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.

[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.

Ultrasound-assisted deep eutectic solvents extraction of glabridin and isoliquiritigenin from Glycyrrhiza glabra: Optimization, extraction mechanism and in vitro bioactivities.

Licorice (Glycyrrhiza glabra) is extensively used owing to the superior pharmacological effects. However, its maximum application potential has not been fully exploited due to the limitation of currently available extraction solvent and methods. In this study, an eco-friendly deep eutectic solvent (NADESs) based ultrasound-assisted extraction (DES-UAE) method was applied to prepare licorice extracts. The DES-UAE using choline chloride and lactic acid as solvent was optimized and modeled by using response surface methodology to maximize the extraction yields of glabridin (GLA) and isoliquiritigenin (ISL). The optimized extracts possessed higher contents of GLA and ISL than available extraction methods, and the enriched products showed superior pharmacological activities in vitro. Furthermore, scanning electron microscopy (SEM) and molecular dynamic simulation analyses were performed to deeply investigate the interaction between solvent and targeted compounds. This study not only provides an eco-friendly method for high-efficient extraction of GLA and ISL from licorice but also illustrates the mechanism of the increased extraction efficacy, which may contribute to the application of licorice and deep insight into extraction mechanism using DES.

[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.

[Moxibustion promoted wound healing in rats with full-thickness cutaneous wounds].

OBJECTIVE: To explore the effects of moxibustion intervention on wound healing in rats with full-thickness cutaneous wounds. METHODS: A total of 28 adult SD male rats were randomly assigned to model group and moxibustion group (n＝14 in each one). The skin wound model was established by removal of a piece of full-thickness skin from the median line of the rats' back (about 2 cm below the shoulder blade). Moxibustion intervention was applied to the surrounding area of the focus for 25 min, once daily for 6 days. The wound healing was observed and photographed after each moxibustion intervention. The wound tissues were harvested after transcardiac perfusion with 4% paraformaldehyde solution on the 2nd and 7thday after modeling, and stained with H．E. and Masson methods for displaying histopathological changes and collagen fiber growth status, respectively. RESULTS: After modeling, the cutaneous wound was healed gradually in both groups, and the wound area was significantly smaller from the 2nd day to the 5th day in the moxibustion group than in the model group (P<0.01, P<0.05). Correspondingly, the wound closure area was significantly bigger from the 2nd to the 5th day in the moxibustion group than in the model group (P<0.01). H．E. and Masson staining showed that the number of the inflammatory cells (monocytes, macrophages, neutrophile, etc.) and the collagen fiber area in the local wound tissue of the moxibustion group were significantly bigger than those of the model group on the 2nd day after intervention (P<0.01). After 6 sessions of moxibustion intervention, the number of fibroblasts and collagen fibers in the moxibustion group were obviously increased than that in the model group, characterized by closer arrangement of fibroblasts and collagen fibers, more and larger of the new-born blood vessels, and thicker of the scab in the wound area. CONCLUSION: Moxibustion can promote the wound healing by enhancing the growth of collagen fibers and cell proliferation in the traumatic cutaneous area in full-thickness cutaneous wound rats.

[Application of Laser Scanning Confocal Microscope to Morphologic Research of Acupuncture and Moxibustion].

Laser scanning confocal microscope (LSCM) has been used in the field of morphological research of acupuncture and moxibustion for more than 20 years. It is one of the important tools for revealing the structure of acupoints and the effect of acupuncture from histological perspective. With the help of technical advantages of LSCM, the quality of morphological research of acupuncture and moxibustion has been greatly improved, helping us gain a deeper understanding about the structure of acupoints and meridians as well as histochemical changes induced by acupuncture/moxibustion intervention. In order to promote the application of LSCM in acupuncture and moxibustion, we simply reviewed some recent studies in this field and combined them with our experience, trying to provide some technical suggestions. We expect that the technique of LSCM could be integrated into more experiments in acupuncture medicine to provide more powerful morphological evidence for exploring the underlying mechanisms of acupuncture and moxibution therapies.

Neuropeptide Initiated Mast Cell Activation by Transcutaneous Electrical Acupoint Stimulation of Acupoint LI4 in Rats.

Transcutaneous electrical acupoint stimulation (TEAS) has been consistently used clinically for its ease of operation, non-invasiveness and painlessness, in contrast to the characteristics of inserted needles. However, the mechanism remains unknown. The aim of this study was to investigate the local response of TEAS at Hegu acupoint (LI4). Immunohistochemistry was used to measure the expression of tryptase-positive mast cells, neuropeptides of the calcitonin gene-related peptide (CGRP) and substance P (SP) in LI4. Mast cells were also labelled with serotonin (5-HT), neurokinin-1 receptor (NK-1R) and toluidine blue. The results showed that cutaneous CGRP and SP immune-positive (CGRP-IP or SP-IP) nerve fibres in LI4 were more highly expressed. There were high degrees of mast cell aggregation and degranulation with release of 5-HT near the CGRP-IP or SP-IP nerve fibres and blood vessels after TEAS. The degranulation of mast cells (MCs) was accompanied by expression of NK-1R after TEAS. Either mast cell membrane stabilizer (Disodium cromoglycate) or NK-1R antagonist (RP 67580) diminished the accumulation and degranulation of MCs induced by TEAS. Taken together, the findings demonstrated that TEAS induced sensory nerve fibres to express CGRP and SP, which then bound to the NK-1R on MCs, after which MCs degranulated and released 5-HT, resulting in TEAS-initiated acupuncture-like signals.

A rational design to enhance the resistance of Escherichia coli phytase appA to trypsin.

Escherichia coli phytase appA, which hydrolyzes phytate, has been widely applied as an important feed supplement, but its resistance to trypsin needs to be improved. Six putative solvent-accessible amino acid residues (K74, K75, K180, R181, K183, and K363), which could be easily attacked by trypsin, were selected to improve trypsin tolerance of Escherichia coli phytase appA. Inspection of the three-dimensional structure and computational design via hydrogen bond analysis, six optimal mutation sites of K74D/K75Q/K180N/R181N/K183S/K363N, which strengthened the hydrogen bonding, were performed to generate three mutants. Results showed that the most beneficial mutant appA-M6 had a specific activity of 3262 U/mg with molecular weight of approximately 52-55 kDa. Similar to appA-WT, the optimal pH (4.5) and temperature (60 °C) of appA-M6 were unchanged. Compared with appA-WT, appA-M6 showed a significant enhancement (p < 0.05) in resistance to trypsin and a 3.8 °C increase in melting temperature (Tm). We concluded that introduction of hydrogen bonds and N-glycosylation modification resulted in decreased enzyme flexibility and increased the enzyme stability against proteolysis and thermal denaturation. The mutant appA-M6 generated in this study could be applied for the large-scale commercial production of phytase and thus could benefit the food and feed industry.

[Neuronal Correlation Between Acupoint BL 23 and the Adrenal Gland in Rats].

OBJECTIVE: To explore the neuroanatomical basis of acupoint-visceral correlation by studying the distribution of the neurons associated with acupoint "Shenshu" (BL 23) area and adrenal gland in rats. METHODS: AF 488-CTB and AF 594-CTB were injected into the left side of BL 23 area and adrenal gland in the same rat respectively. Three days after injection, the dorsal root ganglions (DRG), sympathetic chain, and spinal cord were dissected out from the perfused rats. The neuronal labeling with AF 488/594-CTB was directly observed on the sections under a fluorescent microscope or a laser scanning confocal microscope. RESULTS: All neural labeling was observed in the injection side. The sensory neurons associated with both acupoint BL 23 and adrenal gland distributed from thoracic (T) 10 to lumbar (L) 2 DRG with high concentration in T 12-T 13 and T 11-T 12, respectively, in which some of them were simultaneously labeled with both AF 488/594-CTB and located in T 12-L 1 DRG. For the sympathetic innervation, the postganglionic neurons correlated with BL 23 and adrenal gland were labeled with AF 488/594-CTB separately in the sympathetic chain at the lumbar segments, while the labeled preganglionic neurons were only observed at the lateral horn of T 11-T 13 spinal segments in the cases of adrenal gland. In addition, the labeled motor neurons were mainly detected in the spinal ventral horn at cervical (C) 7-C 8 and T 11-L 1 segments. CONCLUSION: These results indicate that there are segmental correlation between BL 23 and adrenal gland on the sensory and sympathetic innervations, and this correlation might be a neural pathway for modulating the function role of adrenal gland through BL 23 needling.

[Distribution of Substance P-positive Nerve Fibers in Local Skin Tissues of Five Shu-points of the Pericardium Meridian in the Rat].

OBJECTIVE: To investigate the distribution of substance P (SP)-positive nerve fibers in local skin tissues of the five shu-points of the Pericardium Meridian in rats for revealing the differential innervations of the five acupoints. METHODS: Six adult male SD rats were used in the present study. Under deep anesthesia, the rats received transcardiac perfusion with 4% polyformaldehyde+phosphate buffered solution, followed by dissecting the local skin tissues from the sites corresponding to five shu-points "Zhongchong" (PC 9), "Laogong" (PC 8), "Daling" (PC 7), "Jianshi" (PC 5), and "Quze" (PC 3) in the human body. The tissues were sagittally cut into sections (20 µm) using a freezing microtome, followed by fluorescent immunohistochemi-cal and histochemical staining with SP, phalloidin and DAPI, respectively. The distribution of SP-positive fibers were observed under fluorescent microscope and laser confocal microscope. RESULTS: In general, SP expressed on the thin sensory fibers in the skin tissue. SP-positive nerve fibers distributed in a free single style in the epidermis and dermis, and gathered together to form a bundle in the subcutaneous layer. After counting the number and length of free single fibers in the epidermis and dermis of each of the five shu-points, we found that no significant differences existed in the density between the PC 9 and PC 8 regions, as well as among the PC 7, PC 5, and PC 3 regions (P>0.05). In contrast, the density of SP-positive nerve fibers in the former two acupoints (PC 9 and PC 8) was significantly higher than that of anyone of the latter three acupoints (P<0.01). In addition, SP positive nerve fibers mainly ran in parallel to the vessel-like structure. CONCLUSIONS: The local skin tissues of acupoint PC 9, PC 8, PC 7, PC 5, and PC 3 of the Pericardium Meridian express SP-positive fibers, which presents a decreasing tendency in the density from the distal to the proximal part of the forelimb, reflecting a relative specificity of the innervations of the five shu-points.

Callicarpa japonica Thunb. attenuates cigarette smoke-induced neutrophil inflammation and mucus secretion.

ETHNOPHARMACOLOGICAL RELEVANCE: Callicarpa japonica Thunb. (CJT) is traditionally used as an herbal remedy for the treatment of inflammatory diseases in Korea, China, and Japan. In this study, we evaluated the effects of C. japonica Thunb. (CJT) on the development of COPD using a Cigarette smoke (CS)-induced murine model and cigarette smoke condensate (CSC)-stimulated H292 cells, human pulmonary mucoepidermoid cell line. MATERIAL AND METHODS: C. japonica Thunb. was isolated from the leaves and stem of C. japonica. The methanol extract profile was obtained by UPLC Q-TOF-MS analysis. In in vivo experiment, the mice received 1h of cigarette smoke for 10 days. C. japonica Thunb. was administered to mice by oral gavage 1h before cigarette smoke exposure for 10 days. In in vitro experiment, we evaluated the effect of C. japonica Thunb. on the expression of MUC5AC and proinflammatory cytokines in H292 cells stimulated with CSC. RESULTS: CJT treatment effectively suppressed the infiltration of neutrophils, and decreased the production of ROS and the activity of neutrophil elastase in the bronchoalveolar lavage fluid (BALF) induced by CS. CJT also significantly attenuated production of proinflammatory cytokines such as IL-6 and TNF-α in the BALF, and reduced the infiltration of inflammatory cells and the production of mucus in lung tissue induced by CS. In in vitro experiments, CJT decreased the expression of MUC5AC and proinflammatory cytokines in CSC-stimulated H292 cells. Furthermore, CJT attenuated the phosphorylation of ERK induced by CSC in H292 cells. Taken together, CJT effectively reduced the neutrophil airway inflammation and mucus secretion induced by CS in murine model, and inhibited the expression of MUC5AC in CSC-stimulated H292 human lung cell line. These findings suggest that CJT has a therapeutic potential for the treatment of COPD.

[The Expression of Substance P in Sensory Neurons and Nerve Fibers Associated with "Sanyinjiao" (SP 6) Region in the Rat].

OBJECTIVE: To explore the innervation and biochemical characteristics associated with acupoint "Sanyinjiao" (SP 6) in the rat by using neural tracing and immunofluorescence techniques. METHODS: Five male SD rats were used in the present study. Alexa Fluor 488 conjugated cholera toxin subunit B (AF 488-CTB, 5 L, 1%) was injected into the site superior to the prominence of the medial malleolus, a corresponding site to acupoint SP 6 in the human body. After 3 surviving days, the rat's local tissue of SP 6 region, spinal cord and dorsal root ganglia (DRGs) at lumbar (L) segments (L 1 - S 1) were dissected following perfusion with 4% paraformaldehyde, cut into sections (20 µm for DRG and SP 6, and 40 µm for spinal cord) and observed under light microscope. In addition, substance P (SP) immunoreaction in the spinal cord, DRG and SP 6 region tissues was examined with immunofluorescence. RESULTS: All the labeling of AF 488-CTB appeared on the ipsilateral side of the injection. The labeled sensory and motor neurons distributed in L 3 - L 6 DRGs and spinal ventral horns with high concentration in the L 4 and L 5 segments. In addition, SP-positive neural labeling was found in small sensory neurons in DRG, axonal terminals in lamina I - II of spinal dorsal horns, and free nerve fibers in local subcutaneous tissues. Additionally, 24. 1 % of AF 488-CTB labeled sensory neurons simultaneously presented a SP-positive expression. CONCLUSION: These findings reveal that the sensory and motor neurons associated with acupoint SP 6 distribute in L 3- L 6 DRGs and spinal ventral horns of rats, concentrating in the L 4 and L 5 segments, and 24.1% sensory neurons show SP-positive, suggesting a possible involvement of the primary SP-positive sensory neurons in the transmission of acupuncture stimulation signals.

Supplementation of the maternal diet during pregnancy with chocolate and fructose interacts with the high-fat diet of the young to facilitate the onset of metabolic disorders in rat offspring.

Obesity and non-alcoholic fatty liver disease are the most common metabolic disorders in society today. Previously, we found that supplementing the maternal diet during pregnancy with chocolate and fructose has negative effects on the well-being of the offspring that were ameliorated if the offspring were fed a normal diet during postnatal life. In the present study, we investigated whether feeding offspring a high-fat diet would augment the maternal programming effects and whether extra protein supply can correct the low birth weight resulting from the chocolate-supplemented maternal diet. Pregnant Sprague-Dawley rats were divided into three groups and fed either standard chow (normal nutrition; NN), chocolate- and fructose-supplemented standard chow with casein sodium (overnutrition; ON) or the supplemented standard chow without casein sodium (malnutrition; MN) throughout pregnancy. Male offspring were weaned on either standard or high-fat chow. Dams in the MN group exhibited moderate weight gain, consumed 50% less protein (P < 0.001) but more carbohydrates during gestation and delivered pups with a 12% lower birth weight (P < 0.05) than pups in the NN group, results that are consistent with previous findings. When fed on a high-fat diet after birth, pups from dams in the MN group (MNHD) had 30% more body fat (P = 0.023) and liver triglyceride (TG) levels that were double (P < 0.01) those in offspring in the other groups, leading to fatty livers in these offspring at 14 weeks of age. Hepatic expression of the PPARα, ApoB100, MTTP, CPT1 and SREBP1c genes was significantly downregulated in the MNHD group (P < 0.05 for all), indicating changes in lipid metabolism. Although dams in the ON group exhibited marked gestational weight gain (P < 0.01), they gave birth to normal weight pups that only manifested mild increases in body fat and liver TG content (P < 0.05), without significant changes in the expression of most genes when fed with the high-fat diet. The results suggest that the extra protein supply in the form of casein sodium was able to correct some negative programming effects of the chocolate and fructose supplementation of the maternal diet, which, in conjunction with a high-fat diet in the offspring, may facilitate the onset of metabolic disorders, with impaired liver gene expression possibly a key contributor.

Effects of a maternal diet supplemented with chocolate and fructose beverage during gestation and lactation on rat dams and their offspring.

1. Consumption of a high-fat and high-energy diet during pregnancy leads to a risk of long-term consequences on fetal development, as well as on the postnatal health of offspring. To investigate the effects of such a diet on fetal programming, we established a high-energy intake pregnant rat model using chocolate and fructose beverage as supplements to a normal chow diet. 2. Pregnant Sprague-Dawley rats were assigned to either chow (control) or a diet supplemented with chocolate and fructose beverage throughout gestation and lactation. The male F(1) pups received normal chow diet after weaning. Physiological or pathological changes in dams and pups (e.g. glucose and lipid metabolism) were evaluated. 3. The results showed that dams offered the high-fat (mainly from chocolate) and high-calorie diet during gestation consumed more energy and gained more weight than chow-fed dams. Over-consumption of chocolate reduced chow intake in dams, leading to low maternal protein supply. As a result, pups from these dams exhibited reduced birth weight that lasted until adulthood. The high-energy diet during lactation led to increased total body fat, as well as impaired liver function, in offspring; thus, the lactational diet is suggested to be a stronger determinant of offspring fat metabolism than gestational diet. 4. The results of the study suggest that over-supply of carbohydrates, such as chocolate and fructose, either during gestation or lactation has a negative impact on the well-being of offspring.