The mechanism of Simiaotang and Tufuling Fang(四妙汤加土茯苓方)for treatment of acute gouty arthritis:a Toll-like receptor/myeloid differentiation primary response protein 88 signaling pathway-based experimental study
Objective:To explore the mechanism of Simiaotang and Tufuling Fang(四妙汤加 土茯苓方,SMTTFLF)in treatment of acute gouty arthritis(AGA)based on the Toll-like receptor(TLR)/myeloid differentiation primary response protein 88(MyD88)signaling pathway.Methods:Forty-eight specific pathogen-free(SPF)-grade male Sprague-Dawley(SD)rats were selected and randomized into blank group,model group,colchicine group,low-dose SMTTFLF(L-SMTTFLF)group,medium-dose SMTTFLF(M-SMTTFLF)group,and high-dose SMTTFLF(H-SMTTFLF)group,8 cases in each group.All rats but the ones in blank group were intervened by intragastric administration with potassium oxonate(PO)and hypoxanthine(Hx))suspension,followed by intra-articular injection of urate suspension into the right ankle joint for inducing AGA;while the ones in blank group by intragastric administration and intra-articular injection with the same dosage of nor-mal saline.After the end of AGA modeling,the right ankle joint of the rats was observed for assessing the inflammation index,and the ankle swelling index was calculated for confirming whether the AGA models were built successfully.Twenty-four hours after the end of AGA mo-deling,the successfully modeled rats in L-,M-,and H-SMTTFLF group were further intragastric administrated with SMTTFLF in correspond-ing concentration(consumpting the crude drug as 5,10,and 20 g/kg,respectively),the ones in colchicine group with colchicine suspen-sion,whereas,the ones in blank group and model group with the same dosage of normal saline,once a day for consecutive 7 days.Twenty-four hours after the end of drug intervention,the blood was drawn from the abdominal aorta of rats in each group for detecting the serum le-vels of urea nitrogen and creatinine.After phlebotomizing,the synovial tissues were harvested from the right ankle joints,and the mRNA le-vels of TLR/MyD88 signaling pathway-related genes,including TLR2,TLR4,MyD88,nuclear factor-κB inhibitor kinase-α(IKK-α)and nu-clear factor-KB inhibitor-α(IκB-α),were detected by using real-time fluorescence quantitative polymerase chain reaction(qPCR)technolo-gy.The protein expression levels of IKK-α and IκB-α were detected by using Western blotting assay,and the expression of TLR2 and TLR4 proteins,as well as the aggregation of macrophages,were observed by using immunohistochemistry(IHC).Results:① After injection of urate suspension,the swelling gradually appeared in the right ankle joints of all rats but the ones in blank group,and peaked after 24 hours,with the inflammation index being grade 2 or above.At hour 4,8,12,and 24 after successful modeling,the ankle swelling indexes of the modeled rats were all higher in model group,colchicine group,L-SMTTFLF group,M-SMTTFLF group,and H-SMTTFLF group compared to blank group(at hour 4 after successful modeling:P=0.000,P=0.000,P=0.002,P=0.004,P=0.031;at hour 8 after successful modeling:P=0.001,P=0.000,P=0.002,P=0.007,P=0.002;at hour 12 after successful modeling:P=0.000,P=0.000,P=0.004,P=0.010,P=0.006;at hour 24 after successful modeling:P=0.004,P=0.000,P=0.003,P=0.001,P=0.006).②There was no statisti-cal difference in the serum levels of urea nitrogen and creatinine among the 6 groups in general.③The difference was not statistically signifi-cant among the 6 groups in the mRNA level of MyD88 in the synovial tissues of ankle,while,was statistically significant in the mRNA le-vels of TLR2,TLR4,IKK-α and IκB-α.The mRNA levels of TLR2,TLR4,IKK-α and IκB-α were higher in model group compared to blank group(P=0.000,P=0.000,P=0.000,P=0.020),while,were lower in colchicine group and M-SMTTFLF group compared to model group(TLR2:P=0.000,P=0.005;TLR4:P=0.018,P=0.000;IKK-α:P=0.003,P=0.004;IKB-α:P=0.008,P=0.010).The mRNA levels of TLR2 and IκB-α were lower in L-SMTTFLF group compared to model group,while,the differences were not statistically sig-nificant between the 2 groups in the mRNA levels of TLR4 and IKK-α(P=0.000,P=0.020;P=0.564,P=0.884).The mRNA level of TLR4 was lower in H-SMTTFLF group compared to model group(P=0.024),while,the differences were not statistically significant be-tween the 2 groups in the mRNA levels of TLR2,IKK-α and IκB-α(P=0.349,P=0.320,P=0.579).Furthermore,the differences were not statistically significant between L-SMTTFLF group and colchicine group as well as between M-SMTTFLF group and colchicine group in the mRNA levels of TLR2,TLR4,IKK-α and IKB-α(TLR2:P=0.836,P=0.056;TLR4:P=0.669,P=0.069;IKK-α:P=0.387,P=0.989;IKB-α:P=0.721,P=0.518).The mRNA levels of TLR2 and TLR4 were higher in H-SMTTFLF group compared to colchicine group(P=0.000,P=0.002),while,the comparison of the mRNA levels of IKK-α and IκB-α between the 2 groups revealed no significant differences(P=0.313,P=0.136).The mRNA levels of TLR2,TLR4,IKK-α and IκB-α were higher in L-SMTTFLF group and H-SMTTFLF group compared to M-SMTTFLF group(TLR2:P=0.000,P=0.047;TLR4:P=0.000,P=0.001;IKK-α:P=0.006,P=0.042;IKB-α:P=0.021,P=0.037).The mRNA levels of TLR2 and TLR4 were lower in L-SMTTFLF group compared to H-SMTTFLF group(P=0.001,P=0.006),while,the comparison of the mRNA levels of IKK-α and IKB-α between the 2 groups revealed no significant differences(P=0.394,P=0.068).④The protein expression levels of IKK-α and IκB-α in the synovial tissues of ankle were higher in model group compared to blank group(P=0.000,P=0.000),and were higher in model group compared to colchicine group(P=0.000,P=0.000).The protein expression level of IKK-α was higher in colchicine group compared to L-SMTTFLF group(P=0.000),while the comparison of the protein expression level of IκB-α between the 2 groups revealed no significant differences(P=0.050).The protein ex-pression levels of IKK-α and IκB-α were higher in colchicine group compared to M-SMTTFLF group and H-SMTTFLF group(IKK-α:P=0.000,P=0.000;IκB-α:P=0.000,P=0.000),and were higher in L-SMTTFLF group compared to M-SMTTFLF group and H-SMTTFLF group(IKK-α:P=0.000,P=0.005;IκB-α:P=0.000,P=0.006).The protein expression level of IKK-α was lower in M-SMTTFLF group compared to H-SMTTFLF group(P=0.005),while the comparison of the protein expression level of IκB-α between the 2 groups re-vealed no significant differences(P=0.108).⑤Compared to the blank group,the macrophages in the synovial tissues of ankle were signifi-cantly aggregated in rats of the other 5 groups,with the model group presenting the most in number.Additionally,the macrophages decreased in colchicine group,L-SMTTFLF group,M-SMTTFLF group,and H-SMTTFLF group compared to model group,with the M-SMTTFLF group and colchicine group decreasing more pronounced.⑥Compared to the blank group,the TLR2 and TLR4 proteins in the synovial tissues of ankle were significantly expressed in rats of the other 5 groups,with the model group expressed the most pronounced.Besides,the expression levels of TLR2 and TLR4 proteins decreased in colchicine group,L-SMTTFLF group,M-SMTTFLF group,and H-SMTTFLF group compared to model group,with the M-SMTTFLF group and colchicine group decreasing more pronounced.Conclusion:SMTTFLF may work by redu-cing the aggregation of macrophage and the release of inflammatory factors,as well as down-regulating the expression of TLR/MyD88 signa-ling pathway-related genes in treatment of AGA,with medium-dose acting best in the effects,which are comparable to that of colchicine.
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