Screening And Biological Effects Of MyD88 Related Anti-inflammatory Active Components In Qianjin Wenwu Decoction

Objective:From the original components of Qianjin Wenwu Decoction into blood,multi-chamber electrophoresis combined with LC-MS/MS technology was used to screen the components with anti-inflammatory activity combined with myeloid differentiation factor 88(MyD88);The anti-inflammatory effect was evaluated in a high glucose-induced human renal tubular epithelial cell(HK-2)model,and its mechanism was explored.Method:1.Establish a multi-chamber electrophoresis technology system,optimize the relevant parameters of the multi-chamber electrophoresis screening technology,and screen the active components in the blood components of Qianjin Wenwu Decoction that bind to the target protein MyD88:(1)Optimize the technical parameters of multi-chamber electrophoresis screening(buffer p H,buffer flow rate,electrophoresis time);(2)Using MyD88 as the target protein to screen the multicomponents that bind to it from the test sample;(3)Optimize the liquid phase method of the sample to be tested,and establish a LC-MS/MS MRM mode detection method to qualitatively and quantitatively analyze the active components after screening by multi-chamber electrophoresis technology.2.To explore the anti-inflammatory effect of the blood components of Qianjin Wenwu Decoction and the binding components of the target protein MyD88: HK-2 cells were used as experimental cells,high glucose induced modeling,group administration,and CCK8 kit was used to detect cell activity to determine the administration concentration,and Annexin V-FITC/PI kit was used to detect the level of apoptosis to further validation of the anti-inflammatory effects of active ingredients..3.Based on the TLR4/MyD88/NF-κB signaling pathway to explore the molecular mechanism of the effect of the combined medicinal components: The HK-2 cells were cultured in groups,and the sample RNA was extracted.The relative expression levels of TLR4,MyD88,and NF-κB m RNA genes in the pathway were detected by real-time fluorescence quantitative PCR technology.The relative expression levels of TLR4,MyD88 and NF-κB proteins in the pathway were detected by Western blot.Results:1.Multi-chamber electrophoresis screening technical parameters:The 9prototype blood components of Qianjin Wenwu Decoction and MyD88 protein were incubated in a water bath at 37°C for 30 min,using 10 m M ammonium acetate with p H6.21 as a buffer,the flow rate of the buffer was 4.0 m L/min,and the electrophoresis time was 20 min.,the target protein MyD88 migration rate was 55.04%(RSD=4.4086%).2.Based on the multi-chamber electrophoresis screening technology,3 components that bind to the target protein were screened from Qianjin Wenwu Decoction transmigrated into the blood.The established LC-MS/MS MRM mode was used to characterize the three active ingredients,namely: wogonin,baicalin,and wogonoside.At the same time,the three components were quantified by MRM mode,and the molar ratio was determined to be 4:2:1.3.After the cells were modeled and administered,it was found that When the three active ingredients were administered in the ratio of wogonin: baicalin:wogonoside =4:2:1,the cell survival rate at 28 μM was significantly higher than that of the three ingredients alone,which was 136.17%.4.In the apoptosis experiment,it was found that when the administration ratio of wogonin: baicalin: wogonoside =4:2:1,the effect of inhibiting the apoptosis of HK-2 cells induced by high glucose was more significant.5.The active components of Qianjin Wenwu Decoction that bind to the target protein MyD88 can inhibit the relative expression of TLR4,MyD88 and NF-κB m RNA genes and proteins in the intracellular TLR4/MyD88/NF-κB signaling pathway.Conclusion: The active components of Qianjin Wenwu Decoction that bind to the target protein MyD88 and have anti-inflammatory effects are wogonin,baicalin and wogonoside,and the molar ratio is 4:2:1.After the three components are combined in proportion,there is a significant effect;Its mechanism of action may be related to the activation of TLR4/MyD88/NF-κB signaling pathway and the regulation of TLR4,MyD88,NF-κB gene and protein expression.