Poly(I:C) Induced Zbtb32 Expression: Mechanisms And Signal Transduction Pathways

The innate immune system identifies pathogen-associated molecular patterns and endogenous damage-associated molecular patterns to activate the body’s extensive signaling pathways and to mediate immune system activation through the pattern recognition receptors.The double-stranded RNA is a kind of important molecular pattern.Exogenous ds RNA is widely found in the viral genome and it is produced during the virus replication process.Endogenous ds RNA is derived from damaged or necrotic cells,such as UV-injurious non-coding RNA,necrotic neutrophils and RNA released from RA patients’ articular fluid necrotic cells.These ds RNAs are recognized by TLR3,MDA5 and RIG-I.The pattern recognition receptors which have recognized ds RNA promote the expression of type I interferon to play antiviral function or activate NF-κ B signaling pathway to induce proinflammatory cytokine expression by activation the transcription factor IRF-3 and IRF-7.Zbtb32 belongs to the BTB-ZF family of proteins and is an evolutionary conserved transcriptional regulator.Studies have shown that Zbtb32 plays an important role in the differentiation and proliferation of immune cells.We first discovered that the polyinosinic-polycytidylic acid stimulates the expression of Zbtb32.To research the mechanism of Poly(I: C)/ Zbtb32 can help us to fully understand the mechanism of the immune system to respond to the immune response of the virus and provide the theoretical basis for the development of new antiviral strategy.But also conducive to a comprehensive understanding of the mechanism of recognition of receptor-mediated inflammatory response.Which is of great significance for the pathogenesis of inflammation-related disease and provide the theoretical basis for the corresponding drug development and clinical treatment.ObjectiveObjective We plan to investigate the mechanism and its related signal pathway of Poly(I: C) stimulating the expression of Zbtb32.We further explore the biological effect of Poly(I:C)stimulating FLS expression of Zbtb32.Methods(1)Enzyme digestion was used to isolate mice and human synovial fibroblasts(FLS)to explore the growth characteristics of FLS.(2)q PCR,WB was used to detect the the expression of Zbtb32 of FLS stimulated by Poly(I:C),and to explore the Poly(I: C)stimulation of FLS expression of Zbtb32 optimal stimulation conditions and mediated receptors.(3)Gene silencing and protein synthesis inhibitors were used to explore the receptors of the expression of Zbtb32 by FLS.(4)Signal molecule-specific inhibitors,agonists and small molecule inhibitors were used in combination with q PCR and WB to detect the key signaling molecules involved in the regulation of Poly(I: C)stimulation of FLS expressing Zbtb32.(5)To explore the biological effect of Poly(I: C)stimulating FLS expression of Zbtb32 by gene knockdown method.Results A sufficient amount of FLS can be obtained from human synovial tissue by protease pre-digestion combined with Ⅷ-type collagenase digestion;type IV collagenase digestion can obtain a large amount of FLS from mouse synovial membrane.The FLS culture process was observed.we found that FLS can be cultured in vitro for10 generations.The purity of FLS was obtained after three generations of continuous culture.In this study,FLS was used as experimental material.CCK-8 detects FLS growth cycles with a period of approximately 48 hours.LPS-stimulated TLR4 and Poly(I:C)-stimulated TLR3 mediate the expression of Zbtb32,but the LPS stimulus effect is less than Poly(I:C).The expression of Zbtb32 in human FLS was induced by Poly(I: C)in a time-and dose-dependent manner.Indicating that 25 μg / ml Poly(I: C)stimulated human FLS for 24 h while Zbtb32 gene expression reached the highest.TLR3 and MDA5 were involved in the expression of Zbtb32.he results showed that the key signal factors such as IRF-3,NF-KB,GSK3 and p38 MAPK were involved in the regulation of Poly(I: C)-induced Zbtb32 expression.The expression of Zbtb32 was down-regulated by si-Zbtb32.q PCR was used to detect changes in gene expression after zbtb32 silencing,The results showed that silencing Zbtb32 could decrease the expression of IFN-β gene,but promote the expression of anti-inflammatory factor IL-1Ra and the expression of CD55 surface molecule CD55.Conclusion Zbtb32 expression of FLS stimulated by Poly(I:C)was co-mediated by TLR3 and MDA5.IRF3,NF-KB,GSK3,p38 MAPK and other key signal molecules were involved in the regulation of Poly(I:C)stimulation of FLS expressing Zbtb32.Zbtb32 may play an important role in mediating FLS proinflammatory and antiviral processes.