Targeting At LPS-TLR₄ Signaling Pathway To Evaluate The Anti-inflammatory Mechanism Of Jinying Huanggui Tang

Recognizing the source of stimulation by the receptors on the surface of the cell membrane and stimulus signal conducted into the cell played an important role in the activation of cells.Until now,the research about lipopolysaccharide（LPS）-Toll-like Receptor 4（TLR4）（LPS-TLR4）signalling pathways in extracellular mouse mammary epithelial cells（MECs）in vitro has been reported less.Some importent problems such as stimulation condition of LPS,presence of CD₁₄,MD-2 in MECs,the role of them on LPS-TLR4 signaling pathway in extracellular MECs and influence of Chinese herbal about them urgent need to solve.So,this issue cultured cells in vitro and MTT assay was used to examine the median lethal dose and determine the stimulating time of LPS on mouse MECs.The optimal stimulation time and concentration with LPS binding to cells were determined by flow cytometry.CD₁₄、MD-2 were designed and the genes’ expression were detected by the multiplex PCR on mouse MECs.The expression of the protein of CD₁₄、MD-2 were detected by flow cytometry.The cells binding rate of LPS were determinated by flow cytometry after CD₁₄、MD-2 gene transfection on mouse MECs.The changes of TNF-α、IL-6 and IL-1β secreted by LPS-stimulated mouse MECs after CD₁₄、MD-2 gene transfection were texted by ELISA.Effect of gene expression of CD₁₄、MD-2 by JYT and main components by qPCR.Result showing that:（1）With the increase of LPS concentration（begin with 1μg/mL）,the OD value of living cells in LPS groups were lower than that in control groups,and compared with control groups,the OD value in 50μg/mL LPS group extremely significantly reduced（P<0.01）.After calculation,we could know that the IC50 of LPS on mouse MECs was 76.83 μg/mL.Compared with 1 hour,the cell activity of LPS and control groups were extremely significantly enhanced after 6-48 hours stimulated by LPS（P<0.01）,as a logarithmic relationship.There was no difference between LPS group and blank control group in the whole process（P>0.05）.The detection result by flow cytometry showing that the cells binding rate of LPS with concentration and stimulation time of LPS as a logarithmic relationship.When the concentration of LPS is less than 5μg/mL,the cells binding rate of LPS increased rapidly and slow growth of the rate after the certain concentration of LPS,as like saturation.Similar conditions are to be found in hours stimulated by LPS.When the hours stimulated by LPS is less than 6hours,the cells binding rate with LPS increases rapidly and slow growth of the rate after the certain hours stimulated by LPS.（2）Gel PCR and protein detection results showed that there were the expression of the mRNA of CD₁₄、MD-2 in MECs and there was a certain amount of MD-2 protein expression in the cell membrane,but the expression of CD₁₄ protein was little.（3）The mRNA expression of CD₁₄-mus-905 and MD-2-mus-234 siRNA could respectively significantly inhibit the expression of CD₁₄ and MD-2 in 50 nM、30 nM、20 nM（P<0.01）,in which CD₁₄-mus-905 gene transfection at concentration of 50 nM in the highest inhibition rate,while the MD-2-mus-234 fragment in the concentration of 30 nM transfected with the highest inhibition rate.After gene silencing of CD₁₄,the cells binding rate of LPS were extremely significantly reduced（P<0.01,declined 7.88%）,compared with LPS stimulation group,the TNF-α、IL-6 and IL-1β secreted by MECs were extremely significantly reduced（P<0.01）.After gene silencing of MD-2,the cells binding rate of LPS were extremely significantly reduced（P<0.01,declined 3.00%）,compared with LPS stimulation group,the TNF-α、IL-6 and IL-1β secreted by MECs were significantly reduced（P<0.05）.After gene silencing of CD₁₄ and MD-2,the cells binding rate of LPS were extremely significantly reduced（P<0.01,declined 13.41%）,compared with LPS stimulation group,the TNF-a、IL-6 and IL-1β secreted by MECs were extremely significantly reduced（P<0.01）.（4）The expression of CD₁₄ and MD-2 mRNA in the low（40 μg·mL-1）、middle（400 μg·mL-1）and high（4000 μg·mL-1）dose group of JYT、the low（20μg·mL-1）.middle（200 μg·mL-1）and high（2000 μg·mL-1）dose group of chlorogenic acid（CGA）and the low（20 μg·mL-1）、middle（200 μg·mL-1）and high（2000 μg·mL-1）dose group of b-D-Glucopyranoside were extremely significantly reduced（P<0.05 or P<0.01）.Results suggesting that:the effect of LPS on mouse MECs IC50 was 76.83 μg/mL.When the concentration of LPS>5 μg/mL,stimulation time>6h,the cells binding rate of LPS reached saturation,this condition was the optimum conditions of LPS stimulated MECs.The mRNA of CD₁₄ and MD-2 could expresed in mice MECs,and there was a certain amount of MD-2 protein expresed in the cell membrane,and the expression of CD₁₄ protein was little.After CD₁₄ and MD-2 gene silencing,the binding rate of LPS was significantly inhibited,and the secretion of TNF-α,IL-6and IL-1 were significantly decreased.The expression of CD₁₄ and MD-2 mRNA stimulated by LPS could be significantly or extremely significantly reduced by JYTand it’s main components,which may be the mechanism of anti-inflammation of it.The results of this study explored the mammary epithelial cells extracellular LPS to TLR4 signaling pathway,and revealed the mechanism of traditional Chinese medicine from molecular level,solved the problem which traditional Chinese medicine’s mechanism is not clear,and provided theoretical data for traditional Chinese medicine in the treatment of inflammation.