| Objective:To study the protective effect of hand 12 well point bloodletting on myocardial mitochondrial autophagy mediated by BNIP3 pathway in ahh rats,and to explore the therapeutic effect and target of hand 12 well point bloodletting on ahh myocardial injury,so as to provide a new method for preventing and treating ahh induced myocardial injury.Methods:By simulating the hypoxia environment at 6000 m altitude in a low-pressure oxygen chamber,and comprehensively evaluating the changes of myocardial microstructure and myocardial injury markers,the animal model of hypoxia myocardial injury at high altitude was successfully reproduced.Seventy five male SD rats were randomly divided into control group,model group,Bloodletting acupuncture at Jing points(BAJP)group,Bloodletting acupuncture at Jing points+3-ma(BAJP+3-MA)group and Bloodletting acupuncture at non-acupuncture(BANA)group.Control group and model group were fed routinely,and were grabbed once a day;The rats in BAJP group were treated with bloodletting at twelve well points of hand;The rats in BAJP+3-MA group were treated with bloodletting at twelve well points of hand and intraperitoneal injection of 3-MA solution(0.3mg/100g);Rats in BANA group were cut tail and bled.BAJP group,BAJP+3-ma group and BANA group were treated once a day for 7 days,and the amount of bleeding each time was 15-20 μ L.The model group,BAJP group,BAJP+3-MA group and BANA group were placed in the low-pressure simulation cabin(6000 m above sea level,72h)to replicate the AHH myocardial injury model.He staining was used to observe the microstructure of rat myocardium,transmission electron microscope was used to observe the ultrastructure of myocardial tissue and mitochondrial damage,ELISA was used to quantify the changes of serum markers CK-MB and CTnl of rat myocardial damage,TUNEL method was used to measure myocardial cell apoptosis,ELISA was used to measure the levels of SOD and MDA,JC-1 method was used to measure the mitochondrial membrane potential,and ELISA was used to measure the contents of mitochondrial respiratory chain complex I,III,IV and ATP,Determination of signal pathway protein by Western blot HIF-1α、BNIP3,beclin-1,lc3-II/I,ATG5 level.Results: 1.Preparation and evaluation of acute high altitude hypoxia rat myocardial injury model: HE staining pathology showed that hypobaric hypoxia environment can cause myocardial injury in rats,which aggravates with the prolongation of hypoxia time,and myocardial injury markers CK-MB and CTnl persist.However,there was no significant difference between 6h and control group(P>0.05),and there was a significant difference at 12h(P<0.05),and the damage gradually increased with the passage of hypoxia time,and no peak was seen within72 h.2.The protective effect of bloodletting from Twelve Wells of Hand on myocardial injury in acute plateau hypoxia rats through BNIP3 pathway-mediated mitophagy2.1 Protective effect on myocardial pathological injury in AHH rats: HE staining showed that the model group,BANA group Cell necrosis and hemorrhage were observed in myocardial tissue,and the damage in BAJP+3-MA group was more serious than that in BAJP group.Cardiomyocytes were swollen in the BAJP group,but myocardial necrosis and hemorrhage were not observed.Except for the control group,the trend of myocardial fibrinolysis and mitochondrial swelling in the projection electron microscope was as follows:model group,BANA group>BAJP+3-MA group>BAJP group>control group.The trend of the number of autophagosomes was:BAJP group>model,BANA group>BAJP+3-MA>group>control group.The results of TUNEL staining to observe cardiomyocyte apoptosis showed that compared with control rats,the number of apoptotic cells in the myocardial tissue of AHH rats increased.BAJP treatment reduced the number of apoptotic cells in AHH rats,while BANA treatment did not improve apoptosis in AHH rats,and the apoptosis in BAJP+3-MA group was more serious than that in BAJP group.2.2 Effects on serum markers of myocardial injury in AHH rats: Compared with the control group,CTnl in the BAJP group increased but not statistically significant,and the CK-MB and CTnl in each group after entering the hypobaric oxygen chamber were significantly increased(P<0.05);Compared with the model group,the expressions of CK-MB and CTnl in the BAJP group and BAJP+3-MA group were significantly decreased(P<0.01),and the expression in the BANA group was decreased,but not statistically significant(P>0.05).Compared with BAJP+3-MA group and BANA group,the expressions of CK-MB and CTnl increased(P<0.05).2.3 Effects on oxidative stress indexes of myocardial tissue in AHH rats:Compared with the control group,the SOD in the BAJP group decreased,but there was no statistical significance(P>0.05).Compared with model group,MDA decreased and SOD increased in BAJP group and BAJP+3-MA group(P<0.05),while MDA decreased and SOD increased in BANA group,but there was no statistical significance(P>0.05);compared with the BAJP group,the MDA in the BAJP+3-MA group and the BANA group were significantly increased(P<0.01),and the SOD was significantly decreased(P<0.01).2.4 The effect on mitochondrial function of myocardial tissue of AHH rats:Compared with the control group: the membrane potential,respiratory chain complexes I,III and IV of the model group were significantly decreased(P<0.01),and ATP decreased but not statistically significant(P>0.05);complex III and ATP in BAJP group decreased,but not statistically significant(P>0.05),membrane potential,complex I,IV decreased significantly(P<0.01);BAJP+3-MA group membrane potential,respiration Chain complexes I,III,and IV were significantly decreased(P<0.01),and ATP decreased but not statistically significant(P>0.05);the membrane potential,respiratory chain complexes I,III,and IV were significantly decreased in the BANA group(P<0.05).0.01),ATP decreased but not statistically significant(P>0.05).Compared with the model group: the membrane potential,complexes I,III,IV and ATP in the BAJP group increased(P<0.05),the complexes I,III and IV in the BAJP+3-MA group increased(P<0.05),the membrane potential and ATP increased,but not statistically significant(P>0.05);BANA group membrane potential,complexes I,III,IV and ATP increased,but not statistically significant(P>0.05);Compared with BAJP group: BAJP+3-MA complexes I,IV and ATP decreased(P<0.05),membrane potential and complex III decreased,but not statistically significant(P>0.05);BANA group membrane potential,complexes I,III,IV decreased significantly(P<0.01),ATP decreased,but there was no statistical significance(P>0.05).2.5 Effects on mitophagy signaling pathway in myocardial tissue of AHH rats:Compared with control group: HIF-1α,BNIP3,Beclin-1,LC3II/I,ATG5 in model group increased(P<0.05);HIF-1α in BAJP group,BNIP3,Beclin-1,LC3II/I increased significantly(P<0.01),ATG5 increased(P<0.05);BAJP+3-MA group HIF-1α,BNIP3,Beclin-1(P<0.05),LC3II/I,ATG5 increased,but no statistical significance(P>0.05);BANA group HIF-1α,BNIP3,Beclin-1,LC3II/I increased(P<0.05),ATG5 increased,but no statistical significance(P>0.05).Compared with model group,HIF-1α,BNIP3,Beclin-1,LC3II/I,ATG5 in BAJP group increased(P<0.05);BNIP3 and Beclin-1 in BAJP+3-MA group decreased(P<0.05),HIF-1α,LC3II/I,ATG5 decreased,but not statistically significant(P>0.05);BANA group HIF-1α,Beclin-1,LC3II/I increased,BNIP3,ATG5 decreased,but not statistically significant(P>0.05);Compared with BAJP group,HIF-1α,BNIP3,Beclin-1,LC3II/I in BAJP+3-MA group decreased significantly(P<0.01),and ATG5 decreased,but not statistically significant(P>0.05);BANA group HIF-1α,BNIP3,Beclin-1,LC3II/I decreased significantly(P<0.05),and ATG5 decreased,but not statistically significant(P>0.05).Conclusion:1.by simulating the high altitude hypoxia environment in the low-pressure oxygen chamber,the changes of myocardial tissue structure and the expression level of myocardial injury markers were comprehensively evaluated.The rat model of high altitude hypoxia myocardial injury was successfully reproduced by simulating the low-pressure oxygen chamber at 6000 m altitude for 72 hours.2.in the high altitude hypoxic myocardial injury model,he staining showed necrosis and hemorrhage of rat myocardial cells,swelling of myofibrils and mitochondria under electron microscope,TUNEL showed increased apoptosis,and the markers of myocardial injury CK-MB and CTnl increased.Through well point bloodletting pretreatment,the above indexes were improved to reduce myocardial injury.Well point bloodletting has a protective effect on myocardial injury caused by hypoxia at high altitude.3.in the model of myocardial injury induced by hypoxia at high altitude,the indexes of oxidative stress in rat myocardium decreased,SOD increased,mitochondrial membrane potential decreased,the activities of respiratory chain complexes I,III and IV decreased,and ATP production decreased.The pretreatment of well point bloodletting improved the indexes of oxidative stress and mitochondrial respiratory function.Well point bloodletting could improve the myocardial oxidative stress injury and mitochondrial dysfunction caused by high altitude hypoxia.4.in the model of myocardial injury induced by hypoxia at high altitude,HIF-1α The expression of BNIP3 signaling pathways BNIP3,beclin-1,lc3ii/i and ATG5 increased,which may be a protective mechanism.Well point blood letting pretreatment further promoted the expression of these autophagy proteins,and the same results were obtained by observing mitochondrial autophagy under electron microscope.Well point bloodletting pretreatment can promote mitochondrial autophagy of cardiomyocytes under high altitude hypoxia,and protect them through BNIP3 autophagy signal pathway.5.the pretreatment of hand 12 well point bloodletting can reduce the myocardial damage caused by high altitude hypoxia,reduce oxidative stress damage,improve mitochondrial function,and activate BNIP3 autophagy signal pathway.Thus,it has a protective effect on myocardial injury caused by high altitude hypoxia,and achieves the protective effect through mitochondrial autophagy mediated by BNIP3 signal pathway.Well point bloodletting pretreatment can be used as a new technology for the prevention and treatment of hypoxic myocardial injury at high altitude.At the same time,mitochondrial autophagy mediated by BNIP3 signal pathway can be used as a therapeutic target for the study of hypoxic-ischemic heart disease. |
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