Regulatory Role And Mechanisms Of NLRC5in The Cytokine Secretion Of Macrophages

The liver is an significant organ of our body, responsible for metabolism and biotransformation, rich in blood supply, which vulnerable to a variety of pathogenic factors inside and outside the body, causing inflammation and damage. Liver damage is generated by various liver disease lesions and the mechanism of liver damage rather complicated. With development of further research on the pathogenesis of liver injury, the close link between inflammation and liver damage is causing increasingly attention. Inflammation involves the interaction of a variety of inflammatory cytokines, many studies reported inflammatory cytokines can promote the development of liver injury and suppress the anti-inflammatory immune responses, its level in serum has an obvious correlation with the degree of liver inflammation and hepatic damage. The innate immunity, within which plays significant role in liver injury and inflammation, works as the first defense against pathogen invasion. Moderate innate immune can increase response against pathogenic microorganisms, but excessive innate immune response often leads to release of a large number of inflammatory cytokines, causing some chronic inflammatory diseases including inflammation related liver damage and even tumors. Thus, it is of great theoretical and clinical significance to better understand the molecular mechanism of negative regulation of the innate immunity.In the recent years, studies have found that NLRC5(NLR family, CARD domain containing5), as one of the members of the NOD family, can effectively inhibit the innate immunity and inflammatory, and may be an effective molecular targets for immune related disease. JAK2/STAT3pathway is mediated by a variety of cytokine, and involved in the regulation of cell apoptosis, differentiation and immune responses of physiological processes. Our work observed the expression changes of NLRC5in acute liver injury, as well as further discusses the mechanism of cytokine secretion during RAW264.7activation.This experiment adopts the intraperitoneal injection of CC14to construct the acute liver injury model. Normal group were injected with the same doses of peanut oil solution. After24hours, the pathological and the expression of NLRC5in mice peritoneal macrophage was measured. The expression NLRC5was detected by RT-PCR in RAW264.7stimulated with different dose and time of LPS. To explore the effects of expression change of NLRC5on cytokine secretion in LPS-induced RAW264.7by transfecting with the pEGFP-C2-NLRC5and NLRC5siRNA into RAW264.7. Pretreatment with the JAK inhibitor AG490(10μM) before infection of LPS(200ng/ml) in RAW264.7cells for12h. Furthermore, the effects of AG490on the LPS-induced RAW264.7were investigated by RT-PCR and ELISA. Transfecting with the JAK2siRNA into RAW264.7using LipofectamineTM2000to observe the change of NLRC5and cytokine in LPS treatment of RAW264.7.In our study, we founded that NLRC5was upregulated in peritoneal macrophage of acute mice liver injury in response to CCL4stimulation. Moreover, the results indicated that the NLRC5showed a trend of decline after rising in RAW264.7in response to LPS stimulation (0,100ng/ml,200ng/ml,1ug/ml and2ug/ml), and peaked at10h after LPS (200ng/ml) treatment. In addition, RAW264.7transfected with pEGFP-C2-NLRC5significantly suppressed LPS-induced TNF-α and IL-6release compared with the LPS group. Instead transfection of NLRC5siRNA into RAW264.7can obviously increase expression of IL-6and TNF-α treated by LPS. One study shows that NLRC5may be a potential target of JAK2/STAT3pathway. In cells subjected to10μM of AG490revealed that AG490can obviously downregulated NLRC5expression and increase TNF-α, IL-6expression stimulated with LPS. Meanwhile composition proteins p-JAK2 and p-STAT3, decreased too. Suggesting that JAK2/STAT3can regulate the expression of NLRC5. Similarly, transfection JAK2siRNA into RAW264.7can produce a significant decrease in the expression of NLRC5, but a increase in the secretion of IL-6and TNF-a expression treated with LPS. Analogously, the results of Western blots revealed that when the cells were treated with JAK2siRNA, a significant decrease in p-JAK2and p-STAT3was observed. These studies demonstrated that NLRC5negatively mediates cytokine secretion in RAW264.7and modulated by the JAK2/STAT3pathway.