Mechanism Of TLR3 Induces IL-1Ra Expression And Screening Of Traditional Chinese Medicine Monomers That Enhance IL-1Ra Expression

Toll-like receptors?TLRs?recognize pathogen-associated molecular patterns?PAMPs?derived from microorganisms and damage-associated molecular patterns?DAMPs?,mediates the inflammatory response and initiates the body's immune response.TLR3 is a member of the TLRs family of pattern recognition receptors?PRRs?of the innate immune system,recognizing double stranded RNA?dsRNA?to promote an inflammatory response to exert an antiviral effect.However,a large number of recent studies suggest that the biological function of TLR3 is not limited to promoting inflammation and anti-virus,but also can initiate anti-inflammatory signaling pathways and participate in the precise regulation of inflammation.Our previous studies in this paper found that TLR3 ligand Poly?I:C?stimulates Fibroblast-like Synoviocytes?FLS?to secrete Interleukin-1 receptor antagonist protein?IL-1Ra?.IL-1Ra competes with the inflammatory cytokine IL-1 for binding to IL-1R,blocking IL-1 signaling and exerting an anti-inflammatory effect.This paper will delve into the signaling pathways that TLR3 mediates IL-1Ra production and provide a new perspective for understanding the biological functions of TLR3.ObjectiveTo establish an in vitro cell model of TLRs ligand-stimulated cell expression and secretion of IL-1Ra,provide a basis for subsequent research;explore the intracellular signaling pathway of TLR3/IL-1Ra,and hope to find independent regulation of antiviral,anti-inflammatory,pro-inflammatory,the signal molecule of the effect;screening for compounds that regulate the TLR3/IL-1Ra signaling molecule,and provide experimental evidence for comprehensive understanding of the biological function of TLR3 and rational formulation of intervention strategies for inflammatory diseases.MethodsPart ?:Poly?I:C?induced IL-1Ra expression and secretion in human FLS cells1.ELISA was used to detect the secretion of IL-1Ra after TLR1-TLR6 ligand stimulation of human FLS cells for 24 hr,and the dose effect of IL-1Ra secretion by Poly?I:C?and LPS under 0-100?g.2.ELISA was used to detect the secretion of IL-1Ra after stimulation of TLR3 ligand Poly?I:C?for 24 hr in servaral cells3.ELISA and qPCR were used to detect TLR3 ligand Poly?I:C?to stimulate IL-1Ra production in human FLS,and to determining Poly?I:C?stimulation's best time in 0-96hr to detect IL-1Ra optimal gene and protein levels.as well as the optimal stimulation concentration of Poly?I:C?at 0-100?g.Intracellular IL-1Ra was detected by WB detection of Poly?I:C?stimulation of human FLS cells for 24 hr.Part ?:The molecular mechanism of Poly?I:C?-induced IL-1Ra expression1.Using gene silencing siRNA knockdown TLR3,MDA5,RIG-I,ELISA to detect the effect of knockdown TLR3,MDA5,RIG-I on IL-1Ra induced by Poly?I:C?stimulation of human FLS.ELISA assay of Poly?I:C?stimulated IL-1Ra production in FLS of TLR3-/-mice.It was determined whether Poly?I:C?induced FLS secretion of IL-1Ra by TLR3 activation.2.ELISA was used to detect the secretion level of IL-1Ra in Poly?I:C?,IFN-??10 ng/ml?with or without anti-IFN-?mAb?10?g/ml?;ELISA detection of cycloheximide intervention Poly(I:C The protein levels of IFN-?,IL-1Ra and IL-6 in human FLS cells were stimulated,and the relative levels of IL-1Ra mRNA expression were detected by qPCR.3.ELISA was used to detect the effect of BX795 or siRNA IRF3 on the secretion of IFN-?and IL-1Ra induced by Poly?I:C?in human FLS cells.Poly?I:C?stimulated the production of IFN-?by FLF in IRF3-/-mice.IL-1Ra protein levels;and protein levels of IL-1Ra following blocking of IRF3 signaling in other cells.4.Western blotting,ELISA detection of NF-?B,PI3K-Akt,GSK3?,ERK-MSK,p38-MAPK signaling blocked by small molecule compounds or siRNA technology.Poly?I:C?stimulates IL in human FLS cells.-1Ra,IFN-?,IL-6 secretion effects.CCK-8detects the viability of the cells.5.ELISA was used to detect wild-type,TLR3-/-and IRF3-/-mice were injected with Poly?I:C?,and the protein level of serum IL-1Ra.Part ?:Screening of traditional Chinese medicine monomers that enhance IL-1Ra expression and Immunosuppressive activity of benzoxazole derivative PO-2961.ELISA was used to detect the effects of 20 anti-inflammatory Chinese medicine monomers and benzoxazole derivatives PO-296 on the secretion of IL-1Ra and IL-6induced by Poly?I:C?in human FLS cells.2.Flow cytometry was used to detect the inhibition of PO-296 on activated T cell proliferation by IC?50,apoptosis,cytotoxicity,CD25 or CD69 expression level and cell cycle.The effect of PO-296 on the secretion of cytokines IL-2,IL-4,IL-6,IL-10,IL-17A and IFN-?in activated T cells was detected by ELISA.Western blotting was used to detect the effects of JAK3-STAT5,p70S6K,Akt,ERK1/2 signaling.3.The effect of JAK3 inhibitor CP690550 on the expression of IL-1Ra mRNA in human FLS cells induced by Poly?I:C?was detected by qPCR.ResultsPart ?:Poly?I:C?induces IL-1Ra expression and secretion in human FLS cells1.It was observed that only Poly?I:C?and LPS in TLR1-TLR6 ligands stimulated IL-1Ra secretion by human FLS,while the stimulation effect of Poly?I:C?was significantly higher than that of LPS.At the same time,Poly?I:C?was observed to achieve a significant stimulation at 25?g/ml and did not affect FLS cell proliferation.Therefore,the concentration of Poly?I:C?stimulation in the subsequent experiments was 25?g/ml.2.Poly?I:C?stimulates TLR3 activation and induces IL-1Ra secretion in a variety of cells with different genetic backgrounds.The results suggest that TLR3-mediated secretion of the anti-inflammatory cytokine IL-1Ra is also restricted to cell types.It was detected that FLS,AC,NPC,PM and DC can secrete IL-1Ra,but in T lymphocytes,B lymphocytes,DF,BMSC,WI-38,MRC-5,NCI-H358,NCI-H460,SH-SY5Y and BV2are not secreted.Among them,the amount of IL-1Ra secreted by FLS is relatively high.Therefore,in subsequent experiments,FLS cells were selected to study the TLR3/IL-1Ra signaling pathway.3.By using Poly?I:C?to induce the key conditions of IL-1Ra model expression in human FLS cells,it was confirmed that the IL-1Ra gene level detection time was 24 hr after stimulation,and the protein level detection time was 24 hr-48 hr.Part ?:The molecular mechanism of Poly?I:C?-induced IL-1Ra expression1.To detect the effects of TLR3,MDA5 and RIG-I gene silencing and TLR3knockdown on IL-1Ra secretion in human FLS cells stimulated by Poly?I:C?.The results showed that Poly?I:C?induced IL-1Ra expression was dependent on TLR3activation,but not on MDA5 or RIG-I.The secretion of IL-1Ra in serum was significantly decreased after injection of Poly?I:C?in TLR3-/-mice.2.Detection of Poly?I:C?-induced secretion of cytokines and other cytokines by human FLS cells indirectly affects IL-1Ra secretion.The results showed that TLR3 could induce both IFN-?and pro-inflammatory cytokine IL-6,but IFN-?expression was earlier than IL-1Ra.Exogenous IFN-?stimulates cells to secrete small amounts of IL-1Ra,but is far less than the level of Poly?I:C?stimulation,and stimulates IL-expressed IL with Poly?I:C?in the presence of IFN-?-specific antibodies.The level of-1Ra was not significantly reduced.After treatment with FLS by cycloheximide,Poly?I:C?could not stimulate FLS to secrete IFN-?,IL-1Ra and IL-6,but the cells could still transcribe IL-1Ra mRNA in large quantities,even higher than uninhibited cells.3.To detect the effect of IRF3 gene silencing and IRF3 knockout on the secretion of IL-1Ra in human FLS cells stimulated by Poly?I:C?.The results showed that Poly?I:C?induced IL-1Ra expression was dependent on IRF3 activation.IRF3-/-mice were injected with Poly?I:C?in vivo and the secretion of IL-1Ra in serum was significantly reduced.4.To detect the effect of NF-?B signaling on the expression of IL-1Ra induced by TLR3activation.The results showed that BMS-345541 affected Poly?I:C?-induced NF-?B phosphorylation.Blocking NF-?B signaling almost completely abolished Poly?I:C?-stimulated IL-1Ra,IFN-?and IL-6 expression.5.To examine the effect of PI3K-Akt signaling on the expression of IL-1Ra induced by TLR3 activation.The results showed that LY294002 affected Poly?I:C?-induced Akt phosphorylation.Blocking the blockade of PI3K-Akt signaling significantly reduced Poly?I:C?-stimulated IL-1Ra and IFN-?without inhibiting IL-6 secretion.These results indicate that the induction of IL-1Ra expression by Poly?I:C?is dependent on PI3K/Akt signaling.6.To detect the effect of GSK3?signaling on the expression of IL-1Ra induced by TLR3 activation.The results showed that SB415286 affected Poly?I:C?-induced GSK-3?phosphorylation.Inhibition of GSK3?signaling significantly reduced Poly?I:C?-stimulated IL-1Ra and IFN-?without inhibiting IL-6 secretion.7.To examine the effect of ERK-MSK signaling on the expression of IL-1Ra induced by TLR3 activation.The results showed that PD184352 blocked the phosphorylation of ERK1/2.Blocking ERK1/2 phosphorylation significantly increased IL-1Ra and IFN-?expression in Poly?I:C?-stimulated cells,but IL-6 levels were not affected.The results of MSK gene silencing significantly increased the expression of IL-1Ra and IFN-?in Poly?I:C?-stimulated cells,as well as blocking ERK signaling,but IL-6 levels were not affected.SB747651A blocks the phosphorylation of CRK and ATF in the downstream signaling molecules of MSK1/2,but does not affect the expression levels of IL-1Ra,IFN-?and IL-6 in Poly?I:C?-stimulated cells.MSK protein inhibitors are inconsistent with gene silencing.It is possible that SB747651A acts as a multiple protein target and has a lower specific effect on MSK than MSK siRNA.8.To detect the effect of p38 MAPK signaling on the expression of IL-1Ra induced by TLR3 activation.The results showed that SB203580 blocked Poly?I:C?-induced phosphorylation of p38 MAPK.Inhibition of p38 MAPK signaling did not affect Poly?I:C?-stimulated IL-1Ra and IFN-?,but significantly reduced IL-6 expression.9.Serum IL-1Ra levels were measured in TLR3-/-,IRF3-/-mice after injection of Poly?I:C?.The results showed that Poly?I:C?treatment significantly increased IL-1Ra levels in wild-type serum,but IL-1Ra was not significantly elevated in TLR3-/-and IRF3-/-mice.Part ?:Screening of traditional Chinese medicine monomers that enhance IL-1Ra expression and Immunosuppressive activity of benzoxazole derivative PO-2961.The effects of 20 anti-inflammatory Chinese medicine monomers and benzoxazole derivatives PO-296 on the secretion of IL-1Ra and IL-6 in human FLS cells induced by Poly?I:C?were detected.The results showed that the traditional Chinese medicine monomer gentiopicroside,diacerein and scoparone inhibited the secretion of cytokines IL-1Ra and IL-6,but these drugs significantly inhibited the FLS activity in vitro.When luteolin acts alone on cells,the drug is not toxic to cells,but co-stimulation with Poly?I:C?can significantly reduce cell viability and directly inhibit the secretion of cytokines IL-1Ra and IL-6.The benzoxazole derivative PO-296 can promote the secretion of IL-1Ra in human FLS cells stimulated by Poly?I:C?,and the secretion effect of PO-296 on IL-1Ra is positively correlated with the concentration.2.Explore the mechanism of PO-296 inhibition of T cell proliferation.The results showed that PO-296 inhibited the proliferation of T lymphocytes activated in different ways,and the IC50 values were all around 2?M.PO-296 did not increase the apoptosis of activated T lymphocytes within 20?M,and the concentration was not significantly cytotoxic to 80?M.PO-296 did not inhibit the expression of CD25 and CD69 by activated T cells at a concentration of 20?M.PO-296 can block the cell cycle in the G0/G1 phase,and the G0/G1 ratio increases with increasing concentration.PO-296inhibits the production of activated pro-inflammatory factors?IFN-?,IL-6,IL-17?but does not affect the production of anti-inflammatory factors?IL-10?and does not affect the production of IL-2 and IL-4.PO-296 significantly reduced the level of STAT5phosphorylation.3.To detect the effect of JAK3-STAT5 signaling on the expression of IL-1Ra induced by TLR3 activation.The results showed that CP690550 could not promote the expression of IL-1Ra in Poly?I:C?-stimulated FLS cells,but inhibited the expression of IL-1Ra.DiscussionPoly?I:C?stimulates human FLS cells to secrete IL-1Ra more strongly than LPS and induces IL-1Ra expression in various cell types with tissue specificity.The induction of IL-1Ra by Poly?I:C?is dependent on TLR3 but not on MDA5,RIG-1 and Poly?I:C?-induced IFN-?and pro-inflammatory cytokines.Inhibition of IRF3 and NF-?B signaling completely blocked TLR3/IL-1Ra.Inhibition of PI3K-Akt signaling significantly reduced the induction of IL-1Ra by Poly?I:C?.Poly?I:C?is promoted to IL-1Ra by inhibiting ERK-MSK signaling,but p38 MAPK signaling blockade is not associated with IL-1Ra secretion.TLR3^-/-or IRF3^-/-mice further confirmed that the induction of IL-1Ra by Poly?I:C?was dependent on the activation of TLR3 and IRF3.PO-296 promotes the signaling pathway of IL-1Ra by Poly?I:C?independent of JAK3-STAT5 signaling,but JAK3-STAT5 signaling may regulate TLR3/IL-1Ra.