[Electroacupuncture improves ischemic myocardial injury by activating Nrf2/HO-1 signaling pathway to inhibit ferroptosis in rats].

OBJECTIVE: To explore the role of nuclear factor E2 related factor 2 (Nrf2) / heme oxygenase (HO-1) signal pathway in electroacupuncture (EA) induced improvement of acute myocardial ischemia (AMI) and its relationship with ferroptosis in rats. METHODS: Male SD rats were randomly and equally divided into sham operation, model, EA and EA+ML385 (inhibitor of Nrf2) groups (n=8). The rat model of AMI was established by ligating the descending anterior branch of the left coronary artery. EA (2 Hz/100 Hz) was applied to bilateral "Shenmen"(HT7) and "Tongli"(HT5) for 20 min, once daily for 7 days. The electrocardiogram (ECG) of standard Ⅱ (ECG ST) lead and heart rate (HR) in each group was recorded and analyzed before and after modeling and after treatment by using PowerLab physiological recorder system. Histopathological changes of myocardial tissue were observed by H.E. staining, and the ultrastructure of myocardiocytes of cardiac apical tissue was observed under transmission electron microscope. The contents of Fe2+ and glutathione (GSH) in the myocardial tissue were measured by chromato-metry. The protein expression levels of Nrf2, HO-1, glutathione peroxidase 4 (GPX4), ferritin heavy chain polypeptide 1 (FTH1) and long chain acyl CoA synthase 4 (ACSL4) in the myocardial tissue were detected by Western blot. RESULTS: Compared with the sham operation group, the HR, ECG ST, Fe2+ content, expression levels of Nrf2, HO-1, FTH1 and ACSL4 proteins in myocardial tissues were significantly increased (P<0.01), while GSH content and GPX4 protein expression considerably decreased (P<0.01) in the model group. Compared with the model group, both EA and EA+ML385 groups had an obvious decrease in HR, Fe2+ content, and ACSL4 levels (P<0.01), and an increase in the expression levels of GPX4 and FTH1 proteins (P<0.01), EA (rather than EA+ML385) effectively down-regulated ECG ST, and up-regulated GSH, Nrf2 and HO-1 (P<0.01), whereas EA+ML385 apparently down-regulated expression levels of Nrf2 and HO-1 (P<0.01). It shows that ML385 pronouncedly weaken the effects of EA in slowing down ECG ST and HR, down-regulating Fe2+ content and ACSL4 expression (P<0.01), up-regulating GSH content, Nrf2, HO-1, GPX4 and FTH1 expressions (P<0.01). H.E. staining showed disordered arrangement and hyperplasia of myocardiocytes, enlarged myocardial fiber gap, agglomerated and deeply stained myoplasma, and some broken myocardial fibers with irregular mass and local tissue fibrosis in the model group, which was relatively milder in both EA and EA+ML385 groups. Compared with the sham operation group, the model group showed decreased mitochondrial atrophy, increased membrane density, and disappearance or reduction of cristae in myocardial cells，which was improved in the EA group. CONCLUSION: EA of HT7 and HT5 has a protective effect on ischemic myocardium in rats, which may be related to its effects in reducing oxidative stress by regulating Nrf2/HO-1 signaling pathway, and inhibiting "iron death" of myocardial cells.

PC6 electroacupuncture reduces stress-induced autonomic and neuroendocrine responses in rats.

Stress can trigger cardiovascular disease. Both imbalance of autonomic nervous activity and increase of neurohormonal output are core aspects of stress responses and can lead to cardiovascular disease. PC6 as a very important acupoint is used to prevent and treat cardiovascular disease and to improve stress-related activities. We examined the influence of electroacupuncture (EA) at PC6 on stress-induced imbalance of autonomic nervous activity and increase of neurohormonal output. EA at PC6 relieved increased cardiac sympathetic nervous activity and decreased cardiac vagal nervous activity induced by immobilization stress. Also, EA at PC6 reduced immobilization stress-induced increases of plasma norepinephrine (NE) and adrenaline (E) released from sympatho-adrenal-medullary axis. Finally, EA at PC6 reduced immobilization stress-induced increases of corticotropin-releasing hormone (CRH) in paraventricular hypothalamic nucleus and plasma cortisol (CORT) released from hypothalamic-pituitary-adrenal axis. However, EA at tail had no significant effect on the stress-induced autonomic and neuroendocrine responses. The results demonstrate the role of EA at PC6 regulating the autonomic and neuroendocrine responses induced by stress and provide insight into the prevention and treatment of EA at PC6 for stress-induced cardiovascular disease by targeting autonomic and neuroendocrine systems.

[Effect of moxibustion on inflammatory pain and N-methyl-D aspartic acid receptor-nitric oxide-cyclic GMP pathway in spinal cord of adjuvant arthritis rats].

OBJECTIVE: To observe the effect of moxibustion on pain and N-methyl-D aspartic acid receptor/nitric oxide/cyclic guanosine monophosphate (NMDA-NO-cGMP) signaling pathway in the spinal cord of rats with adjuvant arthritis (AA), so as to explore its underlying mechanisms in relieving inflammatory pain of rheumatoid arthritis (RA). METHODS: SD rats were randomly divided into normal, model, moxibustion (Moxi), Moxi +NMDA receptor antagonist AP-5 (Moxi+AP-5) and Moxi +NMDA receptor agonist (NMDA) groups, with 20 rats in each group. The AA model was established by placing the rats in a wind, cold and damp environment for 12 h, once daily for 20 days and by injection of complete Freund's adjuvant into the right hind paw. Rats of the three Moxi groups received ignited moxa-stick stimulation of "Zusanli"(ST36) and "Shenshu"(BL23) alternately for 20 min, once a day for 15 days. The Moxi + AP-5 group and Moxi +NMDA group received intraperitoneal injection of AP-5 (0.7 mg·kg-1·d-1) and NMDA (5 mg·kg-1·d-1), respectively, once a day, for a total of 15 days. Mechanical pain thres-hold (MPT) was measured before and after modeling and interventions. The spinal cord tissue was sampled for detecting the expression of iNOS mRNA and protein, content of cGMP and NO, and the activity of NOS by using fluorescence quantitative PCR, Western blot, ELISA,nitrate reductase method and colorimetric method, respectively. RESULTS: Before modeling, there was no significant difference in MPT among all the 5 groups (P>0.05). After modeling, the MPT was remarkably decreased (P<0.01), the expression levels of iNOS mRNA and protein,the contents of cGMP and NO, the activity of NOS were significantly increased in the model group relevant to the normal group (P<0.01). After the interventions, the MPT was obviously increased (P<0.01), while the expression levels of iNOS mRNA and protein, the contents of cGMP and NO, the activity of NOS were significantly down-regulated in the Moxi, Moxi-AP-5 and Moxi+NMDA groups (P<0.05, P<0.01). The effect of Moxi+AP-5 group was significantly superior to that of Moxi group in raising MPT and down-regulating the expression levels of iNOS mRNA and protein, and the content of NO (P<0.05, P<0.01). The effect of Moxi+NMDA group was obviously inferior to that of Moxi group in up-regulating MPT and down-regulating the levels of iNOS mRNA and protein, and the contents of cGMP and NO, and the activity of NOS (P<0.01), suggesting a reduction of the therapeutic effects in raising MPT and down-regulating expression of iNOS mRNA and protein after administration of AP-5. CONCLUSION: Moxibustion can relieve RA inflammatory pain in AA rats, which may be related to its function in down-regulating the NMDA/NO/cGMP signaling pathway in the spinal cord.

Effect of moxibustion on autophagy and the inflammatory response of synovial cells in rheumatoid arthritis model rat.

OBJECTIVE: To investigate the effect of moxibustion on synovitis and the autophagy of synoviocytes in rheumatoid arthritis (RA). METHODS: Forty Sprague-Dawley rats were randomly divided into a normal group, model group, moxibustion group, cigarette moxibustion group, and medicine group, with eight rats included in each group. The RA model was established by subcutaneous injection of complete Freund's adjuvant into the left posterior toe. Rats in the model group were not interfered with. In the moxibustion group, rats were treated by moxibustion, where a 1-cm diameter moxa stick was applied at the left Zusanli (ST 36) point. The distance of the moxa stick to the skin was 2 cm and moxibustion was completed for 20 min daily for 15 d total. In the cigarette moxibustion group, the moxa stick was replaced by a common cigarette. In the medicine group, rats were treated with a tripterygium glycoside suspension (8 mg/kg) once a day for 15 d total. In each group, the left hind limb toe volume was measured with a toe volume meter; the synovial cells were observed by hematoxylin and eosin staining; the interleukin (IL)-4, IL-6, IL-10, IL-1ß, IL-23, IL-17, and tumor necrosis factor (TNF)-α levels in serum were measured by enzyme-linked immunosorbent assay; the erythrocyte sedimentation rate (ESR) were detected by Westergren sedimentation rate testing; the C-reactive protein (CRP) and rheumatoid factor (RF) levels in serum were detected by rate nephelometry; the expression levels of ULK1, autophagy-associated protein (Atg)3, Atg5, and Atg12 messenger RNA (mRNA) in synovium were detected by real time-quantitative polymerase chain reaction (RT-qPCR); and the protein expression levels of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), LC3-II, beclin-1, phosphorylated-PI3K (p-PI3K), p-Akt, p-mTOR in synovium were detected by Western blotting. RESULTS: Among the RA model rats, joint swelling, an inflammatory reaction, and the proliferation of synovial tissue were obvious and the signal of the PI3K/Akt/mTOR pathway was active, while autophagy was inhibited. Moxibustion at Zusanli (ST36) or intragastric administration of Tripterygium wilfordii glycosides could alleviate the inflammatory reaction of RA rats; relieve the swelling of the toes; downregulate the levels of ESR, CRF, RF; lower the levels of IL-6, IL-1ß, TNF-α, and IL-17; and increase the IL-4 and IL-10. At the same time, the mRNA expression levels of ULK1, Atg3, Atg5, and Atg12 and those of LC3-Ⅱ and beclin-1 were increased, while the PI3K, Akt, mTOR, p-PI3K, p-Akt, p-mTOR were decreased. Cigarette moxibustion did not significantly reduce the swelling of the toe joint in RA rats, and was not as good as that of moxibustion or Tripterygium wilfordii polyglycosides in the effects of inflammation relief and the influences of the levels of ESR, CRF, RF. While cigarette moxibustion has a weak effect to affect the expression of corresponding molecules in autophages and the expression level of the autophagy biomaker in synovial tissue. Moxibustion and tripterygium glycosides can significantly reduce the joint swelling, relieve synovitis and synovial hyperplasia, and inhibit the PI3K/Akt/mTOR signaling pathway to increase autophagy in a manner superior to cigarette moxibustion. CONCLUSION: Moxibustion can limit the proliferation of synoviocytes in RA rats by inhibiting the PI3K/Akt/mTOR signaling pathway, promoting autophagy, effectively reducing synovitis, and alleviating joint swelling.

[Electroacupuncture of acupoints of heart meridian ameliorates acute myocardial ischemia via Akt/mTOR pathway].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of myocardial protein kinase B (Akt) and mammalian target of rapamycin (mTOR) in acute myocardial ischemia (AMI) rats. METHODS: Thirty male SD rats were randomly divided into control, model and EA groups (n=10 in each group). The AMI model was established by occlusion of the descending anterior branch (DAB) of the left coronary artery. EA (2 Hz, 1-2 mA) was applied to bilateral "Shenmen" (HT7) and "Tongli" (HT5) for 20 min, once daily for consecutive 7 days. The electrocardiogram (ECG) of nape-xiphoid lead was recorded for assessing changes of myocardial ischemia. Histopathologic changes of the ischemic myocardial tissue were observed after H.E. staining and ultra-microstructural changes of cardiomyocytes observed by transmission electron microscopy (TEM). The expression levels of Akt, phosphorylated-Akt (p-Akt), mTOR and phosphorylated-mTOR (p-mTOR) in the myocardium were detected by Western blot, followed by calculating the ratios of p-Akt/Akt and p-mTOR/mTOR. RESULTS: Following ligature of DAB, the ECG-ST level was significantly increased in the model group in comparison with the control group (P<0.01). At 30 min after treatment, the ECG-ST level decreased significantly compared with the model group (P<0.01). At the end of the 7-day treatment period, the ECG-ST level increased compared with the model group (P<0.05). The levels of myocardial p-Akt and p-mTOR protein expression, and the ratios of p-Akt/Akt and p-mTOR/mTOR were significantly lower in the model group than those in the control group (P<0.01), and considerably increased in the EA group than in the model group (P<0.01). No significant differences were found among the three groups in the expression levels of Akt and mTOR proteins (P>0.05). Outcomes of H.E. staining and TEM showed damage of mitochondria and occurrence of a large number of autophagosomes in myocardiocytes in the model group, which was milder in the EA group. CONCLUSION: EA at HT5 and HT7 can improve AMI in AMI rats, which may be related to its effect in facilitating Akt/mTOR signaling.

[Effects of moxibustion on p53， SLC7A11， and GPX4 expression in synovial tissues of rats with adjuvant arthritis].

OBJECTIVE: To observe the effects of moxibustion on p53, cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) in synovial tissues of rats with adjuvant arthritis (AA), so as to explore the mechanism of moxibustion in alleviating rheumatoid arthritis. METHODS: Eighty rats were randomly divided into normal, model, moxibustion, and medication groups (n=20 in each group). The AA model was established by exposure to wind, cold, and damp environmental factors combined with injection of complete Freund's adjuvant. Rats in the moxibustion group received suspended moxibustion at "Shenshu" (BL23) and "Zusanli" (ST36) alternately, while those in the medication group were treated with tripterygium glycoside tablet suspension (8 mg/kg) by gavage, once a day, for 15 successive days. The pathological change in synovial tissue of rat right knee joint was observed by HE staining. The protein and mRNA expression levels of p53, SLC7A11, and GPX4 in the synovial tissue were detected by Western blot and quantitative real-time PCR, respectively. The se-rum glutathione (GSH) and reactive oxygen species (ROS) contents were measured by colorimetry and fluorescence probe me-thod. RESULTS: Compared with the normal group, the model group exhibited synovial hyperplasia of the right knee joint, massive inflammatory cell infiltration, up-regulated mRNA and protein expression of p53 in synovial tissue, elevated serum ROS content (P<0.01), down-regulated mRNA and protein expression of SLC7A11 and GPX4 in synovial tissue, and lowered serum GSH content (P<0.01). Comparison with the model group showed that the synovial injuries in the moxibustion and medication groups were obviously alleviated. The mRNA and protein expression levels of p53 in the synovial tissues and the serum ROS content declined significantly (P<0.01), while the mRNA and protein expression of SLC7A11 and GPX4 in the synovial tissues and the se-rum GSH content increased (P<0.01). There was no significant difference in histopathological change of synovial tissue between the moxibustion group and medication group. However, the p53 protein expression in the synovial tissue and the level of serum ROS were significantly higher in the medication group than in the moxibustion group (P<0.05), while the GPX4 protein expression and serum GSH content were down-regulated (P<0.05). CONCLUSION: Moxibustion improves the inflammatory response in synovial tissue of AA model rats, which may be closely related to its regulation of the expression of ferroptosis-related factors.

[Effect of inhalation of different-concentrations of moxa smoke on apoptosis and regeneration of olfactory epithelial cells in rats].

OBJECTIVE: To observe the effect of long-term inhalation of moxa-smoke on olfactory epithelial cells in rats, in order to explore the safety of moxa-smoke inhalation (MSI). METHODS: A total of 32 SD rats (half male and half female) were randomly divided into 4 groups: normal, low concentration (LCMSI), medium concentration (MCMSI) and high concentration (HCMSI), with 8 rats in each group. Rats of the LCMSI, MCMSI and HCMSI groups were put into closed boxes which were filled with ignited moxa stick-released smoke at concentrations of (0.11±0.05)mg/m3, (0.23±0.05) mg/m3 and (0.53±0.05)mg/m3, respectively. The treatment was given 4 h each time, twice a day for 90 days. Rats of the normal group were fed routinely. The rats' general state and behavior (including fur appearance, activities in cage, response to external stimuli, spirit, stool, diet and water drinking) were recorded, and the olfactory function was assessed by using latency of finding the buried food pellet (BFP) test. The number of apoptotic olfactory epithelial cells was counted after terminal labeling (TUNEL), and the proliferation of basal cells of the nasal mucosa was detected by BrdU incorporation immunohistochemical technique. RESULTS: The latency of BFP was significantly longer in the MCMSI and HCMSI groups than in the normal and LCMSI groups (P<0.01), and had no significant differences between the LCMSI and normal groups, and between the MCMSI and HCMSI groups (P>0.05). The numbers of the apoptotic olfactory epithelial cells and proliferative basal cell in the nasal mucus tissue were markedly more in the LCMSI, MCMSI and HCMSI groups than in the normal group (P<0.01, P<0.05), and obviously more in the MCMSI and HCMSI groups than in the LMCMSI group (P<0.01), and apparently more in the HCMSI group than in the MCMSI group (P<0.01). The general state observation showed that in the first 45 days, only yellowish fur and water intake increase were seen in rats of the 3 moxa smoke inhalation groups, while no obvious changes in rats of the LCMSI group, and decrease in activities, being sensitive to external stimulation and fiddle-footed, and lower spirit in rats of the MCMSI and HCMSI groups in comparison with rats of the normal group after 90 day's MSI. CONCLUSION: Long-term inhalation of medium and high concentrations of moxa smoke may cause a reduction of the olfactory sensitivity and an increase of apoptosis of olfactory epithelial cells and proliferation of basal cells.

[Both fumigation and heating of moxibustion promote wound healing and tissue repair in rats with full-thickness skin incision].

OBJECTIVE: To investigate the effect of moxibustion on wound healing, new capillaries and tissue repair in rats with full thickness skin excision. METHODS: SD rats were randomly divided into control, moxa-fumigating and moxa-heating groups, with 24 rats in each group. The full-thickness skin excision injury model was replicated according to Balaji's and colleague's methods. Rats in the moxa-fumigating group received fumigation of moxa-smog at the wound surface (25 min/time) immediately after modeling through a smoke-heat separation device, and those of the moxa-heating group received conventional thermal stimulation of the ignited moxa over the wound (25 min/time) without moxa smog, once a day for 10 consecutive days. The wound color, pus, carrion, granulation, and healing status of the rats were observed. H.E. staining and Masson staining were used to observe the local tissues and collagen fibers of the wound on the 3rd and 7th days. Newly born capillaries at the wound surface were observed on the 10th day. RESULTS: Following modeling, the wound area of the moxa-fumigating group on the 1st and 7th day, and those of both moxa-fumigating and moxa-heating groups on the 3rd and 5th day were significantly reduced compared with the model group (P<0.001), and the wound area of the moxa-fumigating group was obviously smaller than that of the moxa-hea-ting group (P<0.01，P<0.001). On the 3rd and 7th day after modeling, the re-epithelialization rates were significantly increased (P<0.001), and the amounts of inflammatory cells were significantly reduced in both moxa-fumigating and moxa-heating groups (P<0.001). At the same time, the re-epithelialization rate of the moxa-fumigating group was higher than that of the moxa-heating group (P<0.05，P<0.001), and the inflammatory cell count of the moxa-fumigating group was lower than that of the moxa-hea-ting group (P<0.001). On the 10th day after the modeling, the number of new capillaries and capillary density in the wound basal layer were significantly increased in both moxa-fumigating and moxa-heating groups (P<0.05, P<0.001, P<0.01), and were notably higher in the moxa-fumigating group than in the moxa-heating group (P<0.01, P<0.05). CONCLUSION: Both fumigation and heating of moxibustion can promote wound healing in rats with full-thickness skin excision injury，which may be related to their effects in controlling the inflammatory response and promoting the production of collagen fibers, granulation tissue and capillaries.

[Effect of different concentrations of moxa smoke exposure on nasal mucosal injury and expression of serum IL-1, IL-6 and TNF-α in rats].

OBJECTIVE: To observe the effect of long-term moxa smoke exposure of different concentrations on olfactory function in rats, and provide experimental basis of safety study of moxa smoke produced by moxibustion. METHODS: Forty SD rats were randomly divided into a normal control group, a low-concentration moxa smoke group, a moderate-concentration moxa smoke group and a high-concentration moxa smoke group, 10 rats in each one. The rats in the moxa smoke groups were put into three plexiglass moxibustion boxes with different moxa smoke concentrations, 4 hours per times, twice a day for 90 days. The general state of rats was evaluated before and during the experiment. After the intervention, the olfactory function was evaluated by two-bottle experiment (TBE); the morphology of nasal mucosa was observed by HE staining; the apoptosis of olfactory epithelial cells in nasal mucosa was detected by TUNEL method; the serum levels of interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected by ELISA method. RESULTS: In the late stage of moxa smoke exposure (45-90 days into intervention), the behavioral activity of rats in the moderate-concentration moxa smoke group and the high-concentration moxa smoke group was weaker than that in the normal control group, and their response to stimulation was strong, and their mental state was worse. After intervention, the drinking rate of vinegar-water mixture in the moderate-concentration moxa smoke group and the high-concentration moxa smoke group was higher than that in the normal control group and the low-concentration moxa smoke group (P<0.01). The hierarchical structure of nasal mucosa in the moderate-concentration moxa smoke group and the high-concentration moxa smoke group was unclear, disordered, necrotic and inflammatory cell infiltration was serious; the number of apoptotic cells in olfactory epithelium of nasal mucosa in the moderate-concentration moxa smoke group and the high-concentration moxa smoke group was more than that in the normal control group and the low-concentration moxa smoke group (P<0.01), that in the high-concentration moxa smoke group was more than the moderate-concentration group (P<0.01). The serum levels of IL-1, IL-6 and TNF-α in the low-concentration moxa smoke group, the moderate-concentration moxa smoke group and the high-concentration moxa smoke group were higher than the normal control group (P<0.01), and those in the moderate-concentration moxa smoke group and the high-concentration moxa smoke group were higher than the low-concentration moxa smoke group (P<0.01), and those in the high-concentration moxa smoke group were higher than moderate-concentration moxa smoke group (P<0.01). CONCLUSION: The long-term exposure to low, moderate and high concentrations of moxa smoke could cause pathological changes in nasal mucosa and increase the serum levels of IL-1, IL-6 and TNF-α; the moderate and high concentrations of moxa smoke exposure could cause a series of damage to olfactory function and reduce olfactory sensitivity in rats.

[Implication and implementation path of the ideological and political elements in acupuncture- moxibustion courses].

To explore the implementation path of the ideological and political education according to the characteristics of teaching sections in acupuncture-moxibustion courses. Excavating the traditional Chinese culture and medical ethics contained in acupuncture-moxibustion courses helps strengthening the ideological and political quality of medical students and noble medical ethics, strengthening self-confidence in both professions and culture, and also helps students establishing a correct outlook on life, world and value. The moral education integrated with the professional teaching will helps explore ideological and political education path in acupuncture-moxibustion courses, so as to solidify them into each teaching sections and improve the teaching effect.

Roles of central orexinergic system on cardiovascular function and acupuncture on intervention of cardiovascular risk: Orexinergic system mediate the role of acupuncture?

Central orexinergic system contributes to the regulation of cardiovascular function. Orexinergic neurons receiving projections of nerve fibers from multiple structures of brain which involved in control and regulation of cardiovascular function locate in hypothalamus, and their axon terminals widely project to various central structures where orexins receptors are expressed. Here, we summarize the present knowledge that describes the influence of central orexinergic system on cardiovascular activity, the relevance of dysfunction in central orexinergic system with hypertension and psychological stress induced cardiovascular reactivity which are serious risk factors for cardiovascular disease and cardiovascular death. We propose that central orexinergic system may be potentially important targets for the prevention of cardiovascular disease and cardiovascular death, and different orexinergic system involved neuronal circuits may be involved in distinct cardiovascular functions. Acupuncture having bidirectional regulatory ability and a much lower incidence of side effects can prevent disease. We review the improvement of acupuncture on hypertension and psychological stress induced cardiovascular reactivity. We think that acupuncture intervenes hypertension and psychological stress induced cardiovascular reactivity to prevent cardiovascular disease and cardiovascular death. We also summarize relation between acupuncture and central orexinergic system. We propose a hypothesis that acupuncture improve hypertension and psychological stress induced cardiovascular reactivity through regulating central orexinergic system. The knowledge is beneficial for the development of potential therapeutic targets and methods to prevent cardiovascular disease and cardiovascular death.

Mechanisms of Electroacupuncture Pretreatment in Alleviating Myocardial Ischemia Reperfusion Injury: Interactions between the Cerebellar Fastigial Nucleus and Lateral Hypothalamic Area.

Background: Myocardial ischemia reperfusion injury (MIRI) is an important mechanism of post-myocardial infarction injury and a main cause of death in patients with ischemic heart disease. Electroacupuncture (EA) pretreatment is effective for the prevention and treatment of MIRI, but mechanisms mediating the effects of cardiovascular disease EA treatments remain unclear. Objectives: To determine whether the lateral hypothalamus (LHA) and the cerebellar fastigial nucleus (FN) are involved in the protective effects of EA stimulation on MIRI. Methods: EA pretreatment was performed for 7 days before the establishment of the MIRI model. ST-segment changes on electrocardiograms were recorded and the Curtis-Walker arrhythmia score was used to evaluate changes in reperfusion injury. Hematoxylin-eosin staining was applied to evaluate the pathological and morphological changes in myocardial tissue. c-fos expression in the LHA and FN was determined by immunofluorescence staining. Glutamic (Glu) and γ-Aminobutyric acid (GABA) levels were measured using a high-performance liquid chromatography-electrochemical method. Results: EA pretreatment reduced ST-segment elevation, arrhythmia scores, and morphological changes in MIRI myocardial cells in rats, and decreased the c-fos protein expression in LHA/FN nuclei. MIRI was associated with an imbalance between GABA and Glu levels, whereas EA pretreatment increased GABA levels and decreased Glu levels in the LHA/FN. Conclusion: FN and LHA are involved in the EA-mediated attenuation of MIRI. Pretreatment with EA plays a protective role in the myocardium by regulating Glu and GABA release in the LHA and FN.

[Effect of electroacupuncture preconditioning on the expressions of NF-κB p65, IκBα and IKKß in myocardial tissue of the rats with acute myocardial ischemia-reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) preconditioning on the expressions of nuclear transcription factors-kappa B (NF-κB) p65, NF-κB inhibitor (IκB) α and IκB kinase (IKK) ß in rats with acute myocardial ischemia-reperfusion injury (MIRI) and to explore the mechanism of EA on heart meridian in relieving MIRI. METHODS: A total of 40 SD rats were randomized into a sham-operation group, a model group, an EA heart meridian group and an EA lung meridian group, 10 rats in each one. In the EA heart meridian group, acupuncture was applied to "Shenmen" (HT 7) and "Tongli" (HT 5). In the EA lung meridian group, acupuncture was applied to "Taiyuan" (LU 9) and "Lieque" (LU 7). In these two groups, EA was exerted for 20 min each time, 1 V in voltage and 2 Hz in frequency once a day. A total of 7-day EA stimulation was required before model duplication. In the model group, the EA heart meridian group and the EA lung meridian group, using ligating left anterior descending coronary artery to establish the acute MIRI models. In the sham-operation group, the chest was open, but no ligation was exerted, just the needle was penetrated through the corresponding sites for one time. The electrocardiogram (ECG) was detected and ST segment displacement was analyzed. Using Western blot method, the relative expressions of NF-κB p65, IκBα and IKKß in myocardial tissue were determined in each group. Using ELISA method, the levels of serum IL-1ß and IL-10 were determined in each group. RESULTS: Compared with the sham-operation group, ST segment displacement value was elevated 30 min after ligating and reperfusion for 120 min in the model group (P<0.05), and the value in the EA heart meridian group was lower than the model group and the EA lung meridian group (P<0.05). Compared with the sham-operation group, the expressions of NF-κB p65 and IKKß in myocardial tissue were increased (P<0.05) and the expression of IκBα reduced in the rats of the model group (P<0.05). Compared with the model group, the expressions of NF-κB p65 and IKKß in myocardial tissue were reduced (P<0.05) and the expressions of IκBα increased in the rats of the EA heart meridian group and the EA lung meridian group (P<0.05). Compared with the EA lung meridian group, the expressions of NF-κB p65 and IKKß in myocardial tissue were reduced (P<0.05) and the expression of IκBα increased in the rats of the EA heart meridian group (P<0.05). Compared with the sham-operation group, the serum level of IL-1ß was increased (P<0.05) and IL-10 reduced in the model group (P<0.05). Compared with the model group, the serum level of IL-1ß was reduced (P<0.05) and IL-10 increased in the EA heart meridian group and the level of IL-1ß was was reduced in the EA lung meridian group (P<0.05). Compared with the EA lung meridian group, the serum level of IL-1ß was reduced (P<0.05) and IL-10 increased in the EA heart meridian group (P<0.05). CONCLUSION: Electroacupuncture preconditioning at heart meridian acupoints obviously alleviates acute MIRI. IKK/IκB/NF-κB signaling pathway possibly participates in the protective mechanism of electroacupuncture preconditioning on acute MIRI.

[Design and application of a moxibustion device with separated moxibustion smoke and heat].

In the view of the defects of the commonly used moxibustion instruments and moxa heating instruments, such as the moxa ash cannot be removed automatically, the temperature of moxibustion and moxibustion smoke is difficult to be stabilized and adjusted, and the instruments are complex and expensive, a moxibustion device with separated moxibustion smoke and heat is designed. This device can automatically remove the moxa ash and keep it on the isolation net; the temperature of the moxibustion outlet is maintained at 43-48 ℃ (effective moxibustion temperature) for more than 40 minutes, and there is no visible moxa smoke; the temperature of the moxa smoke outlet is controlled between 28-75 ℃, and the effective discharge of moxa smoke can be realized without external power equipment. This device has the advantages of stable and controllable temperature of moxibustion outlet and moxa smoke outlet, automatic removal and collection of moxa ash, separation of moxa smoke without additional power, etc., which can be used in clinical and animal experiments for moxa heating, moxa smoke removal, etc.

[Influence of moxibustion with moxa sticks with different diameters and at different distances from surface of "Zusanli"(ST36) on local skin temperature in rats].

OBJECTIVE: To observe the impact of moxa-stick with different diameters and at different distances on skin temperature in local "Zusanli"(ST36) region, so as to select suitable specifications for moxibustion. METHODS: A total of 120 male SD rats were randomly divided into diameters of 0.5, 0.9, 1.2 and 1.8 cm, and distances of 1, 2, 3, 4 and 5 cm groups, with 6 rats in each group. Moxa-stick with different diameters mentioned above was applied to the right ST36 (right hind limb) for 10 min every time at different distances (between the ignited moxa-stick tip and the skin) mentioned above, and the left ST36 was used as the control point. The skin temperature was detected by using an infrared thermometer. RESULTS: After application of moxibustion to ST36 region, the skin temperature was increased gradually along with the increased diameter of moxa-sticks and decreased along with the increased distance from the ignited moxa-stick tip to the skin. There were no significant changes in the skin temperature of the left control acupoint ST36. The skin temperature was below 40 ℃, between 43 to 55 ℃, over 43℃ and between 43 to 61 ℃, when the moxa-stick was 0.5 cm, 0.9 cm, 1.2 cm and 1.8 cm in diameter, and was kept 1, 2, 3 and 3 to 5 cm away from the skin surface, respectively. When the moxa-stick with a diameter of 1.8 cm was kept at a distance of 1 to 2 cm, the skin temperature reached 71 to 93 ℃ to cause obvious local burn lesion. CONCLUSION: During moxibustion, the ignited moxa-sticks with diameters of 0.5, 0.9, 1.2 and 1.8 cm are suitable to be kept less than 1, 1 to 2, 2 to 3, and 3 to 5 cm away from the skin surface of ST36, respectively.

[Effect of moxibustion on expression of autophagy related molecules in synovial tissues of rheumatoid arthritis rats].

OBJECTIVE: To observe the effect of moxibustion on expression of autophagy related gene(Atg), serine/threonine protein kinase-uncoordinated 51 like kinase-1 (ULK1), Beclin1 and microtubule associated proteins light chain 3 (LC3) and ultrastructure of synovium in rheumatoid arthritis (RA) rats, so as to explore its mechanisms underlying improvement of RA. METHODS: Forty SD rats were randomly divided into normal control, RA model, moxibustion, cigarette-roasting and medication groups (n=8 rats in each group). The RA model was established by keeping the rats in wind, cold and wet environment for 12 h, once a day for 20 days and subcutaneous injection of Freund's adjuvant complete into the sole of the left hind paw. Moxibustion was applied to the left "Zusanli" (ST36) for 20 min, once a day for 15 days. Rats of the cigarette-roasting group was treated by ignited cigarettes instead of moxa strips. Rats of the medication group was treated by gavage of Tripterygium wilfordii polyglycoside tablet suspension (0.8 mg/100 g) once a day for 15 days. The rats' paw volume of the left hindlimb was measured by using a water-based paw plethysmometer. The synovial tissue of the left plantar joint was harvested at the end of experiments for observing changes of the ultrastructure with transmission electron microscope, and the expression of ULK1, Atg3, Atg5, and Atg12 mRNAs was detected with quantitative real-time PCR and the expression of LC3-Ⅱ and Beclin-1 proteins were detected with Western blot. RESULTS: Following modeling, the paw volume of the left hindlimb was significantly increased (P<0.01), while the expression levels of Atg3, Atg5, Atg12 and ULK1 mRNAs, and LC3-Ⅱ and Beclin-1 proteins of the synovial tissue were notably down-regulated in the model group relevant to the normal control group (P<0.01). The increase of the paw volume in the moxibustion and medication groups and the down-regulation of synovial Atg3, Atg12 and ULK1 mRNAs in the 3 intervention groups, and Atg5 mRNA , and LC3-Ⅱ and Beclin-1 proteins in both moxibustion and medication groups were considerably suppressed (P<0.01, P<0.05). The therapeutic effect of moxibustion was apparently superior to that of cigarette-roasting in down-regulating the paw volume, and up-regulating the expression levels of Atg3, Atg5, Atg12 and ULK1 mRNAs, and LC3-Ⅱ and Beclin-1 proteins (P<0.05, P<0.01), and notably weaker than that of medication in up-regulating Atg3 and ULK1 mRNAs (P<0.01), but was comparable to that of medication in up-regulating the expression levels of Atg5 and Atg12 mRNAs, LC3-Ⅱand Beclin-1 proteins (P＞0.05). Results of the ultrastructural observation showed an obvious injury of synovial cells, such as unclear and incomplete nuclear membrane, chromatin condensation, swollen mitochondria with broken crests, cavitation-like degeneration of cytoplasma, and appearance of autophagosomes and lysosomes in the model group, which was relatively milder in the 3 intervention groups. CONCLUSION: Moxibustion can reduce the paw edema and inflammatory injury of the plantar synovial tissue in RA rats, which may be related to its effects in up-regulating Atg3, Atg5, Atg12 and ULK1 mRNAs, and LC3-Ⅱ and Beclin-1 proteins to enhance the cellular autophagy. The therapeutic effect of moxibustion is obviously superior to that of cigarette-roasting and medication in relieving swelling.

[Effect of moxibustion on PI3K/Akt/mTOR signaling pathway in foot-pad synovium in rats with rheumatoid arthritis].

OBJECTIVE: To observe the effect of moxibustion on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin protein (PI3K/Akt/mTOR) signaling pathway in the foot-pad synovial tissue in rats with rheumatoid arthritis (RA), and to explore the mechanism of moxibustion for treating RA. METHODS: Forty healthy SD rats were randomly divided into a control group, a model group, a moxibustion group, a cigarette-moxibustion group and a medication group, 8 rats in each group. The RA model was established with subcutaneous injection of complete Freund's adjuvant (CFA) in the left hind foot-pad under wind, cold and wet environment in the model group, the moxibustion group, the cigarette-moxibustion group and the medication group. The rats in the moxibustion group were treated with moxibustion at "Zusanli" (ST 36) for 20 min; the rats in the cigarette-moxibustion group were treated with moxibustion of ordinary cigarette at "Zusanli" (ST 36) for 20 min; the rats in the medication group were treated with tripterygium glycosides suspension (0.8 mg/100 g) by gavage. All the intervention was given once a day for 15 days. The left hind foot-pad volume was measured before and after modeling and after 15-day intervention. After 15-day intervention, the serum levels of IL-17 and IL-23 were detected by ELISA method, and the expression levels of PI3K, Akt and mTOR in synovial tissue of left hind foot-pad were detected by Western blot method. RESULTS: The volume of left hind foot-pad, the serum levels of IL-23 and IL-17 and the expression of PI3K, Akt and mTOR in synovial tissue of left hind foot-pad in the model group were higher than those in the control group (P<0.01). After intervention, the volume of left hind foot-pad and the expression of PI3K, Akt and mTOR protein in synovium tissue in the moxibustion group and medication group were lower than those in the model group (P<0.01, P<0.05), while the serum levels of IL-17 and IL-23 in the moxibustion group were lower than those in the model group (P<0.01). The volume of left hind foot-pad, the serum levels of IL-23 and the expression of mTOR protein in synovial tissue in the moxibustion group were lower than those in the medication group (P<0.01, P<0.05), while the volume of left hind foot-pad, the serum levels of IL-23 and the expression of PI3K, Akt and mTOR protein in synovium tissue in the moxibustion group were lower than those in the cigarette-moxibustion group (P<0.01). CONCLUSION: Moxibustion may play a therapeutic effect on RA by inhibiting the level of IL-23, IL-17 and the activity PI3K/Akt/mTOR, and regulating inflammatory response and autophagy.

[Effect of Electroacupuncture at "Shenmen" (HT 7)- "Tongli" (HT 5) of Heart Meridian on Neuronal Activities in Paraventricular Nucleus of Hypothalamus in Myocardial Ischemia Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of "Shenmen" (HT 7) - "Tongli" (HT 5) segment of the Heart Meridian on neuronal electrical activities of hypothalamic paraventricular nucleus (PVN) in rats with myocardial ischemia (MI), so as to investigate its possible mechanism underlying improvement of MI. METHODS: Thirty-two SD rats were randomly divided into sham control, model, HT 7-HT 5 and "Taiyuan" (LU 9)- "Lieque" (LU 7) groups (n=8 in each group). EA preconditioning (2 Hz, 1 V, 20 min) was applied to bilateral HT 7-HT 5 and bilateral LU 9-LU 7, respectively, once everyday for 7 days. The electrical activities of the right PVN region were recorded by the implanted microelectrode array(2×4)and Plexon multi-channel acquisition system. Cluster analysis of neuronal signals was carried out by Offline Sorter software. The discharge waveforms, autocorrelation and cross-correlation of neuronal activities were analyzed by using Neuro Explorer software. RESULTS: Cluster analysis of neuronal signals showed that 2, 2, 1 and 1 interneuron in the sham, model, HT 7-HT 5, and LU 9-LU 7 groups, and 3 pyramidal neurons in the HT 7-HT 5 were acquired. Cross correlation analysis showed that the SPK 02 a and SPK 02 b neurons of the HT 7-HT 5 group had an inhibitory relationship. The total discharge frequency was significantly increased in the model group relevant to the sham group (P<0.01), and was markedly lower in the HT 7-HT 5 group than in the model group and LU 9-LU 7 group (P<0.01). Real-time spectrum analysis showed that the local field potential spectrum energy of the HT 7-HT 5 group was significantly lower than that of the model group and the LU 9-LU 7 group. CONCLUSION: EA of HT 7-HT 5 segment of the Heart Meridian can inhibit the electrical activity of interneuron and activate the electrical activity of pyramidal neuron in PVN region, and an inhibitory relationship exists between the interneuron and pyramidal neuron in MI rats, which may be a mechanism of EA in regulating activities of the ischemic heart.

[Effect of Electroacupuncture at Different Acupoints on Expression of NGF and TrkA in Cerebral Cortex in Rats with Myocardial Ischemia].

OBJECTIVE: To compare the therapeutic effect of electroacupuncture (EA) of "Shenmen" (HT 7, Yuan point of the Heart Meridian), "Zhizheng" (SI 7, Luo point of the Small Intestine Meridian) and "Xinshu" (BL15, Back-shu point) on the expression of nerve growth factor (NGF) and its receptor, tyrosine kinase A (TrkA) in the cerebral cortex of myocardial ischemia (MI) rats. METHODS: A total of 70 male SD rats were randomized into sham operation (sham, n=10), model, HT 7, SI 7, and BL15 groups (n=15 in each of the latter 4 groups). The MI model was established by ligation of the anterior descending branch of the left coronary artery. EA (1 mA, 2 Hz) was respectively applied to HT 7, SI 7 and BL15 for 15 min, once per day for 1 week. Immunohistochemistry and real-time fluorescent quantitative PCR were used to detect the expression of NGF and TrkA proteins and genes in the cerebral cortex. RESULTS: Compared with the sham group, no significant changes were found in the number of NGF immune-reaction (IR)-positive and TrkA IR-positive cells, and the expression levels of NGF mRNA and TrkA mRNA in the model group (P>0.05). After EA intervention, the number of NGF and TrkA IR-positive cells and the expression of NGF mRNA and TrkA mRNA in each of the 3 EA groups were significantly increased relevant to the model group (P<0.01, P<0.05). The effect of EA at BL 15 was significantly superior to that of EA at HT 7 and SI 7 in increasing the number of NGF and TrkA positive cells and up-regulating the expression of their mRNAs (P<0.01, P<0.05). CONCLUSION: EA at HT 7, SI 7 and BL 15 can increase the levels of expression of NGF and TrkA proteins and mRNAs in the cerebral cortex of subacute MI rats and the effects of EA-BL 15 are obviously superior to those of EA-HT 7 and EA-SI 7, suggesting a relative specificity of the effect of EA at different acupoints.

[Effect of Electroacupuncture Stimulation of Heart and Lung Meridians on Expression of Myocardial HCN 2 in Acute Myocardial Ischemia Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of Heart and Lung meridians on the expression of hyperpolarization-activated cyclic nucleotide gated potassium channel 2 (HCN 2) mRNA and protein in the myocar-dium of the left ventricle in acute myocardial ischemia (AMI) rats, so as to underlying its mechanism in improving ischemic myocardial injury. METHODS: A total of 120 male SD rats were randomly divided into normal control, model, EA-Heart Meridian (HT) and EA-Lung Meridian (LU) groups (n＝30 rats in each group). The AMI model was established by occlusion of the anterior descending branch of coronary artery. EA (1 mA, 10 Hz) was applied to bilateral "Shenmen" (HT 7) or "Taiyuan" (LU 9) and the spot about 1 mm apart from the HT 7 or LU 9 on the axopetal end for 15 min on the second day after modeling, once a day for 7 days. The left ventricular myocardium tissue was sampled for determining the expression levels of HCN 2 mRNA and protein with qPCR and Western blot, separately. RESULTS: After AMI, both HCN 2 mRNA and protein expression levels in the model group were significantly decreased in comparison with the normal control group (P<0.01). Following EA intervention, the expression levels of HCN 2 mRNA and protein expressions were significantly up-regulated relevant to the model group (P<0.01)．The effects were more obvious in the EA-HT group than in the EA-LU group, and the expression levels were higher in the left ventricular myocardium tissue sampled on the next day after the treatment than those in the tissue sampled immediately after the treatment (P<0.01). CONCLUSION: Both EA stimulation of Heart Meridian and Lung Meridian can improve the expression of HCN 2 mRNA and protein in the myocardium in AMI rats, which existing a delayed effect and meridian specificity.