| Objective:Based on the research of theory about the mechanism and treatment of gouty arthritis(GA) with Guizhishaoyaozhimu Decoction(GD), to study the effect of GD on the expression of inflammatory signal in synovial tissues of joint of GA rats with ankle joint injection of monosodium urate crystal and rat ′ s neutrophils and macrophage induced with sodium urate suspension by detecting Receptors,signaling adaptor proteins,inflammatory factors related to GA based on NLRP3 inflammasomes and Toll-My D88 signaling pathway in three experiments and explore the anti inflammation mechanism. Methods: In first experiment the effect of colchicine and GD on the expression of inflammatory signal factors including TLR-2, TLR-4,NLRP3,My D88,ASC,Capase-1,Capase-12,IKK- β,I κB- α,PPAR-γ,IL-1β,TNF-α,IL-2,IL-6,IL-10,COX-2,TGF-β1 and NF-κB in synovial tissues of joint of GA rats by ankle joint injection of monosodium urate crystal. In another two experiments of cellular pharmacology TLR-2,TLR-4,NLRP3,My D88,ASC,Capase-12,IKK- β,I κ B- α,IL-1β,IL-6,IL-8,TNF- α,S100A8 and NF- κ B in rat ′ s neutrophils and macrophage induced with sodium urate suspension was observed and The main observation indexes and detection methods are as follows:Experiment one:180 male SD rats were randomly divided into 3 experiments(the IHC, ELISA, Westblot experiment)with 60 rats each experiment divided into 6 groups with 10 rats each group according to weight.The high, medium and low dose group of GD(4, 8, 16 g?kg-1)and colchicine group(3×10-4g?kg-1) were treated with medicine by gastric administration, the normal and model group were given equal volume of distilled water. Medicine or distilled water was given once daily for seven consecutive days throughout the experiment.On the fifth day,GA model was made by injection of monosodium urate in the ankle joint cavity of rat.The synovial tissues of the rats were taken. and expression of TLR-2,TLR-4 and NLRP3 inflammasomes were detected with immunohistochemistry and integrated option density(IOD) was measuring with Image-Pro Plus 6.0 analysis system. The expression levels of signaling proteins including My D88,ASC,Capase-1,Capase-12,IKK-β,IκB-α,PPAR-γ were detected by Western blot. Expression of Inflammatory cytokine such as IL-1β,TNF-α,IL-2,IL-6,IL-10,COX-2,TGF-β1 were detected with Enzyme linked immunosorbent assay(ELISA)and activity of NF- κ B was detected with DPI-ELISA.Experiment two and three:30 male SD rats were randomly divided into 5 groups with 6 rats each group according to weight.The high, medium,low dose group of GD(4, 8, 16 g?kg-1)and colchicine group(3×10-4 g?kg-1) were treated with medicine by gastric administration, the normal group were given equal volume of distilled water. Medicine or distilled water was given once daily for seven consecutive days throughout the experiment. One hour after the last gastric administration,all rats were anesthetized with ether, and the serum was collected.Neutrophils and macrophages were respectively isolated from 20 male SD rats and cultured according to the methods from literatures. Cell experiments were randomly divided into 2 experiments( no receptor inhibitors,added NALP3 receptor inhibitors)with 6 groups each experiment.The normal was added to normal rat serum,the model group, the high, medium and low dose group of GD and colchicine group were added uric acid sodium suspension of 200mg?L-1 to induce into cell model with serum containing medicine.Neutrophils and macrophages were put into the CO2 cell incubator for 12 and 2 hour After administration.The expression of Inflammatory cytokine such as IL-1β,IL-6,IL-8, TNF-α,S100A8 were detected with Enzyme linked immunosorbent assay(ELISA), and activity of nuclear factor-κB(nuclear factor kappa B(NF- κ B) was detected with DPI-ELISA. The expression levels of signaling proteins as My D88,ASC,Capase-12,IKK- β,I κ B- α were detected by Western blot. The m RNA expression levels of TLR-2, TLR-4,NLRP3 was detected with reverse transcription-PCR(RT-PCR). Results:Experiment one: Expression of related factors in TLRs-My D88 signaling pathway about the experiment with rat joint synovial tissue:Compared with normal group after 72 hours,the IOD of TLR-2, TLR-4 and the expression level of My D88,IKK-β,IL-2,COX-2,IL-10, NF-κB in synovial tissues of the of GA rats significantly increase-ed,whereas IκB-α,PPAR-γ,TGF-β1significantly decreased. the IOD of TLR-2,TLR-4 and the expression of My D88,IL-2,COX-2,IL-10, NF-κB in colchicine group and medium and high dose group of GD significantly increased than the model group,whereas,IκB-α, PPAR-γand TGF-β1 in medium and high dose group of GD increased(P<0.05) and there was no significant change in IKK-β in all dose group of GD. The expression of My D88 in medium and high dose group of GD was more inhibited than in colchicine group.Expression of related factors in NLRP3 inflammasomes signaling pathway about the experiment with rat joint synovial tissue: Compared with normal group,the expression of NLRP3 inflammasomes,ASC,Capase-1 and IL-1β,IL-6,TNF-α,NF-κB in synovial tissues of the joint of GA rats significantly increased(P<0.05),whereas Capase-12 significantly decreased( P<0.05). the expression of NLRP3 inflammasomes,ASC in medium and high dose group of GD and Capase-1,IL-1β,IL-6,TNF-α,NF-κB in all group of GD significantly decreased than the model group(P<0.05),whereas Capase-12 increased in medium and high dose group of GD(P<0.05). The expression of Capase-12,TGF-β1 significantly increased in medium and high dose group of GD than in colchicine group.Experiment two :Expression of related factors in TLRs-My D88 signaling pathway about the neutrophils experiment: Compared with normal group after 12 hours,the expression of TLR-2,TLR-4 m RNA, My D88,IKK-β,IL-1β,IL-6,IL-8,TNF-α,S100A8 and NF-κB in neutrophils of the model group significantly increased(P<0.05), whereas I κ B- α significantly decreased(P<0.05). With no receptor inhibitor,the expression of TLR-2,TLR-4 m RNA My D88,IKK-β,IL-1, IL-6,IL-8 and TNF-α,in all drug delivery groups, NF-κB in the high dose group of GD and S100A8 in the group of Colchicine significantly decreased than the model group( P<0.05),whereas S100A8 and IκB-α in high dose group of GD increased(P<0.05). The expression of S100A8,IκB-α significantly increased in all dose group of GD than in colchicine group.TLR receptor inhibitor significantly inhibited the expression of TLR-2,TLR-4 m RNA,IKK-β,IκB-α,IL-1β,IL-6, IL-8,NF-κB in neutrophils。Expression of related factors in NLRP3 inflammasomes signaling pathway about the neutrophils experiment: Compared with normal group,the expression of NLRP3 inflammasomes, ASC(with no receptor inhibitor),IL-1 β,IL-6,TNF- α,NF- κ B and in neutrophils of the model group significantly increased(P<0.05), whereas Capase-12 significantly decreased(P<0.05).The expression of NALP3 m RNA,IL-1β,IL-6,TNF-αin all drug delivery groups,and ASC in group of Colchicine and high dose group of GD, NF-κB in medium and high dose group of GD significantly decreased than the model group(P<0.05),whereas Capase-12 in medium and high dose group of GD increased(P<0.05) and there was no significant change in group of Colchicine( P>0.05). The expression of Caspase-12 significantly increased in all medium and high group of GD than in colchicine group.NLRP3 receptor inhibitor significantly inhibited the expression of NLRP3 m RNA,IL-1βIL-6,TNF-αin neutrophils and have no significant effect on the expression of NF-κB.Experiment three:Expression of related factors in TLRs-My D88 signaling pathway about the macrophage experiment: Compared with normal group after 2 hours,the expression of TLR-2,TLR-4 m RNA My D88,IKK-β,IL-1β,IL-6, IL-8,NF-α,S100A8 and NF-κB in macroph-age of the model group significantly increased(P<0.05),whereas IκB-α signi-ficantly decreased(P<0.05).With no receptor inhibit-or,The expression of TLR-2,TLR-4 m RNA,My D88,IKK-β in all drug delivery groups and IL-1β,IL-6,IL-8,TNF-α and NF-κB in the high dose group of GD and Colchicine group significantly decreased than the model group(P<0.05),whereas S100A8,IκB-αand TGF-β1 in high dose group of GD increased(P<0.05). The expression of S100A8〠IκB-α significantly increased in all dose group of GD than in colchicine group.TLR receptor inhibitor significantly inhibited the expression of TLR-2,TLR-4 m RNA,I κ B- α,IL-1 β and TGF- β 1 in macrophage. Expression of related factors in NLRP3 inflammasomes signaling pathway about the macrophage experiment: Compared with normal group after 2 hours,the expression of NLRP3 inflammasomes, ASC,IL-1β,IL-6,TNF-αand NF-κB in macrophage of the model group significantly increased(P<0.05), whereas Capase-12 significantly decreased( P<0.05).The expression of NALP3 m RNA,TNF-αin all drug delivery groups,and ASC,IL-1β,IL-6 and NF-κB in high dose group of GD significantly decreased than the mode group( P<0.05),whereas Capase-12 in high dose group of GD increased more than in group of Colchicine(P<0.05).NLRP3 receptor inhibitor significantly inhibit-ed the expression of NLRP3 m RNA,ASC,Caspase-12,IL-1βin macroph-age and have no significant effect on the expression of NF-κB. Conclusion: The results of the three experiments showed that The anti inflammation mechanism of GD is related to decreasing the expression levels of TLR-2,TLR-4,NLRP3 and My D88,ASC and increasing the expression of PPAR-γ,IκB-αand accordingly inhibiting the maturation of IL-1βand the activation of NF-κB, and reducing the expression of inflammatory factors in Toll-My D88 and NLRP3 inflammasomes signaling pathway.Analysis of the experimental results showed that GD significantly increased the expression of anti inflammatory cytokines such as IκB-α,PPAR-γ,TGF-β1 and Capase-12 to inhibit the GA inflammatory reaction caused by inflammatory signal pathway. |
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