Molecular mechanism of basolateral amygdala involved in electroacupuncture-induced amelioration of cancer pain and concomitant depression based on transcriptomics techniques. / åŸºäºŽè½¬å½•ç»„å­¦æŠ€æœ¯æŽ¢è®¨åŸºåº•å¤–ä¾§æä»æ ¸å‚ä¸Žç”µé’ˆæ²»ç–—ç™Œç—›åŠå ¶è¯±å‘çš„æŠ‘éƒæƒ ç»ªçš„åˆ†å­æœºåˆ¶.

OBJECTIVES: To observe the effect of electroacupuncture (EA) of "Zusanli" (ST36) and "Sanyinjiao" (SP6) on cancer pain and concomitant negative emotion in cancer pain model mice, and to explore its molecular mechanisms in the basolateral amygdala (BLA) by using transcriptomics techniques. METHODS: C57BL/6 mice were randomized into sham operation, model and EA groups, with 10 mice in each group. The cancer pain model was established by injecting PBS suspension containing Lewis lung cancer cells into the femur. The mice in the EA group received EA stimulation(1 mA, 2 Hz) on ST36 and SP6 from the 10th day after modeling, 20 min per day for 12 successive days. The bone damage of the distal femur was observed with X-ray and H.E. staining, respectively. The mechanical pain threshold (MPT) was detected by using von Frey. The depression-like behavior was detected by using sucrose-preference test (sucrose preference index in 12 h), and the immobility (feeling of despair) duration of forced swimming within 4 min. The BLA tissue was extracted for RNA sequencing (RNA library construction, and screening differential gene profiling by transcriptomic sequencing) and bioinformatics analysis. The real-time PCR was used to validate the mRNA expression of differentially expressed genes：tumor necrosis factor superfamily 8 (Tnfsf8), bone marrow stromal cell antigen 1 (Bst1), prodynorphin (Pdyn) and voltage-gated sodium channelß4 (Scn4b). RESULTS: H.E. staining and X-ray showed significant bone damage in the distal femur in cancer pain mice. In contrast to the sham operation group, the MPT on the 1st , 4th, 7th , 10th, 14th and 21st day after modeling and sucrose preference index were significantly decreased (P<0.001, P<0.000 1), and the immobility time of the forced swimming was considerably increased in the model group (P<0.001). In contrast to the model group, the MPT values on the 14th and 21st day and sucrose preference index were obviously increased (P<0.000 1, P<0.05), and the immobility time was strikingly decreased in the EA group (P<0.01). RNA sequencing showed that a total of 404 differentially expressed genes (205 up-regulated, 199 down-regulated) were screened in the model group compared with the sham operation group, and a total of 329 differentially expressed genes (206 up-regulated and 123 down-regulated) were screened in the EA group compared with the model group. Venn diagram analysis of the differentially expressed genes showed that 45 up-regulated and 28 down-regulated genes in the model group were completely reversed by EA. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the screened differentially expressed genes revealed that the above differential genes were mainly enriched in the ligand receptor activity, cytokine receptor binding, and cytokine activity related to neuro-inflammation, as well as in neuropeptide signaling pathways related to neuronal excitability, and calcium ion mediated signal transduction. The analysis of KEGG pathway showed that the differentially expressed genes were mainly enriched in the inflammation-related pathways, such as interleukin-17 pathway. Validation analysis of the differentially expressed genes showed that the expression levels of Tnfsf8 and Bst1 were significantly up-regulated in the model group compared with the sham operation group (P<0.01, P<0.05), and down-regulated by EA (P<0.01, P<0.05), while the expression levels of Pdyn and Scn4b were down-regulated in the model group in comparison with the sham operation group (P<0.01), and up-regulated by EA (P<0.05, P<0.01), which was consistent with the changing trend of the gene sequencing results. CONCLUSIONS: Acupuncture of ST36 and SP6 can significantly relieve cancer pain and concomitant negative emotion in cancer pain mice, which may be related to its functions in alleviating neuro-inflammation and relieving the abnormal activities of specific neurons in the BLA.

Effect of electroacupuncture stimulation of "Sibai"(ST2) and "Quanliao"(SI18) on excitatory neurons of sensorimotor cortex in mice. / 电针"四白"和"颧髎"æ¿€æ´»å°é¼ èº¯ä½“æ„Ÿè§‰è¿åŠ¨çš®å±‚çš„å ´å¥‹æ€§ç¥žç»å ƒ.

OBJECTIVES: To observe the activation state and neuronal types of somatosensory cortex and the primary motor cortex induced by electroacupuncture (EA) stimulation of "Sibai" (ST2) and "Quanliao" (SI18) acupoints in mice. METHODS: Male C57BL/6J mice were randomly divided into blank control and EA groups, with 6 mice in each group. Rats of the EA group received EA stimulation (2 Hz, 0.6 mA) at ST2 and SI18 for 30 minutes. Samples were collected after EA intervention, and immunofluorescence staining was performed to quantify the expression of the c-Fos gene (proportion of c-Fos positive cells) in the somatosensory cortex and primary motor cortex. The co-labelled cells of calcium/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) and gamma-aminobutyric acid (GABA) in the somatosensory cortex and primary motor cortex were observed and counted by using microscope after immunofluorescence staining. Another 10 mice were used to detect the calcium activity of excitatory neurons in the somatosensory cortex and primary motor cortex by fiber photometry. RESULTS: In comparison with the blank control group, the number of c-Fos positive cells, and the proportion of c-Fos and CaMKⅡ co-labelled cells in both the somatosensory cortex and primary motor cortex were significantly increased after EA stimulation (P<0.05). No significant changes were found in the proportion of c-Fos and GABA co-labeled cells in both the somatosensory cortex and primary motor cortex after EA. Results of fiber optic calcium imaging technology showed that the spontaneous calcium activity of excitatory neurons in both somatosensory cortex and primary motor cortex were obviously increased during EA compared with that before EA (P<0.01), and strikingly reduced after cessation of EA compared with that during EA (P<0.05). CONCLUSIONS: Under physiological conditions, EA of ST2 and SI18 can effectively activate excitatory neurons in the somatosensory cortex and primary motor cortex.

[Empirical analysis on lumbar disc herniation treated with "sinew-bone three needling technique" of Chinese medicine].

The paper presents professor WU Han-qing's experience in treatment of lumbar disc herniation (LDH) with "sinew-bone three needling technique" of Chinese medicine. Based on the theory of meridian sinew, the points are located by "three-pass method" in terms of the distribution of meridian sinew and syndrome/pattern differentiation. The cord-like muscles and adhesion are relieved by relaxing technique to work directly on the affected sites and alleviate the local compression to the nerve root. The needle technique is operated flexibly according to the affected regions involved, due to which, the needling sensation is increased while the safety ensured. As a result, the meridian qi is enhanced, the mind and qi circulation is regulated; and the clinical effect is improved.

Low-frequency electroacupuncture improves disordered hepatic energy metabolism in insulin-resistant Zucker diabetic fatty rats via the AMPK/mTORC1/p70S6K signaling pathway.

BACKGROUND AND AIM: Disordered hepatic energy metabolism is found in obese rats with insulin resistance (IR). There are insufficient experimental studies of electroacupuncture (EA) for IR and type 2 diabetes mellitus (T2DM). The aim of this study was to probe the effect of EA on disordered hepatic energy metabolism and the adenosine monophosphate (AMP)-activated protein kinase (AMPK)/mammalian target of rapamycin complex 1 (mTORC1)/ribosomal protein S6 kinase, 70-kDa (p70S6K) signaling pathway. METHODS: Zucker Diabetic Fatty (ZDF) rats were randomly divided into three groups: EA group receiving EA treatment; Pi group receiving pioglitazone gavage; and ZF group remaining untreated (n = 8 per group). Inbred non-insulin-resistant Zucker lean rats formed an (untreated) healthy control group (ZL, n = 8). Fasting plasma glucose (FPG), fasting insulin (FINS), C-peptide, C-reactive protein (CRP) and homeostatic model assessment of insulin resistance (HOMA-IR) indices were measured. Hematoxylin-eosin (H&E) staining was used to investigate the liver morphologically. The mitochondrial structure of hepatocytes was observed by transmission electron microscopy (TEM). Western blotting was adopted to determine protein expression of insulin receptor substrate 1 (IRS-1), mTOR, mTORC1, AMPK, tuberous sclerosis 2 (TSC2) and p70S6K, and their phosphorylation. RT-PCR was used to quantify IRS-1, mTOR, mTORC1, AMPK and p70S6K mRNA levels. RESULTS: Compared with the ZF group, FPG, FINS, C-peptide, CRP and HOMA-IR levels were significantly reduced in the EA group (p < 0.05, p < 0.01). Evaluation of histopathology showed improvement in liver appearances following EA. Phosphorylation levels of AMPK, mTOR and TSC2 decreased, and IRS-1 and p70S6K increased, in hepatocytes of the ZF group, while these negative effects appeared to be alleviated by EA. CONCLUSIONS: EA can effectively ameliorate IR and regulate energy metabolism in the ZDF rat model. AMPK/mTORC1/p70S6K and related molecules may represent a potential mechanism of action underlying these effects.

[Acupoint selection for cancer pain: based on current evidence and Delphi method].

Based on literature research and Delphi expert consensus method, the important acupoints for cancer pain was summarized to provide evidence basis for the formulation of Clinical Practice Guide of Acupuncture in the Treatment of Cancer Pain. Through systematic search of Chinese and English databases, 28 clinical studies regarding acupuncture for cancer pain were included. The acupoint selection methods and high-frequency acupoints were summarized and analyzed. Based on this, a Delphi questionnaire was designed and two rounds of questionnaire survey on 30 experts in acupuncture and tumor related fields in China and abroad were conducted. As a result, it was suggested that the individualized acupoint selection should be adopted for acupuncture treatment of cancer pain, with Zusanli (ST 36), Hegu (LI 4), Taichong (LR 3), Sanyinjiao (SP 6), Yanglingquan (GB 34) and ashi points as the main acupoints. Combined with clinical research evidence and expert consensus, the important acupoints for cancer pain were identified. However, clinical acupoint selection still needed further research and refinement.

Electroacupuncture Attenuates Inflammation after Ischemic Stroke by Inhibiting NF-κB-Mediated Activation of Microglia.

Microglial activation and microglia-mediated inflammation play an important role in the occurrence, development, and outcome of stroke. Brain injury induces the activation and release of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin- (IL-) 1ß, and IL-6. Many studies have confirmed that acupuncture is effective in treating ischemic stroke. However, its protective mechanism against ischemic brain injury is complex and multifactorial. In this study, we observed the effects of electroacupuncture at Baihui (GV20) and Dazhui (GV14) on microglial activation and inflammation in the cortical ischemic penumbra (IP) of permanent middle cerebral artery occlusion (pMCAO) rats. It was found that electroacupuncture inhibited the degeneration and necrosis of microglia in the cortical IP and ameliorated mitochondrial damage. Immunofluorescence and western blot analysis showed that microglia were in a resting state or weakly activated in the normal brain. After cerebral ischemia, the expression of microglial markers (Iba-1 and CD11b) increased, and NF-κB p65, IL-1ß, and TNF-α expression gradually increased. The dynamic changes were generally temporally consistent. Electroacupuncture downregulated the expressions of Iba-1 and CD11b. Additionally, it inhibited the expression of NF-κB p65, IL-1ß, and TNF-α and reduced the conversion of microglia to the M1 phenotype after ischemia. Electroacupuncture regulated the activation of microglia and microglia-mediated inflammation after cerebral ischemia, confirming the relevant theories regarding the effect of acupuncture treatment on cerebral ischemia and guiding clinical practice.

CBS-Induced H2S Generation in Hippocampus Inhibits EA-Induced Analgesia.

Hydrogen sulfide (H2S) is an important mediator participating in both physiological and pathological systems and related to the inflammatory process. Acupuncture has a therapeutic effect on inflammatory pain. However, whether H2S generated in the central nervous system (CNS) is a mediator of electroacupuncture (EA) treatment for inflammatory pain is unknown. We injected complete Freund's adjuvant (CFA) to induce inflammatory pain and applied EA treatment as an interventional strategy for pain relief. The results presented here show that S-adenosyl-l-methionine (SAM), an allosteric activator of cystathionine-ß-synthetase (CBS), may reverse the therapeutic effect of EA. CBS-induced H2S generation might get involved in the mechanism of EA-induced analgesia in the hippocampus on chronic inflammatory pain.

Role of 5-HT1A in the nucleus of the solitary tract in the regulation of swallowing activities evoked by electroacupuncture in anesthetized rats.

Somatic stimulation therapy, such as electroacupuncture (EA), has been widely applied in the clinic to treat dysphagia. However, its underlying mechanism has remained unknown. In the present study, the effect of EA at acupoints Fengfu (DU16) and Lianquan (RN23) on swallowing activities and the involvement of 5-HT1A in the nucleus of the solitary tract (NTS) were examined in anesthetized rats. EA at DU16 and RN23 significantly evoked myoelectric activity of the mylohyoid muscle, which was attenuated by injection of 10 nmol 5-HT1A antagonist (WAY-100635) into the NTS. Meanwhile, 5-HT1A expression in the NTS increased following EA. The results suggested that EA at DU16 and RN23 promotes swallowing activity, and 5-HT1A in the NTS may play an important role in the excitatory effects.

Neuroprotective effects of electroacupuncture on hypoxic-ischemic encephalopathy in newborn rats are associated with increased expression of GDNF-RET and protein kinase B.

OBJECTIVE: To explore the neuroprotective effects of electroacupuncture (EA) on hypoxic-ischemic encephalopathy (HIE) and to further investigate the role of glial cell line-derived neurotrophic factor (GDNF) family receptor member RET (rearranged during transfection) and its key downstream phosphatidylinositol 3 kinase (PI-3K)/protein kinase B (Akt) pathway in the process. METHODS: A total of 220 seven-day-old SD rats (of either sex, from 22 broods) were randomly divided into two groups, one (30 rats) for sham-surgery group and the other (190 rats) for HIE model group. The HIE model was established using the left common carotid artery ligation method in combination with hypoxic treatment. The successfully established rats were randomly divided into five groups, including control model group, EA group, sham-EA group, antagonist group and antagonist plus electroacupuncture group, with 35 rats in each group. Baihui (GV 20), Dazhui (GV 14), Quchi (LI 11) and Yongquan (KI 1) acupoints were chosen for acupuncture. EA was performed at Baihui and Quchi for 10 min once a day for continuous 1, 3, 7 and 21 days, respectively. The rats were then killed after the operation and injured cerebral cortex was taken for the measurement of neurologic damage by hematoxylin-eosin (HE) staining and the degenerative changes of cortical ultrastructure by transmission electron microscopy. RET mRNA level and Akt protein level were detected by real-time reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis, respectively. RESULTS: EA could ameliorate neurologic damage of the first somatic sensory area (S1Tr) and alleviate the degenerative changes of ultrastructure of cortical neurons in rats subjected to HIE. And the longer acupuncture treatment lasted, the better its therapeutic effect would be. This was accompanied by gradually increased expression of GDNF family receptor RET at the mRNA level and its downstream signaling Akt at the protein level in the ischemic cortex. CONCLUSION: EA has neuroprotective effects on HIE and could be a potential therapeutic strategy for HIE in the neonate. Activation of RET/Akt signaling pathway might be involved in this process.

[Clinical application regularity of Fengfu (GV 16) acupoint: research on ancient Chinese medical literature].

OBJECTIVE: To sum up the application regularity of Fengfu (GV 16) acupoint in clinical practice through studying the ancient literature from the early stage of the Qin Dynasty to the Qing Dynasty. METHODS: Chinese ancient medical literature relevant to Fengfu (GV 16) was searched to establish a database containing information of clinical indications of GV 16, supplementary acupoints, relevant needling techniques, moxibustion methods, etc. RESULTS: A total of 277 articles about Fengfu (GV 16) for 61 types of clinical disorders or diseases involving internal medicine, surgery, paediatrics, five sense organs, etc. were obtained from 2,200 ancient Chinese medical books. Fengfu (GV 16) alone was most frequently used for treatment of disorders caused by exogenous factors, such as common cold, neck pain, headache, epilepsy, mania, dizziness, throat dumb, leg-foot problems, etc. , with the auxiliary acupoint being Fengchi (GB 20). In addition, 147 articles relevant to needling and moxibustion stimulation of GV 16 (7 types of methods) were found. CONCLUSION: Fengfu (GV 16) is mainly used for local problems in the human body, and also for problems occurred along the meridian trace, aiming at the pathogenesis. When employed, GV 16 is often stimulated by acupuncture needle.

Study on the correlation between synaptic reconstruction and astrocyte after ischemia and the influence of electroacupuncture on rats.

OBJECTIVE: To observe the effects of electroacupuncture (EA) on the structure parameters of synapse and reactive changes of astrocyte in the marginal zone of focal cerebral ischemia in rats at different time zones so as to further explore its underlying mechanisms in the treatment of cerebral ischemia. METHODS: Ninety male Wistar rats were randomly assigned to sham-operation, model, and EA groups, with 30 animals in each group. Each group was subdivided into 1 h, as well as 1, 3, 7, and 21 days post-operation groups, with 6 animals assigned to each time point subgroup. Heat coagulation-induced occlusion of the middle cerebral artery was performed to establish a model of focal cerebral ischemia. EA was applied immediately following surgery to the EA group [4/20 Hz, 2.0-3.0 V, 1-3 mA, to Baihui (GV20) and Dazhui (GV14)] for 30 min. Treatment was performed once a day, and experimental animals were sacrificed at 1 h, as well as 1, 3, 7 and 21 days postoperation. The ultrastructure changes in synapse and astrocytes were observed by using transmission electron microscopy. Glial fibrillary acidic protein (GFAP) expression and Ca(2+) of astrocytes were measured by using laser confocal scanning microscope. Excitatory amino acid transporters-2 (EAAT2) and connexin 43 (CX43) expressions were assayed with immunohistochemical method. Canonical correlation analysis was conducted between structure parameters of synapse and parameters of astrocyte in the same time and group. RESULTS: Broken synapses were observed following cerebral ischemia, and the numbers of synapses were significantly decreased. Compared with the model group, synaptic ultrastructure was significantly improved in the EA group. Compared with the sham-operation group, synaptic number density was significantly decreased, as were postsynaptic density thickness, synaptic cleft width and synaptic interface curvature in the EA and model groups. However, compared with the model group, postsynaptic density thickness was significantly increased in the EA group at the same time points post-operation (P <0.05, P<0.01). In addition, synaptic cleft width, synaptic number density and synaptic interface curvature were significantly increased with the passage of time (P <0.05, P<0.01). The expression of GFAP in the EA group were significantly lower than those in the model group at all the time points (P <0.05, P<0.01). OD values of EAAT2 in the EA group were significantly higher than those in the model group at the same time (P <0.05, P<0.01). Compared with that in the model group, the expressions of CX43 in the EA group increased significantly at 3 days and 7 days (P <0.05, P<0.01). Ca(2+) average fluorescence intensity of astrocytes in the EA group was significantly lower than those in the model group at 1 h, 1 day, 3 days and 7 days (P <0.05, P<0.01). The changes in structure parameters of synapse were closely related to the changes of CX43, EAAT2, GFAP, Ca(2+) of astrocytes by EA treatment at all the time points. CONCLUSIONS: EA is helpful for synaptic reorganization, which may be related to its effect on intervening the activation state of astrocytes and promoting the beneficial interaction between astrocytes and synapses. Acupuncture could start the adjustment of neuron-glial network so as to promote the synaptic reorganization, which may be the key mechanism of treating cerebral ischemia.

[Effects of acupuncture on serum insulin antibody and tumor necrosis factor alpha in the experimental rat with insulin resistance].

OBJECTIVE: To observe effects of acupuncture and diet on insulin resistance (IR) and to probe the mechanism. METHODS: Forty SID rats were equally divided into 5 groups: blank group (group I), model group I (group II), model group II (group III), acupuncture group I (group IV) and acupuncture group II (group V). The groups II, III, N and V were fed with high-fat-sugar-salt forage to made IR model, then the groups I, III and V were fed with normal forage, and the groups II and IV with the high-fat-sugar-salt forage, and the acupuncture groups IV and V received acupuncture treatment. Two weeks later, the fasting blood glucose (FBG), plasma insulin (INS), insulin sensitivity index (ISI), INS antibody and tumor necrosis factor alpha (TNF-alpha) were detected. RESULTS: As compared with group I, FBG and INS increased, ISI decreased in the group II (all P < 0.01); as compared with the group II , FBG and INS decreased (all P < 0.01) and ISI increased (P < 0.05, P < 0.01) in the group [II, IV, V; no case with INS antibody (+) in all groups; TNF-alpha in the group II increased compared with that of the group I (P < 0.01), and TNF-alpha in the group III, IV, V decreased compared with that of group II (P < 0.01). CONCLUSION: Acupuncture exerts a reversal effect on insulin resistance, and diet can promotes this effect. The mechanism is carried out possibly through decreasing the secretion of TNF-alpha.