Ferritinophagy and Ferroptosis in Cerebral Ischemia Reperfusion Injury.

Cerebral ischemia-reperfusion injury (CIRI) is the second leading cause of death worldwide, posing a huge risk to human life and health. Therefore, investigating the pathogenesis underlying CIRI and developing effective treatments are essential. Ferroptosis is an iron-dependent mode of cell death, which is caused by disorders in iron metabolism and lipid peroxidation. Previous studies demonstrated that ferroptosis is also a form of autophagic cell death, and nuclear receptor coactivator 4(NCOA4) mediated ferritinophagy was found to regulate ferroptosis by interfering with iron metabolism. Ferritinophagy and ferroptosis are important pathogenic mechanisms in CIRI. This review mainly summarizes the link and regulation between ferritinophagy and ferroptosis and further discusses their mechanisms in CIRI. In addition, the potential treatment methods targeting ferritinophagy and ferroptosis for CIRI are presented, providing new ideas for the prevention and treatment of clinical CIRI in the future.

A preliminary investigation of the regulatory effect of acupuncture on hippocampal circHDAC2/miR-3065/SLC30A3 axis in CIRI rats. / å¤§é¼ æµ·é©¬ç»„ç»‡circHDAC2/miR-3065/SLC30A3è½´å‚ä¸Žé’ˆåˆºè°ƒèŠ‚è„‘ç¼ºè¡€å†çŒæ³¨æŸä¼¤çš„åˆæ­¥ç ”ç©¶.

OBJECTIVES: To explore the mechanisms of acupuncture against cerebral ischemia/reperfusion injury (CIRI) through observing the expression of circular RNA HDAC2 (circHDAC2) in the hippocampus of rats. METHODS: SD rats were randomly divided into sham-operation, model and acupuncture groups, with 13 rats in each group. The rat model of CIRI was established by middle cerebral artery occlusion. In the acupuncture group, acupuncture was delivvered at "Dazhui" (GV14), "Shuigou" (GV26) and "Baihui" (GV20), and the needles were retained for 30 min each time and acupuncture was conducted once every 12 h for a total of 7 sessions. Before and after intervention, using modified Garcia scale, the neurological function of the rats were evaluated, and TTC staining was employed to determine the cerebral infarct area. Gene chip technology was used to screen the circRNAs with differential expressions in the ischemic hippocampus, and the circRNAs with co-differential expression (co-DE circRNAs) in the model group/sham-operation group, and the acupuncture group/model group separately. Among those circRNAs, the core circRNAs were screened according to P value, fold change (FC) and gene ontology (GO) analysis；and their expressions in the ischemic hippocampus were determined using quantitative real-time PCR (qPCR). Based on the verification results, a competing endogenous RNA (ceRNA) prediction network was constructed. The expression levels of microRNA (miRNA) and mRNA with high node centrality in the prediction network were validated by qPCR. RESULTS: Before intervention, compared with the sham-operation group, the modified Garcia score of each modeling group decreased (P<0.01). After intervention, the modified Garcia score was reduced and the cerebral infarct area ratio increased (P<0.01) in the model group when compared with the sham-operation group. In the acupuncture group, the modified Garcia score was higher and the cerebral infarct area ratio lower (P<0.01) than those of the model group. The microarray results of gene chip showed that 16 co-DE circRNAs were down-regulated in the model group and up-regulated in the acupuncture group, and 7 co-DE circRNAs up-regulated in the model group and down-regulated in the acupuncture group. The core circHDAC2 and circNTRK2 were screened according to P value, FC and the enrichment number of GO entries. QPCR results showed that, compared with the sham-operation group, the expression of circHDAC2 and circNTRK2 of the ischemic hippocampal tissue was down-regulated in the model group (P<0.01)；and that of circHDAC2 and circNTRK2 up-regulated in the acupuncture group when compared with the model group (P<0.01). The relevant ceRNA regulatory network was constructed for circHDAC2 and the prediction results showed that the regulatory networks contained 12 miRNAs and 31 mRNAs. Results of verifying miRNA with high network node centrality and mRNA relevant with nerve regulation showed that, when compared with the sham-operation group, the expression levels of miR-29a, miR-29b and the solute carrier family 30 member 3 (SLC30A3) mRNA in the ischemic hippocampus were down-regulated (P<0.01)；and those of miR-3065 and mercaptopyruvate sulfurtransferase (MPST) up-regulated (P<0.01) in the model group. Compared with the model group, the expressions of miR-29a, miR-29b and SLC30A3 mRNA in the ischemic hippocampus were up-regulated (P<0.01, P<0.05), while that of miR-3065 down-regulated (P<0.05) in the acupuncture group. CONCLUSIONS: Acupuncture significantly improves the neurological function and reduces the cerebral infarct area in CIRI rats, which may be related to the regulation of hippocampal circHDAC2/miR-3065/SLC30A3 axis.

[Acupuncture regulates autophagy in hippocampal neurons of cerebral ischemia/reperfusion injured rats by activating type â ¢ PI3K/Beclin-1 pathway].

OBJECTIVE: To observe the effects of acupuncture on the expression of type Ⅲ phosphatidylinositol 3-hydroxykinase (PI3K) and Beclin-1 in hippocampus of rats with cerebral ischemia/reperfusion injury (CI/RI), so as to explore the mechanism of acupuncture in regulating type Ⅲ PI3K pathway to activate autophagy in the hippocampal neurons of CI/RI rats. METHODS: SD rats were randomly divided into sham operation group (n=11) and operation group. Then after successful modeling, rats in the operation group were randomly divided into model, acupuncture, model+3-MA and acupuncture+3-MA groups, with 11 rats in each group. The model of CI/RI was established by occlusion of the middle cerebral artery. Rats in the model+3-MA and acupuncture+3-MA groups were injected with 3-MA (400 nmol/ 5 µL) 5 µL into the lateral ventricle 30 min before reperfusion. Rats in the acupuncture and acupuncture+3-MA groups were punctured with filiform needles at "Dazhui" (GV14), "Shuigou" (GV26) and "Baihui" (GV20) and stimulated manually once every 15 min. The acupuncture intervention was conducted for 30 min each time, once every 12 h for a total of 7 times. The degree of neurological impairment was evaluated 2 h after reperfusion and after intervention by Garcia score. After intervention, the percentage of cerebral ischemic area was observed by TTC staining, the protein expression levels of type Ⅲ PI3K, Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B), lysosome associated membrane protein 2 (Lamp2) and P62 in ischemic hippocampal tissue were detected by Western blot, the ultrastructure of neurons in ischemic hippocampus was observed by transmission electron microscopy (TEM). RESULTS: Compared with the sham operation group, the Garcia score was decreased (P<0.01), the percentage of cerebral ischemic area was increased (P<0.01), the expression levels of type Ⅲ PI3K, Beclin-1, LC3B-Ⅱ/Ⅰ, Lamp2 proteins were decreased (P<0.01), and the expression level of P62 protein was increased (P<0.01) in ischemic hippocampal tissue in the model group. Compared with the model group, the Garcia score was increased (P<0.01), the percentage of cerebral ischemic area was decreased (P<0.01), the expression levels of type Ⅲ PI3K, Beclin-1, LC3B-Ⅱ/Ⅰ, Lamp2 proteins were increased (P<0.01, P<0.05) and the expression level of P62 was decreased (P<0.01) in ischemic hippocampal tissue in the acupuncture group； the percentage of cerebral ischemic area was increased (P<0.05), the expressions of type Ⅲ PI3K and Beclin-1 were decreased (P<0.01) and the expression level of P62 protein was increased (P<0.05) in ischemic hippocampal tissue in the mo-del+3-MA group. Compared with the model +3-MA group, the Garcia score was increased (P<0.05), the percentage of cerebral ischemic area was decreased (P<0.01), the expression levels of type Ⅲ PI3K, Beclin-1, LC3B-Ⅱ/Ⅰ in ischemic hippo-campal tissue were increased (P<0.01, P<0.05) in the acupuncture+3-MA group. Compared with the acupuncture group, the Garcia score was decreased, the percentage of cerebral ischemic area was increased (P<0.05, P<0.01), the expression levels of type Ⅲ PI3K, Beclin-1, Lamp2 proteins were decreased (P<0.01, P<0.05) and P62 protein was increased (P<0.05) in ischemic hippocampal tissue in the acupuncture+3-MA group. The results of TEM showed that the edema of neurons was heavier, and few hypolysosomes existed in the model group; there was no obvious damage to neuronal structure, intracellular matrix was abundant, and a few lysosomes existed in the acupuncture group; the neuronal cells had mild edema and primary lysosomes were present in the acupuncture +3-MA group. CONCLUSION: Acupuncture can improve the symptoms of neurological impairment and reduce the percentage of cerebral ischemic area in rats with CI/RI. The mechanism may be related to regulating type Ⅲ PI3K/Beclin-1 pathway, up-regulating the expressions of autophagy related factors LC3B-Ⅱ and Lamp2, and down-regulating the expression of P62.

[Acupuncture mediated miR-34c-5p regulates the autophagy of hippocampal neurons in rats with cerebral ischemia/reperfusion injury].

OBJECTIVE: To observe the effect of acupuncture on the expression of miR-34c-5p, autophagy-related proteins and apoptosis rate in hippocampus of rats with cerebral ischemia/reperfusion injury (CI/RI), so as to explore its mechanism in regulating autophagy in hippocampal neurons in CI/RI rats. METHODS: SD rats were randomly divided into sham operation, model, medication and acupuncture groups, with 20 rats in each group. The rat model of CI/RI was established by occlusion of the middle cerebral artery. In the acupuncture group, "Dazhui" (GV14), "Baihui" (GV20) and "Shuigou" (GV26) were punctured with filiform needles and stimulated manually once every 15 min, for 30 min. The rats of the medication group were intraperito-neally injected with edaravone (5 mg/kg). The treatment was conducted once every 12 h for a total of 7 times. The neurological de-ficit score of all the rats were evaluated according to Garcia's methods, and TTC staining was employed to assess the cerebral ischemic area (percentage of cerebral infarct area, CIA). Transmission electron microscopy (TEM) was used to observe the ultrastructural changes of hippocampal neurons. The expression of hippocampal miR-34c-5p was measured by real-time PCR, and the protein expressions of hippocampal LC3B, Beclin1 and p62 were measured by Western blot. The apoptosis rate of ischemic brain tissue was observed by TUNEL staining. RESULTS: Compared with the sham operation group, the neurological deficit score, the expressions of miR-34c-5p, Beclin1, p62 and LC3B-Ⅱ/LC3B-Ⅰ were significantly decreased (P<0.01, P<0.05), and the CIA and the apoptosis rate significantly increased (P<0.01) in the model group. Compared with the model group, the neurological deficit scores, the expressions of miR-34c-5p, Beclin1, p62 and LC3B-Ⅱ/LC3B-Ⅰ were significantly increased (P<0.01, P<0.05), and the CIA and the apoptosis rate significantly decreased (P<0.01) in the medication and acupuncture groups. Compared with the medication group, the expression of miR-34c-5p was significantly increased (P<0.01). The results of electron microscope showed that the neurons in the acupuncture and medication groups were less damaged than those in the model group, the cells showed mild edema, and the structures were relatively complete. Some normal organelles could be seen, and autophagy bodies, autophagy lysosomes and their encapsulated organelles could still be observed. CONCLUSION: Acupuncture can improve the neurological deficit and reduce the area of cerebral infarction in CI/RI rats, which is closely with its effect in promoting hippocampal neuronal autophagy and anti-apoptosis via up-regulating the expression of miR-34c-5p.

[Effect of acupuncture technique of Tiaoxin Tongdu on learning-memory ability and expressions of hippocampal VEGF and Ang-1 in rats with vascular dementia].

OBJECTIVE: To observe the effect of acupuncture technique of Tiaoxin Tongdu on learning-memory ability and expressions of hippocampal vascular endothelial growth factor (VEGF) and angiogenin-1 (Ang-1) in rats with vascular dementia (VD), and to explore the mechanism of acupuncture technique of Tiaoxin Tongdu for VD. METHODS: A total of 24 male SD rats were randomly divided into a sham operation group, a model group, a medication group and an acupuncture group after Morris water maze test, 6 rats in each group. VD model was established by permanent ligation of bilateral common carotid arteries in the model group, the medication group and the acupuncture group. Treatment was given on the next day after successful modeling. The rats in the acupuncture group were treated with acupuncture at "Baihui" (GV 20), "Shenting" (GV 24), "Shuigou" (GV 26), "Dazhui" (GV 14), "Fengfu" (GV 16), "Mingmen" (GV 4), "Neiguan" (PC 6), "Daling" (PC 7) and "Laogong" (PC 8) for 30 min; the rats in the medication group were treated with nimodipine solution (0.0625 g/kg) by gavage, once a day, for 2 weeks. Morris water maze test was used to detect the behavior of rats before modeling, 2 weeks after modeling and after intervention; after intervention, the expressions of VEGF and Ang-1 protein in hippocampus were detected by Western blot. RESULTS: Compared with the sham operation group, the average escape latency of rats in the model group was prolonged (P<0.01), and the times of crossing the original platform were reduced (P<0.01). Compared with the model group, the average escape latency of rats in the medication group and acupuncture group was significantly shortened (P<0.01), and the times of crossing the original platform were increased (P<0.01, P<0.05). Compared with the sham operation group, the expressions of VEGF and Ang-1 protein in hippocampus in the model group were increased (P<0.05, P<0.01). Compared with the model group, the expressions of VEGF and Ang-1 protein in the hippocampus in the medication group and acupuncture group were significantly increased (P<0.01, P<0.05). CONCLUSION: The acupuncture technique of Tiaoxin Tongdu can significantly improve the learning and memory ability of VD rats, and its mechanism may be related to up-regulating the expressions of VEGF and Ang-1 protein in hippocampus and inducing angiogenesis.

Cross Talk Between Ferroptosis and Cerebral Ischemia.

Recently, ferroptosis has been revealed as a new form of regulated cell death. Distinct from apoptosis and necrosis, ferroptosis is evoked by iron-dependent lipid peroxidation. Furthermore, the metabolism of iron, lipids, and amino acids plays a significant regulatory role in ferroptosis, which can be reversed by glutathione peroxidase 4 and ferroptosis suppressor protein 1. Ferroptosis is implicated in the onset and development of numerous neurological diseases. Emerging studies have reported that ferroptosis induces and aggravates brain tissue damage following cerebral ischemia, whereas inhibition of ferroptosis dramatically attenuates induced damage. In this review, we have summarized the mechanistic relationship between ferroptosis and cerebral ischemia, including through iron overload, downregulation of glutathione peroxidase 4, and upregulation of lipid peroxidation. Although considerable attention has been paid to the effect of ferroptosis on cerebral ischemic injury, specific mechanisms need to be experimentally confirmed, including how cerebral ischemia induces ferroptosis and how ferroptosis deteriorates cerebral ischemia.

[Design and implementation of capacity-oriented curriculum modules of Acupuncture-Moxibustion Medicine].

In order to improve the teaching quality of Acupuncture-Moxibustion Medicine, the author valued the students' ability as guidance and purpose, analyzed the characteristics of the curriculum design and the selection of teaching materials, and proposed 3 teaching modules consisted of theory, practical training and application of acupuncture- moxibustion. Hereafter, it was constantly summarized and improved to realize the goal of curriculum knowledge, ability and quality. The reform method of the curriculum design, teaching content and method of Acupuncture-Moxibustion Medicine in traditional Chinese medicine was preliminarily explored.

[Effect of acupuncture combined with hypothermia on MAPK/ERK pathway and apoptosis related factors in rats with cerebral ischemia reperfusion injury].

OBJECTIVE: To observe effect of acupuncture combined with hypothermia therapy on MAPK/ERK pathway and apoptosis related factorsin rats suffered cerebral ischemia reperfusion and to explore underlying mechanisms.
 Methods: Middle cerebral artery ischemia model were established.Ninety SD rats were randomly assigned into a blank group, a control group, a model group, an acupuncture group, a mild hypothermia group, and an acupuncture with hypothermia group. After 72 h treatment, nerve function defect scores were observed, and infarction area percent was detected by 2, 3, 5-triphenyl-2H-tetrazolium chloride (TTC) staining; expressions of Bcl-2 and Bax were examined by immunohistochemistry; apoptotic cells were detected by TUNEL assay; and expression levels of phospho-mitogen-activated protein kinase(p-MEK2) and phospho-extracellular signal regulated kinase 1/2 (p-ERK1/2) in the rats' hippocampus ischemic side were determined by Western blot.
 Results: In the rats of the model group, the neural function defect scores, the infarction area percent, the expression level of Bax, and apoptotic cells increased, while the level of Bcl-2 decreased significantly. The level of p-MEK2 and p-ERK1/2 increased obviously compared with the blank and control groups (P<0.05 or P<0.01). After treatment with acupuncture and hypothermia, the neural function defect scores, infarction area percent, and the level of Bax, apoptotic cells and the levels of p-MEK2 and p-ERK1/2 were significantly decreased, while the level of Bcl-2 in the treatment group was significantly elevated (P<0.05 or P<0.01) compared with the model group. Compared with the acupuncture group or the hypothermia group, the neural function defect scores and the levels of p-MEK2 and p-ERK1/2 in the acupuncture combined with hypothermia group were significantly reduced (P<0.05 or P<0.01).
 Conclusion: Acupuncture and hypothermia therapy can improve cerebral function, and reduce the cerebral injury through down-regulation of Bax level, and up-regulation of Bcl-2 level, which is related to reducing the levels of p-MEK2 and p-ERK1/2. The therapeutic effects on cerebral ischemia reperfusion injury for combination of acupuncture with hypothermia are better than those with single application of acupuncture or hypothermia.

[Effect of Acupuncture on Neurological Function and Related Differentially-depressed Profiles in the Brain of Middle Cerebral Artery Occlusion Rats Using Antibody Chips Technique].

OBJECTIVE: To observe the effect of acupuncture on changes of neurological function and expression of proteins in the ischemic brain region in middle cerebral artery occlusion (MCAO) rats with protein chip technique, so as to reveal the profiles of cerebral proteins related to its effectiveness in improving cerebral ischemia (CI). METHODS: SD rats were rando-mized into sham operation (sham), CI model, non-acupoint and acupoint groups (n=10 in each group). The CI model was established by MCAO according to modified Longa's method. For rats of the acupoint group, "Dazhui" (CV 14), "Baihui" (GV 20) and "Shuigou" (GV 26) were punctured by twirling the filiform needles for about 1 min, and repeated once again during 30 min of need-le retention, which was conducted once every 12 h, and 6 times altogether. The non-acupoints were about 3 mm away from the above-mentioned acupoints and stimulated with the same method and same procedures. The neurological deficit score was scaled according to Longa's method. The differentially-expressed proteins (≥ 1.5 folds in up- and down-regulation) in the ischemic region of the brain were detected by using Springbio 720 antibody chip technology. RESULTS: Compared with the sham group, the neurological deficit score was significantly higher in the model group (P<0.01), while compared with the model group, the neurological deficit scores were considerably in both acupoint and non-acupoint groups (P<0.05, P<0.01). The therapeutic effect of acupoint group was evidently superior to that of the non-acupoint group in improving neurological function (P<0.05). Compared with the sham group, the differentially-expressed proteins in the ischemic brain including 33 up-regulated and 12 down-regulated were found in the model group, mainly functioning in cell apoptosis, cell cycle regulation, cell differentiation and proliferation, cell cytoskeleton and connection, cell signal transduction, DNA repair and transcription factors. Compared with the model group, the differentially-expressed proteins including 12 up-regulated and 31 down-regulated in the acupoint group, and 15 up-regu-lated and 17 down-regulated in the non-acupoint group were detected, functioning being the same as those mentioned above in the model group. CONCLUSIONS: Manual acupuncture stimulation of GV 14, GV 20 and GV 26 can improve neurological function in CI rats, which may be associated with its function in regulating the expression of many proteins in the ischemic region of the brain.

[Impacts of collaborative teaching method on the teaching achievement of Acupuncture and Moxibustion].

OBJECTIVE: To explore the impacts of collaborative teaching method on the teaching achievement of Acupuncture and Moxibustion. METHODS: Six classes in Hunan University of CM of 2012 grade Chinese medicine department were randomized into an observation group and a control group, 3 classes in each one. In the observation group, the collaborative teaching method was adopted, in which, different teaching modes were used according to the characteristics of each chapter and the study initiative of students was predominated. In the control group, the traditional teaching method was used, in which, the class teaching was the primary and the practice was the secondary in the section of techniques of acupuncture and moxibustion. The results of each curriculum and the total results were compared between the two groups during the whole semester. RESULTS: Compared with the control group, in the observation group, the total achievements of curriculum and case analysis combined with the total result of the theory examination were apparently improved (both P<0.01). CONCLUSIONS: The collaborative teaching method improves the comprehensive ability of students and provides a new approach to the teaching of Acupuncture and Moxibustion.

Acupuncture activates signal transduction pathways related to brain-tissue restoration after ischemic injury.

A middle cerebral artery occlusion-model was established in rats using the improved thread embolism method. Rats were treated with acupuncture at either Dazhui (DU14), Renzhong (DU26), Baihui (DU20), or a non-meridian point. Detection with protein-chip technology showed that the level of protein phosphorylation in both groups was upregulated or downregulated depending on the signaling pathway compared with the model group that did not receive acupuncture. Analysis of proteins showing downregulated phosphorylation revealed that five signaling pathways were activated in the acupuncture-treatment group, while only two were activated in the acupuncture- control group. In contrast, analysis of proteins showing upregulated phosphorylation revealed only one pathway was activated in the acupuncture-treatment group, whereas four pathways were activated in the acupuncture-control group. Furthermore, the number of activated proteins in the acupuncture-treatment group was not only higher than the acupuncture-control group, but unlike the acupuncture-control group, the majority of activated proteins were key proteins in the signaling pathways. Our findings indicate that acupuncture at specific points can activate multiple signaling pathways to promote the restoration of brain tissue following ischemic injury, and that this is based on a combination of effects resulting from multiple pathways, targets, and means.

[Observation on changes of the intragastric protein phosphorylation level involving electroacupuncture-induced improvement of the injured gastric mucosa in the rat].

OBJECTIVE: To study the effect of electroacupuncture (EA) of Stomach Meridian acupoints on changes o of the related protein phosphorylation in the gastric mucosa involving the repair of the injured gastric mucosa in the rat. METHODS: A total of 20 SD rats were divided into control, model, acupoint and non-acupoint groups, with 5 cases in each. Gastric mucosa injury model was established by intragastric perfusion of dehydrated alcohol (0.8 ml/100 g). EA (4 Hz/50 Hz, 1-3 mA) was applied to "Zusanli" (ST 36), "Liangmen" (ST 21) and "Sibai" (ST 2), and the corresponding non-acupoints, once daily for 7 days. Gastric mucosal ulcer index (UI) was measured, and the profiling of the protein phosphorylation of interest in gastric mucosa cells was detected by antibody microarrays. RESULTS: In comparison with control group, the gastric mucosal UI of model group was significantly higher than that of control group (P < 0.01), while the UI of EA group was significant lower than those of model group and non-acupoint group (P < 0.01), suggesting a recovery of the injured mucous membrane. Findings of protein array analysis indicated that a total of 720 kinds of phosphorylated proteins (> or = 1.5 folds) were found in the injured gastric mucosa. Compared to model group, the phosphorylation levels of 100 and 20 proteins were up-regulated, and 16 and 9 proteins down-regulated in acupoint and non-acupoint groups, respectively. The up-regulated proteins involve cell cycle mediation, cell proliferation, anti-apoptosis, and the regulation of the cell adhesion, cytoskeleton protein, inflammation, immune activities, etc. while the down-regulated proteins involve the down-regulation of the phosphorylation levels of the apoptosis proteins, adhesion proteins and cytoskeleton proteins, etc. In comparison to non-acupoint group, the phosphorylation levels of 100 proteins were up-regulated and 16 proteins down-regulated in acupoint group. CONCLUSION: EA of acupoints of the Stomach Meridian can promote repair of the injured gastric mucosa, which may be related to its effects in regulating the levels of phosphorylation of many signaling proteins.