Effects Of Curcumin On Cultured Human Cells Circulating Fiber Migration

Fibrosis is a pathological status characterized by distorted matrix deposition in ECM in the presence of various internal or external pathogenic factors including injury, infection, inflammation, hemodynamic disorder and immunization. Fibrosis is a reaction to damage by organism. Fibrosis could lead to severe damage of tissues and organs, even life-threatening diseases. It is widely acknowledged that (myo)fibroblasts are the main components of extracellular matrix. Circulating fiobrocytes were discovered by Bucala in1994, and recent studies confirm that circulating fiobrocytes may play important roles in the formation and development of fibrosis. Derived from bone marrow, circulating fibrocytes exist in the peripheral circulation and could migrate with the peripheral blood to wound chambers in response to chemokines. Activated circulating fibrocytes could not only secrete extracellular matrix that are essential for the process of fibrosis, but also lead to physiological or pathological fibrosis by differentiated to (myo)fibroblasts. Curcumin, a polyphenolic compound isolated from turmeric (Zingiberaceae), has been shown to exhibit the anti-fibrosis function in various organs.ObjectiveThe purpose of the research is to optimize the technique of culture and identification of human circulating fibrocytes from human peripheral blood and to explore their viability, cytoactive, differentiation and migration pattern. The effects and mechanism of curcumin on circulating fibrocytes have also been studied in the research. The results will provide foundations for the future in vivo study of renal interstitial fibrosis including recruitment and cytoactive of circulating fibrocytes and the effect of curcumin.Methods1. The culture and identification of human circulating fibrocytes The cells were isolated and purified by density gradient centrifugation, identified by flow cytometry and immunocytochemistry and observed at different culturing times by inverted microscope.2. The effect of curcumin on the cytoactive of human circulating fibrocytes CCK-8testing, flow cytometry and ELIS A were used to study the effect of curcumin on cell viability, cell surface phenotype (COL I, CD34, CD45, CCR7, a-SMA) expression and TGF-β1secretion of human circulating fibrocytes respectively.3. The effect of curcumin on the migration of human circulating fibrocytes The experiment was used to explore CCL21/CCR7-mediated recruitment of human circulating fibrocyte and the influence of curcumin by transwell. Results1. The results of immunocytochemistry showed that COL I, CD45and CCR7strongly coexpressed in human circulating fibrocytes while CD34expression was weaker. Flow cytometry indicated that approximately99.8%of the human circulating fibrocytes were COL I positive,77.8%were CD45positive and only1.0%were CD34positive.2. Curcumin could exert regulatory effects on viability of human circulating fibrocytes. Exposure to curcumin for as short as24hours promoted cell growth while prolonged treatment (72h) significantly inhibited cell viability, and this inhibition was elevated with the increase of curcumin concentration. When treated for48hours, curcumin at lower doses (1μmol/L,3μmol/L) could improve cell viability, but higher doses(10μmol/L,20μmol/L) acted as a brake.3. Curcumin blocked the expression of cell surface phenotype (COL I, CCR7, a-SMA) and these inhibitory action were exhibited in a dose-dependent manner. Positive cells manifested at a concentration of curcumin as high as20umol·L-1.4. Curcumin induced a slight increase in CD34and CD45expressions. The ratios of cells (CD34+or CD45+) were increased with the increase of curcumin concentration.5. Curcumin reduced the TGF-β1secretion of human circulating fibrocytes. The effect was descended along with the elevation of curcumin concentration.6. CCL21promoted the migration of human circulating fibrocytes and such effect was blocked by curcumin in a dose-dependent manner. Cells migration decreased significantly after treated for72hours with curcumin. And the effect reached the highest level at doses of10or20μmol/L curcumin.Conclusion1.77.8%of cells were both CD45and COL I positive, typical of human circulating fibrocytes.2. Curcumin could mediate the viability of human circulating fibrocytes and block the transdifferentiation of circulating fibrocytes into (myo)fibroblasts.3. Curcumin showed the capacity of anti-fibrosis by inhibiting the secretion of fibrogenic factor TGF-β1secreted by human circulating fibrocytes.4. Strongly expressing chemokine receptor CCR7, human circulating fibrocytes were recruited by CCL21. The result showed that CCR7/CCL21could induce cells migration.5. Curcumin could hold the migration of human circulating fibrocytes via blocking the signal pathsway, chiefly, via reducing CCR7expression.