Study On The Polarization Of Bone Marrow-derived Macrophages Modulated By 810nm Low-level Laser Therapy And Its Mechanism

Spinal cord injury(SCI)is a serious trauma to the central nervous system.Once it occurs,it often results in permanent disability for the patient,putting a heavy burden on families and society.Secondary inflammatory responses play an important role in the course of spinal cord injury,and a series of pathological reactions triggered by it severely hinder the progress of injury repair.Among the numerous cells involved in the secondary inflammatory response,bone marrow-derived macrophages(BMDM)play a key role: occupying the central area of injury,continuing inflammatory secretion,and ultimately leading to irreversible tissue necrosis and apoptosis.Macrophages have polarization characteristics.Under different stimuli,macrophages can be polarized into cytotoxic M1 cells or M2 macrophages with anti-inflammatory and repair-promoting effects,exerting different biological effects.In the course of spinal cord injury,the cytotoxicity M1 macrophages predominate,while M2 cells only transiently increase.This special polarization state further leads to irreversible damage to the spinal cord.A large number of previous studies have shown that by changing the balance of macrophage polarization during spinal cord injury,inhibiting the dominance of M1 cells can effectively promote the repair of spinal cord injury.Low-level laser therapy(LLLT)refers to the use of low-irradiation laser’s biological effects,producing a non-invasive treatment of anti-inflammation and repair.It has been widely used in many clinical fields.A large number of previous studies have shown that 810 nm LLLT can significantly improve the functional recovery of spinal cord injured animals,promote neuron survival,and inhibit the secretion of inflammatory factors such as TNF-α and IL-6.Related studies also found that 810 nm LLLT can significantly decrease the number of M1 macrophages in the injured area and upregulate the proportion of M2 macrophages.However,whether the LLLT can directly affect BMDM,the key role during the course of spinal cord injury,regulate its polarization phenotype,and change its secretion status,these problems are not yet clear.To address the above issues,the following two experimental studies were conducted.Experiment 1: Effect of Low-Level Laser Therapy on Viability and Polarization of M1 Bone Marrow-Derived MacrophagesObjective: To study the effect of low-level laser therapy on viability and polarization of M1 bone marrow-derived macrophagesMethods: Primary bone marrow-derived macrophages were extracted from Balb/c mice and stimulated with 100 ng/ml LPS and 20ng/m L IFN-γ.Polarization was observed by flow cytometry to obtain mature M1 cells.The M1 cells were randomly divided into irradiation group and control group: the irradiation group used fixed power density parameters(2m W/cm2)and irradiation area(4.5cm2),and obtained 0.4J(44s)and 4J(440s)And 10J(1111s)three energy groups through different irradiation durations.;control group was placed in a dark box without irradiation.2 and 24 hours after irradiation,cell viability was measured using the CCK-8 method.6 hours after irradiation,m RNA expression of i NOS,the surface marker of M1 macrophage,was detected by RT-q PCR,and 24 hours after irradiation,the protein expression of i NOS was detected by western blot and immunofluorescence staining.Results: There was no significant change in cell viability between the groups at 2 h after irradiation.At 24 h after irradiation,the viability of M1 BMDM in the 4J group was significantly higher than that in the control and other irradiation groups.6 hours after laser irradiation,the expression of i NOS m RNA in the irradiation group was significantly lower than that in the control group.At 24 hours after irradiation,the i NOS expression in the 0.4J group and the 4J group was significantly lower than that in the control group,but no significant difference was found in the 10 J group.Conclusion: Low-level laser therapy can increase the viability of M1 bone marrow-derived macrophages and inhibit its polarization expression,and this effect has a certain time-dose dependence.Experiment 2: Effect of Low-Level Laser Therapy on the Secretion of M1 Bone Marrow-derived Macrophages and Its MechanismObjective: To study the effect of low-level laser therapy on the secretion of M1 bone marrow-derived macrophages and the corresponding mechanismMethods: The mature M1 BMDM was randomly divided into irradiation group and control group.The laser parameters were the same as experiment one.6 hours after irradiation,RT-q PCR was used to detect the proinflammatory cytokines TNF-α,IL-1β and MCP-1 m RNA.Enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of TNF-α,IL-1β,MCP-1,and neurotrophins BDNF and NGF 24 hours after irradiation.The level of ROS in M1 cells was measured using ROS probes at 2 and 24 hours after irradiation.24 hours after irradiation,immunofluorescent staining was used to stain the axons of dorsal root ganglion cells that had been adoptively cultured in supernatants of LLLT-M1 macrophages.Western blotting was used to detect the expression of classic polarization transcription factor NF-κB p65 and Phospho NF-κB p65.Results: 6 h after irradiation,compared with the control group,the m RNA expression of TNF-α and IL-1β decreased significantly in the 0.4J and 4J groups,while the irradiation did not significantly alter the m RNA expression of MCP-1.24 hours after irradiation,it was found that compared with the control group,irradiation in the 0.4J group significantly inhibited the secretion of IL-1β and increased the secretion of MCP-1;in the 4J group,the secretion of TNF-α and IL-1β was significantly downregulated.MCP-1,BDNF and NGF secretion increased significantly,the expression of the classical polarized transcription factor NF-κB p65,Phospho NF-κB p65 was also significantly inhibited;10J group irradiation can significantly inhibit the secretion of TNF-α.ROS probe detection found that after 2 hours of irradiation,10 J group ROS content increased significantly compared with other groups,and 24 hours later,4J group ROS content was significantly lower than the control group and 10 J group.After 24 hours of adoptive culture of dorsal root ganglion cells,compared to the control group,the supernatants of 4J and 10 J groups significantly promoted the growth of axons of dorsal root ganglion cells.Conclusion: Low-level laser therapy used in this experiment can directly affect on M1 bone marrow-derived macrophages,inhibit their polarization state and inflammatory secretion,down-regulate oxidative stress,up-regulate the secretion of neurotrophic factors,and promote axons growth and the effect of low-level laser therapy on macrophage polarization may be related to the down-regulation of classical polarized transcription factor NF-κB p65/Phospho NF-κB p65.