| Gout is a disease due to the sustained metabolism or excretion of purine and(or) uric acid,urate crystals(monosodi-um urate, MSU) precipitated from the organization and organ. The pathogenesis of gout has not been fully elucidated, but the domestic and foreign research shows that gout the inflammatory pathogenesis of TLRS/MyD88 signal transduction pathways are closely related. Toll receptors(TLRs) are recognition receptors of the innate immune system of the body, which can recognize pathogen-associated molecular model(PAMPs) and damage associated molecular model(DAMPs), mainly express in immune cells, including monocytes, DCs and granulocytes, macrophages. THP-1 cells is a cell lines established by utilize leukemia patients blood monocytes, express all TLRS receptor except TLR3, a good alternative of monocyte – macrophage in vitro study of human. Sodium urate(MSU) can be specifically recognize by TLR2, TLR4 and mediates its signal transduction to regulation of inflammation.Polygonum cuspidatum is commonly used in clinical medicine, high medicinal value, which is derived from herb Polygonum cuspidatum families and genera(Polygonum cuspidatum Sieb.et Zucc) of dry rhizome and roots with a more extensive pharmacological activity, bitter cold, mainly for joint Bitongkang, heat jaundice and other diseases. Polydatin is the main ingredient of Polygonum cuspidatum. Cassia twig is lauraceae trees drying plant cinnamon twigs, heart, lung, bladder, and other functions have diaphoresis, commonly used in common cold, cold blood by the closure, through joint paralysis and other symptoms. Cassia twig mainly contain volatile content, coumarins, steroids and other different types of chemical composition, cinnamic aldehyde is cassia twig the effect of the best compounds in essential oil. The study found cassia twig and its extract can be able to treat a variety of cinnamic aldehyde and other acute and chronic inflammation, good anti-inflammatory and antibacterial effect. Chinese medicine has a cold, hot, warm, cool four different herbs. Instead compatibility potency herbs such as heat and cold, cold temperature is difficult, hot and the core of traditional Chinese medicinal theory. Our previous study showed that: Rover Tongfengke has a significant clinical improvement for acute gouty arthritis patients, Cold-temperate medicine of giant knotweed- cassia twig is rover at the core of medicine for gout, and giant knotweed, cassia twig is 6:1 is optimal proportion.Based on these findings we have, we observe the giant knotweed- cassia twig intervention effect of acute gout inflammation in rats in this experiment, analysis of giant knotweed- cassia twig medicine for TLRS/MyD88 signaling pathways. In addition,we will use the THP-1 cells inflammatory model as the objects, analysis of giant knotweed glycosides and cinnamic aldehyde of TLRS/MyD88 signaling pathways. Delve into Giant knotweed- cassia twig therapeutic mechanism of acute gouty arthritis, to learn more about it possible target for the treatment of gout. 1 Giant knotweed-cassia twig effect the TLRs / MyD88 signaling pathway of acute gout inflammation induced by MSU. Objective Study on giant knotweed- cassia twig drugs in rats with acute gout TLRs/MyD88 molecular pathways of inflammation and to probe its mechanism of the treatment of gout. Methods 48 male SD rats were randomly divided into control group, model group, positive group of colchicine(0.1g?kg-1), polygonum cuspidatum-Cassia twig low dose group(3.5g?kg-1),polygonum cuspidatum-Cassia twig middle dose group(7g?kg-1), polygonum cuspidatum-Cassia twig high dose group(14g?kg-1). The normal control group and model group were given physiological saline, other each group to give the corresponding drug was administered by gavage for 7 days(1 times a day), the model group and medicine group rats were injected of sodium urate(MSU) 0.2 mL(25 mg?mL-1) in posterior ankle after one hour on the fourth day, and normal control group were injected normal saline with the volume.To observe the Digit volume change of each group in different time. After 72 hours to take blood,ELISA was applied to determine the changes of peripheral blood IL-1β、TGF-β; expression of peripheral blood mononuclear cells expressing TLR2, TLR4 and MyD88 by RT-qPCR. The animal were sacrificed after taking blood, the ankle for pathological observation. Result Compared with model group, two doses of the herbal medicine on group can reduce rats toes volum significantly in 2 hour and 6 hour(p < 0.05). 3 digit volume herbal medicine on group were reduced with significant difference in 48 hour and 72 hour(p<0.05).3 digit volume herbal medicine on group can reduce inflammatory cell infiltration and blood-vessel to hyperaemia differing in degree.Compared with model group,the significantly decrease levels of IL-1β, promoted the expression of TGF-β with no significant difference, significant inhibition of the expression of TLR2 and TLR4 and MyD88 gene level(p < 0.05, p < 0.01). Compared with the positive group, the middle dose of giant knotweed- cassia twig had a similar inhibitory effect on the expression of MyD88、TLR4 gene. Conclusion From the experiment acute gouty arthritis in rats,it shows that 3 dose of medicine suits of Polygonum cuspidatum- Cassia twig have a significant improvement on gouty by reducing the expression of TLR2、TLR4 and MyD88 in Different degrees,and the middle dose(7g?kg-1)may do the better work in gouty. 2 Polydatin and Cinnamic aldehyde effect on TLRs / MyD88 signaling pathway of gout with THP-1 cell acute gout inflammation caused by MSU. Objective Study the giant knotweed glycosides, cinnamic aldehyde suppress the THP 1 cells induced by MSU inflammatory reaction mechanism. Methods THP-1cells were randomly divided into control group, model group, positive group of colchicine(0.1g?kg-1)),polydatin group(15ug?mL-1), cinnamaldehyde group(5 ug?mL-1) and polydatin-cinnamaldehyde group(20ug?mL-1).Dose groups were given the corresponding drugs,and the control group and model group were polishing by DMSO after 24 h cultivated in the condition of 5% CO2, 37 ℃. 2 h later,except the control group were given MSU which final concentration was 100ug?mL-1,and the control group was polishing by PBS. Collected supernatant on cells and the the rest of cells after 24 h cultivated in the condition of 5% CO2, 37 ℃. ELISA was applied to determine the changes of interleukin 1 beta(IL- 1 beta) and tumor necrosis factor(TNF- a) in cultural supernatants. the expression of TLR2,TLR4,MyD88 NF-κBand TRIF gene were tested by qPCR. Testing the expression of protein TLR2、TLR4、MyD88 and TRIF by Western blotting. Result Compare with the control group,the THP-1cells’ supernate of the model group had a significant increase in IL-1βand TNF-a(p < 0.01);and the TLR2、TLR4、MyD88、NF-κB and TRIF gene expressed significant rise.Compare with the model group, the dose group can significantly decrease levels of IL-1β and TNF-a in THP-1cells’ supernate(p < 0.01),and reduce the expression of TLR2,TLR4, MyD88, NF-κB and TRIF gene in different degrees(p <0.05,p <0.01), and the expression of TLR2 and TLR4, MyD88, TRIF protein. Conclusion From the experiment acute gouty arthritis in THP-1 cells, polydatin group,cinnamaldehyde group and polydatin-cinnamaldehyde group also have a significant reduce the MSU-induced TNF-a, IL-1β content and down the signaling pathway TLR2, TLR4, MyD88, NF-κB and gene expression of TRIF,and the decrease of TLR2 and TLR4, MyD88, TRIF protein expression,show that Polydatin, cinnamic aldehyde may play anti-inflammatory effects by influencing and inhibiting TLRs / MyD88 pathway. |
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