Study On The Anti-Cerebral Ischemia/Repersusion Injury Effect Of Luteolin Micelles

Ischemic cerebrovascular disease is a common type of neurological disease in the clinic.Studies have shown that luteolin can effectively alleviate the symptoms of cerebral ischemia,which is related to its anti-inflammatory,oxidative stress and apoptosis reduction.However,its poor water solubility and fast metabolism have greatly limited the clinical application of luteolin.Therefore,the nano drug delivery system came into being.In this study,a biocompatible and degradable mPEG-PLGA was used to successfully prepare luteolin nanomicelles?M-Lu?by membrane hydration,and luteolin was dissolved in DMSO as a control?Free Lu?.And its anti-I/R efficacy was tested in vitro and in vivo.Methods:?1?Preparation and characterization of M-Lu:Lu and mPEG-PLGA were co-dissolved in 4 mL of absolute ethanol,and the solvent was evaporated at about 60?to form a film,which was resuspended in dd H₂O and then freeze-dried to obtain M-Lu powder.The shape,particle size,drug loading and encapsulation efficiency,stability in different temperatures and media,and in vitro release behavior were investigated.?2?In vitro biological activity of M-Lu:The luteolin was replaced by the fluorescent probe coumarin?C6?,and the uptake ability of PC12 cells to M-Lu and Free Lu was detected by immunofluorescence technique.The H₂O₂-induced oxidative stress injury model of PC12cells in vitro was established and the activity and morphology were detected,investigating the neuroprotective effect of M-Lu.The Bcl2/Bax,NF-?B and MAPK related protein content were detected by Western-blot,thus the mechanism of M-Lu antioxidant damage was systematically evaluated.At the same time,immunofluorescence detection of three neuronal marker proteins NSE,GFAP and Nestin expressed by M-Lu-induced bone marrow mesenchymal stem cells?BMSCs?was performed to explore a new mechanism of M-Lu nerve repair.?3?The anti-cerebral ischemia efficacy study of M-Lu:The rat model of right cerebral ischemia-reperfusion injury was established.The cerebral infarct volume,water content,histopathology,inflammation and oxidative stress were investigated in each group,and the pharmacokinetic parameters of M-Lu in rats were detected.Results:?1?M-Lu is spherical,with a particle size of 30 nm and a PDI of 0.07±0.03.The dispersion is uniform and has good stability.The drug loading and encapsulation efficiency were 5%and 97.20±2.34%,respectively.Within 24 h,the cumulative release of luteolin from M-Lu was about 58%in the buffer solution with pH=7.4,while the cumulative release of Free Lu was 92%,indicating that luteolin nanomicelles have sustained release properties.?2?The cell uptake experiments showed that the uptake ability of PC12 cells to M-Lu was significantly enhanced.After M-Lu pre-protection,the cell viability and cell morphology were also significantly improved after H₂O₂ induced.Compared with Free Lu group,the expression of Bcl2/Bax protein in M-Lu group was significantly increased,and the level of NF-?B is significantly reduced.The phosphorylation levels of JNK,P38 and ERK1/2,the three major proteins of MAPK,were significantly decreased.Nanomicelles can effectively reduce apoptosis by inhibiting NF-?B and the activation of MAPK signaling pathways.At the same time,M-Lu can induce BMSCs highly express neural marker proteins NSE,GFAP and Nestin,this indicates that M-Lu has the possibility of inducing the differentiation of BMSCs into neuron-like cells.In summary,indicting that luteolin nanomicelles have a neuroprotective effects in vitro.?3?Compared with Free Lu,M-Lu can significantly reduce the volume of cerebral infarction,brain water content,alleviate Nissl body damage,and significantly reduce the number of TUNEL positive cells and reduce apoptosis.At the same time,the expression of TNF-?,IL-1?and IL-6 was significantly reduced,which effectively alleviated the inflammatory response.M-Lu also significantly increased the expression of SOD and GSH-PX in rat brain tissue,reduced MDA content and alleviated oxidative stress damage.The pharmacokinetic parameters showed that M-Lu significantly increased the blood concentration of luteolin in rats compared with Free Lu,prolonged the half-life,showing a sustained release effect.Conclusions:Nanomicelles formulation effectively improved the medicine defects of luteolin,it effectively reduces cell apoptosis,inhibits inflammation and oxidative stress by up-regulating Bcl2/Bax,down-regulating NF-?B expression,and decreasing MAPK phosphorylation activity,thus significantly alleviating cerebral ischemia-reperfusion injury.