| Sodium(±)-5-bromo-2-(?-hydroxypentyl)benzoate(trade name: brozopine,BZP)was a new compound with independent intellectual property rights,which was synthesized at the Department of Chemistry and Molecular Engineering in Zhengzhou University based on 1-3-n-Butylphthalide(NBP)by the evaluation of structure-activity relationship,pharmacological toxicology,mechanism of action.Currently,BZP was used in the stage ? of clinical trials with encouraging efficacy results.Our previous studies had fully demonstrated the protective effect of BZP on ischemic stroke in rats by intravenous administration,but its targets had not been elucidated.Therefore,we intended to elucidate the regulatory targets and mechanisms of BZP against cerebral ischemia at the molecular level.Through chemical docking simulation in Tsinghua University Academy of Life Sciences,the binding free energies of BZP with 15-LOX-1 and 15-LOX-2 indicated the strong binding affinity of BZP with 15-LOX-2.15-LOX-1 was excluded from the possible target proteins.Based on the results,we further explored the interactions between BZP and 15-LOX-2,found that BZP and C8E4,an inhibitor of 15-LOX-2,had similar effects and inhibited the formation of 15-LOX-2 product(HETE).?t was suggested that BZP was an inhibitor of 15-LOX-2 and 15-LOX-2 might be the regulatory target of BZP against cerebral ischemia.?n this paper,we intended to expound the regulatory targets and mechanisms of BZP against cerebral ischemia in vivo and in vitro at the molecular level.Part ? Effect of BZP on 15-LOX-2 Metabolism Pathway after Cerebral ?schemia in RatsObjective:We established MCAO model in rats to observe the preventive effect of BZP enteric-coated pellets on focal cerebral ischemia-reperfusion injury and to explore the effect of BZP enteric-coated pellets on the metabolic pathway of 15-LOX-2 after cerebral ischemia.Methods:1.Establishment of MCAO Model The MCAO model in rats was established by Zea-longa method.2.Determination of the dose of BZP pellets The dose-effect curve of BZP enteric-coated pellets were made by the action of BZP pellet at different dose in MCAO rats,and the dose of BZP enteric-coated pellets was determined.3.Grouping and administration Divided into 4 groups of Male SD rat: Sham group;Model group;BZP enteric-coated pellets group;C8E4 group.4.Observation index To observe the protective effect of BZP enteric-coated pellets on focal cerebral ischemia-reperfusion injury in MCAO rats,including neurobehavioral score,cerebral infarction volume and cerebral swelling.EL?SA kit was used to detect the contents of 15-LOX-2 metabolite 15-HETE,rat nuclear factor ?B subunit p65(NF-?B p65),tumor necrosis factor(TNF-?),interleukin-6(?L-6)and intercellular adhesion molecule-1(?CAM-1).Results:1.According to the dose-effect curve of BZP enteric-coated pellets on focal cerebral ischemia-reperfusion injury in MCAO rats,the dose of BZP enteric-coated pellets(0.6,1.2,2.5,5.0,10,20 mg/kg)was used as transverse coordinates,neurobehavioral score,cerebral infarction volume and cerebral swelling as longitudinal coordinates to draw dose-response curves of BZP enteric-coated pellets.?nfarction volume was the gold index to evaluate cerebral infarction,the minimum effective dose of BZP enteric-coated pellets was 1.2 mg/kg.There was no significant difference between 20 mg/kg BZP enteric-coated pellets group and 10 mg/kg group of BZP,so 10 mg/kg was the maximum effective dose of BZP enteric-coated pellets.The BZP enteric-coated pellets with the final dose of 10 mg/kg were selected for the mechanism research.2.The tissue structure was clear,the cells were dense,arranged neatly,the nucleus was in the middle,the nucleolus was clear,and the cell space was dense and no edema in the cortex ?n the sham-operated group,hippocampal and striatum? Compared with sham-operated group,marked morphological changes were visualized in ischemic region of model group: neuronal cell loss,nuclei shrinkage,and dark staining of neurons.Compared with model group,BZP(1.2,2.5,5,10,20 mg/kg)significantly decreased neuronal necrosis [(50.76±3.57)%,(39.31±4.96)%,(29.14±4.96)%,(19.31±4.51)% and(20.97±2.25)% respectively;(P< 0.05,P< 0.01)] in cortex.Compared with model group,BZP(1.2,2.5,5.0,10,20 mg/kg)significantly decreased neuronal necrosis [(47.49±9.08)%,(36.29±7.85)%,(24.04±6.42)%,(15.03±2.40)% and(14.20±5.06)% respectively;(P< 0.05,P< 0.01)] in hippocampal.Compared with model group,BZP(1.2,2.5,5.0,10,20 mg/kg)significantly decreased neuronal necrosis [(34.57±7.09)%,(27.13±7.21)%,(19.02±5.73)%,(12.32±1.77)% and(12.66±3.87)% respectively;(P< 0.05,P< 0.01)]in striatum.The results showed BZP had potent protective effect of neurons,the effect of BZP enteric-coated pellets(10mg/kg)group was better than that of C8E4 group with equal molar dose in the cortex,hippocampal and striatum.(P< 0.01).3.Compared with the sham-operated group,the animal behavioral score(neurobehavioral deficit)in the model group was higher,and the contralateral shoulder rotation and adduction of the forelimb appeared.When promoting the shoulder,the contralateral resistance of the operation decreased,walking around the circle,a few animals appeared no spontaneous activity,consciousness disorder,with a larger volume of cerebral infarction and severe brain edema(P< 0.01).Compared with the model group,BZP enteric-coated pellets(0.6,1.2,2.5,5.0,10,20 mg/kg)groups could improve the symptoms of nerve loss,reduce the volume of cerebral infarction and reduce the brain edema in a dose-dependent manner(P<0.05;P<0.01).There was no significant difference between the C8E4(10mg/kg)group and the model group(P>0.05),The effect of BZP enteric-coated pellets(10mg/kg)group was better than that of C8E4 group with equal molar dose(P < 0.01).4.The results of immunohistochemistry showed that the expression of 15-LOX-2 in cortex,hippocampus and striatum of model group was significantly higher than that of sham group(P<0.01),the expression of 15-LOX-2 in BZP(10mg/kg)enteric-coated pellets group was significantly lower than that in model group(P<0.01).There was no significant difference in the expression of 15-LOX-2 between 10mg/kg enteric-coated pellets group and equal molar dose C8E4 group(P> 0.05).?t was shown that BZP was an inhibitor of 15-LOX-2.5.The content of 15-HETE in the model group was significantly higher than that in the sham-operated group(93.94±0.49 pg/ml;P< 0.01).Compared with model group,BZP(10mg/kg)enteric-coated pellets group decreased 15-HETE production(56.67±1.2pg/ml;P<0.05),while C8E4(10mg/kg)group significantly decreased 15-HETE content(52.65±0.95pg/ml;P<0.01).There was no significant difference between BZP enteric-coated pellets(10mg/kg)group and C8E4 group of equal molar dose(P>0.05),indicating that BZP was an inhibitor of 15-LOX-2.6.The content of NF-?B in the model group was significantly higher than that in the sham operation group(9.93 ±0.48ng/mg;P < 0.01).Compared with the model group,BZP(10mg/kg)enteric-coated pellets group and C8E4(10mg/kg)group significantly decreased the content of NF-?B(11.63±0.96ng/mg,22.89±0.35ng/mg;P<0.01).The effect of BZP enteric-coated pellets group of 10mg/kg was better than that of C8E4 group with equal molar dose(P < 0.05).7.The content of TNF-? in the model group was significantly higher than that in the sham operation group(10.52±0.14 ng/mg;P < 0.01).Compared with model group,BZP(10mg/kg)enteric-coated pellets group decreased the content of TNF-?(12.2±0.71 ng/mg;P<0.05).The content of TNF-? in C8E4(10mg/kg)group was 21.87±1.17 ng/mg,and the BZP enteric-coated pellets(10mg/kg)group was better than that of C8E4 group with equal molar dose(P < 0.05).8.The content of ?L-6 in the model group was significantly higher than that in the sham operation group(15.33±0.17 ng/mg;P<0.05).Compared with the model group,BZP(10mg/kg)enteric-coated pellets group could significantly reduce the content of ?L-6(17.87±0.97ng/mg;P<0.01).The content of ?L-6 in C8E4(10mg/kg)group was 27.95±1.1 ng/mg,and the BZP enteric-coated pellets(10mg/kg)group was better than that of C8E4 group with equal molar dose(P< 0.05).9.The content of ?CAM-1 in the model group was significantly higher than that in the sham operation group(73.63±0.46 ng/mg;P<0.01).Compared with the model group,BZP enteric-coated pellets(10mg/kg)group significantly decreased the content of ?CAM-1(97.36±0.52ng/mg;P<0.01),while C8E4(10mg/kg)group also decreased the content of ?CAM-1(116.6±0.22ng/mg;P<0.05).The effect of BZP enteric-coated pellets(10mg/kg)group was better than that of C8E4 group with equal molar dose(P < 0.05).Conclusion:BZP enteric-coated pellets have preventive effect on focal cerebral ischemia-reperfusion injury in rats by inhibiting the production of 15-LOX-2,15-HETE,and then inhibiting the expression of NF-?B,?L-6,TNF-?,?CAM-1.The results suggested that BZP was an inhibitor of 15-LOX-2 and 15-LOX-2 might be the regulatory target of BZP against cerebral ischemia.Part ? Effect of BZP on 15-LOX-2 Metabolism Pathway in PC12 cells after hypoxia injury induced by Na2S2O4Objective:To explore the preventive mechanism of(Brozopine,BZP)against PC12 cell injury induced by Na2S2O4,so as to provide relevant evidence for clinical application.Methods:1.Establishment of OGD/R damage Model PC12 cells were induced by hypoglycemic medium containing 30 mmol Na2S2O4 for 2 h,then reoxygenated and resuscitated for 24 h.A stable OGD/R damage model of PC12 cells was established.2.Grouping and administration Divided into 4 groups: Control group;Model group;BZP group;C8E4 group.The BZP and C8E4 groups were treated with OGD/R damage after 24 hours of pretreatment with corresponding concentration of BZP,C8E4.3.Observation index To observe the protective effect of BZP on PC12 cell injury induced by Na2S2O4;EL?SA kit was used to detect the contents of 15-LOX-2 metabolite 15-HETE,rat nuclear factor k B subunit p65(NF-?B p65),tumor necrosis factor(TNF-?),interleukin-6(?L-6)and intercellular adhesion molecule-1(?CAM-1).Results:1.Compared with control group,the survival rate of PC12 cells in OGD/R injury group was significantly lower(49.5%;P<0.01).Compared with the OGD/R injury group,the survival rate of the PC12 cells pretreated with BZP for 5,10,15,20,30,40 ?mol/L was significantly different(55.5%,61.7%,69.6%,77.8%,85.0%,81.3 %;P<0.05,P<0.01).The survival rate of PC12 cells pretreated with BZP was significantly higher than that of the OGD/R injury group(P < 0.01,P < 0.01).According to the concentration-effect curve of BZP on PC12 cells injury induced by Na2S2O4,the survival rate of PC12 cells pretreated with 40?mol/L BZP decreased slightly compared with BZP of 30?mol/L,so 30?mol/L was the maximum effective concentration of BZP.?n conclusion,mechanism research was carried out by treatment of 30?mol/L BZP in PC12 cells treated with OGD/R.The survival rate of PC12 cells pretreated with C8E4(30 ?mol/L)group was 62.8%,and the final dose of 30 ?mol/L,was calculated according to the equal molar concentration of BZP and C8E4,and the survival rate of PC12 cells pretreated with C8E4(30 ?mol/L)group was 62.8%.The effect of BZP(30 ?mol/L)group was better than that of C8E4 group with equal molar concentration(P < 0.01).2.?n the Control group,there were longer dendritic processes with polygonal or fusiform shape,while the number of neurons in OGD/R group decreased and the number of neurons decreased,some cells showed no protuberance and some neurons showed round shape.Compared with the OGD/R injury group,after treatment of 30 ?mol/L BZP/30 mol/L C8E4,the nerve cell injury was improved,the cell body was larger and the dendritic process was found.Compared with C8E4 group,the effect of BZP(30 ?mol/L)group was better than that of C8E4 group with equal molar concentration(P < 0.01).3.The content of 15-HETE in OGD/R injury group was significantly higher than that in Control group(73.79±0.82 pg/ml;P< 0.01).Compared with OGD/R injury group,BZP(30?mol/L)group significantly decreased 15-HETE production(47.71±0.87 pg/ml;P<0.01),and C8E4(30 ? mol/L)group also decreased 15-HETE content(45.79±0.16 pg/ml;P<0.05).There was no significant difference between the 30 ?mol/L BZP group and the equal molar concentration C8E4 group(P > 0.05).4.Compared with Control group,the content of NF-?B in OGD/R injury group was significantly higher(178.36±1.72 pg/ml;P<0.01).Compared with OGD/R injury group,BZP(30?mol/L group significantly decreased NF-?B content(104.79 ±8.59 pg/ml;P < 0.01),while C8E4(30 ? mol/L)group also decreased NF-?B content(113.43±3.43 pg/ml;P<0.05).There was no difference between the 30?mol/L BZP group and the equal molar concentration C8E4 group(P > 0.05).5.Compared with Control group,the content of TNF-? in OGD/R injury group was increased(94.82±1.03 pg/ml;P<0.05).Compared with the OGD/R injury group,the,BZP(30 ?mol/L)group significantly decreased the content of TNF-?(33.68 ±1.99 pg/ml;P < 0.01),while the C8E4(30 ?mol/L)group also decreased the TNF-? content(76.18±1.67 pg/ml;P<0.05).The effect of BZP(30 ?mol/L)was better than that of C8E4 with equal molar concentration(P<0.01).6.Compared with Control group,the content of ?L-6 in OGD/R injury group was increased(112.92±3.89 pg/ml;P<0.05).Compared with OGD/R injury group,BZP(30?mol/L)group decreased the content of ?L-6(60.92±1.30 pg/ml;P<0.05).The content of ?L-6 in C8E4(30 ?mol/L)group was 98.83 ±2.60 pg/ml,the effect of BZP(30 ?mol/L)was better than that of C8E4 with equal molar concentration(P < 0.05).7.Compared with Control group,the content of ?CAM-1 in OGD/R injury group was significantly higher(372.5±5.15 pg/ml;P< 0.01).Compared with OGD/R injury group,both,BZP(30?mol/L)group and C8E4(30?mol/L)group significantly decreased ?CAM-1 content(83.64±4.55 pg/ml,213.29±2.42 pg/ml;P<0.01).The effect of BZP(30 ?mol/L)was better than that of C8E4 with equal molar concentration(P < 0.01).Conclusion:BZP have preventive effect on PC12 cell injury induced by Na2S2O4,and its mechanism was to inhibit the expression of NF-?B,?L-6,TNF-?,?CAM-1 by inhibiting the production of 15-LOX-2 metabolite 15-HETE.The results suggested that BZP was an inhibitor of 15-LOX-2 and 15-LOX-2 may be the regulatory target of BZP against cerebral ischemia. |
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