Study Of Neuroprotective Mechanism Of Xiao-Xu-Ming Decoction On Cerebral Ischemia/Reperfusion Rats

Part Ⅰ:Metabonomic Analysis of Protective Effect of Xiao-Xu-Ming Decoction on the Rat Model of Cerebral Ischemia/Reperfusion.Objectives:To investigate the effects of Xiao-Xu-Ming Decoction（XXMD）on neurological deficits,cerebral infarct volume,and neuronal cells injury in cerebral ischemia/reperfusion（I/R）model rats.Using 1H NMR-based metabolomics techniques to explore the metabolic effects of Xiao-Xu-Ming Decoction on the cortex of ischemia/reperfusion rats.Methods:All the healthy Sprague-Dawley（SD）rats of 250-280g were randomly divided into three groups:sham+saline group（Sham）;cerebral ischemia/reperfusion model+saline group（MCAO）;and cerebral ischemia/reperfusion model+Xiao-Xu-Ming decoction group（XXMD）.The I/R model was established by the Middle cerebral artery occlusion（MC AO）method,and the sham group only isolated the vessels without nylon fish line insertion.Seven days before modeling,XXMD group was administered intragastrically 30 mL/kg/day（drug concentration 2 g/ml）of XXMD twice daily,while the corresponding volume of saline was gavaged in both the Sham and MCAO groups.The Modified Neurological Severity Scores（mNSS）was used to assess the neurological function of each group at 24h after ischemia/reperfusion;2,3,5Triphenyte-tetrazoliumchloride（TTC）staining was used to stain the cerebral infarct area.Pathological staining of each experimental group of rats was performed using HE staining,Nissl staining and NeuN immunofluorescence staining.Brain area samples were collected from each group of rats at 24 hours of ischemia-reperfusion,and the cortical samples were examined by NMR using a 600 MHz superconducting high-resolution NMR spectrometer.The metabolic differences between the experimental rats in each group were analyzed using multivariate analysis to find and quantify the differential metabolites.Results:mNSS scores showed that the neurological function of the rats in the MCAO group was significantly impaired,while that was restored after the treatment with XXMD.TTC staining showed XXMD treatment reduced the infarct volume percentage compared with the MCAO group.The MCAO group displayed neuronal cell loss,nuclear pyknosis,and neuronal atrophy when compared with the sham group.After XXMD treatment,the number of neurons in the cortex and hippocampus tissues increased,and the ischemic neuropathological abnormalities were alleviated.Cell nuclei in the sham group exhibited typical morphologies in the cortex and hippocampus.In the MCAO group,several neuro-morphological abnormalities were identified,including an increase in pyknotic cells and a notable decrease in Nissl body content,indicating that neurons were damaged.However,the reduction in Nissl-stained nuclei was retained following XXMD treatment.As seen in immunofluorescence staining,the NeuN protein,a neuronal marker,was abundantly expressed in the cortex and hippocampus in the sham group.In the MCAO group,the number of NeuN-positive cells was significantly decreased;when treated with XXMD,the expression of NeuN was reversed,indicating that XXMD treatment alleviated the neuron damage.PLS-DA results showed there were metabolic differences in the cerebral cortex of rats in each group at 24 hours after ischemia/reperfusion.The OPLS-DA results showed that metabolic differences were observed between the MCAO group and the sham group or XXMD groups,and the differential metabolites were obtained by VIP plots;after relative quantification,it was concluded that the XXMD could significantly improve the significantly decreased metabolic levels of creatine,taurine and glutamine and decrease the significantly increased metabolic levels of choline and O-Phosphocholine.The lactate level was significantly increased in the MCAO group,but there was only a decreasing trend of lactate metabolism level without significant difference after the intervention of XXMD.Conclusion:XXMD played a neuroprotective role by improving neurological deficits induced by I/R,reducing the infarct volume,and alleviating the damage of cortical and hippocampal neuronal cells in the infarct area.Additional,Xiao-Xu-Ming Decoction could change the metabolic pattern of cortex in rats and improve the metabolism of energy,phospholipids and amino acids.Part Ⅱ:The Neuroprotective Effect of Xiao-Xu-Ming Decoction on Cerebral Ischemia/Reperfusion via Modulating Astrocyte-Neuron Lactate Shuttle and Activating AMPK/SIRTl/FoxO1A Signaling Pathway.Objective:The objective of the study was to investigate whether XXMD exerts neuroprotective effects on cerebral ischemia/reperfusion（I/R）by regulating astrocyte-neuron lactate shuttle to improve energy metabolism and activating the AMPK/SIRT1/FoxO1A signalling pathway to anti-oxidant and anti-apoptosis.Methods:The 250-280g SPF adult-male Sprague-Dawley rats were randomly divided into Sham,middle cerebral artery occlusion（MCAO）,and the MCAO plus XXMD（XXMD）group.The modelling and administration methods were the same as the previous ones.Brain areas of rats in each group were collected 24hours after I/R,ATP,NAD+/NADH ratio,and lactate and glucose levels were determined.Real-time PCR was used to measure the critical enzymes of the Tricarboxylic acid（TCA）cycle and glycolysis to investigate the effect of XXMD on energy metabolism.Moreover,the Astrocyte-Neuron Lactate Shuttle related protein was detected by western blot to explore the effect of lactate transportation after XXMD treatment.The expression.of AMPK,SIRT1,and FoxO1A was estimated using real-time PCR,western blot,and immunofluorescence analyses.Furthermore,the cortical levels of MDA,GSH,and CAT were determined,and expression of proteins related to oxidative stress was determined using real-time PCR and western blot.TUNEL staining was performed to assess apoptosis,and realtime PCR and western blot were performed to measure the expression of apoptosis-related proteins,including Bax,Bcl-2,caspase3,and cleaved caspase3.Results:Furthermore,the ATP content,NAD+/NADH ratio,glucose uptake and use,glycolytic flux and lactate transport from astrocyte to neuron were increased after XXMD therapy.XXMD enhanced the conversion of lactate to pyruvate and the TCA cycle’s ability to generate ATP.The AMPK/SIRT1/FoxO1A signalling pathway was activated by XXMD therapy,as revealed by real-time PCR and western blot.Moreover,the expression of SIRT1 was elevated after treatment with XXMD,according to immunohistochemical analyses.Meanwhile,XXMD lowered the MDA level while increasing CAT and GSH.XXMD reduced the number of TUNEL-positive cells with the decreased expression of Bax and Cleaved Caspase3/Caspase3 and increased Bcl-2 level.Conclusion:XXMD could stimulate the astrocyte-neuron lactate shuttle to improve energy metabolism and activate AMPK/SIRT1/FoxO1A-mediated anti-oxidant and anti-apoptosis,thereby protecting neurons.