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 major problem to be solved in the world.Researches showed that alleviating secondary injury is the key point to promote SCI repair and improve the clinical prognosis of patients.Macrophages(M)are the most important inflammatory cells involved in SCI secondary injury.In order to response to different cytokines in the environment of the injury area,macrophages show two polarization states which called M1 and M2,and the cytotoxic M1 dominates the process for a long time,while the neurotoxic M2 only increases temporarily.This polarization imbalance further leads to the irreversible injury of the spinal cord.Regulating the proportion of M1/M2after SCI,especially inhibiting the expression of M1 which can alleviate the inflammatory response and significantly promote the functional recovery of injured spinal cord.Drug intervention,cell transplantation and other methods can adjust the polarization of macrophages to a certain extent,but there are many deficiencies,such as narrow drug safety window,immune rejection,and difficulties in effective clinical implementation.Therefore,it is urgent to find a safe,effective,simple and feasible treatment.Low-level laser therapy(LLLT),as a classical physical therapy method,showed the advantagesofsafety,non-invasive,simple implementation and significant anti-inflammatory and repair effect.It has been widely used in clinical fields such as skin lesions and peripheral nerve injuries.Our previous studies have found that LLLT could reduce the inflammatory reaction at the injured part of SCI rats,inhibit the formation of glial scar,and promote the recovery of motor function.Preliminary clinical trial results also confirmed the role of LLLT in promoting nerve repair.We further found that LLLT can change the polarization status of macrophages in SCI area,down-regulate the number of M1 and inhibit the secretion of pro-inflammatory factors TNF-?,IL-13 and IL-1.The up-regulatedproportion of M2 also could promote the secretion of anti-inflammatory factor IL-4 and neurotrophic factor BDNF,increase neuron survival and axon regeneration,and promote the repair of SCI.However,can LLLT directly act on M1-type macrophages in vitro and play a role in regulating the polarization and secretion of M1 phenotype?And in what way is this regulation achieved?What role does it play in promoting the repair of spinal cord injury?These points are not yet resolved clearly.Previous studies have shown that NF-?B signaling pathway plays an important role in regulating the polarization of M1-type macrophages.Activation of NF-?B can promote polarization of macrophages toward M1.So,does the NF-?B signaling pathway participate in the regulation of M1 phenotypic polarization by LLLT and how doesNF-?B regulate macrophage polarization under LLLT?With the above problems,we carried out the following researches.Experiment 1:The effect of 810nm low-level laser on the activity and phenotypic polarization of M1-type bone marrow derived macrophages(BMDM).Objective:To study the effect of low-level laser on the activity and polarization of M1-type BMDMin vitro.Methods:Primary macrophages were extracted from the bone marrow of Balb/c mice.The phenotype polarization of macrophages M1 was induced by the culture medium supplemented with 100ng/ml LPS+20ng/ml IFN-?.The culture and polarization were demonstrated by flow cytometry.Mature M1-type macrophages were randomly divided into two groups:the low-level laser irradiation group and the control group.In the laser irradiation group,the output power was 2mW/cm~2,and the spot area was 4.5cm~2.The irradiation duration was 44s,440s,and 1111s to obtain 0.4J,4J,and 10J,respectively energy parameter groups.The control group was placed in a dark box and not exposed to laser radiation.After irradiatedfor 2 and 24 h,CCK-8 test was used to detect the cell activity in each group,and RT-qPCR was used to detect the mRNA expression of iNOS,a cell specific marker of M1-type macrophage.The expression of iNOS protein and the proportion of iNOS~+cells were detected by western-blot and immunofluorescence staining after irradiated for 24 h.Results:The cell activity of each group did not show significantly change 2 h after irradiation.After irradiated for 24 h,the cell activity of 4J group was significantly enhanced,higher than that of the control group and other irradiation groups.TheiNOS mRNA expression of cells in each group did not change significantly 2 h after the irradiation with low-level laser.After irradiated for 24 h,the expression of iNOS in each group was significantly lower than that in the control group,and the decreased degree in the 4J group was more significant than that in other irradiation groups.After 24 hours of low-level laser irradiation,the expression of iNOS in the 0.4J group and the 4J group decreased significantly compared with the control group,and the decreased level in the 4J group was more significant than that in the 0.4J group,while no significant changes were found in the 10J group.The change of iNOS~+cell proportion was consistent with the protein level.Conclusion:Low-level laser irradiation can enhance the activity and inhibit the phenotypic polarization of M1-type BMDM in a time-dependent and dose-dependent manner.Experiment 2:The effect of 810nm low-level laser on the secretion of M1-type BMDM on neuronal axons.Objective:To study the effect of low-levellaser on the secretion of M1-type BMDM on the axon of the radicular ganglion.Methods:The cell grouping and low-level laser irradiation were same as experiment1.The RT–qPCR was used to detect the phenotypic pro-inflammatory factor M1 TNF-??IL-1?and IL–6 after the laser irradiation for 24 hwere used to detectThe secretion ofTNF-??IL-1??IL-6 andneurotrophic factors BDNF and NGF were demonstrated by ELISA.The axon growth of dorsal root ganglion cells cultured with supernatant of LLLT-M1 macrophages were observed byimmunofluorescencestaining.Therotein expression of transcription factors NF-?Bp65 and Phospho-NF-?Bp65 were detected by Western-blot.ROS probes were used to detect the ROS content of M1-type cells after irradiated for 2 and 24 h.Results:After irradiated for 24h,the expression of TNF-?and IL-1 was significantly down-regulated and the secretion of IL-1 was significantly inhibited,compared with the control group.The secretion levels of TNF-?and the protein expressions of NF-?B p65and Phospho-NF-?B p65 in the 4J and 10J irradiation groups were significantly down-regulated.The mRNA expression of IL-6 were significantly inhibited in the 4J irradiation group,and the secretion levels of neurotrophic factors BDNF and NGF were significantly up-regulated.The ROS probe test found that the ROS content in the 10J group was significantly higher than that in other groups after irradiated for 2 h,while the ROS content in the 4J group was significantly lower than that in the control group and the10J group after irradiated for 10 h.After 24 hours of adoptive culture of dorsal root ganglion cells,the supernatant of 4J group and 10J group can significantly promote the growth of dorsal root ganglion cells'axons compared with the control group.Conclusion:The low-level laser used in this experiment can act directly on the M1-type BMDM,inhibit the secretion of inflammatory,down-regulate oxidative stress reaction,increase neurotrophic factor secretion,promote the growth of axons.And the effect of low-level laser on the polarization and secretion of macrophages may be related to the down-regulatedexpression of NF-?B p65/Phospho-NF-?Bp65 in the classical polarization pathway.Experiment 3:The effect of 810nm low-level laser on phenotypic polarization of M1-type BMDM by regulating Nrf2/NF-?B signaling pathway.Objective:To study the signal pathway of low-level laser regulating phenotypic polarization of M1-type BMDMMethods:The cells were divided into control group,M1 group,M1+LLLT group,M1+LLLT+Nrf2-si-RNA group,and 4J energy parameter 810nm low-level laser was selected for M1+LLLT group and M1+LLLT+Nrf2-si-RNA group to irradiate.The other two groups were placed in a black box and were not irradiated.The expression levels of iNOS,Nrf2,NF-?bp65,p-NF-?bp65,p-Ikk-?,I?B-?and p-I?B-?were detected by Western-blot 24 hours after irradiation.The expression levels of iNOS,Nrf2,NF-?bp65,TNF-?,IL-1?and IL-6 in each group were detected by RT-qPCR.The levels of TNF-?,IL-1?and IL-6 in each group were detected by ELISA.Immunofluorescence staining and Western-blot were used to detect the nuclear levels of Nrf2 and NF-?bp65.Results:Compared with the control group,the protein expression levels of iNOS,Nrf2,NF-?B p65,p-NF-?Bp65,p-Ikk-?,p-I?B-?and the nucleation levels of Nrf2 and NF-?B p65 in the M1 group were significantly up-regulated,the protein expression level of I?B-?were significantly down-regulated,and the expression levels of iNOS?Nrf2?NF-?bp65?TNF-??IL-1?and IL-6 were significantly increased.Compared with the M1group,the protein expression levels of iNOS?NF-?B p65?p-NF-?B p65?p-Ikk-??p-I?B-?in the M1+LLLT group were significantly down-regulated,and the nuclear level of NF-?B p65 was significantly decreased.In addition,the expression levels of I?B-?protein,Nrf2 protein,NF-?B p65,TNF-?,IL-1 and IL-6 were significantly increased.The expression levels of iNOS,NF-?B p65,TNF-?,IL-1 and IL-6 were significantly decreased.Compared with the M1+LLLT group,the protein expression levels of iNOS?Nrf2?p-NF-?B p65?p-Ikk-??p-I?B-?in the M1+LLLT+Nrf2-si-RNA group were significantly up-regulated andthe nuclear level of NF-?B p65 was significantly increased,the expression level of I?B-?protein was significantly decreased,the expression levels of iNOS,Nrf2,TNF-?,IL-1 and IL-6 were significantly increased.Conclusion:low-level laser regulates the Nrf2/Ikk-?/Ikb-?/NF-?bp65 signaling pathway and inhibits phenotypic polarization and pro-inflammatory factor secretion of m1-type macrophages.