Bloodletting Acupuncture at Jing-Well Points on Hand Induced Autophagy to Alleviate Brain Injury in Acute Altitude Hypoxic Rats by Activating PINK1/Parkin Pathway.

OBJECTIVE: To explore the protective effect of bloodletting acupuncture at twelve Jing-well points on hand (BAJP) on acute hypobaric hypoxia (AHH)-induced brain injury in rats and its possible mechanisms. METHODS: Seventy-five Sprague Dawley rats were divided into 5 groups by a random number table (n=15), including control, model, BAJP, BAJP+3-methyladenine (3-MA), and bloodletting acupuncture at non-acupoint (BANA, tail tip blooding) groups. After 7-day pre-treatment, AHH models were established using hypobaric oxygen chambers. The levels of S100B, glial fibrillary acidic protein (GFAP), superoxide dismutase (SOD), and malondialdehyde (MDA) in serum were measured by enzyme-linked immunosorbent assay. Hematoxylin-eosin staining and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method were used to assess hippocampal histopathology and apoptosis. Transmission electron microscopy assay was used to observe mitochondrial damage and autophagosomes in hippocampal tissues. Flow cytometry was used to detect mitochondrial membrane potential (MMP). The mitochondrial respiratory chain complexes I, III and IV activities and ATPase in hippocampal tissue were evaluated, respectively. Western blot analysis was used to detect the protein expressions of Beclin1, autophagy protein 5 (ATG5), microtubule-associated protein 1 light chain 3 beta (LC3B), phosphatase and tensin homolog induced kinase 1 (PINK1), and Parkin in hippocampal tissues. The mRNA expressions of Beclin1, ATG5 and LC3-II were analyzed by quantitative real-time polymerase chain reaction. RESULTS: BAJP treatment reduced hippocampal tissue injury and inhibited hippocampal cell apoptosis in AHH rats. BAJP reduced oxidative stress by decreasing S100B, GFAP and MDA levels and increasing SOD level in the serum of AHH rats (P<0.05 or P<0.01). Then, BAJP increased MMP, the mitochondrial respiratory chain complexes I, III and IV activities, and the mitochondrial ATPase activity in AHH rats (all P<0.01). BAJP improved mitochondrial swelling and increased the autophagosome number in hippocampal tissue of AHH rats. Moreover, BAJP treatment increased the protein and mRNA expressions of Beclin1 and ATG5 and LC3-II/LC3-I ratio in AHH rats (all P<0.01) and activated the PINK1/Parkin pathway (P<0.01). Finally, 3-MA attenuated the therapeutic effect of BAJP on AHH rats (P<0.05 or P<0.01). CONCLUSION: BAJP was an effective treatment for AHH-induced brain injury, and the mechanism might be through reducing hippocampal tissue injury via increasing the PINK1/Parkin pathway and enhancement of mitochondrial autophagy.

Bloodletting Acupuncture at Jing-Well Points Alleviates Myocardial Injury in Acute Altitude Hypoxic Rats by Activating HIF-1α/BNIP3 Signaling-Mediated Mitochondrial Autophagy and Decreasing Oxidative Stress.

OBJECTIVE: To explore the protective effect and possible mechanisms of bloodletting acupuncture at Jing-well points (BAJP) pre-treatment on acute hypobaric hypoxia (AHH)-induced myocardium injury rat. METHODS: Seventy-five rats were randomly divided into 5 groups by a random number table: a control group (n=15), a model group (n=15), a BAJP group (n=15), a BAJP+3-methyladenine (3-MA) group (n=15), and a BANA (bloodletting at nonacupoint; tail bleeding, n=15) group. Except for the control group, the AHH rat model was established in the other groups, and the corresponding treatment methods were adopted. Enzyme-linked immunosorbent assay (ELISA) was used to detect creatine kinase isoenzyme MB (CK-MB) and cardiac troponins I (CTnI) levels in serum and superoxide dismutase (SOD) and malondialdehyde (MDA) levels in myocardial tissue. Hematoxylin-eosin (HE) staining was used to observe myocardial injury, and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was used to observe cell apoptosis. Transmission electron microscopy detection was used to observe mitochondrial damage and autophagosomes in the myocardium. The mitochondrial membrane potential of the myocardium was analyzed with the fluorescent dye JC-1. Mitochondrial respiratory chain complex (complex I, III, and IV) activities and ATPase in the myocardium were detected by mitochondrial respiratory chain complex assay kits. Western blot analysis was used to detect the autophagy index and hypoxia inducible factor-1α (HIF-1α)/Bcl-2 and adenovirus E1B 19k Da-interacting protein 3 (BNIP3) signaling. RESULTS: BAJP reduced myocardial injury and inhibited myocardial cell apoptosis in AHH rats. BAJP pretreatment decreased MDA levels and increased SOD levels in AHH rats (all P<0.01). Moreover, BAJP pretreatment increased the mitochondrial membrane potential (P<0.01), mitochondrial respiratory chain complex (complexes I, III, and IV) activities (P<0.01), and mitochondrial ATPase activity in AHH rats (P<0.05). The results from electron microscopy demonstrated that BAJP pretreatment improved mitochondrial swelling and increased the autophagosome number in the myocardium of AHH rats. In addition, BAJP pretreatment activated the HIF-1α/BNIP3 pathway and autophagy. Finally, the results of using 3-MA to inhibit autophagy in BAJP-treated AHH rats showed that suppression of autophagy attenuated the treatment effects of BAJP in AHH rats, further proving that autophagy constitutes a potential target for BAJP treatment of AHH. CONCLUSION: BAJP is an effective treatment for AHH-induced myocardial injury, and the mechanism might involve increasing HIF-1α/BNIP3 signaling-mediated autophagy and decreasing oxidative stress.

[Effect of blood-letting puncture of "Well-points" on hippocampal mitophagy-related protein expression in rats with acute brain injury due to hypobaric hypoxia].

OBJECTIVE: To study the effect of blood-letting puncture at "Well-points" of the twelve meridians on hippocampal mitophagy of hypobaric hypoxia-induced brain injury (HHIBI) rats, so as to explore its biological mechanisms underlying improvement of high altitude hypoxia-induced brain injury. METHODS: Male SD rats were randomly divided into normal control group （n=9）， and model and blood-letting groups which were further divided into 6, 12, 24, 48 and 72 h subgroups (n=9 in each subgroup). The HHIBI model was established by putting the rats into a hypobaric hypoxia chamber (equivalent to 5 000 m above sea level).The blood-letting groups were given blood-letting therapy at "Shaoshang"(LU11), "Shangyang"(LI1), "Zhongchong"(PC9), "Guanchong"(SJ1), "Shaochong"(HT9), "Shaoze"(SI9), once a day for 7 days. H.E. staining was used to observe the histopatholo-gical changes of hippocampus tissue. Serum hypoxia inducible factor(HIF)-1α and vascular endothelial growth factor(VEGF) contents were assayed using ELISA, and the expression levels of hippocampal Beclin-1 and LC3-Ⅱ proteins detected using Western blot. RESULTS: Compared with the normal control group, the levels of serum HIF-1α and VEGF at each time point, and the expressions of hippocampal Beclin-1 at 12 and 24 h, LC3-Ⅱat each time point were significantly increased in the model group (P<0.05, P<0.01); while in comparison with the model group, the levels of serum HIF-1α and VEGF contents, and the expressions of Beclin-1 at 12 h, LC3-Ⅱ at 24, 48 and 72 h were further significantly up-regulated in the blood-letting group (P<0.01, P<0.05). H.E. staining revealed that the pyramidal cells in the hippocampal CA1 region had a disordered arrangement, and some of them presented swelling with loose and pale cytoplasm or vacuolation at 6, 12 and 24 h, and showed indistinct nucleolus, irregular shape, pyknosis and deep staining and an obvious edema at 48 and 72 h, which was relatively milder in the blood-letting group. CONCLUSION: Blood-letting of "Well-points" can up-regulate serum HIF-1α and VEGF contents and hippocampal Beclin-1 and LC3-Ⅱ (mitophagy related proteins) expressions in HHIBI rats, which may contribute to its effect in reducing hypoxic brain injury.