Effect Of Melatonin On The Inhibition Of Matrix Metalloproteinase-9and Its Protection Mechanism Of Endothelial Cell Barrier

ObjecivePart one:To establish the method of primary isolation, cultivation and identification of rat brain microvessel endothelial cells as well as pericytes.Part two:To explore the effect of melatonin on the inhibition of matrix metalloproteinase-9(MMP-9) activity and expression, and then detect its protection mechanism of vascular endothelium barrier.MethodsPart one:10brains from3week-old Wistar rats were isolated. Two-step enzyme digestions with one-step gradient centrifugation were used to isolate brain microvessel fragments. The microvessel fragments were seeded and cultured on35mm dishes with different culture methods. The brain microvessel endothelial cells (BMEC) were identified according to the morphological observation under the Phase-Contrast microscopy and the detection of vWF associated antigen by immunofluorescence method. The tight junction protein claudin-5was demonstrated by immunocytochemical method. The brain microvessel pericytes (BMPC) were identified according to the morphological observation under the Phase-Contrast Microscopy and the detection of a-smooth muscle actin (a-SMA), NG2, vWF and glial fibrillary acidic protein (GFAP) associated antigens by immunofluorescence method. MTT assay was applied to examine the growth curves of BMPC.Part two:Primary HUVECs were isolated from fresh human umbilical veins and cultured after immunomagnetic separation by anti-CD31antibody coated Dynabeads. For identification of endothelial cells, the cells were incubated with mouse anti-von Willebrand Factor (vWF) and FITC-conjugated goat anti-mouse secondary antibody.The mRNA and proteins expression of MMP-9, MMP-2, TIMP-1and TIMP-2were determined by RT-PCR and Western blot respectively. For assay of MMP-2and MMP-9enzymatic activity, the conditioned media of HUVECs was analyzed by gelatin zymography. Paracellular permeability was studied in a Transwell system by measuring the flux of Na-F across the endothelial monolayer. To detect VE-cadherin, occludin, claudin-5and ZO-1, the cells were incubated with anti-VE-cadherin, anti-occludin and anti-claudin-5and anti-ZO-1antibodies, and then observed by fluorescence microscope. The cellular location changes of NF-κB/p65were analyzed by immunostaining method. The expressions of VE-cadherin, occludin and p65were detected by Western blot.ResultsPart one:The BMECs and BMPCs were isolated successfully from microvessels. BMECs presented'cobblestone'shape under phase-contrast microscopy. BMECs express factor Ⅷ-related antigen, while markers of astrocytes (GFAP) are negative. The tight junction protein claudin-5was completely located at the border of confluent BMECs.Migrating pericytes from cerebral microvascular fragments displayed by spreading with irregular projections and became confluent after12-14days. Pericyte markers a-SMA and NG2associated antigens by immunofluorescence method displayed positive, while vWF and GFAP by immunofluorescence method were negative. The result proved that the cells were identified as BMPCs. The growth rate of primary cells was relatively slow. Passaged cells reached logarithmic growth phase after36～60hours and plateau phase after72-108hours.Part two:(1) Primary HUVECs were isolated sucessfully from fresh human umbilical veins and cultured after purified by immunomagnetic separation. The endothelial origin was confirmed by the positive labeling of vWF with immunofluorescence staining.(2) Comparing with the control group, IL-1β-treated group exhibited higher expression of MMP-9mRNA and protein (P<0.001) and TIMP-1mRNA and protein expression were decreased significantly (P<0.001), but the expression of MMP-2and TIMP-2mRNAs and proteins did not change (P>0.05). Comparing with IL-1β-treated group, Mel pretreatment group significantly inhibited the expression of MMP-9mRNA and protein (P<0.001) and TIMP-1mRNA and protein expression were increased significantly (P<0.001), but the expression of MMP-2and TIMP-2mRNAs and proteins did not change (P>0.05).(3) Gelatin zymography:Comparing with the control group, MMP-9enzymatic activity in IL-1β-treated group obviously increased(P<0.001); Comparing with IL-1β-treated group, Mel pretreatment group significantly inhibited the activity of MMP-9(P<0.001). However, MMP-2enzymatic activity did not change among three groups (P>0.05).(4) Transwell system assay:The permeability of the endothelial monolayers in IL-1β-treated group was obviously increased at different time points compared with control group. However, the permeability to Na-F in the group of pretreatment with melatonin for30min was significantly reduced.(5) Fluorescence microscope:By VE-cadherin, occludin, claudin-5and ZO-1staining, IL-1β-treated group caused a clear disruption of adherens junctions and tight junctions. Melatonin pretreatment attenuated the disruption of VE-cadherin, occludin, claudin-5and ZO-1. Western blott:The level of VE-cadherin and occludin proteins were reduced in IL-1P-treated cells, while that of the reduction was attenuated by melatonin.(6) Western blot and immunostaining results showed that the activated NF-KB/p65was increased in IL-1β-treated group, while melatonin pretreatment could obviously inhibit the nuclear translocation of NF-KB/p65.Conclusions:Part Ⅰ:The above method could isolate high purity of rat brain microvessel endothelial cells and pericytes from rat brain microvessel successfully. Part Ⅱ:(1) MMP-9could disrupt the adherens junctions and tight junctions, enlarge cells fissure, and increase the permeabilty.(2) Melatonin pretreatment in HUVECs has a significant protective effect on HUVECs barrier via inhibiting MMP-9through NF-κB pathway.