Protective Effect And Mechanisms Of Dipfluzine Hydrochloride On Cerebral Ischemia-reperfusion Injury

At present, the clinical treatment of acute stroke is thrombolytic therapy,but the reperfusion after thrombolysis will intensify the brain damage, so it isurgent to research and develop an effective drug against the damage inducedby ischemia-reperfusion. Dipfluzine hydrochloride, a new compound ofdiphenylpiperazines, was first developed by Hebei Medical University. Ourlab has showed that Dip is an effective drug against ischemia-reperfusioninjury by a lot of methods such as physiological, biochemical andmorphological technologies. But because dipfluzine is insoluble in water, it isnot convenient use in clinical practice. So we changed it into dipfluzinehydrochloride, and increased the solubility. The injection of dipfluzinehydrochloride can be conveniently used. The present research was to explorethe protective effect and mechanisms of dipfluzine hydrochloride on ratcerebral ischemia-reperfusion injury in intact animal and cellular level.Part one Protective effect and mechanisms of dipfluzine hydrochlorideon focal cerebral ischemia-reperfusion injury in ratsObjective: To study the protective effect and mechanisms of dipfluzinehydrochloride on focal cerebral ischemia-reperfusion injury in ratsMethods: SD rats, female, weighting280to300g, were randomlydivided into six groups: sham operation; solvent; dipfluzine hydrochloride2.5μmol/kg,1.3μmol/kg, and0.7μmol/kg; Flu1.3μmol/kg. The rat focalcerebral ischemia-reperfusion model was prepared by thread embolismmethod. Drugs were given by tail vein injection in dose of0.2ml/100g. rats insham operation group were performed only with operation pathway. Afterischemia for2h and reperfusion for4h (I2hR4h), we evaluated the neuralsymptoms, water content in brain, SOD activity and MDA content, thechanges of histopathological by HE staining, and the expression of AQP-4by IHC, to study the protective effect and mechanisms of dipfluzinehydrochloride on focal cerebral ischemia-reperfusion injury in rats.Result:(1) Rats in sham operation group had no damage on neuralfunction, but the neurobehavioral score of rats in solvent group was9.50±0.22,which suggested that the neural function of rats in solvent group wereremarkably injured (P＜0.01). The neurobehavioral scores in dipfluzinehydrochloride2.5μmol/kg group (6.50±0.96),1.3μmol/kg group (7.33±0.94)and Flu group (7.66±0.42) were remarkably decreased compared to solventgroup (P＜0.01).(2) Water content in brain was remarkably increased to82.24±0.23%after focal cerebral ischemia-reperfusion injury. Dipfluzinehydrochloride and Flu could significantly decrease water content, which were78.96±0.95%in2.5μmol/kg group,79.95±0.47%in1.3μmol/kg group,81.08±0.40in0.7μmol/kg group, and79.41±0.65%in Flu group, respectively.(3) After ischemia-reperfusion, SOD activity was decreased and MDA contentwas increased obviously. Dipfluzine hydrochloride could relieve the injure,Dipfluzine hydrochloride2.5group increased SOD activity from24.86±0.86U/ml to61.79±1.09U/ml and decreased MDA content from6.11±0.05nm/mlto4.64±0.05nm/ml.(4) In HE staining, brain tissue structure was normal insham group. Brain tissue in solvent group showed ischemic damage in cortex,striatum and hippocampus characterized by condensed nuclei, dark staining,cell body pyknosis and vacuolization. Dipfluzine hydrochloride in three dosegroups could significantly improve the lesions.(5) AQP-4studied byimmunohistochemical staining was mainly expressed in the membrane of glialcell in the surrounding of vessels. Sham group had a small amount of theexpression. After ischemia-reperfusion, the expression was obvious increased(P＜0.01). Dipfluzine hydrochloride2.5μmol/kg group (4.60±0.31),Dipfluzine hydrochloride1.3μmol/kg group (6.10±0.37) and Flu group(5.70±0.47) decreased the expression of AQP-4remarkably (P＜0.01).Conclusion: Dipfluzine hydrochloride reduced cerebral edema degree byscavenging free radicals, resisting lipid peroxide, reducing the expression ofAQP-4around infarction area, to develop neuroprotective effect. Objective: To study the protective effect and mechanisms of dipfluzinehydrochloride on cultured hippocampal neurons after oxygen-glucosedeprivation/oxygen supplyMethods: Cultured hippocampal neurons,7-8days, were randomlydivided into four groups: I2h-R24h; I4h-R24h; I6h-R24h; I8h-R24h. Thesurvival rates of hippocampal neurons in four groups were measured by MTTassay and I6h-R24h was selected as an optimal oxygen-glucosedeprivation/oxygen supply modelfor further researches. Cultured hippocampalneurons suffered from I6h-R24h were randomly divided into six groups: blank;solvent; dipfluzine hydrochloride10μmol/L;1μmol/L;0.1μmol/L; andFlu1.3μmol/L. We determined the MTT OD, LDH leaking, intracellularcalcium content, NO content, NOS activity, and the apoptotic rate, to study theprotective effect and mechanisms of dipfluzine hydrochloride on culturedhippocampal neurons after oxygen-glucose deprivation/oxygen supply.Result:(1) The different concentration of dipfluzine hydrochloride couldincrease the activity of cultured hippocampal neurons sufferred fromoxygen-glucose deprivation/oxygen supply, which was represented a risen ODvalue from MTT assay. And the LDH leakage was decreased significantly,which indicated the decrease of damage degree of cells.(2) Theoxygen-glucose deprivation/oxygen supply induced intracellular calciumoverload (319.40±10.79)in cultured hippocampal neurons. Dipfluzinehydrochloride10μmol/L could significantly lighten the increase ofintracellular calcium content (238.50±7.73)(P＜0.01).(3) NO content andNOS activity of cultured hippocampal neurons after oxygen-glucose eprivation/oxygen supply were increased significantly. Differentconcentration of Dipfluzine hydrochloride and Flu improved the change of thedamage.(4) The apoptotic cells showed by TUNEL after oxygen-glucosedeprivation/oxygen supply were remarkably increased to152.20±2.29insolvent group compared to blank group (41.30±1.78, P＜0.01). Dipfluzinehydrochloride could siginificantly reducethe apoptotic cells induced byoxygen-glucose deprivation/oxygen supply to60.70±1.61in10μmol/Ldipfluzine hydrochloride group,100.10±1.94in1μmol/L dipfluzinehydrochloride group, respectively (P＜0.01).Conclusion: Dipfluzine hydrochloride could block the increases ofintracellular calcium content, NO content and NOS activity of culturedhippocampal neurons induced by oxygen-glucose deprivation/oxygen supply,improves the damage of cultured hippocampal neurons caused byoxygen-glucose deprivation/oxygen supply, and possesses the anti-apoptoticeffects.