Study On The Regulatory Effects Of Human Umbilical Cord Mesenchymal Stem Cell Exosomes On LPS-Induced Inflammatory Factors In RAW264.7 Cells

Objective:The mouse macrophage inflammation model was established by means of Lipopolysaccharide（LPS）induction in RAW264.7 cells to study the regulatory effects of human umbilical cord mesenchymal stem cells exosomes（Human Umbilical Cord Mesenchymal Stem Cells Exosomes,huMSCs-exo）on inflammatory factors in the LPS-induced RAW264.7 cellular inflammation model.Methods:Human umbilical cord mesenchymal stem cells were cultured in vitro,and the serum-free supernatant was collected.The exosomes in the supernatant were extracted and identified by differential centrifugation;Establish LPS-induced RAW264.7 cellular inflammation model,experimental grouping:The experiment was divided into 3 groups:I.RAW264.7 group（normal RAW264.7 cells）,II.LPS Group[RAW264.7+LPS（1μg/ml）],III.The EXO group[RAW264.7+LPS+huMSCs-exo（50μg/ml）].Tested the activity of RAW264.7 cells in groups 0h,24h,48h,and 72h through CCK8 cell proliferation experiments.Based on the CCK8 results,it was determined that the time point of subsequent experiments was 24h after addition to LPS.Each group of RAW264.7 cells was tested by an enzyme linked immunosorbent assay（ELISA）to test the proinflammatory factor tumour necrosis factorα（Interleukin 1β,IL-1β）,interleukin 1β（Interleukin 1beta,IL-1β）,interleukin 6（Interleukin 6,IL-6）,and the anti-inflammatory factor interleukin 4（interleukin 6,IL-6）,and the anti-inflammatory factor interleukin 4（interleukin 6,IL-6）,and the anti-inflammatory factor interleukin 4（interleukin 4）Expression level of Interleukin 4,IL-4);Real-time quantitative polymerase chain reaction（q PCR）detects TNF-α,IL-1β,IL-6,and IL-4 m RNA expression levels in various groups of cells;Use immunohistochemistry（IHC）to test the expression of TNF-α,IL-1β,IL-6,and IL-4in cells in each grpoup;Western Blot detects the protein expression levels of TNF-α,IL-1β,IL-6,and IL-4 in each group of cells.Results:1 huMSCs-exo extraction and identification results:（1）Observation results under transmission electron microscopy showed that the size is uniform,round or oval,with a low density light area visible in the center of the vesicle.The vesicle diameter is about 120 nm,which is in line with the morphological size characteristics of mesenchymal stem cell exosomes;（2）NTA tested the particle size and concentration of huMSCs-exo,and the results showed that the average concentration of huMSCs-exo in the undiluted original solution was 2.5×10¹¹Particles/ml,the peak value was 126.6 nm,which was consistent with the diameter of mesenchymal stem cell exosomes;（3）Western Blot protein gel electrophoresis tested for huMSCs-exo membrane protein markers CD9 and CD63.The results showed that the membrane protein markers CD9 and CD63 were expressed positively.The extracted vesicles met the basic characteristics of exosomes.According to the above results,it was proved that the extracted vesicles were huMSCs-exo.2 The results of CCK8 cell proliferation experiments showed that compared with0h,LPS group and EXO group RAW264.7 cells all increased significantly in 24h after adding LPS（P<0.05）,and cell activity decreased significantly in 48h and 72h（P<0.05）;compared with RAW264.7 group,cell activity of the LPS group increased at 24h（P<0.05）,and decreased significantly at 48h and 72h（P<0.05）;compared with the LPS group,EXO group 244.7h cells decreased significantly（P<0.05）.,48h,72h cells Activity increased significantly（P<0.05）,so 24h was selected as the time point for subsequent experiments.3 ELISA test results showed that compared with RAW264.7 group,the levels of TNF-α,IL-1β,IL-6 and IL-4 in the LPS group and EXO cell supernatant increased significantly（P<0.05）;compared with the LPS group,TNF-α,IL-1β,IL-6 levels in the EXO cell supernatant decreased significantly（P<0.05）,and IL-4 levels increased significantly（P<0.05）.4 qPCR test results showed that compared with RAW264.7 group,TNF-α,IL-1β,IL-6 and IL-4 m RNA expression levels in the LPS group and EXO group cells increased significantly（P<0.05）;compared with the LPS group,TNF-α,IL-1β,and IL-6 m RNA expression levels in EXO group cells decreased significantly（P<0.05）,and IL-4 m RNA expression levels increased significantly（P<0.05）.5 Cellular immunohistochemical staining results showed that very few brown and brownish-yellow particles were seen in the nuclei and cytoplasm of RAW264.7cells,and a large number of brown and brownish yellow particles were seen in the nuclei and cytoplasm of the LPS group and EXO group cells;compared with the RAW264.7 group,the LPS group,the TNF-α,IL-1β,IL-1β,IL-6 and IL-4 positive particles were higher than the RAW264.7 group;compared with the LPS group,the EXO group 24h RAW264.7 cells and nuclei were found in the nuclei and cytoplasm of the LPS group,the EXO group TNF-α,IL-1β,IL-6 in matter There were fewer positive particles of compared to the LPS group,and there were more positive particles of IL-4 compared to the LPS group.6 Western Blot results showed that the expression levels of TNF-α,IL-1β,IL-6and IL-4 proteins in the LPS group and EXO group cells increased significantly（P<0.05）;compared with the LPS group,the expression levels of TNF-α,IL-1β,and IL-6 proteins in EXO cells decreased significantly（P<0.05）,and IL-4 protein expression levels increased significantly（P<0.05）.Conclusions:1 huMSCs-exo can improve the cellular activity of RAW264.7 cells after LPS induction and increase the proliferative activity of RAW264.7;2 huMSCs-exo can regulate the inflammatory effect,down-regulating the expression of anti-inflammatory factors TNF-α,IL-1βand IL-6,up-regulating the expression of anti-inflammatory cytokine IL-4,and alleviating the inflammatory effect of RAW264.7 cells induced by LPS.