Anti-inflammatory Effects Of Dandelion Extract In RAW264.7 Macrophages And Zebrafish Larvae

Objective:The anti-inflammatory impact of dandelion extract（DE）and its mechanism were investigated using lipopolysaccharide（LPS）-induced RAW264.7 macrophage and copper sulfate（CuSO₄）-induced inflammation model in zebrafish larvae.Methods:In this experiment,groupings were set up:control group,LPS group/CuSO₄ group,DE group.1.Extraction of dandelion and analysis of DE components by ultra-performance liquid chromatography（UPLC-QE-Orbitrap-MS）;2.In vitro culture of RAW264.7 macrophages,MTT method to detect the cytotoxicity of DE on RAW264.7 cells,inverted microscopy The changes of cell morphology were analyzed,and the amount of nitric oxide（NO）production in RAW264.7 cells was detected by enzyme standardization;3.Fluorescence microscopy was used to detect the fluorescence intensity of reactive oxygen species（ROS）and apoptosis in RAW264.7 cells,flow cytometry was used to analyze the RAW264.7 cell cycle,and quantitative reverse transcription-polymerase chain reaction（RT-q PCR）was used to detect the RAW264.7 cell M1 phenotypic gene inducible nitric oxide synthase（i NOS）,interleukin（IL）-6,IL-1βand M2 phenotypic gene IL-10,CD206 expression levels;4.Observation of the effect of DE on zebrafish larval survival,fluorescence microscopy for zebrafish larval reactive oxygen species（ROS）fluorescence intensity and neutrophil aggregation,RT-q PCR for zebrafish larval inflammatory genes The expression levels of i NOS,tumor necrosis factor（TNF）-α,IL-6,IL-10,etc.were examined by RT-q PCR.Results:1.UPLC-QE-Orbitrap-MS analysis yielded 406 possible DE components;2.MTT results showed that DE at concentrations below 75μg/m L had no toxic effect on RAW264.7 cells（P<0.05）,and further experiments showed that DE inhibited LPS-induced cell morphological changes,while reducing LPS-induced RAW264.7 DE inhibited LPS-induced reactive oxygen species（ROS）production and apoptosis in RAW264.7 cells（P<0.05）,and flow cytometry revealed that LPS stimulation for 24 h induced macrophage G0/G1phase block and DE relieved macrophage G0/G1 phase block（P<0.05）,whereas LPS stimulation for 6 h failed to induce macrophage G0/G1 phase block,but promoted G2/M phase proliferation（P<0.05）.RT-q PCR results showed that DE significantly decreased LPS-induced M1 phenotype（IL-1β,i NOS and IL-6）m RNA expression levels in RAW264.7 cells（P<0.05）,while DE significantly increased M2 phenotype（CD206,IL-10）m RNA expression levels（P<0.05）;4.In zebrafish larvae experiments,DE at concentrations below25μg/m L was not toxic to zebrafish larvae（P<0.05）,while DE was found to inhibit CuSO₄-induced ROS production and neutrophil aggregation in zebrafish larvae（P<0.05）,and RT-q PCR results showed that DE reduced the expression levels of m RNA of inflammatory genes（i NOS,TNF-α,IL-6 and IL-10）in zebrafish larvae（P<0.05）.Conclusion:DE reduces the LPS-induced inflammatory response by regulating the polarization and apoptosis of RAW264.7 cells,while DE reduces the CuSO₄-induced inflammatory response in zebrafish larvae.