[Baimai Ointment relieves chronic pain induced by chronic compression of dorsal root ganglion in rats by regulating neuroactive ligand-receptor interaction and HIF-1 signaling pathway].

The Baimai Ointment with the effect of relaxing sinew and activating collaterals demonstrates a definite effect on Baimai disease with pain, spasm, stiffness and other symptoms, while the pharmacodynamic characteristics and mechanism of this agent remain unclear. In this study, a rat model of chronic compression of L4 dorsal root ganglion(CCD) was established by lumbar disc herniation, and the efficacy and mechanism of Baimai Ointment in the treatment of CCD were preliminarily explored by behavioral tests, side effect evaluation, network analysis, antagonist and molecular biology verification. The pharmacodynamic experiment indicated that Baimai Ointment significantly improved the pain thresholds(mechanical pain, thermal pain, and cold pain) and gait behavior of CCD model rats without causing tolerance or obvious toxic and side effects. Baimai Ointment inhibited the second-phase nociceptive response of mice in the formalin test, increased the hot plate threshold of normal mice, and down-regulated the expression of inflammatory cytokines in the spinal cord. Network analysis showed that Baimai Ointment had synergistic effect in the treatment of CCD and was related to descending inhibition/facilitation system and neuroinflammation. Furthermore, behavioral tests, Western blot, and immunofluorescence assay revealed that the pain-relieving effect of Baimai Ointment on CCD may be related to the regulation of the interaction between neuroactive ligand and receptors(neuroligands) such as CHRNA7, ADRA2A, and ADRB2, and the down-regulation of the expression of NOS2/pERK/PI3K, the core regulatory element of HIF-1 signaling pathway in spinal microglia. The findings preliminarily reveal the mechanism of relaxing sinew and activating collaterals of Baimai Ointment in the treatment of Baimai disease, providing a reference for the rational drug use and further research of this agent.

[Therapeutic effect of Baimai Ointment on peripheral sensitization of chronic inflammatory pain in mice].

Chronic inflammatory pain is mainly manifested by peripheral sensitization. Baimai Ointment(BMO), a classical Tibetan medicine for external use, has good clinical efficacy in the treatment of chronic inflammatory pain, while its pharmacodynamics and mechanism for relieving peripheral sensitization remain unclear. This study established an animal model of chronic inflammatory pain induced by complete Freund's adjuvant to explore the mechanism of BMO in the treatment of chronic inflammatory pain by behavioral test, side effect assessment, network analysis, and experimental verification. The pharmacodynamics experiment showed that BMO increased the thresholds of mechanical pain sensitivity and thermal radiation pain sensitivity of chronic inflammatory pain mice in a dose-dependent manner, and had inhibitory effect on foot swelling, inflammatory mediator, and the expression of transient receptor potential vanilloid-1(TRPV1) and transient receptor potential A1(TRPA1). The results of body weight monitoring, pain sensitivity threshold detection in normal mice, rotarod performance test, and forced swimming test showed that BMO had no obvious toxic or side effect. The network analysis of 51 candidate active molecules selected according to the efficacy of BMO, content of main components, and ADME parameters showed that the inhibitory effect of BMO on chronic inflammatory pain was associated with the core regulatory elements of tumor necrosis factor(TNF) and T cell receptor signaling pathways. BMO down-regulated the protein levels of mitogen-activated protein kinase 14(MAPK14), MAPK1, and prostaglandin-endoperoxide synthase 2(PTGS2), and up-regulated the phosphorylation le-vel of glycogen synthase kinase 3 beta(GSK3 B) in the plantar tissue of mice. In conclusion, BMO can effectively relieve peripheral sensitization of chronic inflammatory pain without inducing tolerance and obvious toxic and side effects. The relevant mechanism may be related to the regulation of BMO on core regulatory elements of TNF and T cell receptor signaling pathways in surrounding tissues.

Total saponin from Anemone flaccida Fr. Schmidt abrogates osteoclast differentiation and bone resorption via the inhibition of RANKL-induced NF-κB, JNK and p38 MAPKs activation.

Osteoclasts, bone-specialized multinucleated cells, are responsible for bone destructive diseases such as rheumatoid arthritis and osteoporosis. Natural plant-derived products have received substantial attention given their potential therapeutic and preventive activities against bone destructive diseases. In the present study, we investigated the effects of total saponin (TS) from Anemone flaccida Fr. Schmidt, on receptor activator of nuclear factor-κB ligand (RANKL)-induced in vitro osteoclast differentiation. We observed that TS concentration-dependently inhibited RANKL-induced osteoclast formation from RAW 264.7 cell and bone marrow-derived macrophages (BMMs), as well as decreased extent of actin ring formation and lacunar resorption. The RANKL-stimulated expression of osteoclast-related transcription factors were also diminished by TS. Moreover, TS blocked the RANKL-triggered TRAF6 expression, phosphorylation of mitogen-activated protein kinases (MAPKs) and IκB-α, and inhibited NF-κB p65 DNA binding activity. Furthermore, TS almost abrogated the nuclear factor of activated T cells (NFATc1) and c-Fos expression. Taken together, our results demonstrated that TS suppresses RANKL-induced osteoclast differentiation and inflammatory bone loss via the down-regulation of TRAF6 level, suppression of JNK and p38 MAPKs and NF-κB activation, and subsequent decreased expression of c-Fos and NFATc1. Therefore, TS may be a potential agent and needs to be more evaluated in vivo or in clinical trials to become a therapeutic for lytic bone diseases.

[Effect of traditional Chinese medicines with different properties on thermoregulation and temperature-sensitive transient receptor potentialion channel protein of rats with yeast-induced fever].

OBJECTIVE: To compare the intervention effects of four traditional Chinese medicines (TCMs) with typical cold or hot property on body temperature and temperature-sensitive transient receptor potential ion channel proteins (TRPs) of rats with yeast-induced fever. METHOD: The pyrexia model was induced by injecting yeast suspension subcutaneously. Totally 108 male SD rats were randomly divided into the normal group, the model group, the Rhei Radix et Rhizoma treated group, the Coptidis Rhizoma treated group, the Euodiae Fructus treated group, and the Alpiniae Officinarum Rhizoma treated group, with 18 rats in each group. At the 4 h, 8 h and 12 h after injection of yeast, the rats were sacrificed to collect their hypothalamus and dorsal root ganglion. The expressions of TRPV1 and TRPM8 were detected by immunohistochemistry and Western blot method. RESULT: Compared with the normal group, after injection of yeast, the temperature of rats in the model group notably increased, and reached the peak at 8 h (P < 0.01). The TRPV1 level in hypothalamus and dorsal root ganglia (DRG) of the model group significantly increased, whereas the TRPM8 level significantly reduced. Compared with the model group, the Rhei Radix et Rhizoma group and the Coptidis Rhizoma group showed significant decrease in the high body temperature of rats caused by yeast, down-regulation in the expression of TRPV1, and up-regulation in the expression of TRPM8 (P < 0.05 or P < 0.01). Euodiae Fructus and Alpiniae Officinarum Rhizoma had no significant effect on either temperature or TRPs of fever rats. CONCLUSION: Rhei Radix et Rhizoma and Coptidis Rhizoma, both are TCMs with cold property, can reduce the temperature of fever rats induced by yeast, which may be related to their effective regulation of TRPV1 and TRPM8 in hypothalamus and DRG, while Euodiae Fructus and Alpiniae Officinarum Rhizoma had no relevant effect.

Inhibitory effect of acupoint electrostimulation with different layers and intensities on muscular inflammatory pain and spinal WDR neuron activity. / ç©´ä½ä¸åŒå±‚æ¬¡å’Œå¼ºåº¦ç”µåˆºæ¿€ç¼“è§£è‚Œè‚‰ç‚Žæ€§ç—›åŠå¯¹è„Šé«“èƒŒè§’å¹¿åŠ¨åŠ›ç¥žç»å ƒçš„æŠ‘åˆ¶ä½œç”¨.

OBJECTIVES: To observe the analgesic effects of different levels and intensities of electrical stimulation on the local acupoints in the pain source area and their impact on wide dynamic range (WDR) neurons in the spinal dorsal horn, in order to provide a basis for selecting appropriate parameters for electroacupuncture (EA) stimulation. METHODS: Wistar rats were used in 3 parts of the experiment. Complete Freund's adjuvant was used to establish a model of inflammation-induced pain in the gastrocnemius muscle. After modeling, 6 rats were randomly selected for multi-channel extracellular electrophysiological recording of the electrical activity of WDR neurons, to determine the threshold for activating the A-component (Ta) and the C-component (Tc), which were used as the intervention intensities for skin transcutaneous electrical acupoint stimulation (TEAS) or EA. Thirty-six rats were randomly divided into normal , model , TEAS-Ta , TEAS-Tc, EA-Ta , and EA-Tc groups, with 6 rats in each group. In the pain source area , Ta or Tc intensity of TEAS or EA intervention at"Chengshan"(BL57) was performed for 30 min each time, once a day, for 3 consecutive days. A small animal pressure pain measurement instrument was used to measure the mechanical pressure pain threshold of the gastrocnemius muscle in rats, and the Von Frey filament was used to measure the mechanical pain threshold of the footpad. Thirteen rats were randomly selected to observe the immediate responsiveness of WDR neurons to Ta/Tc intensity of EA or TEAS in BL57. RESULTS: The thresholds of TEAS to activate WDR neuron A-component or C-component were (2.43±0.57) mA and (7.00±1.34) mA, respectively, while the thresholds for EA to activate muscle WDR neuron A-component or C-component were (0.72±0.34) mA and (1.58±0.35) mA, respectively. After injection of CFA into the gastrocnemius muscle, compared with the normal group both the mechanical pressure pain threshold of the gastrocnemius muscle and the mechanical pain threshold of the footpad of rats in the model group were significantly decreased (P<0.001). After TEAS-Ta, TEAS-Tc or EA-Ta intervention in the BL57, both the mechanical pressure pain threshold of the gastrocnemius muscle and the mechanical pain threshold of the footpad were significantly higher than those in the model group (P<0.05, P<0.001). Compared with the normal group, the electrical threshold for evoking WDR neuron C-component discharge was significantly decreased (P<0.001) in the model group, while increased after TEAS-Ta, TEAS-Tc, or EA-Ta intervention (P<0.01) compared with the model group. The evoked discharge frequency of muscle WDR neurons decreased significantly after immediate intervention with TEAS-Ta, TEAS-Tc, or EA-Ta (P<0.01, P<0.05). EA-Tc had no significant improvement on the evoked electrical activity of WDR neurons or pain behavior. CONCLUSIONS: TEAS-Ta, TEAS-Tc, or EA-Ta can all alleviate the local and footpad mechanical pain in rats with muscle inflammation and inhibit the responsiveness of WDR neurons, indicating that different intensities are required for analgesic effects at different levels of acupoints in the pain source area.

Electroacupuncture activated local sympathetic noradrenergic signaling to relieve synovitis and referred pain behaviors in knee osteoarthritis rats.

Introduction: Recent research has focused on the local control of articular inflammation through neuronal stimulation to avoid the systemic side effects of conventional pharmacological therapies. Electroacupuncture (EA) has been proven to be useful for inflammation suppressing and pain reduction in knee osteoarthritis (KOA) patients, yet its mechanism remains unclear. Methods: In the present study, the KOA model was established using the intra-articular injection of sodium monoiodoacetate (MIA) (1 mg/50 µL) into the knee cavity. EA was delivered at the ipsilateral ST36-GB34 acupoints. Hind paw weight-bearing and withdrawl thresholds were measured. On day 9, the histology, dep enrichment proteins, cytokines contents, immune cell population of the synovial membrane of the affected limbs were measured using HE staining, Masson staining, DIA quantitative proteomic analysis, flow cytometry, immunofluorescence staining, ELISA, and Western Blot. The ultrastructure of the saphenous nerve of the affected limb was observed using transmission electron microscopy on the 14th day after modeling. Results: The result demonstrated that EA intervention during the midterm phase of the articular inflammation alleviated inflammatory pain behaviors and cartilage damage, but not during the early phase. Mid-term EA suppressed the levels of proinflammatory cytokines TNF-α, IL-1ß, and IL-6 in the synovium on day 9 after MIA by elevating the level of sympathetic neurotransmitters Norepinephrine (NE) in the synovium but not systemic NE or systemic adrenaline. Selective blocking of the sympathetic function (6-OHDA) and ß2-adrenergic receptor (ICI 118,551) prevented the anti-inflammatory effects of EA. EA-induced increment of the NE in the synovium inhibited the CXCL1-CXCR2 dependent overexpression of IL-6 in the synovial macrophages in a ß2-adrenergic receptor (AR)-mediated manner. Discussion: These results revealed that EA activated sympathetic noradrenergic signaling to control local inflammation in KOA rats and contributed to the development of novel therapeutic neurostimulation strategies for inflammatory diseases.

Local analgesia of electroacupuncture is mediated by the recruitment of neutrophils and released ß-endorphins.

ABSTRACT: The efficacy of acupuncture in treating pain diseases has been recognized in clinical practice, and its mechanism of action has been a hot topic in academic acupuncture research. Previous basic research on acupuncture analgesia has focused mostly on the nervous system, with few studies addressing the immune system as a potential pathway of acupuncture analgesia. In this study, we investigated the effect of electroacupuncture (EA) on the ß-endorphins (ß-END) content, END-containing leukocyte type and number, sympathetic neurotransmitter norepinephrine (NE), and chemokine gene expression in inflamed tissues. To induce inflammatory pain, about 200 µL of complete Frester adjuvant (CFA) was injected into the unilateral medial femoral muscle of adult Wistar rats. Electroacupuncture treatment was performed for 3 days beginning on day 4 after CFA injection, with parameters of 2/100 Hz, 2 mA, and 30 minutes per treatment. The weight-bearing experiment and enzyme-linked immunosorbent assay showed that EA treatment significantly relieved spontaneous pain-like behaviors and increased the level of ß-END in inflamed tissue. Injection of anti-END antibody in inflamed tissue blocked this analgesic effect. Flow cytometry and immunofluorescence staining revealed that the EA-induced increase in ß-END was derived from opioid-containing ICAM-1 + /CD11b + immune cells in inflamed tissue. In addition, EA treatment increased the NE content and expression of ß2 adrenergic receptor (ADR-ß2) in inflammatory tissues and upregulated Cxcl1 and Cxcl6 gene expression levels. These findings provide new evidence for the peripheral analgesic effect of acupuncture treatment by recruiting ß-END-containing ICAM-1 + /CD11b + immune cells and increasing the ß-END content at the site of inflammation.

Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches.

Skin innervation is an important part of the peripheral nervous system. Although the study of the cutaneous nerve fibers has progressed rapidly, most of the understanding of their distributional and chemical characteristics comes from conventional histochemical and immunohistochemical staining on thin tissue sections. With the development of the tissue clearing technique, it has become possible to view the cutaneous nerve fibers on thicker tissue sections. The present protocol describes multiple fluorescent staining on tissue sections at a thickness of 300 µm from the plantar and dorsal skin of rat hindfoot, the two typical hairy and glabrous skin sites. Here, the calcitonin gene-related peptide labels the sensory nerve fibers, while phalloidin and lymphatic vessel endothelial hyaluronan receptor 1 label the blood and lymphatic vessels, respectively. Under a confocal microscope, the labeled sensory nerve fibers were followed completely at a longer distance, running in bundles in the deep cutaneous layer and freestyle in the superficial layer. These nerve fibers ran in parallel to or surrounded the blood vessels, and lymphatic vessels formed a three-dimensional (3D) network in the hairy and glabrous skin. The current protocol provides a more effective approach to studying skin innervation than the existing conventional methods from the methodology perspective.

[Effect of electroacupuncture on spontaneous pain during the synovial inflammatory response stage and allodynia at the later stage in rats with knee osteoarthritis].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on pain behavior, synovial inflammatory response and demyelination of saphenous nerve in the rats modeled with knee osteoarthritis (KOA) and explore the effect mechanism of EA for reliving allodynia. METHODS: Eighty-four male SD rats were randomly divided into a control group, a model group and an EA group, 28 rats in each one. Intra-articular injection of sodium monoiodoacetate (MIA) was administered in right knee joint of each rat in the model group and the EA group to establish the KOA model. In the EA group, separately, on day 5, 7 and 9 after modeling, EA was applied at "Zusanli" (ST 36) and "Yanglingquan" (GB 34) on the right side, with disperse-dense wave (2 Hz/15 Hz), 1 mA in current intensity, for 30 min in one intervention, once a day, and 3 interventions were required. On the 9th day after modeling, the weight-bearing rate was calculated for the affected limbs of the rats in each group, the synovial morphological changes were observed using HE and Masson staining, flow cytometry was adopted to detect the synovial immunocyte counts, and MSD multi-spot assay was used to detected the synovial inflammatory cytokine content. On the 14th day after modeling, the hind-paw mechanical withdrawal threshold was observed in each group and the ultrastructure of the saphenous nerve was observed under transmission electron microscopy. RESULTS: On the 9th day after modeling, compared with the control group, the weight-bearing rate of the affected limb was reduced (P<0.01), the synovial hyperplasia, inflammatory cell infiltration and synovial fibrosis occurred in the affected limb; the counts of synovial CD11b+ cells and M1 macrophages (CD11b+CD86+) were increased (P<0.01), the contents of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1ß, IL-10 and IL-13 in the synovial tissue were elevated (P<0.01, P<0.05) for the rats of the model group. Compared with the model group, the weight-bearing rate of the affected limb was increased (P<0.05), the synovial hyperplasia, inflammatory cell infiltration and synovial fibrosis were mitigated, the counts of CD11b+ cells and M1 macrophages (CD11b+CD86+) in the synovial tissue, and the contents of TNF-α and IL-6 were reduced (P<0.01, P<0.05) in the EA group. On the 14th day after modeling, the hind-paw mechanical withdrawal threshold was reduced in the model group when compared with the control group (P<0.01), and it was increased in the EA group when compared with the model group (P<0.05). Besides, in the model group, obviously, the myelin sheath structure was destroyed, the myelin layer was disintegrated and loosened, the axon was extruded or the layer thicken and cracked. Compared with the model group, the injury of saphenous nerve was alleviated remarkably in the EA group. CONCLUSION: The intervention with EA may attenuate the synovial inflammatory response and the injury of saphenous nerve in the affected limb of the rat with KOA, so that the spontaneous pain during the synovial inflammatory response stage and allodynia at the later stage are relieved.

Electroacupuncture and moxibustion-like stimulation activate the cutaneous and systemic hypothalamic-pituitary-adrenal axes in the rat.

OBJECTIVE: To determine whether electroacupuncture (EA) or moxibustion-like stimulation (MLS) can affect the cutaneous and/or systemic hypothalamic-pituitary-adrenal (HPA) axes. METHODS: Rats were divided into Control, EA, 37°C MLS and 43.5°C MLS groups. EA and MLS were performed at bilateral ST36 or LI4. The expression of corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH) and the glucocorticoid receptor (GR) was detected in local cutaneous tissues at the site of ST36 and LI4 by immunohistochemical staining. In addition, levels of CRF, ACTH and corticosterone (CORT) in cutaneous tissue and plasma were determined. RESULTS: Cutaneous expression of CRF, ACTH and GR significantly increased after EA at ST36, while only GR increased after 43.5°C MLS at ST36. The results of EA and MLS at LI4 were in parallel with those at ST36. In plasma, compared with the control group, the level of CORT increased after EA at ST36, while both ACTH and CORT were markedly increased after 43.5°C MLS. For LI4, plasma CRF and CORT increased after EA, while the levels of all three hormones increased following 43.5°C MLS. Notably, compared with the effect of EA, 43.5°C MLS at ST36 produced a more substantial increase in plasma CORT, and 43.5°C MLS at LI4 induced a more dramatic increase in plasma CRF and CORT. CONCLUSION: Both EA and 43.5°C MLS can activate the cutaneous and systemic HPA axes of the rat. EA tended to activate the local cutaneous HPA, while 43.5°C MLS was more likely to activate the systemic HPA axis.

Acupuncture Enhances Signals at Sensitized Acupoints to Elevate Pressure Pain Threshold in Knee Osteoarthritis Patients.

OBJECTIVE: To observe the pressure pain threshold (PPT), skin conductance (SC) and blood perfusion (BP) of the sensitized acupoints in patients with knee osteoarthritis (KOA), and explore the mechanism of acupuncture at the sensitized acupoints for treating diseases. METHODS: Eleven healthy subjects and 11 unilateral KOA patients were recruited from July 2020 to March 2021 in this study. The PPT, SC and BP of control acupoints in healthy controls, and non-sensitized and sensitized acupoints in KOA patients were measured and compared between baseline and after manual acupuncture (MA) treatment. RESULTS: Before MA treatment, lower PPT was observed at the sensitized acupoints compared with non-sensitized and control acupoints (P<0.05). After MA treatment, PPT at the sensitized acupoints increased significantly in KOA patients (P<0.05). Before MA treatment, there was no statistical difference in SC and BP among control, non-sensitized and sensitized acupoints (P>0.05). Compared with the control and non-sensitized acupoints, there were significant increases of SC and BP in sensitized acupoints of KOA patients after MA treatment (P<0.05 or P<0.01). CONCLUSION: MA at sensitized acupoints could elevate PPT of KOA patients, which may be associated with the increment of SC and BP.

[Mechanisms of electroacupuncture at "Zusanli"(ST36) in delaying colon "inflammation-cancer transformation"].

OBJECTIVE: To investigate the mechanism of electroacupuncture (EA) "Zusanli" (ST36) in delaying colon "inflammation-cancer transformation" in mice by anti-inflammatory. METHODS: C57BL/6J mice were randomly divided into normal, model and EA groups, with 12 mice in each group. The mouse model of colorectal cancer (CRC) was established by intrape-ritoneal injection of azomethane (AOM) and feeding dextran sodium sulfate (DSS). At the beginning of the 2nd cycle, EA was applied to bilateral ST36 for 30 min once every other day for 12 times. The number of colon tumors in each group was observed, and the weight and length of colon were recorded. The contents of interleukin (IL)-1ß, IL-6, IL-17A, IL-23, tumor necrosis factor (TNF)-α and CXC chemokine ligand 1 (CXCL1) of serum and colon tissue were detected by MSD multifactorial assay.The apoptosis of local cells in colon tumor was observed by TUNEL staining. Cell proliferation in colon tumor was observed by immunohistochemistry. RESULTS: Compared with the normal group, the colon length was significantly shortened (P<0.05) and the colon mass was significantly increased (P<0.001) in the model group, the contents of IL-1ß, IL-6, TNF-α, IL-17A and CXCL1 of serum and colon tissue were significantly increased (P<0.05, P<0.01, P<0.001), and the content of IL-23 was increased in colon tissue (P<0.05) in the model group. Compared with the model group, the colon mass was decreased (P<0.05) and the contents of IL-1ß, IL-6, TNF-α and IL-17A in serum were decreased (P<0.05), while the contents of IL-17A, CXCL1 and IL-23 in colon tissue were decreased (P<0.05) in the EA group, the percentage of local apoptotic cells in the EA group was increased (P<0.001), the percentage of PCNA positive cells was decreased (P<0.001), the number of tumors and the tumor volume were significantly decreased (P<0.01, P<0.05). The contents of IL-6, TNF-α, IL-17A and IL-23 in serum of CRC mice were positively correlated with tumor burden (P<0.05).The contents of IL-1ß, TNF-α, CXCL1 and IL-23 in colon tissue of CRC mice were positively correlated with tumor burden (P<0.05). CONCLUSION: Electroacupuncture at ST36 can inhibit the inflammatory response of AOM/DSS inflammatory associated CRC mice and delay the "inflammation-cancer transformation" of colon.

Electroacupuncture and Moxibustion-Like Stimulation Relieves Inflammatory Muscle Pain by Activating Local Distinct Layer Somatosensory Afferent Fibers.

Recent studies have shown that both superficial and deep acupuncture produced clinically relevant and persistent effect on chronic pain, and several subtypes of somatic primary afferents played critical roles in acupuncture and moxibustion analgesia. However, which kind of primary afferents in the superficial and deep tissue of the acupoint is activated by acupuncture or moxibustion to relieve pain persistently remains unclear. The aim of this study is to investigate the roles of distinct peripheral afferents in different layers of the tissue (muscle or skin) in the acupoint for pain relief. Muscular A-fibers activated by deep electroacupuncture (dEA) with lower intensity (approximately 1 mA) persistently alleviated inflammatory muscle pain. Meanwhile, cutaneous C-nociceptors excited by noxious moxibustion-like stimulation (MS) and topical application of capsaicin (CAP) on local acupoint area produced durable analgesic effect. Additionally, spontaneous activity of C-fibers caused by muscular inflammation was also inhibited by dEA and CAP. Furthermore, decreases in pain behavior induced by dEA disappeared after deep A-fibers were demyelinated by cobra venom, whereas CAP failed to relieve pain following cutaneous denervation. Collectively, these results indicate that dEA and MS ameliorate inflammatory muscle pain through distinct primary afferents in different layers of somatic tissue; the former is achieved by activating muscular A-fibers, while the latter is mediated by activating cutaneous C-fibers.

Electroacupuncture regulates inflammatory cytokines by activating the vagus nerve to enhance antitumor immunity in mice with breast tumors.

AIMS: The aim of this study was to explore the potential effect of electroacupuncture (EA) at ST36 on mice bearing breast tumors by regulating inflammatory cytokines to enhance antitumor immunity via vagus nerve. MATERIALS AND METHODS: Female BALB/c mice were implanted with 4T1-luc2 breast tumor cells to establish a murine mammary cancer model. Tumor growth was evaluated by tumor volume, weight and bioluminescence imaging. Inflammatory conditions in serum and tumor tissue were assessed by cytokines (IL-1ß, TNF-α and IL-10) and HE staining. Proportions and functions of CD8+ T cells, NK cells and MDSCs were identified by flow cytometry and western blot. Involvement of vagal efferent components was confirmed by ChAT and c-Fos double labeling immunohistochemistry in dorsal motor nucleus of vagus (DMV). Subdiaphragmatic vagotomy was employed to determine if the effect of EA was mediated by vagus nerve. KEY FINDINGS: EA at ST36 reduced the volume and weight of tumors within 22 days after implantation. Proinflammatory cytokines IL-1ß and TNF-α in serum, tumor and local inflammatory infiltration were obviously attenuated after EA. Meanwhile, EA intervention significantly augmented the proportion and cytolytic function of CD8+ T cells and NK cells, along with a decline in the accumulation and immunosuppressive activities of MDSCs. Finally, c-Fos expression in ChAT+ neurons in DMV increased following EA, and the ameliorating effect of EA was obviously blocked by subdiaphragmatic vagotomy. SIGNIFICANCE: EA intervention relieved tumor progression in breast tumor-bearing mice by alleviating inflammation and enhancing antitumor immunity, which was mediated by eliciting efferent vagus nerve activity.

Electroacupuncture-Induced Muscular Inflammatory Pain Relief Was Associated With Activation of Low-Threshold Mechanoreceptor Neurons and Inhibition of Wide Dynamic Range Neurons in Spinal Dorsal Horn.

Acupuncture is an effective alternative therapy for pain management. Evidence suggests that acupuncture relieves pain by exciting somatic afferent nerve fibers. However, the mechanism underlying the interaction between neurons in different layers of the spinal dorsal horn induced by electroacupuncture (EA) remains unclear. The aim of this study was to explore the mechanism of EA relieving inflammatory muscle pain, which was associated with activation of the spontaneous firing of low-threshold mechanoreceptor (LTM) neurons and inhibition of wide dynamic range (WDR) neuronal activities in the spinal dorsal horn of rats. Inflammatory muscle pain was induced by injecting complete Freund's adjuvant into the right biceps femoris muscle. EA with intensity of threshold of A fibers (Ta) in Liangqiu (ST34) muscle considerably inhibited the abnormal spontaneous activities of electromyography (EMG) due to muscle inflammation. While EA with intensity of C-fiber threshold (Tc) increased the abnormal activities of EMG. EA with Ta also ameliorated the imbalance of weight-bearing behavior. A microelectrode array with 750-µm depth covering 32 channels was used to record the neuronal activities of WDR and LTM in different layers of the spinal dorsal horn. The spontaneous firing of LTM neurons was enhanced by EA-Ta, while the spontaneous firing of WDR neurons was inhibited. Moreover, EA-Ta led to a significant inverse correlation between changes in the frequency of WDR and LTM neurons (r = -0.64, p < 0.05). In conclusion, the results indicated that EA could alleviate inflammatory muscle pain, which was associated with facilitation of the spontaneous firing of LTM neurons and inhibition of WDR neuronal activities. This provides a promising evidence that EA-Ta could be applied to relieve muscular inflammatory pain in clinical practice.

[Electroacupuncture and transcutaneous electrical acupoint stimulation relieve muscular inflammatory pain by activating afferent nerve fibers in different layers of "Liangqiu"(ST34) in rats].

OBJECTIVE: To compare the effect of electroacupuncture (EA) and transcutaneous electrical acupoint stimulation (TEAS) of skin and muscle layers of "Liangqiu" (ST34) area on inflammatory muscular pain in rats, so as to study the role of different-layer afferent nerve fibers in acupuncture analgesia. METHODS: A total of 120 male SD rats were used in the present study, including 8 rats used for determining the excitatory threshold of Aδ(Ta) and C (Tc) afferent nerve fibers, 48 employed for comparing the analgesic effect of EA and TEAS at intensities of Ta and Tc, and 64 for observing the effect of EA and TAES stimulation of ipsilateral (Ipsi), contralateral (Contra) ST34 and ipsilateral LI4 at Ta and Tc intensities. Inflammatory muscle pain was induced by injection of complete Freund's adjuvant (CFA) into the right biceps femoris muscle. In the second part of the present study, 48 rats were randomly and equally divided into control, model, TEAS-Ta, TEAS-Tc, EA-Ta and EA-Tc groups, while in the 3rd part, 64 rats were randomly and equally divided into control, model, Ipsi-ST34-TEAS, Contra-ST34-TEAS, Ipsi-ST34-EA, Contra-ST34-EA, Ipsi-LI4-TEAS and Ipsi-LI4-EA groups. TEAS or EA was applied to the skin and muscle layers, respectively. Before and after modeling, the animal was forced to stand on a bipedal equilibrator, the difference in body mass distribution of both feet (bearing difference) was used to assess the pain severity. The frequency of myoelectrical discharges of the right bicep femoris muscle in responding to electrical stimulation of the spot between the 4th and 5th toes of the ipsilateral hindlimb at an intensity of two-folds of C-fiber excitatory threshold was recorded. RESULTS: 1) The bearing difference between the bilateral hindlimbs was markedly higher in the model group than in the control group (P<0.01), and significantly lower at the 2nd and 3rd day in the EA-Ta and EA-Tc, and TEAS-Tc groups relevant to the model group (P<0.05, P<0.01). 2) The frequency of C-fiber reflex induced electromyogram (EMG) activities were significantly decreased at 0 and 1 min after TEAS of both ipsilateral ST34 at Tc, and 0 min after TEAS of the contralateral ST34 at Tc (rather than at Ta and not LI4 even at Tc) in comparison with pre-TEAS, and 0, 1 and 2 min after TEAS of ipsilateral ST34 at Tc, and 0 min after TEAS of contra-ST34 at Tc compared with the model group, respectively (P<0.01, P<0.05). In comparison with pre-EA, the frequency of C-fiber reflex induced EMG activities were significantly decreased at 0 and 1 min after EA of the ipsilateral ST34 at Ta, and 0 min after EA of the contra-ST34 at Ta. In addition, the frequency of C-fiber reflex induced EMG activities were decreased at 0, 1 and 2 min after EA of the ipsilateral ST34 at Tc, and 0 and 1 min after EA of the contra-ST34 at Tc, as well as 0 min after EA of LI4 at Tc (P<0.01, P<0.05).In comparison with the model group, the frequency of C-fiber reflex induced EMG activities are significantly decreased at 0, and 1 min after EA of the ipsilateral ST34 at Ta, and 0 min after EA of the contra-ST34 at Ta. In addition, the frequency of C-fiber reflex induced EMG activities were decreased at 0, 1 ,2,and 3 min after EA of the ipsilateral ST34 at Tc, and 0 and 1 min after EA of the contra-ST34 at Tc, as well as 0 min after EA of LI4 at Tc, respectively (P<0.01, P<0.05). CONCLUSION: TEAS-ST34 at Tc and EA-ST34 at both Ta and Tc can alleviate pain behavior in inflammatory pain rats, which may be related to its effect in activating the afferent nerve fiber in different layers of ST34 area.

[Discussion on the effect pathways of preventing and treating coronavirus disease 2019 by acupuncture and moxibustion from the regulation of immune inflammatory response].

The effect of acupuncture-moxibustion on respiratory system and systemic immune inflammatory response were reviewed to explore the possible role of neuroimmunomodulation in the control of inflammatory response and the effect mechanism of cholinergic anti-inflammatory pathway on coronavirus disease 2019 (COVID-19). Acupuncture-moxibustion could produce the local and systemic anti-inflammatory effect on COVID-19 through the activation of cholinergic anti-inflammatory pathway. Compared with humoral anti-inflammatory pathway, the neuronal anti-inflammatory pathway has earlier initiation, rapider action, and more localization, which play a more important role in the initial stage of inflammatory response. This may be an important basis for acupuncture-moxibustion intervention in the early stage of COVID-19. In addition to cholinergic anti-inflammatory pathway, acupuncture-moxibustion may also play an anti-inflammatory role in activating sympathetic nerve, hypothalamic-pituitary-adrenal axis and other neural anti-inflammatory pathways. How acupuncture-moxibustion play its role in stimulating the vagus nerve and sympathetic nerve in different periods of inflammatory response, and whether the effect is based on the selection of acupoints and the methods of stimulation, will be the research direction of the transformation from basic research to clinical research for acupuncture-moxibustion.

[Electroacupuncture and transcutaneous electrical acupoint stimulation with different intensities relieve muscular of inflammatory pain of the rats].

OBJECTIVE: To observe and compare the ameliorate effects of electroacupuncture (EA) and transcutaneous electrical acupoint stimulation (TEAS) with different intensities on inflammatory muscle pain, so as to confirm the role of different afferent nerve fibers in different layers (skin or muscle) in pain relief of acupuncture. METHODS: The intensities of the thresholds of A (Ta) and C (Tc) were selected for TEAS and EA. TEAS and EA were applied on the layer of skin and muscle of "Liangqiu" (ST34), respectively. Forty-eight rats were randomly divided into control, model, TEAS-Ta, TEAS-Tc, EA-Ta and EA-Tc groups (n=8 in each group). The inflammatory muscle pain model was established by complete Freund's adjuvant (CFA) injection into the right biceps femoris muscle. EA or TEAS was applied once a day for 3 days. The weight difference between the hind limbs and the abnormal electromyography (EMG) activities were observed as an index for pain of the rats. RESULTS: After modeling, the weight difference between the hind limbs was increased markedly in the model group relevant to the control group (P<0.01). Compared with the model group, the weight difference between the hind limbs in the TEAS-Tc, EA-Ta, and EA-Tc groups was significantly decreased (P<0.05, P<0.01). Compared with that before intervention, the area under the curve and discharge frequency of abnormal EMG of rats in the TEAS-Tc and EA-Ta groups were significantly reduced after intervention (P<0.01, P<0.05), while those in the EA-Tc group were significantly increased (P<0.05). After the intervention, compared with the TEAS-Ta group, the inhibition rate of the area under the curve and the discharge frequency of the abnormal EMG in the TEAS-Tc group increased significantly (P＜0.05); and those were also increased in the EA-Ta group when compared with the EA-Tc group (P<0.01). CONCLUSION: TEAS with Tc or EA with Ta in the muscle layer can alleviate the pain and inhibit the abnormal EMG in inflammatory muscle pain rats. It is indicated that local pain relief by acupuncture was related to the afferent nerve fiber in different layer with different intensities.

Antiangiogenesis-Combined Photothermal Therapy in the Second Near-Infrared Window at Laser Powers Below the Skin Tolerance Threshold.

Photothermal agents with strong light absorption in the second near-infrared (NIR-II) region (1000-1350 nm) are strongly desired for successful photothermal therapy (PTT). In this work, titania-coated Au nanobipyramids (NBP@TiO2) with a strong plasmon resonance in the NIR-II window were synthesized. The NBP@TiO2 nanostructures have a high photothermal conversion efficiency of (93.3 ± 5.2)% under 1064-nm laser irradiation. They are also capable for loading an anticancer drug combretastatin A-4 phosphate (CA4P). In vitro PTT studies reveal that 1064-nm laser irradiation can efficiently ablate human lung cancer A549 cells and enhance the anticancer effect of CA4P. Moreover, the CA4P-loaded NBP@TiO2 nanostructures combined with PTT induce a synergistic antiangiogenesis effect. In vivo studies show that such CA4P-loaded NBP@TiO2 nanostructures under mild 1064-nm laser irradiation at an optical power density of 0.4 W cm-2, which is lower than the skin tolerance threshold value, exhibit a superior antitumor effect. This work presents not only the development of the NBP@TiO2 nanostructures as a novel photothermal agent responsive in the NIR-II window but also a unique combined chemo-photothermal therapy strategy for cancer therapy.