Study On The Effect Of Total Saponins From Rhizoma Dioscorea Nipponica On Gouty Arthritis Based On "TLR2/4-NF-κB Signal Pathway"

Objectives:This project is based on the previous study of gouty arthritis(GA)in our laboratory.Lactic acid,amino acid and other indicators were selected for pharmacodynamic studies.The Toll-like receptors and relatively TLR2/4-NF-κB receptor singnal patheway which plays important role in the happen and development of gout are introduced to study the intervention of total saponins from Rhizoma Dioscoreae Nipponicae(RDN)in order to clarify the molecular mechanism of RDN in treating GA.All above is used to explore single drug of safety,effectivity and clear mechanism.Method:1.MSU was injected into the knee-joint to induce gouty arthritis rat models in 5 consecutive days.They were normal group,model group,total saponins of high,middle and low groups as well as positive colchicine group.Whole blood were collected two hour after the model were induced on the seventh day was drawn through the abdominal aorta.The content of uric acid,creatinine and lactic acid were detected by UV spectrophotometry.High performance liquid chromatography were used to test the level of glycine and tryptophan.2.Urine were collected after the model were induced on the fifth day by metabolic cage.The content of uric acid and creatinine were detected by UV spectrophotometry.High performance liquid chromatography were used to test the level of guanosine.3.Whole blood and the synovial tissue were collected two hour after the model were induced on the seventh day was drawn through the abdominal aorta and knee joint.Realtime PCR and Western Blot methods were used to test the mRNA and protein levels of TLR2、TLR4、TRAF6、IKKα、IκBα and NF-κB in the synovial tissue.The content of TNF-α、IL-6 and IL-1β were tested by Elisa.Results:1.The content of uric acid,creatinine and lactic acid in serum of gouty arthritis could be reduced by high,middle and low dose of RDN.The dose of high and middle of RDN both could significantly increase the value of glycine and tryptophan in serum while low dose had no effect.2.The content of guanosine,creatinine and uric acid in urine of gouty arthritis could be increased by the dose of high and middle of RDN.There were no significantly effect of low dose RDN.3.The content of IL-6 and IL-1β could be reduced by the dose of high of RDN compared with model rats.The other doses of RDN have no effect.The content of TNF-α could be reduced by the dose of high and middle of RDN.4.The mRNA expression level and the protein expressions of TLR4、TRAF6、IκBα、IKKα in the synovial tissue could be reduced by high,middle and low dose of RDN compared with model rats.High,middle and low dose could reduce the mRNA expression level of TLR2 in the synovial tissue while only low dose decreased the protein expression of TLR2.High,middle and low dose could reduce the mRNA expression level of NF-κB in the synovial tissue while only high dose decreased the protein expression NF-κB.Conclusion:1.Total Saponins of RDN could promote the excretion of uric acid and creatinine by improve kidney function.It could treat GA by regulating the expression of uric acid and creatinine.It is conductive to the control of gout by regulating the expression of lactic acid,glycine and tryptophan in order to affect the occurrence and development of inflammatory reactions.2.Total Saponins of RDN inhibited the patheway of TLR2/4-NF-κB by down-regulation of the mRNA and protein expressions of TLR2、TLR4、TRAF6、IKKα、IκBα and NF-κB as well as the content of inflammatory factors such as TNF-α、IL-6 and IL-1β in the blood.Through the regulation of this pathway to achieve the suppression of inflammatory responses.