Interaction Between Macrophages And Myoblasts Under Debris Intervention Myotubes

Objective: To treat RAW264.7 macrophages in vitro using myotube cell debris and establish a co-culture system of macrophages and myoblasts through Transwell.We study the effect of muscle cell debris on macrophage polarization after skeletal muscle injury and macrophage polarization.The co-existence of phagocytes and myoblasts on myoblast differentiation and macrophage polarization provides a theoretical guide for the mechanism of muscle cell repair and remodeling after skeletal muscle injury.Methods: Mouse C2C12 myoblasts and RAW264.7 macrophages were used as research objects;(1)Macrophages were treated with myotube cell debris for different lengths of time(4h,4h × 2,24h).Cell morphology,CCK-8 Detection of cell viability,flow cytometry detection of F4 / 80,CD86,CD206 cells polarized marker proteins explore the best time to deal with debris.(2)Fragmented macrophages and myoblasts were divided into three groups for co-culture,group C: control group;CM group:C2C12 + macrophage group;CMP group: C2C12 + cell debris treated macrophage group(Prefabricated myotube fragments were intervened for 4h at a ratio of 5: 1);Cell morphology was observed during co-culture,CCK-8 was used to detect cell proliferation in each group after 5 days of co-culture,Myogenin staining was used to count myotube area,and Western Blotting was used to detect C2C12Myoblast-related proteins MyoD and Myogenin are expressed.Macrophages are divided into 4 groups for co-culture,M group: control group;Mp group: macrophage group treated with cell debris;MC group: macrophage + C2C12 group;Mp C group:macrophage treated with cell debris Cell group + C2C12 cells;After 2 days of co-culture,cells were collected for Western Blotting to detect the expression of inflammatory factors IL-1β,IL-10 protein,M1M2 type marker protein iNOS,Arg-1protein expression and Akt phosphorylation level.Results:(1)After treatment of macrophages by myotube cell debris for 4h,4h × 2,and 24 h,observe the cell morphology and flow cytometry to detect the macrophage polarization marker protein.The cell morphology is most activated in the 4h group,M1 type polarization The degree of activation of the landmark protein was the most significant(P <0.05 vs C group).(2)By co-culturing macrophages phagocytic myofibroblast cell debris with C2C12 cells,microscopic observation and immunofluorescence Myogenin staining revealed that the myotube area and degree of fusion of CM and CMP groups co-cultured with macrophages increased significantly,CCK-8 detection and Western Blotting results showed that after co-culture with myofibroblast-activated macrophages,C2C12 cell proliferation significantly increased(P <0.05 vs group C),and MyoD and Myogenin protein expressions significantly increased(P <0.05 vs group C).(3)After co-culture with C2C12 cells,the secretion of macrophage pro-inflammatory factor IL-1β and anti-inflammatory factor IL-10 was significantly increased(P <0.05 vs M group).INOS was used as a M1 macrophage marker protein.The expression was significantly increased(P <0.05 vs M group),while Akt phosphorylation was not increased.Conclusion:(1)After 4h of myotube cell debris intervention,the ratio of macrophage polarization to M1 type reached a peak.(2)Activated macrophages can promote the proliferation and differentiation of myoblasts.At the same time,myoblasts promote the activation of macrophages to continue to polarize to M1 type.Together,the two can promote the regeneration and remodeling of skeletal muscle cells.