Study On Anti-inflammatory Effects And Mechanism Of Dioscorea Nipponica Makino Total Saponins In Vitro And In Vivo

Dioscorea nipponica Makino is the dried rhizome of Dioscorea nipponica,family Dioscoreaceae,which is widely available and relatively cheap.,and has pharmacological effects such as anti-inflammatory,hypoglycemic,regulating immune function,improving cardiovascular function and hepatoprotective,etc.Its main components are saponins,flavonoids and phenanthrenes.The previous research showed that saponins have good anti-inflammatory activity.Inflammation can be induced and produced by bacteria,viruses and parasites,which affects the health of livestock and poultry and can even lead to death,reducing the economic benefits of breeding enterprises and affecting the sustainable development of the livestock industry.At present,steroidal anti-inflammatory drugs,non-steroidal anti-inflammatory drugs and Chinese medicine are mainly applied clinically for the prevention and treatment of inflammation.Chinese medicine has the advantages of low toxicity,small adverse reactions and many pharmacological targets,which has more ideal development and application prospects.In this experiment,the total saponins of DNM were taken as the research object,and the extraction process,chemical composition,anti-inflammatory effect and mechanism in vitro and in vivo were investigated to provide certain research ideas and methods for the research and development of veterinary clinical anti-inflammatory drugs.In this study,single-factor and response surface tests were used to optimize the optimal ultrasonic extraction process of Dioscorea nipponica Makino total saponins;the main chemical components of Dioscorea nipponica Makino total saponins were initially analyzed by ultra-high liquid chromatography coupled with quadrupole tandem time-of-flight mass spectrometry(UPLC-QTOF-MS);lipopolysaccharide(LPS)-induced RAW 264.7 cells and acute lung injury(ALI)mouse models were used for enzyme-linked immunosorbent assay(ELISA)to determine the levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-10(IL-10)and interferon β(IFN-β)in cell supernatant and mouse lung lavage fluid;the Griess method,neutral red phagocytosis assay and HE staining were used to detect the levels of inflammatory mediators NO phagocytosis and lung tissue damage,respectively,to reveal the anti-inflammatory activity of Dioscorea nipponica Makino total saponin in vitro and in vivo;real-time fluorescence quantitative PCR(RT-PCR)and immunoblotting(WB)were also used to detect the m RNA and protein expression levels of TLR4,TRAF6,p65,p-p65,IκB,p-IκB and TRIF in My D88-dependent and non-dependent signal transduction pathways,to reveal the anti-inflammatory mechanism of D.nipponica total saponins.In this study,the optimal extraction process for the ultrasonic extraction of Dioscorea nipponica Makino total saponin was ethanol concentration of 50%,extraction time of 25 min,liquid to material ratio of 55:1(m L/g),and the content of Dioscorea nipponica Makino total saponin was 1.72% under these conditions;18 chemical components in Dioscorea nipponica Makino total saponin were initially inferred;in vitro and in vivo anti-inflammatory the results showed that Dioscorea nipponica Makino total saponin significantly inhibited the phagocytic activity of RAW264.7 cells,reduced the wet/dry weight ratio of lung in ALI mice,was able to siginificantly reduce the levels of pro-inflammatory cytokines TNF-α,IL-6,IFN-β and inflammatory mediator NO,promoted the secretion of anti-inflammatory cytokine IL-10,and dose-dependently down-regulated the TRAF6,TLR4 and TRIF m RNA and protein expression levels,and inhibited the phosphorylation of p65 and IκB proteins,revealing that Dioscorea nipponica Makino total saponin could exert anti-inflammatory effects through mediating My D88-dependent and non-dependent signaling pathways.In summary,this experiment successfully optimized the best extraction process of Dioscorea nipponica Makino total saponin and clarified its main chemical components;it also clarified that Dioscorea nipponica Makino total saponin has good anti-inflammatory effects in vitro and in vivo;and further revealed that Dioscorea nipponica Makino total saponin can exert anti-inflammatory effects through mediating My D88-dependent and non-dependent signaling pathways,providing an important theoretical basis for the research and development of veterinary clinical anti-inflammatory Chinese medicine.