The Effect Of FK506on Angiogenesis After Spinal Cord Injury

Spinal cord injury (SCI) is a severe trauma to the central nervous system. Mostpatients can not take care of themselves, resulting in a heavy family and social burden.In recent years, the immunosuppressant treatment for SCI has brought great hope. Ithas been confirmed that the tacrolimus (FK605), as a new type of immunosuppressant,can reduce nerve cell apoptosis, inhibit the immune cell activation, reduce theinflammatory response, and promote functional recovery after SCI.Early angiogenesis can provide nutritional support for damaged tissue, promotemacrophage infiltration and necrotic tissue clearance after SCI. Vascular injury canbe regenerated, the drug can also promote the angiogenesis. But it has not beenreported that whether FK506have effect on angiogenesis after SCI. Astrocytes arewidely distributed in the nervous system, it plays an important role to maintain thestability of the microenvironment around the nerve cells, and regulate the metabolicprocesses. Early proliferating glial cells in vivo after spinal cord injury, not onlysecrete the growth factor, but also can reduce bleeding, and inhibit the inflammatoryresponse.Change of many physiological or pathological conditions can cause the endoplas-mic reticulum stress (ER stress). In order to eliminate this stress, the endoplasmicreticulum immediately start the unfolded protein response (UPR), it reduce thefunction of protein folding in the endoplasmic reticulum load to a certain extent, earlyin the stress is an adaptive protective mechanism of cells. It is reported that, in theXBP1knockout mouse, the recovery of motor function is diminished after SCI; otherresearch indicates that the IRE1pathway may regulate angiogenesis.Therefore, we designed the following in vivo and vitro experiments to study theeffect and mechanism of FK506on angiogenesis after SCI:Firstly, to investigate the effect of FK506on motor function and angiogenesis,the surgeries were performed on male SD rats weighing280g~320g, using Allen’s method to estabish a contusion model. FK506were administrated intravenously at asingle dose of0.5mg/kg body weight at30min after SCI. We did BBB scores andinclined plane tests in the3,7,14,21,28,35,43day, and use the immunofluo-rescence to observe the vessels in the1,3,7,14day. Compared with the controlanimals, rats from the treatment group showed significantly better locomotorfunctional outcomes after7days, and more blood vessels in one day; three daysreached a peak; then after14days unchanged.Secondly, the vascular endothelial cells (Eahy926) and astrocytes (U251) werecultured to study the mechanisms.(1): The Eahy926cells were stimulated withdifferent concentration of FK506, and the CCK8result showed no direct effect on cellviability.(2): Use different concentration of FK506to treat the U251cells, thenCCK8was used to detect the cell proliferation. We choose20μM concentration ofFK506to treat cells for8hours, and after ultrafiltration, use the conditioned mediumto culture Eahy926cells. The CCK8result showed the increase in cell viability, thewound healing experiment showed an increase in cell migration, and the tubeformation experiment showed the increased capacity of cells to form blood vessels.The results of DNA chip showed that angiogenesis-related factors in the U251cellshave a significant increase, including VEGFA and EGF.(3): After treatment ofFK506on U251cells in different concentration or time, we used western-blot andRT-PCR to determine the expression of protein correlate to UPR. FK506significantlyenhanced expression of GRP78in a dose dependent manner. With20μM concen-tration of treatment, the expression of GRP78was highest in four hours; the phosph-orrrrylation of JNK had a highest increase in eight hours; and the splicing of XBP1increased in two hours.These findings suggest that, FK506may promote early angiogenesis after SCI,and thus promote the late functional recovery. The promotion of angiogenesis-relatedfactor such as VEGF and EGF, which were secreted by the treatment of FK506onastrocyte, promote the function of endothelial cell, it may related to the activation ofIRE1pathway in astrocyte.