Hydroxysafflor Yellow A Nanoemulsion Protects Mice From Focal Cerebral Ischemia-reperfusion Injury

Part Ⅰ Preparation and pharmacokinetic study of HC@HMCObjective:To prepare HYA nanoemulsion preparation and study its physical and chemical properties.And to investigate the pharmacokinetics of HC@HMC in SD rats.Methods:1.Preparation of hyaluronic acid modified multi-walled carbon nanotubes and chitosan modified hydroxysafflor yellow A-hydroxypropyl-β-cyclodextrin phospholipid complex water-in-oil nanoemulsion（HC@HMC）by titration.2.The particle size and Zeta potential of HC@HMC were measured by Malvern Laser Particle Size Potentiometer.The pH of HC@HMC was measured by PH meter measures.The conductivity of HC@HMC was measured by conductivity meter.3.A reversed-phase HPLC method was established to determine the concentration of HYA in rat plasma after oral administration of HC@HMC 60mg·kg^-1 and HYA 60 mg·kg^-1,and the pharmacokinetic parameters and bioavailability of HYA and HC@HMC were investigated.Results:1.The appearance of HC@HMC was a clear and uniformly dispersed liquid.2.The average Zeta potential of HC@HMC was about 0 m V,and the particle size chart had no results.3.The maximum blood concentration（Cmax）of HYA was only 1.25±0.33mg·L-1,while the Cmax of HC@HMC was 20.14±4.23 mg·L-1,which was 16.11times that of HYA.In addition,the AUC_0-72h,Tmax and MRT_（0-96h）of HC@HMC were higher than those of HYA,The AUC_{0-96 h} and Cmax of HC@HMC were about10 times and 16 times of HYA.Besides,the Cl of HYA was more than double that of HC@HMC,indicating that HC@HMC could prolong the residence time in rats.Compared with HYA,the relative bioavailability of HC@HMC was increased by about 9 times.Conclusion:1.Hyaluronic acid modified multi-walled carbon nanotubes and chitosan modified hydroxysafflor yellow A-hydroxypropyl-β-cyclodextrin phospholipid complex water-in-oil nanoemulsion（HC@HMC）were prepared by titration.2.HC@HMC could promote the absorption of HYA in rats,and obviously improve the blood peak concentration and the bioavailability of HYA in rats.Part Ⅱ Determination of intracranial concentration of HC@HMC by HPLC/MSObjective:In order to observe the effect of HC@HMC on cerebral ischemia-reperfusion injury in mice,this part intends to establish a method for measuring the concentration of HYA in mouse brain tissue.Methods:1.Animal groups:18 mice were randomly divided into 6 groups.Intravenous injection of HYA 40mg·kg^-1 0.5h,1h,1.5h group,intragastric administration of HC@HMC 160mg·kg^-1（The absolute bioavailability of HC@HMC is 23%of HYA）0.5h,1h,1.5h group.2.Determining method:The concentration of HYA in brain tissue of mice was detected by ultra performance liquid chromatography-tandem mass spectrometry.ACE Excel 2C18-PFP column（2.1×100 mm,2.0μm）for separation,0.1%formic acid-methanol was used as the mobile phase for gradient elution,the flow rate was 0.2m L·min^-1,and the injection volume was 5μL.ESI+Agilent Jet Stream ion source,multi-reaction monitoring mode（MRM）detection.Rutin was used as the internal standard,and the internal standard method was used for quantification.Results:1.A HPLC/MS method for the determination of HYA was established.The specificity experiment results showed that the endogenous substances in brain tissue had no interference on the determination of HYA,and the method had a good linear relationship in the range of 1～1000 ng·m L^-1 of HYA,good precision,extraction recovery about 50%,and RSD less than 15%.2.Intravenous injection of HYA(40 mg·kg^-1)could penetrate the blood-brain barrier,and the maximum concentration of brain tissue reached 20 ng·m L^-1 at 1 h.Oral administration of HC@HMC 160 mg·kg^-1(equivalent to intravenous injection of40 mg·kg^-1after considering the bioavailability)also reached the maximum concentration of about 40 ng·m L^-1 at 1 h.The brain tissue concentration-time curves of HYA and HC@HMC were both increased at first and then decreased.Conclusion:1.A method for measuring the concentration of HYA in brain tissue was successfully established.2.HC@HMC may penetrate the blood-brain barrier more easily than HYA.Part Ⅲ Effects of HC@HMC on improving cerebral ischemia-reperfusion injury in miceObjective:To observe the protective effect of HC@HMC on cerebral ischemia-reperfusion injury in mice,and to explore its mechanism from the inflammatory related COX-2/PGD2/DPs pathway.Methods:1.Establishment of middle cerebral artery occlusion reperfusion（MCAO/R）mice model.Male,8-10 week old mice,weighing 18-22g.The mice were intraperitoneally anesthetized with pentobarbital sodium(45mg·kg^-1)and then fixed,and the right common carotid artery,internal carotid artery and external carotid artery were separated.A nylon tether was inserted into the right common carotid artery to the beginning of the middle cerebral artery（the insertion depth of the tether was about10mm）to block the blood flow of the right middle cerebral artery for 60 minutes and then reperfused for 24 hours.The sham-operated group was treated with the same methods as the model group except that the bolt was not inserted.2.Experimental groupThe experimental animals were divided into 7 groups:sham group,MCAO/R group,vehicle group,HC@HMC 40mg·kg^-1 gavage group,HC@HMC 80mg·kg^-1gavage group,HC@HMC 160mg·kg^-1 gavage group,HYA 20 mg·kg^-1 tail vein injection group.The drug was administered five days before the operation,once a day,and the model was created immediately after the fifth day.3.Observation indicators and methodsNeurological deficit score 0-5 score to evaluate the changes of neurological deficits in mice;Rotating rod experiment to evaluate postoperative exercise ability of mice;TTC staining was used to detect the cerebral infarction in mice and the infarct volume was calculated;The pathological changes of hippocampus and cortex were observed by HE staining;The expression of COX-2,DP1 and DP2m RNA was detected by q-PCR;Western blot was used to detect the protein expression of COX-2,DP1 and DP2;The contents of PGD2,TNF-αand IL-1βin cortex were detected by ELISA.Results:1.Compared with the normal group,the sham operation group had no obvious infarction,and the neurological deficit score had no significant difference,and the normal group was no longer set up in subsequent experiments.2.Compared with the sham group,the neurological deficit score of the mice in the MCAO/R group was significantly increased;The residence time of the rotating rod experiment was significantly reduced;The cerebral infarction volume was significantly increased,and the cortex and hippocampus vacuolization,nuclear deep staining and pyknosis were significantly significantly increased;The expression of COX-2,DP1,DP2 m RNA and protein in the cortex increased;the content of PGD2,IL-1β,and TNF-αin the cortex significantly increased.Compared with MCAO/R group,80mg·kg^-1 HC@HMC group and 160mg·kg^-1 HC@HMC group and 20mg·kg^-1HYA group significantly decreased the infarct volume and neurological score;The retention time of the rotating rod experiment was significantly increased;The m RNA and protein expressions of COX-2,DP1 and DP2 in cortex were significantly decreased,and the contents of PGD2,IL-1βand TNF-αin cortex were significantly decreased.There was no significant difference between the HYA 20 mg·kg^-1 group and the HC@HMC 80mg·kg^-1 group,but the cerebral infarction volume and the cortical and hippocampal cell mortality were relatively reduced in the HC@HMC80mg·kg^-1 group.Conclusion:1.HC@HMC 80mg·kg^-1 group,HC@HMC 160 mg·kg^-1 group and HYA20mg·kg^-1group have protective effects on cerebral ischemia-reperfusion injury.2.HC@HMC and HYA may protect against cerebral ischemia-reperfusion injury through COX-2/PGD2/DP_S pathway.