| BackgroundStroke is a common clinical disease of cerebrovascular diseases.According to its etiology,it can be divided into two types:ischemic and hemorrhagic,of which ischemic stroke is more common.Rapid restoration of cerebral blood flow is the primary goal of the treatment of ischemic stroke,and also the premise of neuroprotective therapy.However,reperfusion would cause complex ischemia reperfusion injury(CIRI),which involved a series of ischemia cascade reactions including excitatory toxicity,immunity and inflammation,blood-brain barrier,energy metabolism and oxidative stress.Among them,energy metabolism disorder during ischemia and oxidative stress during reperfusion were the initial factors.At present,various neuroprotective agents in the stage of research and development or clinical application only block a certain link of stroke cascade to interfere with cellular pathology and biochemical reactions caused by ischemia and hypoxia,and it is difficult to achieve good efficacy by targeting a single molecular target.The multi-target holistic treatment strategy derived from traditional Chinese medicine may be the breakthrough point for the treatment of stroke.The advantage of TCM compound lies in the integration and regulation of each single effect.The active ingredient group of traditional Chinese medicine plays an integrated role in drug efficacy characterization through multi-target and multi-channel integration and antagonism.The mechanism of action of traditional Chinese medicine is not limited to direct antagonism of focal targets,but also includes indirect regulation across organs and systems through the regulation of intermediary substances.There is a close relationship between the central nervous system and the peripheral immune system after CIRI.In addition to the drastic pathological changes in the brain,cerebral ischemia-reperfusion injury can induce extensive pathophysiological changes outside the brain,and the resulting peripheral disorders can in turn exacerbate brain injury by affecting key aspects of stroke progression(neuroinflammation).With the further development of this research,the "dialogue" between the central nervous system and the peripheral immune system after ischemic stroke has become closer.Shengmai injection(SMI)is a traditional Chinese medicine derived from a traditional formula Shengmai San,consisting of Red ginseng(the root of Panax ginseng),Fructus schisandrae(the fruit of Schisandra chinensis),and Radix ophiopogonis(the root of Ophiopogon japonicus).It is a representative prescription for supplementing qi and nourishing yin.The analysis of clinical medication characteristics based on the hospital system and the consensus of clinical experts showed that SMI is widely used in the treatment of cerebral stroke and cerebral infarction.At present,most studies on SMI in the treatment of cerebral ischemia focus on clinical observation and single link,and further studies are still needed on the scientific characterization of its effects and the explanation of its mechanism.In summary,this study takes the chemical composition of SMI as the starting point to study the exposure of SMI in vivo under the pathological state of cerebral ischemia reperfusion and identify the potential effector organs.Further,combined with multidimensional pharmacodynamic evaluation and protein network,explain the effect effect link of SMI on CIRI.Based on the above two parts of research,combined with the target organ and the peripheral immune organ,the mechanism of the main effect link was discussed,to clarify the overall regulatory role of SMI in CIRI.Contents1 Chemical composition identification and in vivo exposure analysis of Shengmai injection:In this study,multiple analytical techniques of UPLC-LTQOrbitrap-MS and UPLC-QQQ-MS were applied to qualitatively and quantitatively analyze the components of SMI,the systemic exposure of these components to I/R rats.The composition of pharmacodynamic substances,effector organs and target components of Shengmai injection were determined to lay the foundation for the analysis of action mode of Shengmai injection.2 Confirmation of the effect link of Shengmai injection on CIRI:A Transient Middle cerebral artery occlusion(tMCAO)model was constructed by thread embolism.The dose-effect and time-effect relationship of SMI were evaluated by neurological function score,infarct size and pathological staining(HE and Nissl staining).To investigate the protective effect of SMI from different pathological links during cerebral ischemia reperfusion.Secondly,proteomics technology was used to construct the pathological link network of cerebral ischemia reperfusion injury and the characteristic network of SMI intervention.Identify the dominant effect link of SMI intervention on cerebral ischemia reperfusion injury from multi-level and multi-approache.3 Regulatory mechanism of shengmai injection on inflammatory link of cerebral ischemia-reperfusion injury:The mode of action of SMI was analyzed based on direct regulation of central target organ and indirect regulation of peripheral immune organ spleen.Central direct regulation:The characteristic network of SMI intervention in cerebral ischemia reperfusion injury established by proproteomics was analyzed and mined to screen out the driving proteins of oxidative stress-inflammation.Peripheral indirect regulation:In the pathological condition of cerebral ischemiareperfusion injury,the "brain-spleen" communication was determined by dynamic observation of spleen index(spleen mass/body weight),neurological function score,infarct volume and splenectomy;Flow cytometry was used to determine the media of"brain-spleen" communication.The peripheral regulatory mechanism of SMI is analyzed based on the "brain-spleen"interorgan communication.ResultsSection one:Chemical composition identification and in vivo exposure analysis of Shengmai injection(1)Chemical composition analysis of Shengmai injection based on UPLC-LTQOrbitrap-MS technique:In this study,UPLC-LTQ-Orbitrap-MS method was established to analyze the chemical constituents of Shengmai injection.Combined with online and offline databases,a total of 59 prototype compounds were identified from SMI.These compounds were mainly derived from Red ginseng and Fructus schisandrae,including 25 ginsenosides,25 lignans and a few radix ophiopogonis saponins.On this basis,the fragmentation behaviors of representative components were summarized.(2)Study on tissue distribution of Shengmai injection based on LC-QQQ-MS technique:By UPLC-QQQ-MS,23 chemical compounds were then quantitatively identified with their contents in SMI.Ginsenosides,as the main active compounds from Red ginseng,showed the highest contents in SMI.The contents of these components in brain,heart,liver,spleen and kidney tissues under cerebral ischemia reperfusion were further detected.Among them,15 compounds including ginsenosides and Schisandrol A were further found to have considerable exposure in different tissues,and were distributed in the target organs of cardiovascular and cerebrovascular diseases,brain tissue,heart and peripheral immune organ spleen.(3)Pharmacokinetic analysis of SMI:A selective and sensitive UPLC-QQQMS/MS method was developed for the simultaneous quantification of the seven compounds in rat plasma.The established method was then successfully applied to pharmacokinetic analysis in I/R rats receiving an intravenous infusion dose of SMI.The results showed that PPD-type ginsenosides exhibited an extremely high exposure level and a much slower elimination rate,whereas PPT-type ginsenosides were quickly eliminated.Section two:Confirmation of the effect link of Shengmai injection on cerebral ischemia-reperfusion injury(1)Study on neuroprotective effect of Shengmai injection on cerebral ischemia reperfusion injury:After Shengmai injection intervention,the neurological deficit score,infarct volume,the number of neurons and Nissellite bodies in brain tissue were significantly improved.Shengmai injection has neuroprotective effect on cerebral ischemia-reperfusion injury.(2)Study on effects of Shengmai injection on cerebral ischemia reperfusion injury:The results of molecular biology research show that SMI can achieve neuroprotective effects by improving endogenous antioxidant system(ROS,SOD,GSH-Px,MDA),reducing the expression of anti-inflammatory factors(IL-6,IL-β),interfering with oxidative stress and inflammation links,and thereby reducing cell apoptosis(TUNEL staining).(3)Construction of characteristic map of Shengmai injection for cerebral ischemia reperfusion:A total of 5746 proteins were identified by proteomic analysis.Compared with sham operation group,there were 441 differentially expressed proteins in model group,which were mainly concentrated in immune and inflammation,oxidative stress,energy metabolism,neuronal apoptosis,blood-brain barrier,synaptic transmission,ion homeostasis and other pathological processes.Compared with the model group,there were 602 differentially expressed proteins in SMI group.Among them,the proteins in the SMI repair group and the cerebral ischemia-reperfusion injury group that change together regulate the above pathological links.The proteins regulated by SMI repair group are mainly related to immunity,inflammation and oxidative stress.Therefore immune and inflammatory links may be the main effect link of SMI intervention.Section three:Regulatory mechanism of shengmai injection on inflammatory link of cerebral ischemia-reperfusion injury(1)Shengmai injection interferes with central regulation of inflammatory link in cerebral ischemia reperfusion injury:In the pathological state of CIRI,under the stimulation of ROS,the driving protein CLIC1 of oxidative stress-inflammatory link forms chloride channels,the concentration of chloride ions in cells decreases,promote the formation of inflammatory bodies and the release of inflammatory factors.SMI achieves neuroprotective effect by regulating the above indicator.(2)Shengmai injection interferes with the peripheral regulation of inflammation in cerebral ischemia reperfusion injury:Spleen atrophy began on the first day,the atrophy was the most severe from the third day,and returned to normal level on the fifth day after CIRI.The degree of brain injury in rats after splenectomy was significantly reduced.The above results indicate that the spleen plays an important role in the process of cerebral ischemia.After cerebral ischemia,the peripheral immune cells enter the brain to aggravate the inflammatory damage of the brain.The immune cells infiltrated into the brain are classified as Th cells,Tc cells,B cells and NK cells.After SMI intervention,the spleen index,the proportion of immune cells NK and B cells in spleen and brain all returned to normal levels.After the intervention of SMI in the splenectomized model animals,both cerebral infarction size and neurological function score tended to decrease,which also confirmed that SMI play a neuroprotective role partly by controlling the spleen and partly by controlling the center.In summary,through chemical composition identification and in vivo Systemic Exposure,the exposure of major chemical components in SMI in vivo was determined,and the main effective organs of SMI in the treatment of cerebral ischemia reperfusion injury were brain tissue and spleen.Through the overall characterization of pathological processes and disease protein networks in different links,the main effect links of SMI,immunity and inflammation,were identified.Based on the above two parts of theresearch,the central target organ(brain tissue)and peripheral immune organ(spleen)were respectively used to analyze the mode of action of immune and inflammatory links.In the center,brain inflammation can be alleviated by inhibiting ROS production,downregulating the expression of CLIC1,a key protein of oxidative stress-immunity and inflammation,inhibiting intracellular chloride ion outflow,and inhibiting the formation of inflammatory bodies.In the periphery,based on the "brain-spleen" inter-organ communication,it is clear that the process of cerebral ischemia will cause spleen atrophy and spleen immune cells to transfer to the brain,aggravating brain inflammation.SMI can reduce brain inflammation by regulating the spleen,and then regulating the mediators of "brain-spleen" organ communication,immune cells.In this study,from the indirect regulation of peripheral immune cells and the direct regulation of the central nervous system,systematically reveals the action mode of SMI in the inflammatory link of cerebral ischemia reperfusion injury,and provides a reference for the analysis of the complex mechanism of action of traditional Chinese medicine.The innovation point of the study:1 This study is under the guidance of holistic view of traditional Chinese medicine theory.Taking the main effect links and chemical bases of Shengmai injection were taken as the entry point to analyze the action characteristics of the drug by integrating the mode of action of direct regulation of target organs and indirect regulation of peripheral immune organs,and provide research strategies for the analysis of action mode of traditional Chinese medicine compound;2 This study focuses on the "brain-spleen" communication under the cerebral ischemia reperfusion injury,revealing the mediating role of peripheral immune cells in"brain-spleen" communication,and focusing on the regulatory pathways of peripheral immune organs to provide a new research perspective for the treatment of cerebral ischemia inflammation;3 Regulation of central target organs in the pathological state of cerebral ischemia and reperfusion.This study focused on CLIC1,the driving protein of oxidative stressinflammation,and proposed that Shengmai injection might inhibit the formation of chloride ion channels by regulating the expression of CLIC1 and the release of ROS,thus inhibiting the release of downstream inflammatory factors. |
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