| Objective: Subarachnoid hemorrhage(SAH)is an acute severe hemorrhagic apoplexy with high mortality and morbidity.Numerous studies proved that early brain injury(EBI)was the main reason leading to high mortality and poor prognosis after SAH.The conception of the neurovascular unit(NVU)better explained the structural basis and pathophysiology of EBI.In previous studies of cerebral ischemia,we found that ?-caryophyllene(BCP)showed the effect of improving cerebral circulation and reducing neurological impairment.In this study,?-caryophyllene loaded liposomes(BCP-LP)was prepared for injection and their effect on NVU injury in SAH rats was investigated.SAH models in rats were established to evaluate BCP-LP's effects on EBI and NVU injury,and preliminary explored the biological mechanism.Method:1.BCP-LP was prepared by the ethanol injection and ultrasound dispersion method,and its encapsulation efficiency,Microscopic morphology,Particle size distribution,Zeta potential,drug release characteristics and in vivo analysis experiments were performed.2.SAH model was built by injecting non-heparinized arterial blood into prechiasmatic cistern on male SD rats.Experiment rats were randomly divided into Sham group(blank control group),SAH group(model control group),SAH + solvent group(solvent control group),SAH + BCP-LP group(BCP experiment group).The neurobehavioral function test and balance beam experimental test were used to detect the movement ability and balance ability of the rats in each group at 6 h,12 h,24 h,48 h,and 72 h after modeling.The cerebral blood flow(CBF)of experiment rats in each group was monitored to assess brain blood supply at the same time.Permeability of Evans blue(EB)in brain tissue was measured to evaluate the blood brain barrier(BBB)permeability of rats at 72 h after SAH to detect the degree of BBB damage.Wet and dry weight method was used to detect the degree of brain edema in each group.EBI levels of rats in each group were evaluated by the experiments above.3.At 72 hours after modeling,the expressions of tight junction proteins Occludin and Zonula occludens-1(ZO-1)were detected by Western blot(WB)to detect tight junction damage.WB and TUNEL staining were used to detect the expression of Bax,Bcl-2 and Cleaved-caspase3 to reflect the degree of apoptosis in the brain tissue of rats in each group.Expressions of vascular endothelial growth factor receptor 2(VEGFR-2),Glial fabrilary acidic protein(GFAP),and laminin-?(Laminin-?,LN-?)were determined to study the expression of various NVU structural proteins.The distribution and expression of VEGFR-2 and GFAP in brain tissue were observed by immunohistochemical and immunofluorescence staining.The distribution and expression of Cannabinoid receptor type 2(CB2)in rat brain tissue were verified by immunohistochemistry and WB experiments.The peroxisome proliferator-activated receptor-?(PPAR?)and Matrix metalloproteinase 9(MMP-9)expression levels were detected to elucidate the role of BCP-LP in EBI.Result:1.BCP-LP was successfully prepared with an encapsulation efficiency of approximately 81%.The TEM image revealed the formation of nanometer-sized particles.The average size distribution was 189.3±3.8nm,and the average Zeta potential distribution was-13.9±0.3 mV.The drug release profiles of BCP-LP showed a relatively slow release.The in vivo analysis experiments demonstrated that BCP-LP prominently increased the bioavailability of BCP in rats when compared to BCP-OLIVE.2.The SAH model rats in each group showed severe neurological deficits.At 24 hours after modeling,the rats in the BCP-LP intervention group gradually showed better nerve repair ability.At 72 hours after the operation,behavioral ability and balance of SAH rats in the BCP-LP group almost recovered to normal levels,showing better and faster functional recovery compared to the rats in SAH group.CBF was significantly impaired after SAH in rats,until 48 hours after surgery,the CBF of rats in the BCP-LP group showed better recovery in SAH rats compared with rats in the SAH group.Significant BBB destruction and brain edema were detected in rats at 72 hours after SAH,while rats in the BCP-LP intervention group showed less EB exudation,suggesting that BCP-LP can promote BBB repair and reduce brain edema.3.The expressions of tight junction proteins Occludin and Claudin-5 were significantly reduced at 72 h after SAH.The expression of tight junction proteins in the brain tissue of BCP-LP treated rats was significantly higher than that of SAH rats.TUNEL staining showed that apoptosis of neural cells in hippocampus after SAH was obvious increased.WB detection results showed the increased expressions of Bax,Bcl-2 and Cleaved-caspase 3.The treatment of BCP-LP raised the expression of Bcl-2 and reduced the expression of Bax and Caspase3.4.After SAH,the expression of GFAP and VEGFR-2 was significantly increased,as well as proliferation of glial cells and cortical microvessels,while the loss of laminin was increased.The intervention of BCP-LP inhibited the overexpression of GFAP and VEGFR-2,and promoted the expression of LN-?.CB2 is mainly expressed in the cell membrane and cytoplasm in rats' brain tissue.After SAH,the expression of CB2 is compensatory increased,and BCP-LP administration could distinctly raise the expression of CB2.Meantime,the expression of PPAR? and MMP-9 were both increased after SAH in rats.With the up-regulated expression of CB2 by BCP-LP,the expression of PPAR? was increased and the expression of MMP-9 was significantly reduced.Conclusion:1.BCP-LP can be successfully prepared,and BCP-LP can increase the bioavailability of BCP.2.BCP-LP can maintain BBB's integrity by repairing its damage,reducing cerebral edema,and then improving CBF,reducing neurological dysfunction after SAH,and thereby exert the neuroprotective effect on injuries of EBI.3.The effect of BCP-LP in improving EBI may be achieved by regulating the CB2/PPAR?/MMP-9 molecular pathway,reducing apoptosis,protecting tight junction proteins,and repairing NVU damage. |
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