Pharmacokinetic Characteristics And Neuroprotective Effect Against Cerebral Ischemia-Reperfusion Injury Of Main Components In Guhong Injection

Objective1.To develop a high sensitive liquid chromatography-mass spectrometric method（LC-MS/MS）for the determination of the recovery rates of blood and brain microdialysis probes of acetylglutamate（NAG）and its decomposition products—glutamic acid（Glu）andγ-aminobutyric acid（GABA）in vitro.2.To determine the pharmacokinetic characteristics of NAG and its metabolites Glu and GABA in different doses of NAG and Guhong injection（GHI）in rats and to compare thedifferences inpharmacokineticsof NAG alone or in combination with other components.3.To investigate the neuroprotective effect and underlying mechanism of hydroxysafflor yellow A（HSYA）,NAG alone or HSYA together with NAG using a rat model of cerebral ischemia reperfusion injury.Methods1.The separation was accomplished on an Agilent Zorbax SB-C18 column（2.1×100 mm i.d.,3.5μM particle）with the mobile phase composed of acetonitrile-water（70:30,v/v）containing 5 m M ammonium acetate at a flow rate of 0.3 m L/min.N-carbamyl-L-glutamic was used as internal standard.Positive ion electrospray ionization with multiple reaction-monitoring mode（MRM）was employed by monitoring the transitions m/z 189.1/130.0,148.0/84.1,104.1/87.1,191.0/130.1 for NAG,Glu,GABA and the IS respectively.The concentrations of NAG,Glu and GABA in blood and brain microdialysates were determined by LC-MS/MS and the probe recoveries were calculated.The in vitro recoveries of NAG,Glu and GABA were studied using positive dialysis and retrodialysis methods at different flow rates and concentrations.2.Rats were given low（NAG-L,75 mg/kg）,medium（NAG-M,150 mg/kg）,high（NAG-H,300 mg/kg）NAG and Guhong injection（GHI,10 m L/kg）.LC-MS/MS combined with microdialysis technology were performed to determinatethe contents of NAG and the major components of its metabolites Glu and GABA.PKSolver2.0software was used to fit pharmacokinetic parameters with non-compartmental model.3.Male Sprague-Dawley（SD）rats（n=5）were randomly divided into sham group,model group,HSYA low dose（5 mg/kg）,middle dose（10 mg/kg）,high dose（20 mg/kg）groups,NAG（300 mg/kg）,HSYA（10 mg/kg）+NAG（300 mg/kg）,For the intraluminal suture technology to establish a focal cerebral ischemic model,the approach of inserting a monofilament suture into the internal carotid artery to block the origin of the middle cerebral artery（MCAO）has been applied.In the sham group,rats were subjected to the same surgical procedures as above except that no nylon suture was applied.The rats in treated groups were injected with the corresponding drugs in the tail vein,in the other groups,the rats were injected normal saline,once a day for 7 days.At 1,3,7 d after MCAO,the neurological deficit and infarct volume of rets were assessed.At 7 d after MCAO,we used TUNEL to evaluate the apoptosis rate in brain tissue after the model,¹⁸F-FDG-PET to evaluate the regional cerebral metabolic rate of glucose consumption,immunohistochemical analysis to detect the expression of glial fibrillary acidic protein（GFAP）,nerve growth factor（NGF）,B-cell lymphoma-2（Bcl-2）,Bcl-2 associated X protein（Bax）,caspase-3 and intercellular adhesion molecule 1（ICAM-1）in brain tissue at day 7 after cerebral I/R injury.Meanwhile,the m RNA levels of ICAM-1,IL-1?,TNF-αand NF-κB were determined by q RT-PCR,the protein levels of Bcl-2,Bax and caspase-3 were detected by western blot.Results1.LC-MS/MS method determined the NAG,Glu and GABA in vitro recovery rates of blood brain and brain probes indicating that NAG showed a good linear relationship at concentration of 1-10000 ng/m L,the LLOQ of NAG was 1 ng/m L.The linear range was 10-50000 ng/m L for Glu and GABA,the LLOQ of Glu and GABA was 10 ng/m L.The specificity,precision,accuracy,stability of methods were in accordance with Pharmacopoeia regulations.The in vitro recoveries of blood and brain probes of NAG,Glu and GABA were inversely proportional to the perfusate flow rates while independent of drug concentrations.The recovery rates obtained by positive dialysis and retrodialysis methods in vitro were approximately equal under the same condition,and were stable for at least 6 h.At the flow rate of 1.5μL/min,the average in vitro recoveries of NAG,Glu,and GABA in blood and brain microdialysis were 40.31%,42.46%,47.55%and 11.63%,20.66%,34.36%,respectively.2.After administration of low（75 mg/kg）,medium（150 mg/kg）,and high（300mg/kg）NAG and GHI in rats via the tail vein.NAG rapidly passed through the blood-brain barrier and metabolized to Glu and GABA.In the blood test,the values of C_max in the NAG-L group were markedly lower than those of the NAG-M,NAG-H and GHI groups（P<0.01,respectively）.There was no significant difference between NAG-M and GHI groups,while,the C_max of NAG in NAG-H group was significantly upgraded compared with GHI group.In brain microdialysate,Compared with NAG-L and-M groups,the AUC_0-t and MRT values of NAG were significantly increased in GHI group.The T_1/2 value of NAG in GHI group was significantly higher than that in NAG-H group（P<0.05）.After administration,the C_max of Glu in blood was no significant difference between NAG-H and GHI groups;While,in the brain microdialysate,the C_max of Glu in GHI group was significantly lower than that in NAG-H group,conversely,the C_max of GABA in the GHI group was significantly higher than NAG-H group.The drug distribution coefficients of NAG,Glu,GABA in brain and blood in low,medium,high doses of NAG and GHI groups were 13.99,27.43,34.81,31.37;11.04%,59.07%,21.69%,2.69%;212.88%,234.92%,157.59%and 102.65%,respectively.Our results indicated that NAG has easy and dose-dependently access to the blood-brain barrier and exhibits a moderate residence time in rats.3.HSYA、NAG and HSYA+NAG significantly improved neurological function,decreased cerebral infarction volume,inhibited the expression of apoptosis-positive cells in infarcted area.The expressions of GFAP,NGF,and Bcl-2 of HSYA+NAG group were obviously augmented than those of NAG and HSYA treatment alone.The Bax and caspase-3 protein expression in HSYA+NAG group was significantly decreased than 10 mg/kg HSYA and 300 mg/kg NAG group alone.The expression of ICAM-1 protein in high dose HSYA（P<0.01）and HSYA+NAG（P<0.01）groups was significantly inhibited than that in the model group.The expression of ICAM-1protein in HSYA+NAG group was notably suppressed than that in NAG group.QRT-PCR results showed that HSYA,NAG and HAYA+NAG profoundly decreased the m RNA expression of NF-κB,TNF-αand IL-1βafter I/R injury.Conclusion1.Microdialysis can be used for blood and brain pharmacokinetic study of NAG,and retrodialysis method can be used to study the probe recovery of NAG.2.The multi-point synchronous microdialysis technique can simultaneously detect the distribution of multiple components in multiple effect sites,and has the advantages of continuous sampling in vivo.The ground provides certain examinations for the effect material basis of carbon multi-component medicines.The specificity,precision,accuracy,stability of LC-MS/MS method was in accordance with Pharmacopoeia regulations,this method could be successfully used for the pharmacokinetic studies of NAG and its related metabolites in rats,these findings provide an important pharmacokinetic foundation for the effective clinical use of NAG.The good blood-brain barrier permeability and pharmacokinetic properties of GHI provide a theoretical basis for it to treat cerebrovascular diseases.3.HSYA,NAG and HSYA+NAG exerted neuroprotection against cerebral I/R injury by recoverying glucose metabolism,modulating inflammation and apoptosis process,and HSYA in combination with NAG possessed a synergetic effect on protecting cerebral I/R brain injury.