Effect And Mechanisms Of Icariin On Lipopolysaccharide-induced Macrophage Polarization

Objective: To investigate effect and mechanisms of icariin on lipopolysaccharide(LPS)-induced macrophage polarization.Methods:1.Identification of peritoneal macrophages of rats: Immunofluorescence technique was used to identify CD68,a membranal marker of macrophages.2.Effect of ICA on LPS-induced polarization of rat peritoneal macrophages: Rat peritoneal macrophages were randomly divided into control,LPS,and ICA(0.1 μM,1 μM,10 μM)+ LPS groups.ICA was administered according to groups scheme,and after 2 h,LPS was added at a final concentration of 100 ng/m L to continue the treatment for 4 h.MTT assay was used to detect the effect of ICA on the cell viability of rat peritoneal macrophages;light microscopy was used to observe the effect of ICA on the morphology of rat peritoneal macrophages;RT-PCR was used to detect the TNF-α,IL-6 and Arg-1 m RNA levels of ICA on LPS-induced M1 rat peritoneal macrophages;and Western blot was used to detect the protein levels of TNF-α,IL-6,Arg-1,i NOS and CD206 in LPS-induced M1 rat peritoneal macrophages.3.Effect of ICA on TLR4/MyD88 pathway in LPS-induced rat peritoneal macrophages:(1)Effect of ICA on TLR4/MyD88 pathway in LPS-induced rat peritoneal macrophage model: the grouping and administration scheme was the same as above,and the changes in TLR4,MyD88 and NF-κB p65 protein amounts were detected by Western blot.(2)Effect of ICA on TLR4/MyD88 pathway in LPS-induced TLR4 gene mutant RAW264.7 cell model: To further determine the mechanism of ICA,RAW264.7 was selected as the subject cells,and the stable expression of TLR4 mutant gene was used.The specific methods were as follows:(1)Molecular docking: semi-flexible molecular docking of ICA and TLR4(PDB: 5IJC)was performed using Auto Dock Vina software to determine the binding site between ICA and TLR4;(2)Selection of mutation points: the optimal mutation site was selected by DUET database;(3)An overexpression adenoviral vector(OE-S439RTLR4)with S to R amino acid residues at position 439 on the TLR4 amino acid sequence was constructed based on the optimal mutation site determined,while a wild-type overexpression adenoviral vector(OE-WT-TLR4)was constructed;(4)Transfection: the unloaded adenovirus,OE-TLR4 and OE-S439R-TLR4 adenoviruses containing MOI = 500 were used to infect RAW264.7 cell lines;(5)Identification of transfection effect: the transfected groups were control group,unloaded virus(Null)group,OE-WT-TLR4 group,OE-S439R-TLR4 group and the GFP efficiency of adenoviral transfection of RAW264.7 cell lines was observed by fluorescence microscopy Western blot was used to detect the expression of DYKDDDDK Tag and TLR4 protein transfected with tag protein;(6)TLR4function identification of the mutant strain: OE-WT-TLR4 group,OE-WT-TLR4+LPS group,OE-S439R-TLR4 group and OE-S439R-TLR4+LPS group were set up TLR4 and MyD88 protein expression levels were measured by Western blot to determine whether the function of TLR4 was still present;(7)The effect of ICA on the TLR4/MyD88 pathway in the LPSinduced TLR4 gene mutant RAW264.7 cell model: OE-WT-TLR4 group,OE-WTTLR4+LPS group,OE-WT-TLR4+LPS+ICA(10 μM)group and OE-WT-TLR4+LPS+TAK-242(TLR4 inhibitor)group;OE-S439-TLR4 group,OE-S439R-TLR4+LPS group,OE-S439R-TLR4+LPS+ICA(10 μM)group and OE-S439R-TLR4+LPS+TAK-242 group were used to detect TLR4,MyD88,and TNF-α protein expression levels by Western blot.Results:1.Identification of rat peritoneal macrophages: The extracted cells were proved to be rat peritoneal macrophages with good purity.2.Effect of ICA on LPS-induced polarization of Rat peritoneal macrophages: ICA concentration was between 0.1 and 10 μM,and there was no significant toxicity to rat peritoneal macrophages;a large number of irregular pseudopodia appeared in the morphology of rat peritoneal macrophages after LPS induction by morphological observation,and the morphology tended to be oval or round when ICA was treated;after 4h of LPS induction in rat peritoneal macrophages,TNF-α,IL-6 and i NOS protein expression was significantly up-regulated,indicating that M1 macrophages were successfully induced.ICA could significantly inhibit the LPS-induced increase in the protein levels of TNF-α,IL-6 and i NOS and up-regulate the protein levels of CD206 and Arg-1 after treatment.In addition,ICA was able to significantly inhibit LPS-induced increase in m RNA levels of TNF-α and IL-6 and increase Arg-1 m RNA levels in peritoneal macrophages,indicating that ICA can transform LPS-induced M1 macrophages to M2.3.Effect of ICA on TLR4/MyD88 pathway in LPS-induced rat peritoneal macrophage model:(1)TLR4,MyD88 and NF-κB protein levels were significantly increased in rat peritoneal macrophages after 4 h of LPS induction,and ICA was able to significantly inhibit LPS-induced increase in TLR4,MyD88 and NF-κB p65 protein levels in rat peritoneal macrophages,indicating that ICA regulates macrophage polarization by inhibiting TLR4/MyD88 pathway.(2)Effect of ICA on TLR4/MyD88 pathway in LPS-induced TLR4 gene mutation RAW264.7 cell model: According to the docking results of ICA with TLR4 protein molecule,serine(S)at position 439 of TLR4 protein was mutated to arginine(R)by DUET database analysis,and TLR4(S439R)point mutation overexpression adenovirus was constructed;the GFP positive rate = 80% of RAW264.7 cells transfected with null,OE-WT-TLR4 and OES439R-TLR4 adenoviruses at MOI = 500;TLR4 and DYKDDDDK Tag protein levels in OE-WT-TLR4 and OE-S439R-TLR4 groups were significantly increased,up to 1.5-fold of control group,indicating successful TLR4 overexpression;TLR4 and MyD88 protein levels were significantly increased after 4 h of LPS treatment in OE-WT-TLR4 and OE-S439RTLR4 groups,indicating that TLR4 function was not affected in the mutant strain.ICA significantly inhibited LPS-induced TLR4,MyD88 and TNF-α protein levels in OE-WTTLR4 RAW264.7 cell line,but had no significant effect on LPS-induced TLR4,MyD88 and TNF-α protein levels in OE-S439R-TLR4 RAW264.7 cell line,preliminarily indicating that the key binding target of ICA for exerting efficacy with TLR4 may be S439.Conclusion:1.ICA was able to polarize LPS-induced M1 macrophages into M2 macrophages.2.The mechanism by which ICA regulates macrophage polarization is related to the activation of the TLR4/MyD88 signaling pathway and the target at which it acts may be the S439 residue on the TLR4 protein.