| Objective: To investigate the protective effect of bloodletting from well points on brain injury in rats in acute plateau hypoxia model,and to explore its molecular mechanism through PI3K-AKT-m TOR pathway-mediated mitochondrial autophagy,so as to provide new methods and targets for clinical prevention and treatment of acute plateau hypoxia brain injury.Methods: The rats were treated with low-pressure hypoxia for 6h,12 h,24h,48 h and 72 h using a low-pressure oxygen chamber to simulate a plateau environment at6000 m above sea level.The rat model of acute high-altitude hypoxia brain injury was successfully replicated.Seventy-five adult male SD rats were randomly divided into control group(Control group,n=15)and experimental group(n=60)after 1 day of adaptive feeding.The experimental group(n=60)was randomly divided into model group(Model group,n=15),Bloodletting Acupuncture at Jing-well Points of hand(BAJP group,n=15),Bloodletting at Jing-well Points of hand + 3-MA group(BAJP+3-MA group,n=15),and bloodletting acupuncture at non-acupoint group(Bloodletting Acupuncture at Non-Acupoint group,BANA group,n=15).Seven days prior to chamber entry,rats in the BAJP group were bled from the twelve well points in a volume of approximately 15-20 μL,once daily;The rats in the Control and Model groups were kept normally and grasped once a day,and the method was the same as that in the BAJP group;The rats in the BAJP+3-MA group were injected with 3-MA inhibitor at a rate of 0.3 mg/100 g according to their body weight daily,and then bled at Jing-well Points of hand half an hour later;The BANA group was bled daily by tail tip clipping with a volume of approximately 15-20 μL.After 7 days,the rats in the Control group were extracted and placed in a low-pressure oxygen chamber(6000 m above sea level)for 72 h to prepare an acute high-altitude hypoxia brain injury model.The neuronal ultrastructure and autophagosome formation in hippocampus were observed by electron microscopy,and the apoptosis in hippocampus was detected by TUNEL.Mitochondrial complex activity assay kit was used to detect mitochondrial NADH dehydrogenase(complex Ⅰ),cytochrome C reductase Ⅲ and cytochrome C oxidase(complex Ⅳ)activities;ATP synthase kit was used to detect mitochondrial synthase activity;PI3K,LC3-Ⅰ,LC3-Ⅱ,Beclin1,AKT,Atg5 protein expression in rat hippocampal tissue was detected by Western blot;m TOR AKT m RNA expression level in rat brain hippocampal tissue was detected by PCR.Results:1.Using a low-pressure oxygen chamber to simulate a plateau environment at6000 m above sea level,the formation of autophagosomes in hippocampal tissues was used as the observation index,combined with the damage of hippocampal tissues and serological indexes S100 B,GFAP,HIF-1α and VEGF expression levels,which proved that the rat model of acute high-altitude hypoxia brain injury was successfully replicated at 72 h of low-pressure hypoxia.2.HE staining results: the cone cell layer in the hippocampal CA1 area of rats in the Control group was normally arranged,and no obvious cell necrosis was seen.A large number of necrotic cone cells were seen in the hippocampal CA1 area of rats in the Model and BANA groups,and the number of cone cells was significantly reduced.The number of necrotic cone cells in the hippocampal CA1 area of rats in the BAJP group was significantly reduced.The addition of 3-MA inhibited the therapeutic effect of BAJP.3.Electron microscopic results: Acute High-altitude Hypoxia(AHH)model rat hippocampal tissue injury was mainly manifested by neuronal apoptosis,mitochondrial swelling and the appearance of autophagy in the cytoplasm,and the trend according to the severity of injury was: Model group,BANA group >BAJP+3-MA group > BAJP group > Control group,and the trend of autophagosome number was: BAJP group > Model group,BANA group > BAJP+3-MA group >Control group.4.TUNEL results: Almost no green apoptotic cells were seen in the hippocampal tissue of Control group rats.The number of apoptotic cells in the hippocampal CA1 region of AHH rats was significantly increased.BAJP reduced the number of apoptotic cells in the hippocampal CA1 region of AHH rats,and BANA had no significant effect on apoptosis in the hippocampal CA1 region of AHH rats.The addition of 3-MA inhibitor increased the number of apoptotic cells in the CA1 region of the hippocampus of AHH rats compared with the BAJP group.5.ELISA results: Compared with Control group rats,S100 B,GFAP and MDA levels were significantly higher(P<0.01)and SOD levels were significantly lower(P<0.01)in AHH rats,and after BAJP treatment,the elevated levels of S100 B,GFAP and MDA decreased(P<0.05)and SOD levels increased significantly(P<0.01).BANA treatment had no significant effect on these indicators(P>0.05).3-MA significantly inhibited the improvement effect of BAJP on S100 B,GFAP,and SOD indicators in AHH rats(P<0.05).6.Western-blot results: The expression levels of Beclin1 and Atg5 proteins and LC3-Ⅱ/Ⅰ ratio in hippocampal tissues of AHH rats were significantly increased(P<0.01),and the phosphorylation levels of PI3 K,AKT and m TOR were significantly decreased(P<0.01).After BAJP treatment,the expression levels of autophagy-related proteins Beclin1 and Atg5 and LC3-Ⅱ/Ⅰ ratio were further increased in AHH rats(P<0.01),and the phosphorylation levels of PI3 K,AKT and m TOR were further decreased(P<0.01).BANA treatment had no significant effect on these indexes(P>0.05).After the addition of 3-MA inhibitor,Beclin1 and Atg5 expression levels and LC3-Ⅱ/Ⅰ ratio showed a significant decrease compared with the BAJP group(P<0.01),while PI3 K,AKT,and m TOR phosphorylation levels continued to decrease on the original basis(P<0.01).7.Real time PCR results: The expression of AKT and m TOR m RNA in the hippocampal tissue of AHH rats was significantly decreased compared with the Control group(P<0.01),and after BAJP treatment,AKT,m TOR m RNA in hippocampal tissues of AHH rats decreased further(P<0.01),and the addition of3-MA induced AKT,m TOR m RNA expression continued to decrease(P<0.01).BANA had no significant effect on AKT,m TOR m RNA expression in AHH rats(P>0.05).8.Results of mitochondrial membrane potential,mitochondrial respiratory chain complex and ATPase activity: Mitochondrial membrane potential,mitochondrial respiratory chain complex Ⅰ,Ⅲ and Ⅳ and ATPase activity were significantly decreased in AHH rats(P<0.01),BAJP increased mitochondrial membrane potential,mitochondrial respiratory chain complex Ⅰ,Ⅲ and Ⅳ and ATPase activity in AHH rats(P<0.01),none of the BANA had a significant effect on mitochondrial physiology(P>0.05),and the addition of 3-MA inhibitor affected the improvement of mitochondrial physiology in AHH rats by BAJP(P<0.05).Conclusion: A hypobaric oxygen chamber simulating a 6000 m altitude plateau environment for 72 h can successfully replicate the acute high-altitude hypoxia brain injury rat model.BAJP has a protective effect on brain injury induced by acute high-altitude hypoxia,and the mechanism may be related to the effect on the level of mitochondrial autophagy mediated by PI3K-AKT-m TOR signaling pathway;the effect of 3-MA inhibitor on the overall level of autophagy is greater than the effect on PI3K-AKT-m TOR pathway-mediated mitochondrial autophagy. |
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