MMP-9 Gene Targeting Therapy And Mechanism Study Of Herpes Simplex Encephalitis

PARTⅠMechanism Study of Herpes Simplex Encephalitis in vivoExperimentⅠThe Expression of MMP-9 and its Effect on BBB Permeability inHerpes Simplex Encephalitis MiceObjective: To investigate the expression and the roles of matrix metalloproteinase-9(MMP-9) protein in brain edema formation in herpes simplex encephalitis (HSE).Methods: Mice were inoculated intracerebrally with herpes simplex virus type1(HSV-1) to form herpes simplex encephalitis. MMP-9 gelatinolytic activity was assessedby zymography from brain tissues 3 days and 7 days after HSV-1 infected. The expressionof MMP-9 was detected by immunofluorescence staining in the brain slices. Theblood-brain barrier (BBB) permeability was quantitated by Evans blue dye extravasationsand brain water content.Results: We found that HSE mice were associated with significantly raised levels ofMMP-9 at 3 days and 7 days after infection. The results were concordant with the increasedBBB permeability and brain water content. The administration of MMP-9 inhibitor BB-94 (30 mg·kg^-1) significantly attenuated BBB permeability. Double-labeling experimentsshowed that the MMP-9 expression was colocalized with actived microglia cells.Conclusion: This study demonstrates that excessive activation of MMP-9 fromactived microglia is deleterious to the brain, which is associated with the degree of BBBleakage.ExperimentⅡDelayed Dexamethasone Treatment in Combination with AcyclovirProtect Experimental Herpes Simplex Encephalitis in MiceObjective: To evaluate the effects of early versus delayed glucocorticoidadministration in combination with antivirus drug in herpes simplex encephalitis mice.Methods: Mice were randomly assigned to 5 groups with 15 mice in each: normalcontrol group (NC), HSV-1 infected group (HC), Acyclovir treated group (HA), earlydexamethasone administration group (DE), delayed dexamethasone administration group(DD). Flow cytometry was adopted to detect CD11b expression in the brain tissue. Weused semiquantitative reverse transcription polymerase chain reaction (RT-PCR) to detectproinflammatory cytokines TNF-αand IL-6. Pathologic slices of brain tissues of mice wereused to identify tissue morphology changes. Neurological score was quantified by anestablished neurological scale at 5 d after HSV-1 encephalitis. Differences in cumulativesurvival among the groups were analyzed according to Kaplan-Meier followed by alog-rank test 2 weeks after viral infection.Results: After HSV-1 infection, brain tissue CD11b and cytoldnes were significantlyincreased compared with NC group (P＜0.01), suggesting that microglial cells wereactivated. DE group (started immediately after infection) showed still obviously microglialactivation associated with vigorous cytokines production in contrast to NC group. CD11bexpression in HA and DD group were decreased compared with HC group (P＜0.01), cytokines reduced to normal levels. CD11b in HA group was higher than that in DD group(P＜0.01), however there were no significant difference of cytokines expression andcumulative survival (P＞0.05). DD group (started 3 d after infection) inhibited CD11b andattenuated proinflammatory cytokines expression (P＜0.01), and also showed attenuatedbrain tissue pathological changes compared with DE group. Neurological score andcumulative survival were higher in DD group than that in DE group (P＜0.05).Conclusions: Delayed but not early dexamethasone treatment in combination withacyclovir might protect experimental HSV-1 encephalitis in mice.PARTⅡCharacteristic of MMP-9 Activation in Microglia and itsCytokines Secretion after HSV-1 infectedObjective: To investigate the characteristic of MMP-9 activation in microglia and itscytokines secretion.Methods: Balb/c mice were injected intracerebrally with herpes simplex virus type 1(HSV-1) to form herpes simplex encephalitis. BV2 cells were inoculated with HSV-1 tomake cell-infected model. The concentrations of clinical medication for BV2 cells weredetermined by MTT method. The mRNA expressions of MMP-9, iNOS, IL-2, IL-4, IL-10,and TNF-αin mice brain tissues and BV2 cells were measured by semiquantitative reversetranscription polymerase chain reaction (RT-PCR) and their changes characteristics werecompared between mice and BV2 cells.Results: The characteristics of MMP-9 mRNA expression and many inflammatoryfactors are much comparable between BV2 cells and mice model.Conclusion: BV2 cells might become the perfect model in herpes simplexencephalitis study instead of the animal ones. PARTⅢNADPH oxidase activation is essential for the HSV-1-inducedMMP-9 expression in murine microgliaObjective: To investigate the role of NADPH oxidase in the expression of matrixmetalloproteinase 9 (MMP-9) in HSV-1-induced murine microglial cells (BV2).Methods: BV2 cells were randomly assigned to four groups: normal control group,HSV-1 infected group, Apocynin(0.5 mmol/L, 1.0 mmol/L) treated groups after HSV-1infection. We used semiquantitative reverse transcription polymerase chain reaction(RT-PCR) to detect the mRNA of p47phox of NADPH oxdiase and MMP-9. IntracellularROS levels were measured by staining with Dihydroethidium (DHE). MMP-9 gelatinolyticactivity was assessed by zymography in supernatants from cells.Results: HSV-1 Promoted the up-regulation of mRNA of NADPH oxidase subunitp47phox and MMP-9 in murine BV2 cells. HSV-1 infection also resulted in a 2-foldincrease in generation of ROS and was associated with the increased gelatinase MMP-9activity. These effects were inhibited in the presence of Apocynin.Conclusion: HSV-1 may induce MMP-9 activation through NADPHoxidase-dependent ROS generation in microglia.PARTⅣFavorable effects of MMP-9 knockdown in murine herpessimplex encephalitis using small interfering RNAObjective: To investigate the expression of matrix metalloproteinase-9 (MMP-9)protein and the role of MMP-9 gene knockdown in prognosis of herpes simplexencephalitis (HSE). Methods: The animal models of herpes simplex encephalitis were established byintracerebrally inoculated herpes simplex virus type 1(HSV-1) in mice. Pathologic brainslices of mice were used to identify the morphology differences. Mice were inoculatedintracerebrally with MMP-9 targeting siRNA (MMP-9-siRNA). MMP-9 expression wasassessed by RT-PCR and western blot analysis at 3 days and 7 days after HSV-1 infected.The blood-brain barrier (BBB) permeability was quantitated by Evans blue dyeextravasations and brain water content. Immunohistochemistry method was adopted toanalyze the expression of AQP4 protein. Quantitative real-time PCR analysis was used todetect cytokines expression. Neurological Score was quantified using an establishedneurological scale at 7d after HSE.Results: We found that MMP-9-siRNA treated mice were associated with decreasedlevels of MMP-9 at 3 d and 7 d after infection, which concordant with the attenuated BBBpermeability and brain water content. The mice treated with MMP-9-siRNA significantlyreduced AQP4, TNF-αand IL-6 expressions and improved clinical outcome compared withthe control group.Conclusion: Our data reveal MMP-9 is a key pathogenicity factor in the developmentof HSE. Local injection synthetic siRNA could knockdown MMP-9 expression in acutephase of HSE and improved mice outcomes.