[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.

[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.

[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.

[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.

[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.