Mechanism Of HIF-1? Signaling Pathways And Metabonomics Of Brassica Rapa Polysaccharide Against High-altitude Hypoxia

Because the plateau is the thin air,low atmospheric pressure and low partial pressure of oxygen,partial pressure of oxygen(Pa02)inalveolar decreases while the body enters it,resulting in pulmonary vasoconstriction,cardiac output increase,excessive ventilation and tissue metabolic disorders,which distributes to increasing microvascular endothelial permeability and neuronal apoptosis.And thus the body feels headache,dizziness,shortness of breath,palpitations,nausea,lack of appetite etc.Even worse,people may suffer severe head pain,ataxia,dyspnea,and a variety of acute mountain sickness(AMS).HIF-1 is a key medium that specifically regulates oxygen homeostasis.When the body is in a hypoxic environment,HIF-1 can induce transcription of a series of downstream target genes such as vascular endothelial growth factor(VEGF),erythropoietin(EPO),cytochrome oxidase(COX),etc.Tibetan medicine indicates that there are the vulnerable populations of three types of AMS,namely,Long type,Longpei type and Peigen type.When entering the plateau,Long type aggravates the "coarse" and "cold" nature,because the plateau climate has the characteristics of "cold" and "rough";andPeigenype with the weakness of stomach firebecomes more weaker.It leads to the disorder of Qi and blood,resulting in the occurrence of AMS.Tibetan medicine Brassicae Radix is sweet and warm,which has the function of nourishes and detoxifies.After digestion by"three stomach fires",it can cure Long disease and Chiba disease.What's more,"Gan"flavor with the features of "water and soil" can treat the disorder of Qi mechanism caused byLong disease,thus treating AMS.Recent studies have shown thatBrassica rapa polysaccharide(BRP),the main component of "Gan" flavor(sweet)component,has antioxidant and anti-hypoxic effects,and also has protective effects on tissue damage caused by Tibetan "Ladu" disease(AMS).Objective:1.Establish the quality control method and purify and separateBRP by different extraction methods.2.The protective effect of BRP on high-altitude hypoxia was to be evaluated by chronic/acute high-altitude hypoxia models and its mechanism was discussed.3.Metabonomic methods were used to study the biomarkers and key pathways of BRP on the protection of acute high-altitude hypoxia,providing research ideas and a foundation for the prevention of high-altitude hypoxia.Methods:1.Establishment of quality control of Radix Brassicae:to collect samples from different sources,identifyRadix Brassicae by DNA Barcoding technology,identify them by microscopy and TLC,inspect moisture and ash,determine the content of total polysaccharides and glucose respectively by ultraviolet spectrophotometry and HPLC,and establish specific chromatogram.2.Purification and separation of Polysaccharides from Radix Brassicae:the deproteination methods of Sevag and macroporous adsorption resin were studied respectively.DEAE 52 and Sephadex G100 chromatography were used to separate and purify polysaccharides.The purity and fractions of purified polysaccharides are determined by HGPC.3.Protective effect of BRP on chronic high-altitude hypoxia mice model:The CHHmice model was established at simulated altitude of 7000m(for 7days).The changes of body weight,feed consumption were observed.The histopathological changes of heart,brain,lung and kidney were detected.The oxidative stress indexes of various tissues were detected.and the expression of HIF-1?,EPO and VEGF was determined by ELISA.4.Protective effect of BRP on acute high altitude hypoxia rat model:Acute high-altitude hypoxia(AHH,9000m,24h)rats were used to record the behavioral changes during the modeling period.Histopathological changes of hippocampus and cortex were observed by H&E,Nissl,TUNEL staining.ISCU1/2,COX10,Capase-3 were detected by immunohistochemical technique in hippocampus and cortex of rats.Western blot was used to detect the protein levels of HIF-1?,Bax,Bcl-2,caspase-3,and qRT-PCR was used to detect the expression of microRNA-210,ISCU 1/2 and COX 10.5.Using UPLC-MS metabonomics technology,the metabolic differentials in brain tissue of rats in control group,model group and BRP administration group were detected.PCA,PLS-DA and other multivariate statistical methods were used to analyze the differences between groups,identify differentially metabolites,and find out the key metabolic pathways closely related to differentially metabolites.The key metabolic pathway was validated.Results:1.ITS2 sequence could effectively distinguish Radix Brassicae from its common adulterants.12 batches of Radix Brassicaewere determined.The content of water was 10.40-16.32%,total ash content was 7.18-10.22%,acid-insoluble ash content was 0.10-0.59%,extract content was 30.32-53.75%,total polysaccharide content was 8.79-4.76%,and glucose content was 10.32-21.43%2.The macroporous adsorption resin method was studied,which found that dynamic static was better than static adsorption.D301R resin was used and 5 mg/ml polysaccharide solution is slowly added within a diameter-to-height ratio of 1:9 at a flow rate of 1BV/h.The retention rate of polysaccharides was 81.12%,the decolorization rate was 65.96%,and the deproteinized rate was 53.86%After seperating by DEAE-52 cellulose and Sephadex G100 column chromatography,four components were obtained,namely,BRP-3B(Average Weight Mw:14254 Da)and BRP-4B(Average Weight Mw:16312 Da)that were purified by DEAE-52 cellulose of 0.6 mol/L NaCl elution,BRP-1B(Average Weight Mw:380883 Da)and BRP-2B(Average Weight Mw:227089 Da)were separated by Sephadex G-100 of water elution.Five components from BRP(cold extraction Da)were respectivlyseperated by DEAE cellulose of 0.3 mol/ml elution BRP-5C(Average Weight Mw:39606 Da),Sephadex G-100 of distilled water BRP-1C(Average Weight Mw:1540 Da)and BRP-2C(Average Weight Mw:25454 Da),and Sephadex G-100 of 0.1 mol/1 NaCl elutionBRP-3C(Average Weight Mw:74182 Da),BRP-4C(Average Weight Mw:36518 Da).3.BRD had protective effect on CHH mice.Compared with CHH model group,BRP could significantly improve the content of oxidative stress markers in serum and heart,brain,lung and kidney tissues,and regulate the expression of HIF-1a,VEGF and EPO in those tissues4.BRP had protective effect on AHH rats.It could increase the levels of SOD and GSH in serum of AHH rats,decrease the contents of MDA,GSSG and LDH,decrease the apoptosis of neurons in rat brain,decrease the expression of Capase-3 and Bax,increase the expression levels of HIF-1a proteins and microRNA-210 genes,and increase the expression of ISCU1/2 and COX10.These results suggested that BRP could reduce neuronal apoptosis and activate HIF-1a/microRNA 210/ISCU 1/2(COX 10)signaling pathway for improving mitochondrial function to achieve brain protection.5.UPLC-MS was used to detect brain metabolites in rats of control group,AHH model group and high-dose administration group of BRP.By control vs AHH model group,16 biomarkers and related 12 metabolic pathways were identified.By contrasting AHH model with BRP treatment group,41 biomarkers and related 25 metabolic pathways were identified.After BRP administration,there were 7 distinct metabolites,including Psychosine,sphingosine,sphingosine-1-phosphoric acid,myristoyl carnitine,1-palmitoyl lysophosphatidic acid,LypoPE(16:1(9Z)/0:0),and pyruvate.The five related pathways were sphingolipid metabolism,cysteine and methionine metabolism,citric acid cycle(TCA cycle),glycolysis or gluconeogenesis,gluconeogenesis,and pyruvate metabolism.The results showed that the protective mechanism of BRP against brain injury induced by AHHwas closely related tothe metabolism of regulating the expression of HIF-1 by sphingosine-1-phosphoric acid,sphingosine and galactosyl sphingosine.SIP could inhibit the release of cytochrome C from mitochondrial protein and reduce caspase activation through PI3K/AKT signaling pathway;and BRP could regulate citric acid cycle(TCA cycle)and glycolysis.The pathways of glycolysis,gluconeogenesis and pyruvate metabolism were closely related to the activation of HIF-1.HIF-la regulated cell metabolism under hypoxia,reduced TCA cycle and ROS damage to mitochondria,thus achieved hypoxic protection.The validation experiments showed that BRP could activate the expression of PI3K/Akt signaling pathway in sphingolipid metabolism.Conclusions:Combining the Tibetan medicine theory of "Weixing Huawei" and the theory of tinyessential metabolism,the quality control ofRadix Brassicae was studied and its"Gan" flavor components were separated and purified.Based on HIF-1 ? signaling pathways and energy metabolism,the mechanism of BRP for protecting anti-hypoxia at high altitude was explainedfrom the level of protein and metabonomics,which provided theoretical basis and data support for further research on prevention and treatment of related diseases.Innovations:1.For the first time,the content of glucose in Radix Brassiceawas determined by HPLC,and the characteristic chromatograph was established.The polysaccharides of Brassicea Radix(cold extraction)were separated and purified.2.The pharmacodynamics and mechanism of BRP in improving chronic altitude sickness caused by CHH were studied.Because of the long-term compensatory reaction in vivo,the cardiovascular and cerebrovascular systemhas been changed.These studies showed that BRP could improve the antioxidant stress of various tissues and regulate the expression of HIF-1a,VEGF and EPO.3.Neuronal apoptosis was used as a breakthrough point to study the prevention of brain injury caused by AHH.It was clarified that BRP could protect brain by activating HIF-1a/microRNA 210/ISCU 1/2(COX 10)signaling pathway.4.Combining with Tibetan medicine theory that the "Gan" flavor component of BRP can treat the Long disease and Chiba disease after digestion by "three stomach fires",and change the metabolism of the body,UPLC-MS metabonomics method was used to study the differential metabolites and metabolic pathways of BRP in cerebral tissue of rats with CHH.The mechanism of preventing brain injury caused by AHH was revealed from the metabolite level.