Influence And Mechanism Of Macrophage Polarization By Different Mechanical Stimulus

Objective: Macrophages were stimulated by dynamic mechanic(different amplitude cyclic tensile strain)and static mechanic(different elastic modulus)to research the effect and mechanism of mechanical stimulation on the polarization of macrophages.Methods: Cyclic tensile strain dishes and elastic modulus hydrogels were prepared for experiments.Part1: Macrophages from mice were seeded on tensile strain dishes and stimulated with 2%,5% and 12% amplitude cyclic tensile strain 1)Cell growth was detected by live/dead staining and cell count.2).The expression of macrophage cell surface markers was analyzed by flow cytometry assay(FCA).The reactive oxygen species(ROS)detection kit was used to detect the levels of ROS in macrophages using fluorescent probe DCFH-DA.ELISA kit was utilized to examine the pro-inflammatory and anti-inflammatory cytokines in the supernatant which were secreted by mechanical stimulated macrophages.The expressions of pro-inflammatory and anti-inflammation genes were detected by real-time quantitative PCR(RT-PCR)3).Western Blot(WB)was applied to determine the effects of different amplitude cyclic tensile strain on activation of NF-κB signaling in macrophages.Part2: Macrophages from mice were seeded on the different elastic modulus hydrogels.1)Cell growth was detected by live/dead staining,CCK-8 assay.Scanning electron microscopy(SEM)used to observe the morphological characteristics of the cells on different hydrogel elastic modulus.2).The expression of macrophage cell surface markers was analyzed by FCA.The reactive oxygen species(ROS)detection kit was used to detect the levels of ROS in macrophages using fluorescent probe DCFH-DA.ELISA kit was utilized to examine the inflammatory and anti-inflammatory-related cytokine which were secreted by different elastic modulus stimulated macrophages.The expressions of pro-inflammatory and anti-inflammation related genes were detected by real-time quantitative PCR(RT-PCR)3).WB was applied to determine the effects of different elastic moduli on activation of NF-κB signaling in macrophages.Result:Part 11)Macrophages stimulated by different amplitude cyclic tensile strain,the results of live/dead staining showed that the cell treated with 2% and 5% cyclic tensile strain exhibited excellent cell growth same as the ones without cyclic tensile strain,however the 12% cyclic tensile strain induced cell death.2)5% amplitude cyclic tensile strain significantly increased the ratio of CD206/CD86,meanwhile,the level of reactive oxygen species(ROS)in cells decreased significantly,which indicated that macrophages were polarized toward M2.The secretion of inflammatory cytokines and the expression of inflammatory genes were significantly down-regulated,anti-inflammatory cytokines and anti-inflammatory genes were significantly increased;3)5% amplitude cyclic tensile strain decrease the expression of related protein of NF-κB signaling in macrophages,indicating that the activation of NF-κB signaling pathway was inhibited.Part 2: 1)live/dead staining and CCK-8 experiments showed that different elastic modulus hydrogels did not affect cell adhesion and proliferation.2).Macrophages were inoculated onto different elastic modulus hydrogels.With the increase of elastic modulus,the expression of CD86/CD206 on macrophages became lower and lower.The surface of macrophages in the low modulus group was CD206 molecular expression of the lowest,the lowest expression of reactive oxygen species in the cells,indicating that the low modulus group of macrophages polarized to the M1 direction;Macrophages inflammatory cytokines and genes were also downregulated with the increase of elastic modulus.The expression of NF-κB in macrophages of low modulus group was the highest,which indicated that the low modulus could promote the activation of NF-κB signaling pathway in macrophage.Conclusion: Appropriate amplitude cyclic tensile strain could inhibit the activation of NF-κB signaling pathway and causes macrophages to M2.Low modulus of elasticity stimulates macrophages to M1 by activating NF-κB signaling pathway.