Effects Of Sinomenine Combined With Total Glucosides Of Paeony On Expression Of MyD88,NF-kappaB,ASC And Caspase-1 In RAW264.7 Macrophages Induced By Monosodium Urate Crystal

Objective: Acute gouty arthritis is an acute inflammatory rheumatic disease,which caused by the deposition of monosodium urate crystals in the articular joints and surrounding tissues.Monosodium urate crystals are identified and engulfed by the resident macrophages,activing NF-kappaB through the MyD88 dependent signaling pathway,meanwhile,NLRP3 Inflammasome is assembled and activated by various ways.And finally,a large number of IL-1beta,TNF alpha,IL-8 and other inflammatory factors are released,causing joint swelling and unbearable pain.At present,the main treatment drugs of AGA are colchicine,corticosteroids,non steroidal anti-inflammatory drugs,IL-1 inhibitor,etc,however,the clinical efficacy of these drugs is insufficient,and the toxic side effects of them are large or the drugs are too expensive.Thus,clinical applications of these drugs are limited and it is urgent to explore more safe and effective drugs.Traditional Chinese medicine has a long history of gout.In recent years,more and more attention has been paid to researching safe and effective anti-gout drugs from traditional Chinese medicine.Based on the previous research,in this study we established inflammatory model in RAW264.7 macrophages induced by monosodium urate crystal and observed the effect of sinomenine,total glucosides of paeony and its compatibility on the expression of inflammatory cytokines and key proteins in MyD88-NF-kappaB and NLRP3 inflammasome signaling pathway.To explore the mechanism of sinomenine,total glucosides of paeony and its compatibility on AGA,providing the basis for clinical applications.Methods:1Establishment of MSU crystal induced RAW264.7 macrophage inflammatory modelThe expression of IL-1beta,a key inflammatory factor in gouty arthritis,was used as a reference index for successful modeling.Respectively,using 0,10,50,100,200,400,600,1000mg/L monosodium sodium urate crystal suspension to stimulate RAW264.7 cells,collecting culture supernatant at 3,6,12,24,48 h,then,ELISA method was used to detect the expression level of IL-1beta.2 Cell viability was detected by MTT assayLogarithmic growth phase of RAW264.7 cells were seeded into 96 well plate at a density of 5 × 104/mL,200 ? l per well.Cultured overnight in a humidified atmosphere of 5% CO2 at 37°C.Then,the medium was changed to serum-free DMEM.Setting up normal group(Control)(DMEM),model group(Model)(DMEM+MSU,final concentration of 50mg/L),Colchicine+MSU group(DMEM+MSU,final concentration of 50mg/L+Colchicine,final concentration of 1 ? mol/L),SIN+MSU group(DMEM+MSU,final concentration of 50mg/L + SIN,final concentration of 300?mol/L),TGP+MSU group(DMEM+MSU,final concentration of 50mg/L +TGP,final concentration of 40mg/L),SIN+TGP+MSU group(DMEM+MSU,final concentration of50mg/L+SIN,final concentration of 300 ? mol/L+TGP,final concentration of40mg/L),Colchicine group(DMEM+ Colchicine,final concentration of1?mol/L),SIN group(DMEM+SIN,final concentration of 300?mol/L),TGP group(DMEM+final concentration of 40mg/L TGP),SIN+TGP group(DMEM+final concentration of 300 ? mol/L SIN+final concentration of40mg/L TGP).Each group had 6 wells,culturing 24 h in a humidified atmosphere of 5% CO2 at 37°C.In addition,setting zero hole(without cells and DMEM).20 ?l of MTT dye solution(5 mg/ml)were added to each well and the plate was incubated for 4h,Then,the medium was decanted,200 ?l of DMSO was added to the wells and reaction for 10 min under gentle shaking at37°C to dissolve the tetrazolium dye.Read absorbance of each well at 570 nm wavelength.Relative cell viability was calculated by the percentage of OD value of each group and the control group.3 Drug intervention and index detection in each experimental groupLogarithmic growth phase of RAW264.7 cells were seeded into 6 well plate at a density of 1.5 × 106/well,each well with 2ml serum containing DMEM medium,culturing overnight in a humidified atmosphere of 5% CO2 at 37°C.Then,supernatant was abandoned and the cells were washed 1-2times by PBS.The medium was changed to serum-free DMEM.Setting up normal group(Control)(DMEM),model group(Model)(DMEM+MSU,final concentration of 50mg/L),Colchicine+MSU group(DMEM+MSU,final concentration of 50mg/L +Colchicine,final concentration of1 ? mol/L),SIN+MSU group(DMEM+MSU,final concentration of50mg/L+SIN,final concentration of 300?mol/L),TGP+MSU group(DMEM+MSU,final concentration of 50mg/L + TGP,final concentration of 40mg/L),SIN+TGP+MSU group(DMEM+ MSU,final concentration of 50mg/L + SIN,final concentration of 300?mol/L + TGP,final concentration of 40mg/L).The final volume of solution was 2ml in each well,and each group had 3 wells,culturing for 24 h in a humidified atmosphere of 5% CO2 at 37°C.Then,culture supernatant was collected,and after 4 ?,3000 rpm for 10 min collecting culture supernatant again,using for ELISA test.In addition,the cells was collected for western blot test.(1)Detection of NO secretion in macrophages by nitrate reductase method;(2)The levels of IL-1beta,TNF-alpha,IL-8,MCP-1,MIF cytokines in each group measured by ELISA,which secreted by RAW264.7 macrophages;(3)Western blot method was used to detect the expression of MyD88,NF-kappa B,ASC,and Caspase-1 in macrophages.4 Statistical methodsSPSS 21 statistical software was applied.One-way ANOVA was used when the data were in line with the normal distribution,when variance uneven using rank sum test.The differences were considered statistically significant with P < 0.05.Results:1 Establishment of MSU crystal induced inflammation model of RAW264.7 macrophagesAfter 6h,the level of IL-1beta began to increase in the group of 50mg/L MSU and other group with higer dose of MSU,comparing with group 0mg/L.And reaching peak at 24 h,decreasing at 48 h.At 24 h,the level of IL-1beta was significantly higher in the group of 50mg/L MSU,comparing with the 0mg/L MSU,the difference was statistically significant(P < 0.05).And in view of the high concentration of MSU may have an impact on cell viability.Thus,50mg/L MSU crystals stimulating cell 24 h was used.2 Cell viabilityCompared with Control group,the relative viability of the cells were not significantly different in the group of MSU,Colchicine+MSU,SIN+MSU,TGP+MSU,SIN+TGP+MSU,Colchicine,SIN,TGP,SIN+TGP.The results showed that MSU,Colchicine,SIN and TGP had no obvious cytotoxic effect under the experimental conditions.3 The levels of NO in each experimental group of macrophages culture supernatantCompared with Control group,the levels of NO secretion were significantly increased in Model group,the difference was statistically significant(P < 0.05);compared with the Model group,the levels of NO were significantly reduced in each group,the difference was statistically significant(P < 0.05);compared with Colchicine group,the levels of NO were significantly increased in SIN group,TGP group and SIN+TGP group,the difference was statistically significant(P < 0.05);compared with SIN+TGP group,the levels of NO have no significant difference in SIN group and TGP group,the difference was not statistically significant(P > 0.05).4 The levels of IL-1beta,TNF-alpha,IL-8,MCP-1,and MIF in each experimental group of macrophages culture supernatantCompared with the Model group,the expression levels of IL-1beta,TNF-alpha,IL-8,MCP-1 and MIF were significantly decreased in Colchicine group,SIN group,TGP group and SIN+TGP group,the difference was statistically significant(P < 0.05);compared with the Colchicine group,the expression levels of IL-1beta,TNF-alpha,MCP-1,and MIF were nosignificant difference in SIN+TGP group,there was no statistically significant difference(P > 0.05),but the levels of IL-8 increased significantly,the difference was statistically significant(P < 0.05);compared with Colchicine group,the expression levels of IL-1beta,TNF-alpha,IL-8,MCP-1 were increased significantly in SIN group and TGP group,the difference was statistically significant(P < 0.05),the expression level of MIF was no significant difference in SIN group,there was no statistically significant difference(P > 0.05),the expression level of MIF increased significantly in TGP group,the difference was statistically significant(P < 0.05);compared with SIN group,the expression levels of IL-1beta,IL-8 increased significantly in TGP group,the difference was statistically significant(P < 0.05),the levels of TNF-alpha was no significant difference,the difference was not statistically significant(P > 0.05);compared with SIN+TGP group,the level of IL-8 was significantly increased in TGP group,the difference was statistically significant(P < 0.05),the level of IL-8 was no significant difference in SIN group,the difference was not statistically significant(P > 0.05).5 The expression of MyD88,NF-kappaB,ASC,and Caspase-1 in each experimental group of macrophagesCompared with the Model group,the expression levels of MyD88,NF-kappa B,ASC,and Caspase-1 were significantly lower in Colchicine group,SIN group,TGP group and SIN+TGP group,the difference was statistically significant(P < 0.05);compared with Colchicine group,the expression levels of MyD88,NF-kappaB were no significant difference in SIN+TGP group,the difference was not statistically significant(P > 0.05),the expression of MyD88,NF-kappa B were significantly increased in SIN group and TGP group,the difference was statistically significant(P < 0.05);compared with Colchicine group,the expression of ASC,Caspase-1 were significantly increased in SIN group and TGP group and SIN+TGP group,the difference was statistically significant(P < 0.05);compared with SIN group,the expression levels of NF-kappaB increased in TGP group,the difference was statistically significant(P < 0.05);compared with SIN+TGP,theexpression of ASC,Caspase-1 were significantly increased in SIN group and TGP group,the difference was statistically significant(P < 0.05).Conclusions:1 Sinomenine,total glucosides of paeony and its combined application can significantly reduce the levels of NO,IL-1beta,TNF-alpha,IL-8,MCP-1,MIF in MSU crystal induced RAW264.7 macrophage and its anti-inflammatory effect is significant;2 The combined application of sinomenine and total glucosides of paeony could significantly reduce the expression levels of MyD88,NF-kappa B,ASC and Caspase-1 in the inflammatory signaling pathway,and the effect was better than the two alone.3 Sinomenine combined with total glucosides of paeony,the effective components of Chinese herbal medicine,have multi target synergistic anti-inflammatory effect in RAW264.7 macrophage inflammatory model induced by monosodium urate crystal,which may be one of the mechanisms of its treatment of AGA.