Protective Effects And Mechanism Of Luteolin On LPS-induced Injured IEC-6 Cell And Research Of Extraction And Enrichment Of Luteolin From Flos Populi

Luteolin is a natural flavonoid compound,which is widely found in the plantage.It has many pharmacological effects,such as anti-tumor,antioxidation,anti-inflammatory,antibacterial,hypolipidemic,protection of the nervous system and enhancement of immunity.It can protect cells and tissues from injury,but its mechanism of action is not yet clear.In this study,the viability of IEC-6 cell CCK8 was measured by CCK8,and the expression levels of gene and protein of TLR4,MyD88,NF-?B,IL-6,and TNF-?were measured by Real-time PCR,Western blot,ELISA and other molecular biology techniques.The protective mechanism of luteolin on LPS(lipopolysaccharides)induced IEC-6 cell injury model was discussed from the perspective of TLR4/MyD88/NF-?B signaling pathway.Ultrasonic extraction of luteolin in Flos populi and enrichment process was optimized for the first time.On the basis of single-factor test,the response surface method was used to design and optimize the ultrasonic extraction process of luteolin from Flos populi,and macroporous resins were used to study the luteolin enrichment process from Flos populi.The results of the study are as follows:1.Compared with the control group,the viability of IEC-6 cells in 0.1,0.5 and 1.0?g/mL luteolin groups were not significantly different(p>0.05),but the viability of IEC-6 cells in the2.0?g/mL luteolin group decreased significantly(p<0.05),the concentration of luteolin was set to 0.1,0.1,0.5 and 1.0?g/mL.Compared with the control group,there was no significant difference in the viability of IEC-6 cells between the 0.1 and 0.5?g/mL LPS groups(p>0.05),the viability of IEC-6 cells in the 1.0 and 2.0?g/mL LPS groups decreased significantly(p<0.05),the cells were treated with 1.0?g/mL LPS.Compared with the LPS group,0.5 and 1.0?g/mL luteolin significantly increased the viability of IEC-6 cells induced by LPS(p<0.05).Compared with the control group,the mRNA and protein levels of IL-6,TNF-?,TLR4,MyD88 and NF-?B of IEC-6 cells in the LPS group increased with different degrees(p<0.05).Compared with the LPS group,the mRNA and protein levels of IL-6,TNF-?,TLR4,MyD88 and NF-?B in the IEC-6 cells of 0.1,0.5 and 1.0?g/mL luteolin groups decreased significantly(p<0.05);2.The Box-Behnken model was used to optimize the ultrasonic extraction process of luteolin in Flos populi,and the optimal extraction conditions of luteolin were evaluated by response surface method;the extraction process and the extraction rate of luteolin were described with two polynomial model and response surface diagram respectively;the optimum extraction parameters obtained by the above results were as follows:the ethanol concentration was 63%,the extraction temperature was 68~?C,the particle size was 0.18-0.25 mm,the extraction time was 42 min,the liquid-solid ratio was 25 mL/g,the electrical acoustic intensity was 68.12 W/cm~2,and the extraction time was 2;3.On the basis of the results of ultrasonic extraction and optimization,the enrichment of twelve macroporous resins for luteolin was studied,and the results of static adsorption test showed that the SP-825 resin was the most appropriate,and the data were fitted to the Langmuir and Freundlich isothermal;in order to get the best separation process,we further studied the factors such as the rate of sampling,the loading of sample and the concentration of desorption solution,and the optimal adsorption conditions are as follows:the concentration of luteolin sample was16.48?g/ml,pH was 5.0,sample volume was 10 BV,flow rate was 2 BV/h,and temperature was25~?C;the optimal desorption parameters were:distilled water elution was 5 BV,20%ethanol elution was 5 BV,50%ethanol eluted 5 BV,flow rate was 2 BV/h.The results showed that luteolin could protect the viability of LPS induced IEC-6 cells and decreased the gene and protein expression level of TLR4,MyD88,NF-?B,IL-6 and TNF-?.The mechanism of luteolin protection of intestinal mucosal barrier was revealed from the regulation of TLR4/MyD88/NF-?B signaling pathway.The extraction process of luteolin in Flos populi was optimized by response surface method.The extraction rate of luteolin was increased to 8.89±0.34mg/g,and the enrichment process of luteolin was optimized by macroporous resin,and its content was increased by 3.67 times.The results shows theoretical and practical value in the extraction and separation of natural medicines.