Anti Hypoxia Effect Of Yak Blood Antioxidant Peptide And Its Mechanism

Yaks are special livestock scattered over 3000 m above sea level.After generations of natural selection,yaks have showed excellent adaptability to hypoxic environment.It can adapt to the harsh ecological environment while ordinary livestock can not,to which including high altitude,low air pressure,high oxygen deficit and so on.So the whole bodies of yaks including fur,skin,blood,meat,milk,viscera,bone and horn all have deep development value to be researched.In the previous study,yak blood antioxidant peptide with antioxidant activity in vitro was prepared.However,the anti-hypoxia effect and mechanism of yak blood antioxidant peptide have not been reported yet.Therefore,on the basis of previous studies,the preparation process of yak blood antioxidant peptide was simplified at the beginning.Besides,its amino acid composition,peptide composition and its sequence were researched.The physical chemical stability such as thermal stability,acid-base stability,metal ion stability,freeze-thaw stability and the stability of food accessories were also studied.Andthe antioxidant activity of yak blood antioxidant peptide in vivo was clarified furtherly by animal experiments in which the new indications of yark blood antioxidant peptide that we named“anti-hypoxia”were studied on the basis of its definite antioxidant effect.The effect and mechanism of yark blood antioxidant peptide in protecting hypoxic myocardial cells named H9c2 were studied under the animal model of mice which were physically anoxic.The anti-hypoxia effect and mechanism of yark blood antioxidant peptide were furtherly studied under the animal model of mice which were hypxia in normal pressure.The main research results were as follows:1.The·OH scavenging ability of yak blood antioxidant peptide obtained from 1 kDa ultrafiltration classification was similar to that of peak I(P>0.05)obtained from Sephadex G-25 separation and purification.The·OH scavenging ability,the DPPH·scavenging activity,the total antioxidant ability,and the lipid peroxidation inhibition of yak blood antioxidant peptide all were significantly better than that of the commercial soybean peptide(P<0.01).In addition,the·OH scavenging ability,the DPPH·scavenging activity,the total antioxidant ability were also significantly better than that of the commercial fish collagen peptide(P<0.01).The oligopeptide obtained from yak blood protein hydrolysis was the main source of its antioxidant substancesLC-MS/MS was used to detect the yak blood antioxidant peptide with molecular weight<5 kDa.According to the method of qualitative and non-marking quantitative analysis,1159 kinds of peptide segments were detected.2.The·OH scavenging activity of yak blood antioxidant peptide with molecular weight<1 kDa was stable at the temperature range of 20?-60?.It was also stable in weak acid and basic conditionswhile the·OH scavenging activity of yak blood antioxidant peptide reduced as the number of freeze thaw reactions increased High concentrations of Zn²⁺and Cu²⁺inhibited its·OH scavenging activitywhile NaCl had synergistic effect on its·OH scavenging activity.And bothglucose and citric acid had significant inhibitory effect on its·OH scavenging activit(P<0.05).3.The animal experiments about the antioxidant activity of yak blood antioxidant peptide showed that yak blood antioxidant peptide could improve the serum level of mice SOD activity,the GSH-PX activity and the T-AOC activity,and they were significantly different(P<0.05).Furthermore,the MDA content in serum of mice was decreased,but with no statistical significance(P>0.05).In conclusion,yak blood antioxidant peptide had definite antioxidant activity in vivo.4.In the animal experiment of protecting mice's hypoxic myocardial cells named H9c2,the cell survival rate of the hypoxia group significantly decreased(P<0.01),the apoptosis rate increased dramatically(P<0.01),and the LDH release level increased remarkably(P<0.01),comparing with the control group,which indicated that H9c2 myocardial cells were obviously damaged because of hypoxia.In contrast,compared with the hypoxia group,the survival rate of cells increased,cell apoptosis and the release level of LDH in the cells decreased underthe intervention of the yak blood peptide,which showed a good dose-dependent relationship.At the same time,yak blood antioxidant peptide could inhibit the decrease of mitochondrial membrane potential,increase the T-AOC level,decrease the TNF-a level,inhibite the release of cytochrome C mediated by hypoxia,up-regulat the expression of anti-apoptotic proteins(Survivin,p-Akt,Akt),and reduc the activity of apoptotic protein(Caspase-3/9).Yak blood antioxidant peptide can also affect the level of gene encoding of these proteins mentioned.Stated thus,the protective effect of yak blood antioxidant peptide on H9c2 myocardial cellswas closely related to the inhibition of PI3K-Akt signal transduction pathway and the protection of the structural and functional integrity of mitochondria.5.The results of hypoxia tolerance test in mice at atmospheric pressure showed that different doses of yak blood antioxidant peptide could improve the hypoxia tolerance of mice differently in a dose-dependent manner,and the high dose group could significantly prolong the survival time of hypoxia mice at atmospheric pressure(P<0.05).The survival time of mice with sodium nitrite poisoning was prolonged in all dosage groups of yak blood antioxidant peptide,of which the prolongation rate of high and middle dosage groups was extremely significant(P<0.01).Compared with the control group,the intervention of high dose yak blood antioxidant peptide significantly reduced MDA content(P<0.01),H₂O₂content(P<0.01),LDH activity(P<0.01)in brain and myocardium of normobaric hypoxic mic,where the SOD activity(P<0.01),GSH-Px activity(P<0.01 in myocardium and P<0.05 in brain tissue),CAT activity(P<0.01)and ATP content(P<0.01)were also significantly increased.To summarize,theyak blood antioxidant peptide could alleviate oxidative stress the injury in mice caused by hypoxia,whose mechanism were related to a series of activities including scavenging free radicals,inhibiting lipid peroxidation,maintaining the activity of antioxidant enzymes and maintaining the normal process of oxidative phosphorylation in mitochondria.