[Bibliometric analysis on the literature characteristics of animal experiment research with acupuncture and moxibustion].

OBJECTIVE: To compare the diversities in the literature characteristics of animal experiments with acupuncture and moxibustion (acu-moxibustion) published in both Chinese and English, so as to summarize the similarities and differences in the reporting content for the animal experiment research with acu-moxibustion in the journals at home and abroad. METHODS: The articles of animal experiments with acu-moxibustion published from 2016 to 2018 were searched from CNKI, Wanfang, SinoMed, PubMed and Web of Science databases. The articles were screened according to the inclusion and exclusion criteria, and the database was established by importing the essential information, e.g. title, author, journal, impact factor, country, year of publication, citation frequency, funding, disease type, as well as the number of observation indicators and charts. The diversity was initially summarized among this type of articles between China and foreign countries. RESULTS: A total of 7 515 articles of animal experiments with acu-moxibustion were retrieved and 2 458 articles were eligible in compliance with the inclusion and exclusion criteria. Of them, there were 1 827 articles in Chinese and 631 in English. (1) Among those of Chinese-version, 169 articles (9.25%) were published in Acupuncture Research, listed the first of the article publications. Regarding the impact factor of published journal, Acupuncture Research was ranked the highest (3.187). For those published in English, 78 articles (12.36%) were published in Evidence-based Complementary and Alternative Medicine, listed the top of the article publications. Gastroenterology occupied the highest in terms of the impact factor (17.373) of published journal. (2) The first authors of Chinese-version articles were all from China, distributing in 461 institutions; of which, Beijing University of Chinese Medicine occupied the top for article publications (142 articles, 7.77%). For the English articles, 16 countries were involved regarding the first authors, and the most of them were from China (523 articles, 82.88%), followed by South Korea, Brazil, the United States and Japan. (3) The frequency of citations of Chinese articles was 7.50, which was significantly higher than that of English ones (4.61). (4) The funding supported Chinese and English articles were 1 680 (91.95%) and 569 (90.17%) respectively. (5) In the aspects of disease name and animal model, 135 and 220 diseases were included in Chinese and English articles respectively. The common top 10 diseases referred to 8 categories, i.e. stroke-related diseases, arthritis, Alzheimer's disease, depression, diabetes, spinal cord injury, hypertension and obesity. (6) In terms of the number of indicators, the maximum number was 6 for Chinese-version articles, averagely 2.46, while, it was 12 for English-version ones, 4.02 in average. (7) Among the articles of Chinese-version, the maximum number of charts was 17, and 1 028 articles had 2 to 4 charts, accounting the largest proportion (56.27%). Among those of English-version, the top number of charts was 27, and 347 articles had 4 to 6 charts, occupying the largest proportion (54.99%). CONCLUSION: The number of Chinese-version articles for acu-moxibustion experiment research is much higher than that of the English ones, the authorship is led by Chinese and most of the researches are supported by funds. There is less difference in the disease types between Chinese and English articles, but the frequency citation of Chinese articles is obviously higher than that of English ones; while, the numbers of observation indicators and charts in English articles are much more than those of Chinese ones. It is suggested that the great attention has been drawn on the acu-moxibustion experiment researches published in Chinese journals, and the reports of the researches are more complete in English journals.

[κ-opioid receptor is involved in electroacupuncture analgesia via inhibition of spinal microglial Toll-like receptor 4 in neuropathic pain rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of lumbar spinal κ-opioid receptor (KOR) and Toll-like receptor 4(TLR4) in microglia in neuropathic pain rats, so as to explore the role of cross-talk between KOR and TLK4 in EA-induced alleviation of chronic neuropathic pain. METHODS: Wistar male rats were randomized into control, model, EA and EA plus KOR inhibitor (EA+inhibitor) groups (n=18 in each group). The neuropathic pain model was established in rats by ligature of the right sciatic nerve. EA was applied at bilateral "Zusanli"(ST36) and "Yanglingquan"(GB34) for 30 min, once daily for 5 days. JDTic dihydrochloride (a KOR inhibitor) was administrated by intraperitoneal injection before EA intervention. The difference value of paw withdrawal thermal latency (PWLD) of the bilateral hind-limbs was used as the thermal pain reaction level. At the end of experiments, the rat's lumbar spinal cord (L2-L4) was taken for detecting the expression of CD68 mRNA (a marker of the activated microglia) and Iba-1 (a marker for the activated and resting microglia) immunoactivity, and dynorphin content, and KOR mRNA and TLR4 protein (in immunomagnetic microbead method separated microglia) by using fluorescence quantitative PCR, immunofluorescence, radioimmunoassay and Western blot, separately. RESULTS: Compared with the control group, a strong thermal hyperalgesia was induced, the expression levels of Iba-1 and CD68 mRNA in the spinal cord, TLR4 protein of the spinal microglia were significantly increased(P<0.01) in the model group. The microglia were characterized by somatic hypertrophy and thickened branches in the model group. After EA intervention, the PWLD, the expression of Iba-1, CD68 mRNA and TLR4 protein of the microglia were significantly decreased(P<0.05), while the content of spinal dynorphin and the expression of KOR mRNA of the microglia increased in the EA group relative to the model group(P<0.05). The hypertrophic microglia shrinked slightly in the EA group. After injection of KOR inhibitor, the PWLD and expression levels of Iba-1, CD68 mRNA and TLR4 protein were significantly increased(P<0.05), and the expression of KOR mRNA was significantly decreased(P<0.05) in the EA+inhibitor group in comparison with the EA group. CONCLUSION: The analgesia effect of EA may partly mediated by spinal microglial KOR and the activation of KOR of microglia may be a target for inhibition of microglial TLR4-induced pro-inflammatory signaling.

[Spinal cord Toll like receptor 4 and its co-stimulatory molecule heat shock protein 90 may parti-cipate in electroacupuncture analgesia in rats with chronic neuropathic pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of Toll like receptor 4(TLR4)and heat shock protein 90(HSP90) in the spinal cord of rats with chronic constriction injury (CCI) of sciatic nerve, so as to explore the mechanism of spinal cord TLR4 and HSP90 in alleviating chronic neuropathic pain by EA. METHODS: Male Wistar rats were randomized into control, model, EA, HSP90 inhibitor (inhibitor) and EA+ inhibitor groups (n=10 in each group). The neuropathic pain model was established by ligature of the right sciatic nerve to induce CCI. EA (1 mA，2 Hz/15 Hz)was applied at bilateral "Zusanli"(ST36) and "Yanglingquan"(GB34) for 30 min, once daily for 5 days. Rats of the inhibitor and EA+inhibitor groups were given a subcutaneous injection of HSP90 inhibitor geldanamycin (50 µg/kg) at the neck before daily EA. The paw withdrawal latency (PWL) of the bilateral hind-limbs was detected by using an algesia-detector. The contents of interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) in the lumbar spinal cord (L2-L4) tissue were detected by enzyme-linked immunosorbent assay. The relative expression levels of HSP90 and TLR4 proteins in the lumbar spinal cord (L2-L4) were detected using Western blot and immunofluorescence double labeling, respectively. RESULTS: Following CCI, a strong thermal hyperalgesia, an apparent up-regulation of expression of HSP90 and TLR4 proteins and TLR4 in microglia, and increasing levels of IL-1ß and TNF-α in the spinal cord were induced in the model group relevant to the control group (P<0.01，P<0.05). Five sessions of EA intervention or inhibitor injection significantly attenuated hyperalgesia, reversed the increase of IL-1ß and TNF-α, and down-regulated the expression of TLR4 in microglia (P<0.05). Compared with the model group, the expression of HSP90 was further increased (P<0.05), and those of TLR4 in microglia and neurons were significantly decreased and increased, respectively in the EA group (P<0.05). Compared with the EA group, the levels of PWLD,TLR4 and HSP90 expression, and the proportions of neuronal nuclei antigen（NeuN） and TLR4, and ionized calcium binding adapter molecule （Iba1） and TLR4 co-expressed cells were significantly decreased in the inhibitor group and EA+inhibitor group (P<0.05). The proportion of NeuN and TLR4 co-expression cells in the EA+inhibitor group was significantly higher than that of the inhibitor group (P<0.05). CONCLUSION: EA stimulation of ST36 and GB34 can alleviate thermal hyperalgesia in CCI rats, which is closely associated with its effect in regulating the expression of TLR4 in the spinal cord neurons and microglia. HSP90 in the spinal cord may be a co-stimulatory molecule for EA induced relief of neuropathic pain by regulating TLR4.

[How to improve the quality of histological pictures in the manuscripts of Acupuncture Research].

Histological pictures are important parts of medical research articles, and a good histological picture not only visualizes the content of the article, but also makes it easier for readers to understand. With the rapid development of microscopic technology and image processing technology, journals have higher requirements for the quality of histological pictures and more strict requirements for the editing and annotation of pictures. Based on an analysis of the current status of histological pictures in the articles of Acupuncture Research, this article proposes our suggestions and Methods for rational use of histological pictures, so as to provide a reference for improving the quality of histological pictures in the articles published in Acupuncture Research.

[High Mobility Group Box 1/ CD 24 Receptor/ß-EP Signaling in "Zusanli" (ST 36) Region Contributes to Electroacupuncture Analgesia in Rats with Neuropathic Pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of high mobility group box 1 (HMGB 1) and its receptor CD 24 proteins and ß-endorphin (ß-EP) content in "Zusanli" (ST 36) region in rats with chronic constriction injury (CCI), so as to explore its mechanisms underlying pain relief. METHODS: Thirty male Wistar rats were rando-mized into control, CCI model and EA groups (n＝ 10 rats in each). The neuropathic pain model was established by ligature of the left sciatic nerve to induce CCI in the model and EA groups, and sham operation was performed in rats of the control group. Paw with drawal latency (PWL, thermal pain threshold) of the bilateral hind-limbs was detected by using an algesia-detector. Eight days after CCI operation, EA was applied to bilateral "Zusanli" (ST 36) and "Yanglingquan" (GB 34) for 30 min, once daily for 5 days. The acetylated-HMGB 1 expression was determined by immunoprecipitation, and the expression of HMGB 1 and toll like receptor 4 (TLR 4) proteins and CD 24 mRNA were detected using Western blot and fluorescent quantitative real time-PCR, respectively, and the content of ß-EP in the acupoint region was assayed by enzyme linked immunosorbent assay (ELISA). Anti-CD 24 neutralizing antibody (200 µL, 100 µg/mL) was injected into ST 36 region once daily for 3 days for verifying the involvement of HMGB 1/CD 24 signaling in EA analgesia. RESULTS: Compared with the control group, the bilateral PWL difference values in the other two groups were significantly increased (P<0.05), meaning an occurrence of hyperalgesia after CCI. In comparison with the CCI model group, the hyperalgesia in the EA group was obviously decreased (P<0.05). After CCI, the expression levels of HMGB 1 and TLR 4 proteins were considerably increased compared with those in the control group (P<0.05). After 5-times' EA, the acetylated-HMGB 1, the expression of CD 24 mRNA, and the content of ß-EP were notably up-regulated (P<0.05), and there were no obvious changes in the expression levels of HMGB 1 and TLR 4 proteins (P>0.05). After local injection of anti-CD 24 antibody, EA-induced increases of ß-EP content and reduction of thermal pain threshold were significantly suppressed (P<0.05). CONCLUSION: EA of ST 36 and GB 34 can alleviate neuropathic pain in CCI rats, which is associated with its effects in up-regulating ß-EP content, and HMGB 1 protein and CD 24 mRNA expression levels in ST 36 region. The activated HMGB 1/CD 24/ß-EP signaling contributes to EA-ST 36 induced analgesia.

[Effect of Electroacupuncture Intervention on Expression of Pain Sensory and Affective Proce- ssing-related µ-opioid Receptor, etc. in the Amygdala in Chronic Neuropathy Pain Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on expression of pain sensory and affective processing-related µ-opioid receptor (MOR), glutamatergic AMPA receptor subunit GIuA 1, extracellular signal-regulated kinase (ERK 1/2), cAMP response element binding protein(CREB) in the amygdala in chronic constrictive injury (CC) + negative affection(NA) rats, so as to reveal its mechanism underlying pain relief. METHODS: A total of 32 male Wistar rats were randomized into normal control, model, EA, and anesthesia+ EA (AEA) groups (n = 8 in each group). The neuropathic pain NA model was established by ligation of the left sciatic nerve and repeated electrical stimulation of the paw-bottom in the pain-paired compartment. EA was applied to bilateral "Zusanli" (ST 36) and "Yanglingquan" (GB 34) for 30 min, once daily for 7 days. Thermal pain threshold (paw withdrawl latency, PWL) of the bilateral paws was measured by using a Tail-Flick Unit. The expression levels of MOR and p-CREB in the central amygdala (CeA) and those of MOR, GluA 1, p-ERK 1/2 and p-CREB in the right amygdala area were determined using immunofluorescence and Western blot, respectively. RESULTS: in comparison with the normal group, PWL difference (PWLD) values of the model group were significantly increased (P < 0.001), and the time spent in the CPA-paired. compartment was considerably decreased (P < 0.001). After EA, the PWLD levels of both EA and AEA groups were apparently decreased (P < 0.05), showing a pain relief; and the time spent in the CPA-paired compartment was apparently increased in the EA group (P < 0.05) , rather than in the AEA group (P > 0.05). Additionally , compared to the normal group, the expression level of MOR protein in the amygdala was remarkably increased (P < 0.05) and those of GIuA 1, p-ERK 2 and p-CREB proteins were apparently decreased (P < 0.05). After EA intervention for 7 days, the expression levels of these four proteins in the EA group, and those of MOR, p-ERK 2 and p-CREB in the AEA group were significantly up-regulated (P < 0.001, P < 0.01, P < 0.05). The expression level of GIuA 1 was significantly higher in the EA group than in the AEA group (P < 0.05). CONCLUSION: Repeated EA stimulation of ST 36-GB 34 has a definite effect in relieving both sensation and affection dimensions of pain in NA rats, which may be related to its effect in up-regulating the expression of GIuA 1 in the amygdala, but the effects of MOR, p-ERK 2 and p-CREB need being researched further.

Analysis and Thoughts about the Negative Results of International Clinical Trials on Acupuncture.

An increasing number of randomized controlled trials (RCTs) of acupuncture have proved the clinical benefits of acupuncture; however, there are some results that have shown negative results or placebo effects. The paper carried out an in-depth analysis on 33 RCTs in the 2011 SCI database, the quality of the reports was judged according to Jadad scores, and the "Necessary Information Included in Reporting Interventions in Clinical Trials of Acupuncture (STRICTA 2010)" was taken as the standard to analyze the rationality of the therapeutic principle. The difference between the methodology (Jadad) scores of the two types of research reports did not constitute statistical significance (P > 0.05). The studies with negative results or placebo effects showed the following deficiencies with respect to intervention details: (1) incompletely rational acupoint selection; (2) inconsistent ability of acupuncturists; (3) negligible needling response to needling; (4) acupuncture treatment frequency too low in most studies; and (5) irrational setting of placebo control. Thus, the primary basis for the negative results or placebo effects of international clinical trials on acupuncture is not in the quality of the methodology, but in noncompliance with the essential requirements proposed by acupuncture theory in terms of clinical manipulation details.

[On the necessity of intellectual property rights involving standardization of acupuncture and moxibustion therapies].

In the process of working out and implementing standardization of acupuncture-moxibustion (acu-moxi) therapy, the issue of intellectual property rights has been frequently involved. Whether is the standardization inevitably involved in intellectual property rights? A reasonable answer to this question is definitely of important realistic guiding value and significance for acu-moxi standardization work. For this reason, authors of the present paper sum up historical development of correlation between acu-moxi standardization and intellectual property rights, and fully analyze the related causes under the conditions of knowledge economy from 1) increasing protection of acu-moxi intellectual property rights, 2) intrinsic requirements for raising the standardization level of acu-moxi, 3) profits drive of the intellectual property rights owners, and 4) increasing impetuous international economic trade competition.

[Progress of researches on mechanisms of acupuncture underlying improvement of depression in the past five years].

Depression is a state of low mood and aversion to activity that has a negative effect on the patient's thoughts, behavior, feelings, and physical well-being. In the past five years, a significant progress has been made in understanding the mechanism of acupuncture therapy for improving depression. In the present paper, the authors summarize mechanisms of acupuncture for depression from 1) upregulating levels of monoamine neurotransmitters, gamma-aminobutyric acid, 5-HT, receptor binding, and hypothalamic neuropeptide Y, beta-endorphine, and mu-receptor expression, and down-regulating hippocampal glutamate content, etc.; 2) upregulating expression of hippocampal brain-derived neurotrophic factor protein and mRNA and its receptor TrkB, and activating adenyl cyclase-cAMP-PKA-CREB signaling pathway; 3) increasing hippocampal growth-associated protein 43 level and suppressing hippocampal c-Jun N-terminal kinase activation and cellular apoptosis; 4) upregulating expression of glial fibrillary acidic protein (activating glia cells); 5) regulating activities of hypothalamus-pituitary-adrenal axis and hypothalamus-pituitary-sex gland axis; 6) down-regulating serum IL-1beta, IL-6, and tumor necrosis factor-alpha levels. Moreover, the authors also put forward some suggestions about the further research on the mechanisms of acupuncture intervention for depression in the future.

[Involvement of hippocampal NO/PKG signaling pathway in the accumulative analgesic effect of electroacupuncture stimulation of "Zusanli" (ST 36)-"Yanglingquan"(GB 34) in chronic neuropathic pain rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Zusanli"(ST 36)-"Yanglingquan"(GB 34) on pain behavior and expression of hippocampal neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase(iNOS)and cGMP-dependent protein kinase (PKG) mRNA in rats with chronic neuropathic pain so as to analyze its mechanism underlying analgesia. METHODS: Forty male Wistar rats were randomly divided into control, CCI model, EA-2 Hz, EA-2 Hz/15 Hz, EA-100 Hz groups, with 8 cases in each group. Chronic neuropathic pain model was established by ligature of the left sciatic nerve under anesthesia (Urethane + Alpha-Chloralose) except rats in the control group. EA(2 Hz, 2 Hz/15 Hz, 100 Hz, 1 mA) was applied to bilateral "Zusanli"(ST 36)-"Yanglingquan"(GB 34) for 30 min, once each day for 2 weeks. The thermal and mechanical paw withdrawal latencies (pain thresholds) of the bilateral limbs were detected before and after EA interventions. The hippocampal tissue of the rat was collected for detecting the expression levels of nNOS, INOS and PKG genes using quantitative real-time-PCR technique. RESULTS: In comparison with the control group, the thermal and mechanical pain thresholds of the model group were decreased obviously (P<0. 05).Compared with the model group, both thermal and mechanical pain thresholds of the EA-2 Hz, EA-2 Hz/15 Hz and EA-100 Hz groups were markedly increased after EA intervention for 3, 7, 10 and 14 days (P<0. 05). Compared with the control group, the expression levels of hippocampal nNOS and PKG mRNA were significantly and moderately up-regulated in the model group (P<0. 05). While in comparison with the model group, the expression levels of hippocampal nNOS and PKG mRNA in the EA-2 Hz, EA-2 Hz/15 Hz and EA-100 Hz groups were markedly down-regulated (P<0. 05). No significant differences were found among the EA-2 Hz, EA-2 Hz/15 Hz and EA-100 Hz groups in the analgesic effect and in down-regulating hippocampal nNOS and PKG mRNA expression (P>0.05). However, the recovery state of the pain reaction of both EA-2 Hz and EA-2 Hz/ 15 Hz groups was relatively better than that of the EA-100 Hz group from day 3 to 10 after EA intervention. CONCLUSION: EA stimulation of "Zusanli"(ST 36)-"Yanglingquan"(GB 34) at 2 Hz, 2 Hz/15 Hz and 100 Hz can significantly suppress chronic neuropathic pain induced in CCI rats, which may be closely associated with its effects in down-regulating hippocampal nNOS and PKG mRNA expression levels.

[Effects of electroacupuncture at "Futu"(LI 18), etc. on pain threshold and cervico-spinal mGlu receptor 5/cAmp/CREB signaling in rats with neck incision pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Futu" (LI 18),etc. on the pain threshold and expression of metabotropic glutamate receptor subtype 5 (mGluR 5), cyclic adenosine monophosphate (cAMP), mitogen-activated protein kinase (MAPK), cAMP responsive element binding protein (CREB) genes in the cervico-spinal dorsal region in rats with neck incision pain so as to analyze its underlying analgesic mechanism in performing thyroid surgery. METHODS: Forty male Wistar rats were randomly divided into control, model, Futu (LI 18), Hegu (LI 4)-Neiguan (PC 6), Zusanli (ST 36)-Yanglingquan (GB 34) groups, with 8 cases in each group. Neck-incision pain model was established by making a longitudinal incision along the midline of the neck under anesthesia (isoflurane) except rats in the control group. EA(2 Hz/15 Hz, 1-2 mA) was applied to bilateral LI 18, LI 4-PC 6 and ST 36-GB 34 for 30 min, 4 h, 24 h and 48 h after modeling, respectively. The thermal pain threshold was detected before modeling, and before and after each treatment. After removing the ventral part, the spinal tissue of the cervical 1 -4 segments was collected for detecting the expression of mGluR 5, cAMP, MAPK, CREB genes by using quantitative real-time-PCR technique. RESULTS: In comparison with the control group, the pain threshold of the model group was decreased obviously (P<0.05). Compared with the model group, the pain thresholds of the LI 18 and LI 4-PC 6 groups were upregulated markedly after treatment (P<0.05). No significant differences were found between the LI 18 and LI 4-PC 6 groups, and between the model and ST 36-GB 34 groups in pain thresholds (P>0.05). Compared with the control group, the expression le-vels of mGluR 5 mRNA, cAMP mRNA and CREB mRNA in the cervico-spinal cord were upregulated significantly in the model group (P<0.05). While in comparison with the model group, cervico-spinal cAMP mRNA, and CREB mRNA were down-regulated markedly (P<0.05) and mGluR 5 mRNA was down-regulated moderately (P>0.05) in the LI 18 group, rather than in the LI 4-PC 6 and ST 36-GB 34 groups (P>0.05). CONCLUSION: EA-LI 18 can significantly suppress neck incision pain induced pain reaction, which may be closely associated with its functions in down-regulating expression levels of intracellular cAMP mRNA and CREB mRNA in the cervico-spinal cord.

[Effects of electroacupuncture of "Futu" (LI 18), etc. on pain behavior and expression of GABA receptor subunit genes in cervical spinal cord in rats with thyroid regional pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at different acupoints on the expression of gamma aminobutyric (GABA) receptor (R) subunit genes in the cervical spinal cord in rats with thyroid regional inflammatory pain so as to analyze its analgesic mechanism for thyroid surgery. METHODS: A total of 50 Wistar rats were randomized into control, model, Futu (LI 18), Hegu (LI 4)-Neiguan (PC 6, LI 4-PC 6), Zusanli (ST 36)-Yanglingquan (GB 34, ST 36-GB 34) groups, with 10 rats in each group. Thyroid regional pain model was established by subcutaneous injection of 2.5% formalin (100 microL). Ten minutes after modeling, EA (2 Hz/15 Hz, 1 mA) was applied to LI 18, LI 4-PC 6 and ST 36-GB 34 for 30 min, respectively. The animals' face-grooming (FG) times in 5 min and thermal pain threshold (paw withdrawal latency, PWL) were recorded. The expression of GABA(A) R, GABA(B) R1, and GABA(B) R2 genes in the cervical 1-3 segments of the spinal cord 80 min after modeling was detected by using reversed transcription-polymerase chain reaction (RT-PCR). Histological changes of the tissues of the thyroid region were observed by using H. E. staining. RESULTS: After subcutaneous injection of formalin, the animals' FG times in 5 min were increased considerably and the thermal pain threshold was decreased obviously in the model group (P < 0.05). Concomitantly, the expression levels of GABA(B) R1, and GABA(B) R2 genes of the cervical spinal cord were upregulated significantly in the model group (P < 0.05), and GABA(A) R expression was increased slightly (P > 0.05). Compared with the model group, the FG times in 5 min in the LI 18 and LI 4-PC 6 groups at 40 min and 70 min after modeling were decreased significantly (P < 0.05), and their thermal pain threshold values were increased markedly (P < 0.05). The expression levels of GABA(B) R1 mRNA, GABA(B) R2 mRNA and GABAA R mRNA were significantly higher in the LI 18 and LI 4-PC 6 groups than in the model group (P < 0.05). The expression level of GABA(B) R1 mRNA of the ST 36-GB 34 group was obviously higher than that of the model group (P < 0.05). The inflammatory cells in the formalin-injected thyroid region were relatively fewer in the LI 18 and LI 4-PC 6 groups than in the model group. CONCLUSION: Both EA of LI 18 and LI 4-PC 6 can significantly suppress formalin-injection induced pain reactions in the thyroid region, which may be closely associated with its effects in upregulating expression levels of cervico-spinal GABA(B) R1 mRNA, GABA(B) R2 mRNA and GABA(A) R mRNA, and reduce regional inflammatory reactions in the thyroid region.

[Analysis on shortcomings or insufficiencies of some manuscripts for publication in acupuncture research].

Based on the manuscripts delivered to Acupuncture Research, the author of the present paper analyzes some current shortcomings existing in scientific research work of acu-moxibustion. The main shortcomings are 1) insufficient experimental design, 2)inopportune intervention time, 3) indexes unable to explain the underlying mechanism of therapeutic effects, 4) unfounded exaggeration of the roles of acu-moxibustion therapy, and 5) improper conclusion. In addition, the author gives some typical examples to show these shortcomings or insufficiencies, so as to provide references for researchers in avoiding some defects in experimental designs and in giving reasonable explanations about their research findings in the future.

[Effect of acupuncture on contents of beta-endorphin in the plasma and hypothalamus in rats with stress-induced gastric mucosal injury].

OBJECTIVE: To explore the mechanism of acupuncture in preventing and treating stress-induced gastric mucosal injury from the view of brain-gut axis. METHODS: Forty male Wister rats were randomly divided into normal control, model 1, treatment, model 2 and prevention groups (n=8). Gastric mucosa injury model was established by intragastric perfusion of dehydrated alcohol (1.0 mL/rat). Rats of the treatment group were treated with acupuncture after modeling, while those of the prevention group treated first, followed by modeling. The time of modeling in model 1 group and model 2 group was simultaneous with that of the treatment group and prevention group respectively. Acupuncture was applied to "Zusanli"(ST 36), "Zhongwan" (CV 12) and "Neiguan" (PC 6) for 20 min, once daily for 5 days. Before sampling the tissues, 10% charcoal suspension was intragastric perfused 1 h for analyzing the rate of gastrointestinal propulsion(distance from the upper end of the charcoal powder to the cardia/total length of the cardia to the anus x 100%). Gastric mucosal ulcer index was measured by using Guth's method. The contents of beta-endorphin(beta-EP)in plasma and hypothalamus were determined by enzyme-linked immunosorbent assay. RESULTS: Compared with the normal control group, the gastrointestinal propulsion rates were decreased considerably in the two model groups (P < 0.05), while the gastric mucosal ulcer indexes and the contents of beta-EP in both plasma and hypothalamus were increased significantly in the model groups (P < 0.05, P < 0.01). Compared with the corresponding model groups, the gastrointestinal propulsion rate was increased remarkably in the prevention group (P < 0.05), and the gastric mucosal ulcer indexes and the contents of plasma beta-EP level were decreased obviously in both treatment and prevention groups (P < 0.05, P < 0.01). The contents of hypothalamic beta-EP were increased further in the later two groups (P < 0.01). CONCLUSION: Acupuncture of ST 36, CV 12 and PC 6 can promote the repair of gastric mucosal injury and improve gastrointestinal function, which may be related to its effects in reducing plasma beta-EP and upregulating hypothalamic beta-EP level. Acupuncture also has a better effect in preventing gastric mucosal injury.

[Effects of scalp acupuncture on expression of hippocampal MMP-9 in cerebral ischemia injury rats].

OBJECTIVE: To observe the effect of scalp-acupuncture on the expression of matrix metalloproteinase 9 (MMP-9) in the endothelium of the microvessels in the hippocampal CA 3 area in local cerebral ischemia (CI) rats, so as to study its underlying mechanism in improving CI. METHODS: Eighty male Wistar rats were randomized into normal control (n = 8), sham-operation (n = 8), CI model (n = 32), and scalp-acupuncture (n = 32) groups. The later two groups were further equally and respectively divided into 1, 3, 5 and 10 d subgroups. CI model was established by occlusion of the middle cerebral artery (MCAO). "Baihui" (GV 20) and "Qubin"(GB 7) were punctured with filiform needles and stimulated by twirling the needle at a frequency of about 200 revolutions/min for 1 min. The needles were retained for 30 min and re-manipulated once again every 10 min. The treatment was given once daily, continuously for 1, 3, 5 and 10 days respectively in different subgroups. MMP-9 immunoactivity of hippocampal CA 3 area was assayed by immunohistochemistry (SABC). RESULTS: Following CI, the CI rats' neural deficit scores in the model and scalp-acupuncture (treatment) groups had no apparent difference (P > 0.05). After acupuncture, the neural deficit score in the treatment group decreased gradually, and was significantly lower than that in the 10 d subgroup of model group (P < 0.05). Compared with the 1, 3, 5 and 10 d subgroups of control group, the total area and integral optical density (IOD) values of MMP-9 immunoactivity of hippocampal CA 3 area were all increased significantly in the corresponding 4 subgroups of model group (P < 0.01). Compared with the 3, 5 and 10 d subgroups of the model group, the total area and IOD values of MMP-9 immunoreaction positive products in the corresponding 3 subgroups of scalp-acupuncture group were decreased significantly (P < 0.05, P < 0.01). CONCLUSION: Scalp-acupuncture can effectively down-regulate MMP-9 expression in the hippocampal CA 3 area in focal cerebral ischemia rats, which may contribute to its effect in improving the neural deficit.

[Effects of electroacupuncture at "Futu" (LI 18), etc. on expression of spinal 5-HT 1 AR mRNA, 5-HT 2 AR mRNA and protein in rats with neck incision pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at bilateral "Futu" (LI 18), etc. on the expression of 5-HT 1 A receptor (R) mRNA, 5-HT2 AR mRNA and protein in the spinal cord of rats with neck incision pain, so as to explore its underlying mechanism in relieving incision pain. METHODS: A total of 48 Wistar rats were randomly divided into control, model (incision pain), Futu (LI 18), Hegu (LI 4)-Neiguan (PC 6, LI 4-PC 6), Zusanli (ST 36)-Yanglingquan (GB 34) cervical cord (ST 36-GB 34 C) and lumbar cord (ST 36-GB 34 L) groups. A 1.5 cm longitudinal incision was made in the middle of the neck under Isoflurane inhalation anesthesia. Pain threshold (PT) was measured using radiant heat. EA (1-2 mA, 2 Hz/15 Hz) was applied to bilateral LI 18, LI 4-PC 6, ST 36-GB 34 for 30 min. The expression of 5-HT 1 AR mRNA, 5-HT 2 AR mRNA and protein in the cervical spinal cord (C1-C4) tissue, and 5-HT 1 AR mRNA and 5-HT 2 AR mRNA in the lumbar cord (L1- L3) were detected with Real-time PCR and Western blot analysis, respectively. RESULTS: After the operation, the thermal PT was shortened obviously In comparison with pre-operation (P < 0.05). Compared with post-operation, the PT values were increased markedly in the LI 18 group and LI 4-PC 6 group (P < 0.05), rather than in the ST 36-GB 34 group (P > 0.05). The expression levels of 5-HT 1 AR mRNA, 5-HT 2 AR mRNA and 5-HT 2 AR protein in the cervical cord of the model group were increased significantly compared with those of the control group (P < 0.05). In comparison with the model group, the expression level of 5-HT 1 AR mRNA was down-regulated considerably in the LI 18 and LI 4-PC 6 groups (P < 0.05), and those of spinal 5-HT 2 AR mRNA and protein were up-regulated significantly in the LI 18 and LI 4-PC 6 groups (P < 0.05). No significant changes were found in the expression of 5-HT 1 AR mRNA and 5-HT 2 AR mRNA in the ST 36-GB 34 C group in comparison with the model group (P > 0.05). The expression levels of 5-HT 1 AR mRNA and 5-HT 2 AR mRNA in the ST 36-GB 34 L group were significantly lower than those of the ST 36-GB 34 C group (P < 0.05). CONCLUSION: EA of LI 18 and LI 4-PC 6 can significantly suppress pain reaction of neck incision in the rat, which is closely associated with its effects in down-regulating the expression of 5-HT 1 AR mRNA and in up-regulating 5-HT 2 AR mRNA and 5-HT 2 AR protein in the cervical spinal cord.

[Research on basic questions of intellectual property rights of acupuncture and moxibustion].

Along with the modernization and internationalization of acupuncture-moxibustion (acu-moxibustion), the issue of intellectual property rights has been becoming prominent and remarkable increasingly. In the present paper, the authors explain the basic issues of acu-moxibustion learning from the concept, scope, subject, object, contents and acquisition way of intellectual property rights. To make clear these questions will help us inherit and carry forward the existing civilization achievements of acu-moxibustion, and unceasingly bring forth new ideas and further improvement in clinical application, so as to serve the people's health in a better way.

[Effect of electroacupuncture of "Baihui" (GV 20)-"Yintang" (EX-HN 3) on the expression of glucocorticoid and glucocorticoid receptor mRNA of the chronic stress model rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of glucocorticoid (GC) mRNA and GC in the hippocampus, hypothalamus and pituitary in depression rats so as to study its mechanism underlying EA-resisting depression. METHODS: Seventy SD rats were randomized into normal control, model, Fluoxetine (Flu), constraint-stress, EA, RU 486 (an antagonist of GC) and EA+ RU 486 groups (n = 10/group). Chronic depression model was established by lonely raising and chronic unpredictable mild stress for 21 days. EA (2 Hz, 0.6 mA) was applied to "Baihui" (GV 20) and "Yintang" (EX-HN 3) for 20 min, once daily for 21 days. Subcutaneous injection of RU 486 (20 mg/kg) was given to the rats from the 14th day on and con- tinuously for 7 days in order to block the negative feedback reflex of hypothalamus-pituitary-adrenal (HPA) axis. Cortisol (CORT) content of the adrenal gland tissue was detected by radioimmunassay. The expression of GC receptor (GR) mRNA in the hippocampus, hypothalamus and pituitary tissues was determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Compared with the normal control group, adrenal CORT content of model group was increased significantly (P < 0.05), and in comparison with model group, adrenal CORT level of EA group decreased evidently (P < 0.05). Comparison between the RU 486 and EA + RU 486 groups showed that the adrenal CORT content, and hippocampal GR mRNA expression level of the latter were remarkably lower than those of the former (P < 0.05). Compared with the normal control group, the expression level of GR mRNA of the hipppocampal and pituitary tissues in the model, constraint-stress, and RU 486 groups,and those of the hypothalamus in the constraint-stress and RU 486 groups were down-regulated significantly (P < 0.05). In comparison with the constraint-stress group, hippocampal, hypothalamic and pituitary GR mRNA expression level in the EA group were upregulated considerably (P < 0.05). No significant differences were found among model, Flu, constraint-stress, RU 486 and EA + RU 486 groups in the ardenal CORT contents, and hippocampal, hypothalamic and pituitary GR mRNA expression levels (P > 0.05). CONCLUSION: EA can effectively down-regulate adrenal CORT content and hippocampal GR mRNA expression and normalize the function of HPA axis negative feed reflex in the depression rats, which may contribute to its effect in relieving depression.

[Progress in the study on the mechanism underlying the correlation between acupoints/meridians and Zangfu organs].

In the present paper, the authors review development of studies on the mechanism underlying the correlation between acupoints/ meridians and internal organs in recent 10 years from (1) doctrine of "dynamic acupoint"; (2) effect of one acupoint-stimulation on multi-viscera, (3) effect of multi-acupoints/multi-meridians stimulation on one single internal organ, (4) specific reactions of visceral disorders on the body surface, (5) the therapeutic effect of acupoint-injection being better than muscular injection, (6) effect of acupuncture on the expression of some known intracellular genes/proteins of the related viscera, (7) effect of acupuncture on the expression or phosphorylation of whole genes/whole proteins of the related viscera, (8) peripheral pathways connecting acupoints and the associated internal organ, (9) central pathways connecting acupoints and the related viscera, and (10) overview of the similar researches in foreign countries. In addition, the authors also make a preliminary analysis on the research significance, current problems and the thoughts in the coming studies. Utilizing advanced knowledge and techniques of systems biology to study "the correlation between acupoints/meridians and internal organs" will greatly promote the development of acupuncturology.

[Effect of electroacupuncture on hippocampal NO-cGMP signaling pathway in depression rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on hippocampal nitric oxide (NO)-cyclic guanosine 3,5-monophosphate (cGMP) signaling pathway in depression rats in order to explore the underlying mechanism of EA in improving depression. METHODS: Thirty male SD rats were equally randomized into control, model and EA groups. Depression model was established by using chronic unpredictable mild stress stimulation (forced ice-water swimming, electric shock, tail-clamping, etc.) combined with lonely raising for 21 days. EA (2 Hz, 0.6 mA, 20 min) was applied to "Baihui" (GV 20) and "Yintang" (EX-HN 3), once daily for 21 days. The expression of neuronal nitric oxide synthase (nNOS) and the content of cGMP in the hippocampus were determined by immuno-histochemistry and radioimmunoassay separately. RESULTS: Many nNOS immuno-reaction (IR)-positive granular cells were observed in the hippocampus in control group, fewer found in EA group and fewest in model group. Image analysis showed that the grey value of model group was significantly higher than that of control group (P < 0.01), and that of EA group was obviously lower than that of model group (P < 0.01), suggesting upregulation of nNOS expression after EA. Compared with control group the content of cGMP in hippocampus in model group showed a decreasing trend, but without significant difference between two groups (P > 0.05). In comparison with model group, hippocampal cGMP content of EA group increased considerably (P < 0.01), being comparable to that of control group (P > 0.05). CONCLUSION: Electroacupuncture of "Baihui" (GV 20) and "Yintang" (EX-HN 3) can upregulate the expression of nNOS and the content of cGMP in the hippocampus in depression rats, maintaining a normal activity of the NO/cGMP signaling pathway, which may contribute to its effect in relieving depression.