Effect Of Anti-hypoxia,Anti-fatigue And Hypoxic Myocardial Protein Expression Of New Huangqi-fuling Compound Recipe

Oxygen is necessary for the human body.Hypoxia is the pathological process of reducing or unable to make full use of oxygen,which leads to abnormal changes in tissue metabolism,function and morphology.The oxygen consumption of normal adults at rest is about 250 ml/min,which can increase by 8-9 times when strenuous exercise,and the amount of oxygen stored in the human body is only 1500 ml.Once the breath and heart stop,people may die of hypoxia within a few minutes.Hypoxia is the most common stress factor in plateau,aviation,diving and other special environments.It is also a common pathological process of many diseases such as chronic obstructive pulmonary disease,severe acute respiratory syndrome,myocardial infarction,ischemic stroke,hemorrhagic shock,cyanide poisoning and so on.It can lead to many kinds of diseases,and affects many kinds of metabolism of the body,especially on the oxidation and energy supply of the body.Long-term or severe hypoxia can lead to death due to the lack of energy supply of vital organs such as heart and brain.Fatigue,also known as tiredness,is a kind of subjective discomfort.Objectively,when people lose their normal activity or working ability under the same conditions,and when the disease develops to a certain stage,fatigue appears.Fatigue is a common pathophysiological state under the fast-paced lifestyle of modern society,which belongs to sub health category.Fatigue can reduce work efficiency,cause premature senility of the body,various diseases,even karoshi.Huangqi-Fuling prescription?HF?,Chinese medicine compound developed in1970s by Tianjin Institute of Environmental&Operational Medicine,composed of12 species of traditional Chinese Medicine including Astragalus membranaceus,Codonopsis pilosula and so on.According to the internal data of the Institute,HF widely used in Tibetan troops stationed in the plateau,only used within the army,and the symptoms of high altitude disease were relieved or disappeared after the soldiers take HF.New Huangqi-Fuling compound recipe?XHF?is a simple recipe modified from HF.In this paper,the anti-hypoxia and anti-fatigue effects of XHF were verified,by the close normobaric hypoxia and the weight loading swimming experiment respectively.To study the anti-hypoxia mechanism of XHF in the molecular level,the proteomic technology was used as the main research means,providing a good experimental basis for its subsequent development and utilization.Objective:1.To achieve the same or better control effect with less drug composition,the new small prescription group was selected from HF according to the basic principles of the prescription compatibility.2.To provide a theoretical basis for further research on the application and development of XHF,the pharmacodynamic experiments of XHF were carried out to clarify the effects of XHF on anti-hypoxia and anti-fatigue.3.To uncover the anti-hypoxia related molecular mechanism of XHF on hypoixa-induced mice myocardial injuries,TMT quantitative proteomics and bioinformatics technique were employed to contrast differentially expressed protein and statistically analyze.Method:1.Drug screeningTo form a recombinated new prescription?XHF?based on HF,“Inheritance platform software of traditional Chinese Medicine”was employed to analyze the formula compatibility's rule of Chinese medicine compound containing Astragalus membranaceus in the Drug standard of People's Republic of China Ministry of health--Chinese medicine preparation and to screen the medcinal candidate.2.Close normobaric hypoxia experiment in miceA total of 52 mice were used to observe the protective effect of different doses of the XHF on normobaric hypoxia,with 0.5%carboxymethyl cellulose as blank control group,HF as positive control.The levels of T-AOC,CAT,T-SOD,GSH-PX and MDA in serum and Na⁺K⁺-ATPase and Ca²⁺Mg²⁺-ATPase in myocardium and brain tissue of mice were detected by ELISA Kit.3.Weight loading swimming experiment in miceA total of 50 mice were used to observe the effect of different doses of XHF on swimming time in mice,with 0.5%carboxymethyl cellulose as blank control group,HF as positive control.The contents of LA,LDH and BUN in serum were detected by ELISA Kit at 530 nm,440 nm and 520 nm,liver glycogen and muscle glycogen at 620 nm.4.TMT relative quantitative proteomics and bioinformatics analysisTo investigate the differential expression of protein in the heart of mice,protein extraction and peptide hydrolysis from the blank control and XHF high dose group were performed,followed by taking the TMT labeling and chromatography grading.Identification and quantitative analysis of proteins were carried out by Liquid Chromatography Tandem Mass Spectrometry?LC-MS/MS?data acquisition.Subsequently,bioinformatics analysis was used to The differentially expressed proteins was identified by Mascot and Proteome Discoverer software.Finally,cluster analysis,functional annotation and path analysis were performed by bioinformatics analysis.Result:1.Drug screeningXHF composed of six species of traditional Chinese medicine Astragalus membranaceus,including Astragalus membranaceus,including Codonopsis pilosula,Angelica sinensis,Poria cocos,Salvia miltiorrhiza and Polygonum multiflorum,is a simple recipe modified from HF.2.Close normobaric hypoxia experiment in miceThe effect of XHF on the weight of mice:At the middle of the experiment and at the end of the experiment,the average weight of mice in HF and XHF dose groups was not statistically significant,compared with mice in the blank control group?P>0.05?,showing that XHF had no adverse effect on the weight of mice.The effect of XHF on the survival time of hypoxia tolerance in mice:The survival time of HF group,XHF low,medium and high dose group were significantly prolonged?P<0.05?,compared with the blank control group.There was no significant difference between the XHF high dose group and the HF group,which indicated that XHF could prolong the survival time of mice under normal atmospheric pressure and has anti-hypoxia effect.Effects of XHF on antioxidant and antioxidant indices in serum of mice:T-AOC in serum of HF group and XHF high dose group increased?P<0.05?,CAT activity in serum of group HF and XHF increased?P<0.05?,the activity of SOD in serum of HF group,XHF medium and high dose group increased?P<0.05?,the activity of GSH in serum of XHF group was increased?P<0.05?,MDA content in serum of group HF,XHF and high dose group decreased?P<0.05?,compared with blank control group.Effects of XHF on energy metabolism in myocardium and brain tissue of mice:?1?The effect of on myocardial tissue:the activity of Na⁺K⁺-ATPase in HF group and XHF high dose group increased?P<0.05?,and the activity of Ca²⁺Mg²⁺-ATPase in HF group and XHF high dose group increased?P<0.05?,compared with the blank control group.?2?The effect on brain tissue:the activity of Na⁺K⁺-ATPase in XHF high dose group increased?P<0.05?,and the activity of Ca²⁺Mg²⁺-ATPase increased in group HF and XHF in low,medium and high dose groups?P<0.05?,compared with the blank control group.3.Weight loading swimming experiment in miceEffects of XHF on swimming time in mice:The swimming time of HF group,XHF low,medium and high dose group were significantly increased?P<0.01?,compared with the blank control group.The effect of XHF on liver glycogen and muscle glycogen in mice:Liver glycogen storage in group HF,XHF and high dose group increased significantly?P<0.01?,and the storage of muscle glycogen in XHF low,medium and high dose groups was significantly increased?P<0.05?,compared with the blank control group.Effects of XHF on serum indexes in mice:?1?LA content in serum of group HF,XHF middle and high dose group was significantly decreased?P<0.05?,compared with the blank control group.The content of LA in serum of XHF high dose group was still significantly lower than that of HF group?P<0.05?,showing that XHF could reduce the content of LA in the serum of mice.?2?The LDH activity in serum of HF group and XHF low dose group increased significantly?P<0.05?,and the activity of LDH in serum of XHF medium and high dose group increased significantly?P<0.01?,compared with the blank control group.The XHF high dose group still increased significantly?P<0.05?,compared with HF group.The result suggested that XHF could increase the activity of LDH in serum of mice.?3?The level of BUN in serum of group HF was significantly decreased?P<0.05?,and the content of BUN in the serum of XHF low,middle and high dose groups decreased significantly?P<0.01?,compared with the blank control group.The content of BUN in the serum of XHF low dose group was still significantly decreased?P<0.01?,compared with the HF group.The result suggested that XHF could reduce the content of BUN in the serum of mice.4.TMT relative quantitative proteomics bioinformatics analysisThe total number of mass spectrograms of two levels identified in this experiment is 339,987.Among them,the number of peptide spectrum match is 77,963,the total number of peptides identified was 27,558,the total number of unique peptides is 24,749.There were 3790 proteins in the heart of mice were identified.Among them,10 differential expressed proteins were up-regulated and 27 down regulated proteins.There were 330 GO functional annotations by functional enrichment analysis of differentially expressed protein,including 268 important biological processes,containing regulation of eIF2 alpha phosphorylation by heme and regulation of translational initiation by eIF2 alpha phosphorylation etc;52molecular functions,containing oxygen transporter activity,heme binding etc;10cellular component,containing hemoglobin complex,cytosolic part etc.The functions of these differentially expressed proteins are mainly about binding,protein binding,cationic binding,metal ion binding,ion binding,and so on;mainly involved in single cell processes,cell processes,biological regulation,biological process regulation,stimulation reactions,and so on.Up regulation of differentially expressed proteins were mainly involved in 7 pathways,including African trypanosomiasis,Malaria,Autoimmune thyroid disease,Natural killer cell mediated cytotoxicity,Other types of O-glycan biosynthesis,Human papillomavirus infection,Neurotrophin signaling pathway;down regulation of differentially expressed proteins were mainly involved in 7 pathways,including African trypanosomiasis,Malaria,Autoimmune thyroid disease,IL-17 signaling pathway,Natural killer cell mediated cytotoxicity,Graft-versus-host disease,Human papillomavirus infection,Neurotrophin signaling pathway,by enrichment analysis of KEGG pathway about differential expression protein.There was a direct or indirect relationship between 4upregulated and 16 down regulated differentially expressed proteins,according to construction of protein and protein interaction?PPI?network.Gapdh?Hbb-b2?Arhgef6?H2-K1?Ighm?Cav3?Arhgdib?S100a8?S100a9 genes are more active in the network,the corresponding proteins,containing Glyceraldehyde-3-phosphate dehydrogenase,Beta-2-globin,Rho guanine nucleotide exchange factor 6,MHC H-2K antigen,Ig mu chain C region,Caveolin-3,Rho GDP-dissociation inhibitor 2,Protein S100-A8,Protein S100-A9,interact closely with other proteins in the network,suggesting that they play an important role in the protein interaction network.Conclusions:1.XHF could exert anti-hypoxia effect via eliminating excessive oxygen free radical,reducing cellular energy metabolism and lipid peroxidation production in myocardium and brain tissue of mice.2.XHF could alleviate muscle damage and enhance fatigue resistance in mice via improving the storage of liver glycogen and muscle glycogen,decreasing LA and BUN level in serum,and increasing the LDH activity.3.XHF may down-regulated the protein expression of Beta-2-globin,S100-A8and S100-A9 along with the regulation of IL-17 signaling pathway.The underlying mechanisms need to be further investigated.