The Effect Of Vitamin C On Macrophage Proliferation,migration,Phagocytic,and Polarization Activity

Vitamin C(Vitamin C VC)is a strong deoxidizer,and catalyst of metabolic reaction,which participates in a variety of metabolism in the body.VC can promote various hormone synthesis and has special effects on various bioprocesses.Previous literatures indicate that VC can play a role in neuroprotection through antioxidation.It is also reported that VC can promote the recovery of motor function after spinal cord injury.Combined use of VC and VE can reduce the neuralgia caused by the chronic ligation of peripheral nerve.Our previous research has also found that supplementary of VC can promote functional recovery after sciatic nerve injury in mice.Macrophages are a group of classical innate immune cells,widely distributed in organisms.It is well known that macrophages play a important role in the process of nerve injury recovery.When the nerve damage,hematogenous macrophages quickly gathered to injury sites.While resident macrophages rapid proliferation and work together with the migrated hematogenous macrophages to clear the debris of degraded axon and myelin by phagocytosis.By which they can creat a favorable micro-environment for the nerve regeneration.In the nerve injury area,macrophages can be differentiated into M1 and M2 subtypes.M1 is a pro-inflammatory of phagocytosis,while M2 can secrete lots of favorite cytokines to facilitate the functional recovery.In our previous study,we found that VC can significantly promote the morphological and functional recovery after sciatic nerve injury,however the underlying mechanism is still unknown.Therefore,present study aimed to explore whether VC can play effects on macrophages proliferation,migration,phagocytic and polarization activity and then in turns to enhance the peripheral nerve regeneration.1.Materials and methods(1)primary culture and VC treatment of peritoneal macrophages.The peritoneal cavity was exposed and peritoneal fluid was sucked.The primary macrophage was obtained and the culture medium was replaced after 2h to remove the no adherent cells.(2)BrdU and WST methods were used to detect the proliferation of macrophages(3)detection of macrophage phagocytosis by fluorescent microspheres(4)detection of migration by scratch test and Transwell compartment(5)detection of differentiation by immunohistochemical method In vivo:(1)Establish rat sciatic nerve crush injury model(2)Using immunofluorescence to detect macrophage aggregation at the injury site(3)Using immunofluorescence to detect macrophage differentiation at the injury site2.Results2.1 BrdU cell positive rate and WST absorbance showed that VC could increase the proliferation of macrophages compared with the control group that did not add VC.2.2 We calculate phagocytosis by counting the number of Lumispheres phagocytosis in macrophages.We found that VC treatment increase the number of macrophages phagocytosis microspheres compared with the control group without addition to VC.2.3 The healing rate of the scratch and the number of cells migrated from the Transwell chamber evaluated the migration of macrophages.It was found that compared with the control group without VC,the migration ability of VC group increased significantly.2.4 Immunofluorescence can identify the direction of differentiation by counting the number of iNOS and CD 163 positive cells.We can see that compared with the control group without VC,VC treatment reduced the number of M1 macrophages and increased the number of M2 macrophages.2.5 In vivo Macrophage accumulation and differentiation was determined by immunofluorescence counting of ED1,iNOS,and CD 163 positive cell numbers.The experimental results show that VC treatment promotes macrophage aggregation and makes M1 more effective and the number of M2 macrophages increased than the control group without VC.3 Conclusion:Through in vitro experiments,it was found that VC promoted the proliferation,migration,and phagocytosis of macrophages,and promoted the differentiation of macrophages into M2 type and inhibited their differentiation into M1 type.VC was found in vivo to promote the proliferation of macrophages,differentiate into M2 type,and inhibit its differentiation into M1 type.