Based On Exosome-mediated CCL11 Regulation Of Intercellular Signal Transmission In Lung And Intestinal Tissue

Objective:The effects of exosomes from lung structure cells on inflammation of intestinal structure cells and related signaling pathways were studied to explore the mediating role of exosomes in disease transmission,and to provide a new idea for the study of the theory of viscera and the relationship between exterior and interior in traditional Chinese medicine.Methods:The in vitro experiments were investigated that exosomes derived from lung and intestinal structure cells on the functions of intestinal epithelial cells and lung epithelial cells after inflammatory stimulation,and to reveal the mechanism of the CCL11-mediated pathway of exosomes in lung disease and the transmission of intestinal disease and lung disease.First,two groups of lung epithelial cells(A549)and intestinal epithelial cells(Caco-2)were cultured in exosomal-free medium.After stimulating the model group with recombinant human interleukin IL4/IL13,exosome secretion was induced.Exosomes were isolated and extracted by high-speed centrifugation method.Morphological structure of exosomes was observed by transmission electron microscopy,particle size distribution and particle concentration of exosomes were detected by Nanosight,and expression of exosome marker proteins(CD9,CD63,TSG101)was detected by Western blot,and the extracted exosomes were identified.At the same time,the expression of CCL11 in exosome carriers was detected by ELISA and Western blot in order to investigate the changes of CCL11 expression in lung and intestinal tissue cells after inflammatory stimulation.Finally,the obtained lung cell-derived exosomes were co-cultured with intestinal epithelial cells,and the intestinal cell-derived exosomes were co-cultured with lung epithelial cells.Protein-chip technology was used to detect the expression of key nodal proteins in downstream Jak/Stat and PI3K/Akt inflammation-related signaling pathways.To explore the regulation effect of exosome-mediated CCL11 signaling pathway on inflammatory signaling pathways in lung and intestinal epithelial cells,and to reveal the mediating mechanism of exosomal disease transmission between lung and intestinal tissues.RESULTS:The particulate matter extracted by differential and high-speed centrifugal method was identified as exosomes by transmission electron microscopy,nanosight and Western blot.Compared with the blank group,more exosomes were extracted from the model group,indicating that the addition of inflammatory factors could effectively promote the secretion of exosomes.At the same time,the ELISA results showed that the content of CCL11 in the model group was higher than that in the blank group,indicating that the addition of inflammatory factors could promote the secretion of CCL11.Western blot results showed that compared with the blank group,CCL11 was carried on the exosomes of the model group,indicating that the addition of inflammatory stimulation could promote the secretion of cell exosomes and CCL11,and exosomes could be the carrier of CCL11.Protein chip results showed that,after co-culture,compared with the blank group,the A549-derived exosomes in the model group could regulate the expression of Akt and JakStat pathways through the regulation of AMPKA,4E-BP1,STAT1,STAT5,Src and SHP1 in the Akt and JakStat pathways in Caco-2 by CCL11,thus regulating the expression of Akt and JakStat pathways.Caco-2-derived exosomes can regulate the expression of Akt and JakStat pathways by regulating the nodal proteins 4E-BP1,RSK2,ERK1/2,Src and SHP2 in A549 by CCL11,thus regulating the expression of Akt and JakStat pathways.Conclusion:Exosomes carrying CCL11 can be used as an important signal communication medium between cells.Exosome CCL11/CCR3 signaling pathway may be used as one of the molecular basis in the lung and large intestine surface to mediate disease transmission between lung and intestinal tissues.