[Effect of electroacupuncture of "Weizhong" (BL40) on iron metabolism disorder in rats with lumbar multifidus muscle injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Weizhong" (BL40) on the disorder of iron metabolism and the level of oxidative stress after lumbar multifidus muscle injury (LMMI), so as to explore its mechanisms underlying promoting the repair of LMMI. METHODS: Male SD rats were randomly divided into normal, model and EA groups (6 rats in each group). The LMMI model was established by injecting 0.5% bupivacaine (BPVC) solution (400 µL) into the lumbar multifidus muscle with the syringe-needle close to the spinous process (L4-L5). Twenty-four hours after successful establishment of the model, EA (2 Hz/15 Hz, 2 mA) was applied to bilateral BL40 for 30 min, once a day for 2 days. Histopathological changes of the multifid muscle were observed under microscope after H.E. staining, and the iron granules in the multifid muscle tissue observed after Prussian blue staining. The expression of glutathione synthase (GSS) was detected by Western blot, and the expressions of iron regulatory protein 1 (IRP1), ferroportin (Fpn), ferritin heavy chain 1 (FTH1, iron metabolism-related proteins) and gluta-thione peroxidase 4 (GPX4, functions in protecting cells against detrimental lipid peroxidation and governing a novel form of regulated necrotic cell death, called ferroptosis) mRNAs were detected by quantitative real-time PCR. The contents of glutathione (GSH) and malondialdehyde (MDA) were measured by biochemical methods. RESULTS: H.E. staining showed large areas of necrosis and breakage of muscle fibers, disordered arrangement of muscle fibers, widened muscle cell space, accompanying with a large number of inflammatory cell infiltration in the multifidus muscle tissue of the model group, which was relatively milder in the EA group. Outcomes of Prussian blue staining showed that compared with the normal group, there were more iron particles in the multifidus muscle tissue and enlarged muscle fiber gaps, which was also milder in the EA group. Compared with the normal group, the expression level of IRP1 mRNA and content of MDA were significantly increased (P<0.001), the expression levels of Fpn, FTH1 and GPX4 mRNAs and GSS protein, and the content of GSH were considerably decreased (P<0.001) in the model group. In comparison with the model group, the increase of IRP1 mRNA expression and MDA content, as well as the decrease of Fpn, FTH1 and GPX4 mRNAs expressions and GSH content were reversed in the EA group (P<0.001,P<0.05,P<0.01). CONCLUSION: EA of BL40 has a protect effect in BPVC-induced injury of lumbar multifidus muscle in rats, which may be related to its functions in improving iron metabolism to reduce oxidative damage by regulating expression of IRP1, Fpn and FTH1.

[Acupuncture ameliorates negative emotion in PCOS patients: a randomized controlled trial].

OBJECTIVE: To evaluate the effectiveness and possible mechanism of acupuncture treatment for negative emotion in patients with polycystic ovary syndrome (PCOS). METHODS: A total of 40 PCOS patients were randomly divided into an observation group and a control group, 20 cases in each one. Both groups received lifestyle interventions (exercise and diet guidance) on the 5th day of menstruation. On the basis of above treatment, the patients in the observation group received acupuncture at Guanyuan (CV 4), Zhongwan (CV 12), Guilai (ST 29), Futu (ST 32), Liangqiu (ST 34), Sanyinjiao (SP 6), Zusanli (ST 36), Hegu (LI 4), Shenmen (HT 7), Baihui (GV 20) as the main acupoints, and connected the electroacupuncture (continuous wave, 2 Hz, 30 min), once every other day, 3 times a week. The treatment for 1 month was as one course and 4 courses were required totally in both groups. Before and after treatment, the body mass index (BMI), ferriman-gallway (F-G) score, self-rating anxiety scale (SAS) score, self-rating depression scale (SDS) score, PCOS health-related quality of life questionnaire (PCOSQ) score were observed, meanwhile, serum sex hormone, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), estrogen (E2), progestin (P), prolactin (PRL), testosterone (T), sex hormone-binding globulin (SHBG) and free androgen index (FAI) levels, and serumß-endorphin levels were detected. RESULTS: Compared with before treatment, the BMI, F-G score, SAS score, SDS score and serum FAI level were decreased and the PCOSQ score and the levels of serum SHBG andß-endorphin were increased in the observation group after treatment (all P<0.05). Compared with before treatment, the SDS score was decreased in the control group after treatment (P<0.05). Compared with the control group, the F-G score, SDS score, SAS score, and serum FAI level were lower, and the PCOSQ score and serumß-endorphin level were higher in the observation group after treatment (all P<0.05). CONCLUSION: Applying acupuncture to the treatment of patients with PCOS can effectively relieve anxiety and depression, and the mechanism may be related to the regulation on the levels of serumß-endorphin and androgen.

[Effect of electroacupuncture at "Weizhong" (BL40) on expression of collagen I and matrix meta-lloproteinases 2 in rats with lumbar multifidus muscle injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Weizhong" (BL40) on histopathological changes and expression of extracellular matrix (ECM) component Collagen Ⅰ， matrix metalloproteinases 2 (MMP2), MyoD and Pax7 proteins of lumbar muscle tissues in rats with lumbar multifidus muscle injury (LMMI), so as to explore its underlying mechanisms in improving muscular injury. METHODS: A total of 24 male SD rats were equally randomized into blank control, model and EA groups. The LMMI model was established by injection of 0.5% Bupivacaine (100 µL/ point) into bilateral multifidus muscles of lumbar 4 and 5 (4 points). EA (2 Hz/100 Hz in frequency, 1－2 mA) was applied to BL40 for 20 min, once a day for 3 days. The morphological changes of the left lumbar multifidus muscle were observed under microscope after H．E. and Masson staining, and the expression of Collagen Ⅰ， MMP2, MyoD and Pax7 of the right lumbar multifidus muscle was determined by Western blot. RESULTS: H．E. staining showed large areas of degeneration and necrosis of muscle fibers, and vacuolar structure formed by degradation of muscle fibers in the model group, and newborn juvenile muscle fibers with different diameters in the EA group. Masson staining showed a large number of morphologically damaged muscle fibers and blue stained collagen fibers in the model group, and significantly reduced collagen fibers as well as new muscle fibers with uneven diameters in the EA group. The expression levels of Collagen Ⅰ， MMP2 and MyoD proteins were significantly up-regulated (P<0.01, P<0.05), and that of Pax7 was considerably down-regulated in the model group relative to the control group (P<0.01). After EA intervention, the expression levels of Collagen Ⅰ was significantly down-regulated (P<0.01), and those of MMP2, MyoD and Pax7 proteins were obviously or further obviously up-regulated in the EA group compared with the model group (P<0.01, P<0.05). CONCLUSION: EA at BL40 can reduce the degree of skeletal muscle fibrosis to promote the regeneration of the injured multifidus at the early phase, which may be related to its effect in regulating the expression of Collagen Ⅰ and MMP2 proteins.

Protective Effect of Norcantharidin on Collagen-Induced Arthritis Rats.

OBJECTIVE: To observe the effect of norcantharidin (NCTD) on collagen-induced arthritis (CIA) rats. METHODS: Sixty Sprague-Dawley(SD) rats were randomly divided into 6 groups (n=10): normal group, CIA model group(model group), NCTD low-dose group [1.35 mg/(kg•d)], NCTD middle-dose group [2.7 mg/(kg•d)], NCTD high-dose group [5.4 mg/(kg•d)] and methotrexate (MTX) group [1.8 mg/(kg/w)]. Anesthetized rats were sacrificed by luxation of cervical vertebra after 4 weeks of administration. The arthritis scores were evaluated twice a week. The pathological changes in the ankle joints of rats were observed by hematoxylin-eosin (H&E) staining. The serum levels of interleukin (IL) 1ß, IL-6, tumor necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), IL-17 and transform growth factor (TGF) ß were detected by enzyme linked immunosorbent assay (ELISA). The mRNA expression of retinoid-related orphan nuclear receptorγt (RORγt) and forkhead box P3 (Foxp3) in peripheral blood lymphocytes were confirmed by real-time polymerase chain reaction. RESULTS: MTX and high-dose NCTD not only decreased the arthritis scores but also alleviated the pathological changes in CIA rats' ankle joints compared with the model group (P<0.05 or P<0.01). All doses of NCTD significantly inhibited the serum levels of IL-6, IL-17 and TNF-α in CIA rats (P<0.05). Only middle- and high-dose of NCTD prominently decreased serum IL-1ß and TGF-ß levels of CIA rats (P<0.05). However, NCTD has no effect on vascular endothelial growth factor (VEGF) level in CIA rats. The Foxp3 mRNA expression in all NCTD groups were increased significantly than in the model group (P<0.05). The mRNA expression of RORγt in NCTD high-dose group was decreased apparently in comparison with the model group (P<0.05). CONCLUSIONS: NCTD showed therapeutic effect on CIA rats by inhibition of cytokines and regulation of Th17/Treg cells.

Effect of electroacupuncture stimulation on expression of angiotensinogen, angiotensin II type 1 receptor, endothelin-1, and endothelin a receptor mRNA in spontaneously hypertensive rat aorta.

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation on the expressions of angiotensinogen (AGT), angiotensin II type 1 receptor (AT1R), endothelin-1 (ET1), and endothelin A receptor (ETAR) mRNA in spontaneously hypertensive rat (SHR) aorta. METHODS: Eighteen male SHRs were randomly divided into three groups, an SHR group, an SHR Baihui (DU 20) and Zusanli (ST 36) acupoint (SHR-AP) group, and an SHR non-acupoint (SHR-NAP) group, with 6 rats in each group. Six Wistar rats were used as a control. Rats in the SHR-AP group were stimulated by DU 20 and ST 36 acupoints, both of which were connected with EA. EA was handled one time every Monday, Wednesday and Friday, for total 24 times (8 weeks). SHRNAP rats were acupointed at a 15°angle flat into 0.5 cm to two points, which were 1 and 2 cm from rail tip separately. EA parameters were the same as the SHR-AP rats. SHR control rats and Wistar rats were fixed without EA. Real-time quantitative polymerase chain reaction (PCR) was used to measure AGT, AT1R, ET1, and ETAR mRNA expression in rat aorta. RESULTS: EA stimulation significantly reduced rat aorta vascular AGT, ET1, ETAR and AT1R mRNA expressions in the SHR-AP and SHR-NAP groups (P <0.01). Among these four genes, AT1R mRNA expression was significantly lower in the SHR-AP than in the SHR-NAP group (P <0.01). CONCLUSION: EA could reduce the AT1R mRNA expression in SHR-AP rat aorta, indicating a potential mechanism for the hypotensive effects of EA.

[Effect of Electroacupuncture Serum on Proliferation of Cultured Multifidus Muscle Satellite Cells and Expression of Pax-7, MyoD and p-Akt].

OBJECTIVE: To observe the effect of electroacupuncture (EA) serum on proliferation of multifidus muscle sa-tellite cells (SCs) and expression of paired box transcription factor Pax-7, MyoD and protein kinase B (PKB or Akt) proteins of SCs, so as to explore its underlying mechanism in promoting repair of multifidus muscles. METHODS: Thirty-two SD rats were randomly assigned to control, model, EA-Weizhong (BL 40) and EA-Shenshu (BL 23) groups. The multifidus muscle injury (MFMI) model was established by injection of 0.5% bupivacaine hydrochloride (400 µL) into the bilateral L4-L5 paravertebral muscles (4 points, 100 µL for each point). EA stimulation was separately applied to bilateral BL 40 and BL 23 for 20 min, once daily, 4 days altogether. Blood samples of the abdominal artery of rats in the above mentioned 4 groups were separately collected for extracting serum, followed by deactivation and filtration, and then were respectively applied to the Dulbecco's Modified Eagle Media (DMEM) culturing each multifidus muscle SCs of the normal serum, model serum, EA-BL 40 serum and EA-BL 40 serum+LY 294002 (an inhibitor of phosphotidylinsitol-3-kinase, PI 3 K), EA-BL 23 serum and EA-BL 23 serum+LY 294002 groups for ana-lyzing the impact of EA serum on the proliferation state of SCs by Cell Counting Kit-8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine (EdU) methods, respectively. The expression of Pax-7, MyoD and phosphorylated (p)-Akt proteins of the cultured SCs was detected for characterization of SCs by Western blot. RESULTS: Compared with the normal serum group, the proliferation levels (detected by both CCK-8- and EdU) and the expression levels of MyoD and p-Akt proteins of SCs in the model serum group were significantly increased (P<0.05, P<0.01), while in comparison with the model serum group, the proliferation and expression levels of MyoD and p-Akt proteins of SCs were further significantly increased in both EA-BL 23 and EA-BL 40 serum groups (P<0.01, P<0.05), but not in the EA-BL 40 serum+LY 294002 and EA-BL 23 serum+LY 294002 groups (P>0.05), suggesting an involvement of PI 3 K in the proliferation of SCs. No marked differences were found in the proliferation levels between the EA-BL 23 and EA-BL 40 serum groups and in the expression levels of Pax-7 proteins among the 6 serum groups (P>0.05). CONCLUSIONS: Both EA-BL 40 and EA-BL 23 serum can promote proliferation of multifidus muscle SCs, which may contribute to the effect of EA intervention in promoting repair of the injured muscle, partially by way of Akt/PI 3 K signaling.

[Protective effect of acupuncture on heart in mice with hyperlipemia and its mechanism].

OBJECTIVE: To observe the inhibiting effect of acupuncture on blood lipid, myocardial hypertrophy and fibrosis in mice with hyperlipemia, and explore its possible action mechanism. METHODS: Ten inbred mice (C57) were applied. Forty ApoE(-/-) mice who removed gene of apolipoprotein E were randomly divided into a control group, a non-acupoint group, an acupoint group and a medication group. The points 0. 5 cm and 1 cm next to the end of mice tail were respectively punctured in the non-acupoint group; "Neiguan" (PC 6) and "Fenglong" (ST 40) were punctured in the acupoint group; intragastric administration of simvastatin was applied in the medication group. After 8 weeks of treatment, the changes of total cholesterol (TC) and ratio of heart to body mass in each group were measured; changes of cardiac muscle fiber and ventricular wall thickness were observed; enzyme linked immunosorbent assay (ELISA) was used to test the level of angiotensin II (Ang I ) in plasma, and western blotting method was used to test protein content of angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR) in the heart. RESULTS: After 8 weeks of intervention, compared with the control group, rising range of blood lipid was obviously decreased (P<0.01) in the acupoint group and medication group, ratio of P<0.01), myocardial heart to body mass was decreased (P<0.05), thickness of ventricular wall was reduced (P fibrosis was relieved, levels of Ang II and ET-1 in plasma were decreased (P<0. 05), content of NO was increased (P<0. 05), and protein content of AT1R and ETAR was decreased in the heart (P<0. 05). CONCLUSION: 40) could inhibit the rising of blood lipid in ApoE(-/-) mice, lower the levels of Ang II and ET-1 in peripheral blood, increase the content of NO and inhibit the expression of AT1R and ETAR in heart tissue, which could relieve myocardial hypertrophy and fibrosis to play a protective role on heart.

The ameliorating effects of long-term electroacupuncture on cardiovascular remodeling in spontaneously hypertensive rats.

BACKGROUND: The purpose of this study was to investigate the inhibitory effects of long-term electroacupuncture at BaiHui (DU20) and ZuSanLi (ST36) on cardiovascular remodeling in spontaneously hypertensive rats (SHR) and underlying mechanisms. METHODS: 6-weeks-old SHR or Wistar male rats were randomly, divided into 6 groups: the control group (SHR/Wistar), the non-acupoint electroacupuncture stimulation group (SHR-NAP/Wistar-NAP) and the electroacupuncture stimulation at DU20 and ST36 group (SHR-AP/Wistar-AP), 24 rats in each group. Rats were treated with or without electroacupuncture at DU20 and ST36, once every other day for a period of 8 weeks. The mean arterial pressure (MAP) was measured once every 2 weeks. By the end of the 8th week, the left ventricular structure and function were assessed by echocardiography. The content of angiotensin II (Ang II), endothelin-1 (ET-1) and nitric oxide (NO) in the plasma was determined using enzyme-linked immunosorbent assay. Histological studies on the heart and the ascending aorta were performed. The expression of angiotensin II type 1 receptor (AT1R), endothelin-1 type A receptor (ETAR), eNOS and iNOS in rat myocardium and ascending aorta was investigated by Western blotting. RESULTS: The MAP in SHR increased linearly over the observation period and significantly reduced following electroacupuncture as compared with sham control SHR rats, while no difference in MAP was observed in Wistar rats between electroacupuncture and sham control. The aortic wall thickness, cardiac hypertrophy and increased collagen level in SHR were attenuated by long term electroacupuncture. The content of Ang II, ET-1 in the plasma decreased, but the content of NO increased after electroacupuncture stimulation in SHR. Long term electroacupuncture significantly inhibited the expression of AT1R, ETAR and iNOS, whereas increased eNOS expression, in myocardium and ascending aorta of SHR. CONCLUSIONS: The long term electroacupuncture stimulation at DU20 and ST36 relieves the increased MAP and cardiovascular abnormality in both structure and function in SHR, this beneficial action is most likely mediated via modulation of AT1R-AT1R-ET-1-ETAR and NOS/NO pathway.

The Effects of Needling Fenglong (ST40) and Neiguan (PC6) on IL-17 of ApoE-Gene-Knockout Mice's Liver.

The aim of the present paper was to observe the effects of needling ST40 and PC6 on IL-17 of ApoE(-/-) mice with fatty liver. Forty male ApoE(-/-) mice were randomized into Needling-Acupoint Group, Simvastatin Intragastric Administration Group, Needling Nonacupoint Group, and Model Group. Each was fed with high fat diet for 8 weeks since 16 weeks of age; after 8 weeks of intervention, mice were sacrificed and tested for various examinations. Result showed that the body weight, TC, and serum IL-17 in Needling-Acupoint Group decreased. Compared with Model Group, the immunohistochemical expressions of IL-17 in liver tissue were significantly decreased among the other three groups. In conclusion, acupuncture was able to lower the expression of IL-17 level both in serum and liver tissue in ApoE(-/-) mice, which is helpful to reduce the inflammation and defers the progress from fatty liver to cirrhosis.

Long-Term Stimulation with Electroacupuncture at DU20 and ST36 Rescues Hippocampal Neuron through Attenuating Cerebral Blood Flow in Spontaneously Hypertensive Rats.

This study was designed to investigate the effect of long-term electroacupuncture at Baihui (DU20) and Zusanli (ST36) on cerebral microvessels and neurons in CA1 region of hippocampus in spontaneously hypertensive rats (SHR). A total of 45 male Wistar rats and 45 SHR were randomly grouped, with or without electroacupuncture (EA) at DU20 and ST36, once every other day for a period of 8 weeks. The mean arterial pressure (MAP) was measured once every 2 weeks. Cerebral blood flow (CBF) and the number of open microvessels in hippocampal CA1 region were detected by Laser Doppler and immunohistochemistry, respectively. Nissl staining and Western blotting were performed, respectively, to determine hippocampus morphology and proteins that were implicated in the concerning signaling pathways. The results showed that the MAP in SHR increased linearly over the observation period and was significantly reduced following electroacupuncture as compared with sham control SHR rats, while no difference was observed in Wistar rats between EA and sham control. The CBF, learning and memory capacity, and capillary rarefaction of SHR were improved by EA. The upregulation of angiotensin II type I receptor (AT1R), endothelin receptor (ETAR), and endothelin-1 (ET-1) in SHR rats was attenuated by electroacupuncture, suggesting an implication of AT1R, ETAR, and ET-1 pathway in the effect of EA.

Effects of acupuncture with meridian acupoints and three Anmian acupoints on insomnia and related depression and anxiety state.

OBJECTIVE: To assess the clinical therapeutic effect of acupuncture with meridian acupoints combined with three Anmian acupoints. METHODS: Sixty subjects were randomly divided into two groups: the acupuncture with meridian acupoints group (Group A), and the acupuncture with meridian acupoints and three Anmian acupoints group (Group B) with 30 cases in each group. After 4-week treatment, the clinical therapeutic effects and scores for the Pittsburgh Sleep Quality Index (PSQI), depression index and anxiety index were assessed in the two groups before and after treatment. RESULTS: The total effective rate of therapy was higher in Group B (93.3%) compared with that in the Group A (66.7%, P<0.05). When comparing after treatment with before treatment, there were significant differences in the PSQI score of sleep quality, time to sleep, sleep time, sleep disorders in Group A (P <0.05) and significant differences in the PSQI score of sleep quality, time to sleep, sleep time, sleep disorders and daytime disorders in Group B (P <0.01 or P<0.05). There were significant differences in total PSQI score between Group A and Group B (P<0.05). Both groups improved the depression and anxiety state of the patients, but acupuncture with meridian acupoints combined with three Anmian acupoints was more effective than meridian acupoints alone (P<0.05). CONCLUSION: Acupuncture with meridian acupoints combined with three Anmian acupoints can improve the sleeping quality of patients with insomnia.

[Influence of electroacupuncture on spinal TNF-alpha and IL-6 contents in spinal cord ischemia-reperfusion injury rabbits].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Zusanli" (ST 36)-"Quchi" (LI 11) on behavior changes and spinal inflammatory reactions in spinal ischemia-reperfusion injury (SI/RI) rabbits, so as to assess its protective effect on SI/RI. METHODS: Thirty New Zealand rabbits were randomly divided into sham-operation (sham), SI/RI model (model) and EA groups. SI/RI model was established by occlusion of the abdominal aorta for 45 min. EA (2 Hz, 1 mA) was applied to bilateral "Zusanli" (ST 36)-"Quchi" (LI 11) for 30 min after modeling, 4 times (0 h, 12 h, 24 h and 36 h after modeling) altogether. The animals' motor function score of the hindlimb was assessed according to Tarlov's method. The contents of tumor necrosis factor-alpha (TNF-alpha) and Interleukin-6 (IL-6) of the lumbar spinal cord tissue were assayed by ELISA 48 h after modeling. RESULTS: Compared with the sham group, the animals' motor scores at 24 h and 48 h after modeling were decreased, and spinal TNF-alpha and IL-6 contents were increased significantly (all P < 0.05) in the model group. In comparison with the model group, the animals' motor scores at both 24 h and 48 h were up-regulated considerably in the EA group (P < 0.05), suggesting an improvement of the neuronal function after EA intervention. The spinal TNF-alpha and IL-6 contents were obviously lower in the EA group than in the model group (P < 0.05). CONCLUSION: Electroacupuncture has a protective effect on the spinal cord ischemia- reperfusion injury of rabbits probably by reducing the local inflammatory reactions.

Assessment of clinical effect of therapy combining disease with syndrome on rheumatoid arthritis.

OBJECTIVE: To observe the effectiveness and safety of a therapy combining disease with syndrome on rheumatoid arthritis. METHODS: Eighty patients with rheumatoid arthritis belonging to syndrome of damp-heat obstruction were randomly divided into a treatment group and a control group according to stratified blocked randomization method. Forty cases in the control group orally took Loxoprofen Sodium Tablet and Leifumite Tablet and the other 40 cases in the treatment group orally took a Chinese medicine for 12 weeks as a course of treatment. ACR therapeutic effect was used as the standard for evaluating the total therapeutic effect. RESULTS: After 12 weeks of treatment, there was a statistical difference (P < 0.01) in the improvement of VAS score, morning stiffness time, number of swelling joints, index of swelling joints, number of joints with tenderness, index of joints with tenderness, average grip strength of both hands, DSA28 score, HAQ, patient's assessment, physician's assessment, ESR, CRP and RF in both groups. The improvement of morning stiffness time, number of swelling joints, index of swelling joints, grip strength, HAQ and patient's assessment in the treatment group was much better than that in the control groups with statistical difference (P < 0.05). ACR20, ACR50 and ACR70 was 27.5% (11/40), 37.5% (15/40) and 22.5% (9/40) respectively in the treatment group and 40% (16/40), 27.5% (11/42) and 10.0% (4/40) respectively in the control group with statistical difference (P < 0.05) in the superiority of the treatment group over the control group. The incidence of adverse reaction in the control group was higher than that in the treatment group (P < 0.05). CONCLUSION: Definite therapeutic effect and high safety can be achieved in using the therapy combining disease with syndrome to treat rheumatoid arthritis belonging to syndrome of damp-heat obstruction.