Effect Of TYRO3 And AXL On TLR4 Signaling Pathway In Macrophages And Its Mechanism

Objective: This experiment was designed to investigate the role and mechanism of Tyro3,Axl receptor and its ligands Gas6,ProS in inflammatory response,and preparation of CS-CNT targeted drug delivery system.Methods:1.ConstructTyro3-pEGFP-N1 and Axl-pEGFP-N1 plasmids.The mRNA sequences of mouse Tyro3 and Axl genes were searched in GenBank.The company designed primer sequences,and amplified the full-length fragments of Tyro3 and Axl genes by RT-PCR,and ligated them with the empty plasmid pEGFPN1.The identification method was sequenced and double-enzyme digested.2.Preparation of CS-CNT.The CNT was purified and oxidized by the mixture of concentrated nitric acid and concentrated sulfuric acid,and then reacted with CS to synthesize CS-CNT.3.Experimental group3.1(1)experimental group 1 : Tyro3 ? Tyro3+Lps ? Tyro3+Gas6+Lps ?Tyro3+ProS+Lps?Tyro3 +Gas6+ ProS+Lps(2)experimental group 2 : Axl ? Axl+Lps ? Axl+Gas6+Lps ? Axl+ProS+Lps ?Axl+Gas6+ProS +Lps(3)Empty plasmid group:pEGFP-N1?pEGFP-N1+Lps?pEGFP-N1+Gas6+Lps?pEGFP-N1+ProS+Lps?pEGFP-N1+Gas6+ProS+Lps(4)Single ligand group:BC?Lps?Gas6+Lps?ProS+Lps?Gas6+ProS+Lps4.ELISA was used to detect of cytokines IL-6,IL-10,IL-33,TLR4 and TNF-?.5.Expression of TNF-?-TLR4 mRNA in cells of different groups by Real Time Q-PCR6.Expression of SOCS1,SOCS3,TLR4 and NF-?B protein in cells of each group by Western blot7.Observation of CNT Modification by scanning and Transmission Electron Microscopy8.Preparation of CS-CNT complex of Drug delivery system.Results: 1.Construction of Tyro3-pEGFP-N1 and Axl-pEGFP-N1 Plasmids.1.1 Construction and identification of Tyro3-pEGFP-N1 and Axl-pEGFP-N1plasmidsTyro3-pEGFP-N1 and Axl-pEGFP-N1 plasmids were successfully constructed.The two bands were identified by agarose gel electrophoresis after double enzyme digestion.The molecular weight of the bands was the same as the expected size.The sequencing results were consistent with the sequence of mouse Tyro3 and Axl genes in GeneBank.1.2 Expression of Tyro3-pEGFP-N1 and Axl-pEGFP-N1 plasmids in macrophages of RAW264.7 miceAfter the transfer of Tyro3-pEGFP-N1 axl-pEGFP-N1 and empty plasmid pEGFP-N1 into RAW264.7 cells for 24 hours,green fluorescence was detected under fluorescence microscope,indicating that the transfection was successful.2.Effect of Tyro3 AXL on macrophages induced by LPS in RAW264.7 mice2.1 ELISA test results: ligands Gsa6 and ProS pretreated 30 min could significantly reduce the inflammatory factor IL-6,IL-33,TLR4andTNF-?stimulated by Lps,and promote the secretion of anti-inflammatory factor IL-10.2.2Real Time Q-PCR method detection results: compared with the experimental groups,adding ligand Gsa6 and ProS preconditioning 30 min can obviously inhibit the expression of IL-6,MYD88,TLR4 and TNF-?.2.3The results of Western blot showed that the expression of TLR4 protein in the experimental groups was significantly increased after Lps stimulated RAW264.7 cells,but the expression level of TLR4 was significantly decreased with the addition of ligand Gas6 and ProS.At the same time,it could promote the expression of SOCS1 and SOCS3.3.Preparation of CS-CNT The best synthetic method of CS-CNT was obtained by experiments and related literatures.Conclusion:Tyro3 and Axl and their ligand Gas6 and ProS inhibited the activation of the inflammatory response induced by LPS activation of TLR4 signaling pathway and the expression of Gas6 and ProS.The results indicate that the TLR4 signaling pathway is interregulated with Tyro3,Axl and its ligand Gas6/ProS system.