The Regulation Of Th1/Th2 Paradigm By Tripterine

Objective: To investigate the effects of low dose Tripterine on Th1/Th2 paradigm(IL-2,IL-4,IL-6,IL-10,INF-γand TNF-a) in vitro and explore new drugs to improvetransplant outcomes.Materials and Methods:Whole blood cultures: Whole blood was aspirated from the peripheral vein of younghealthy volunteers and collected in sterile heparinized tubes. 300μl of the undilutedblood samples were cultured at 37℃in humidified air supplemented with 5% CO₂. Adose-related response to Tripterine was studied by adding it to the culture with anultimate concentration varied from 10^-11 mmol/L to 10^-5 mmol/L.BD^TM CBA Human Th1/Th2 Cytokine Assay: Incubate the whole blood cells for 9hours, then centrifuge at 200g for 5 minutes. Reconstitute Human Th1/Th2 CytokinesStandards in Assay Diluent. Dilute Standards by serial dilutions with the AssayDiluent. Mix 10μl/test of each Human Cytokines Capture Bead suspension andtransfer 50μl of mixed beads to each assay tube. Add PE Detection Reagent(50μl/test)and Standard Dilutions and test samples to the appropriate samplestubes(50μl).Incubate the assay tubes for 3 hours at RT and protect from directexposure to light. Add Wash Buffer to each assay to re-suspend the bead pellet. Startanalyzing samples on a flow cytometer.Analysis and Statistics of Sample Data:Run the BD 6 Beads Analysis software and acquire the data. The software will makeexamples of standard curves. Enter the FL2 MFI of the samples then it will come tothe concentrations. The difference between control and samples was tested by apaired sample t-test.Results: The cytokines (IL-6,IL-10,INF-γ, and TNF-a) can be measured in most ofthe samples but IL-2 and IL-4,whose contents were too low to be measured. Thesamples added Tripterine show a significant increase of cytokines(IL-6,IL-10,TNF-a)secretion.(P＜0.05)Although the stimulating effects fluctuate as the content of Tripterine varied, its peak appears when the concentration of Tripterine is10^-8mmol/L.At this concentration, the contents of IL-6 and TNF-a are significantlyhigher than that of the control but the INF-γ.The CBA analysis between IL-6,IL-10 and TNF-a indicates that each pair of thethree cytokines has a significant correlation.Conclusions: This study indicates that Tripterine has various effects on the cytokinessecretion of whole blood cells at the concentration of 10^-8mmol/L which is just 1/100of the concentration when it induces apoptosis.Though both the Th1 and Th2cytokines are up-regulated, the stimulating effects is much more inclined toup-regulate the latter.This study also comes to a conclusion that a linear correlationbetween each pair of the three cytokines IL-6,IL-10 and TNF-a. It's possible that twoof the cytokines are regulated by the third, or the three cytokines are all regulated byanother still unknown component.