Research Daphnoretin Anti-inflammatory And Transport Mechanisms

Backgrounds: Arthritis is an inflammation that occurs in human joints and tissues around the joints,There are many factors that cause arthritis,which may be inflammation,infection,trauma,etc.Arthritis is characterized by redness,heat and pain in the joints.According to statistics,the prevalence rate of arthritis in people over 50 years old in China is above 50%.Arthritis has a great impact on the normal life of patients.Disability,etc.Daphnoretin,the chemical name is7-hydroxy-6-methoxy-3,7'-dicoumarin,also known as Daphnoretin,is found in many plants of the family Leguminosae,Rutaceae and Rutaceae.It has a rich content and can be obtained from the extraction and purification process.In traditional medicine,the use of medicinal materials containing daphnoretin,such as Wikstroemia indica and Zushima,are used to treat arthritis and other inflammations.According to the reports in literature,daphnoretin also has anti-tumor,analgesic,antibacterial,anti-anxiety,anti-viral and other effects,so this subject takes daphnoretin as the main research object,to improve the practicability of the daphnoretin.Objective: To verify the anti-inflammatory effects of daphnoretin by measuring the effects of cytokines on the cytokines by THP-1 cell inflammatory model.A Tanswell-simulated small intestine transport model of Caco-2 cells was established to compare the absorption of daphnoretin in the transport model of small intestine in the presence of energy inhibitors and transporter inhibitors.To study the absorption mechanism of daphnoretin in human intestinal tract,to provide a theoretical basis for the future pharmacokinetics and clinical application of daphnoretin.Methods: In the first part of the anti-inflammatory experiment,The effect of different concentrations of daphnoretin on the activity of THP-1 cells was detected by CCK-8 method.The amount of each cytokine secreted by LPS(lip opolysaccharide)-induced THP-1 cells in the THP-1 cell inflammation model wa s examined by ELISA experiment,The effect of daphnoretin on inflammatory cells after treatment with cytokine secretion.In the second part of the study on transport mechanisms,The classical MTT assay was used to detect the toxicity of daphnoretin to Caco-2 cells,and the concentration interval of the experiment was determined.The Transwell transport cell model was established by Caco-2(colon adenocarcinoma),the growth degree of single cell membrane was detected by cell resistance meter,and the differentiation of Caco-2 single cell membrane was observed by detecting the concentration of AKP(alkaline phosphatase)secreted by cells,verification of cell monolayer integrity by phenol red assay.Using the established canal transport model of Caco-2 cells,combined with energy inhibitors and transporter inhibitors,the content of daphnoretin in the upper and lower chambers of Transwell transport cell model was determined by HPLC(high performance liquid phase)method.To study the transport mechanism of daphnoretin absorption through human intestinal epithelial cells.Results: CCK-8 method was used to detect the effect of different concentrations of daphnoretin on the activity of THP-1 cells.The results showed that the secretion of TNF-? inflammatory factors was significantly inhibited when the concentration of daphnoretin was higher than 10?g/ml(P<0.05),at concentrations above 1?g/ml,it can significantly inhibit the secretion of MCP-1,secretion of IL-1? inflammatory factors(P<0.05),50?g/ml,20?g/ml,10?g/ml three concentrations of daphnoretin it has an effect on the secretion of IL-6 inflammatory factors.The secretion level is obviously affected by the concentration of sirrofollin.The P value is less than 0.05,However,when the action time was 12 h,the concentration of daphnoretin was 10?g/ml,which was not significant compared with the LPS group.The cytotoxicity of daphnoretin to Caco-2 was determined by MTT assay.The results showed that the toxicity of Caco-2 was negligible when the concentration of daphnoretin solution was below 20?g/ml.After establishing the Caco-2 cell transport model,the transport mechanism of daphnoretin was studied by different transporter inhibitors.The experimental results show that after the addition of energy inhibitors,the transport AP-BL and BL-AP of daphnoretin are significantly affected(P<0.05).After the addition of P-gp protein inhibitor,daphnoretin's transport has not been significantly affected(P>0.05).After the addition of MRP2 protein inhibitor,the Papp of daphnoretin AP-BL was increased by 7.1%,and the direction of BL-AP was not significantly affected.It is suggested that MRP2 protein may be involved in the transport of daphnoretin.There was no significant difference in AP-BL direction transport and daphnoretin group after adding BCRP protein inhibitor(P>0.05),but the apparent permeability coefficient of BL-AP transport was 23.8% lower than that of daphnoretin group,and the PAB/PBA is 0.75,indicating the participation of BCRP protein in the transport of daphnoretin.Conclusion: It was found that daphnoretin has a strong inhibitory effect on inflammatory factors secreted by cells,but has no significant effect on THP-1 cell viability and cell morphology.The Transwell model of Caco-2 cells established in this experiment can be used to study the absorption and metabolism of daphnoretin in human cells,which lays a solid foundation for the further study of daphnoretin.