PDTC(pyrolidine dithiocarbamate), a specific inhibitor of NF-κB phosphorylation, was employed in LPS-induced primary type II AECs to further analysis. The q RT-PCR and ELISA results indicated that PDTC suppressed the transcription of NLRP3, IL-1β, IL-18, TNF-α and the cytokines secretion. By genetic recombination and transfection of B1 RNA into primary type II AECs, we found that over expression of B1 RNA could increase the transcription and translation of NLRP3, IL-1β, IL-18 and TNF-α dramatically. However this was also could be reversed by PDTC treatment.At last, we measured type II AECs viability by MTS assay after B1 transfection. The results indicated that LPS treatment and B1 RNA transfection both reduced primary type II AECs viability, while the effects were downregulated by PDTC.Part III: The B type RNAs production and signaling pathway in LPS induced ALI mouse model.Mice were intratracheal injection with LPS to build model of ALI. Primary type II AECs from ALI mouse were isolated. The results were consistent with in vitro assay. q RT-PCR assay indicated B1 and B2 RNA were generated in type II AECs. Western blot assay showed that NF-κB phosphorylation and NLRP3 expression were upregulated in type II AECs from ALI mouse. ELISA assays proved the production of IL-1β, IL-18 and TNF-α were augmented in type II AECs. Immunohistochemistry was used to show NF-κB phosphorylation, NLRP3 expression, and ds RNA generation in macrophages and lung tissues AECs from LPS-induced ALI mouse.Conclusion: In this research, we preliminary confirmed that ds RNA appeared in A549 and mouse type II ACEs under the treatment of LPS. These ds RNA mainly origins from B1 RNA in mouse type II ACEs. These results provided evidences that the expression of NLRP3, IL-1β, IL-18 and TNF-α was upregulated by B1 RNA transfection and LPS treatment in mouse type II ACEs. Furthermore, the phosphorylated NF-κB detection confirmed that B1 RNA could promote expression of NLRP3 and inflammatory cytokines by NF-κB phosphorylation, which has been identified as an important role in the regulation of inflammation in LPS-induced mouse ALI. This study not only revealed the mechanisms on NLRP3 activation and inflammatory factor production under B1 RNA regulation, but also provided a new theory for ALI pathogenesis development.