Self-Assembling Sulfated Lactobacillus Exopolysaccharide Nanoparticles as Adjuvants for SARS-CoV-2 Subunit Vaccine Elicit Potent Humoral and Cellular Immune Responses.

Coronavirus disease 2019 (COVID-19) has caused a global pandemic since its onset in 2019, and the development of effective vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to induce potent and long-lasting immunity remains a priority. Herein, we prepared two Lactobacillus exopolysaccharide (EPS) nanoparticle adjuvants (NPs 7-4 and NPs 8-2) that were constructed by using sulfation-modified EPS and quaternization-modified chitosan. These two NPs displayed a spherical morphology with sizes of 39 and 47 nm. Furthermore, the zeta potentials of NPs 7-4 and NPs 8-2 were 50.40 and 44.40 mV, respectively. In vitro assays demonstrated that NPs could effectively adsorb antigenic proteins and exhibited a sustained release effect. Mouse immunization tests showed that the NPs induced the expression of cytokines and chemokines at the injection site and promoted the uptake of antigenic proteins by macrophages. Mechanically, the NPs upregulated the expression of pattern recognition receptors (toll-like receptors and nod-like receptors) and activated the immune response of T cells and the production of neutralizing antibodies. In addition, the NP adjuvants had favorable immune-enhancing effects in cats, which are of great significance for controlling the trans-host transmission and re-endemicity of SARS-CoV-2. Overall, we demonstrated that NP-adjuvanted SARS-CoV-2 receptor binding domain proteins could induce robust specific humoral and cellular immunity.

Assessment of the Potential of Sarcandra glabra (Thunb.) Nakai. in Treating Ethanol-Induced Gastric Ulcer in Rats Based on Metabolomics and Network Analysis.

Gastric ulcer (GU) is one of the most commonly diagnosed diseases worldwide, threatening human health and seriously affecting quality of life. Reports have shown that the Chinese herbal medicine Sarcandra glabra (Thunb.) Nakai (SGN) can treat GU. However, its pharmacological effects deserve further validation; in addition, its mechanism of action is unclear. An acute gastric ulcer (AGU) rat model induced by alcohol was used to evaluate the gastroprotective effect of SGN by analysis of the histopathological changes in stomach tissue and related cytokine levels; the potential mechanisms of action of SGN were investigated via serum metabolomics and network pharmacology. Differential metabolites of rat serum were identified by metabolomics and the metabolic pathways of the identified metabolites were enriched via MetaboAnalyst. Furthermore, the critical ingredients and candidate targets of SGN anti-AGU were elucidated. A compound-reaction-enzyme-gene network was established using Cytoscape version 3.8.2 based on integrated analysis of metabolomics and network pharmacology. Finally, molecular docking was applied to verify the acquired key targets. The results showed that SGN exerted a certain gastroprotective effect via multiple pathways and targets. The effects of SGN were mainly caused by the key active ingredients isofraxidin, rosmarinic, and caffeic acid, which regulate hub targets, such as PTGS2, MAPK1, and KDR, which maintain the homeostasis of related metabolites. Signal pathways involved energy metabolism as well as immune and amino acid metabolism. Overall, the multi-omics techniques were proven to be promising tools in illuminating the mechanism of action of SGN in protecting against diseases. This integrated strategy provides a basis for further research and clinical application of SGN.

Research on the effect and underlying molecular mechanism of Cangzhu in the treatment of gouty arthritis.

OBJECTIVE: We aimed to identify the active ingredients and elucidate the underlying mechanism of action of Atractylodes lancea (Thunb.) DC (namely, Cangzhu) for the treatment of gouty arthritis (GA) based on network pharmacology methods. These findings are expected to provide a theoretical basis for the clinical treatment of GA. METHODS: We used monosodium urate (MSU)-induced GA rats as a model to test the overall efficacy of Cangzhu in vivo. Then, the components of the Cangzhu decoction were analyzed and identified, and we screened the active ingredients and their targets. The GA disease targets were predicted by GeneCards and Disgenet databases and found to overlap in both databases. The STRING database was used to construct a protein-protein interaction network, followed by identification of the hub genes using Network Analyzer. Thereafter, Cytoscape software (version 3.8.2) was applied to construct a network for drug-active ingredient-key targets. Next, we applied cluego, a plug-in of Cytoscape, to perform gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analyses. Additionally, molecular docking was used to verify the characteristics of the key candidate components interacting with the hub therapeutic targets. Finally, we established an inflammatory injury model of LPS using RAW264.7 macrophages and used it to experimentally validate the critical active ingredients. RESULTS: Cangzhu effectively protected against gouty arthritis in vivo, and network pharmacology results revealed various active ingredients in Cangzhu, such as wogonin, atractylenolide I and atractylenolide II. These compounds were found to act on 16 hub targets, including tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), interleukin-1ß (IL-1ß), prostaglandin-endoperoxide synthase 2 (PTGS2), recombinant mitogen-activated protein kinase 14 (MAPK14) and transcription factor p65 (RELA), which have significant effects on regulating inflammatory factors and apoptosis-related pathways to improve the proinflammatory or anti-inflammatory imbalance in the body, and this may be one of the underlying mechanisms of Cangzhu in anti-GA. CONCLUSION: Our findings revealed that Cangzhu comprises multiple active components that exert various targeted effects during GA treatment. These findings provide relevant insights to illuminate the mechanism of Cangzhu in the treatment of GA and provide a reference for further experimental research.

Sargassum fusiforme Fucoidan Alleviates High-Fat Diet-Induced Obesity and Insulin Resistance Associated with the Improvement of Hepatic Oxidative Stress and Gut Microbiota Profile.

Sargassum fusiforme fucoidan (SFF) exhibits diverse biological activities. Insulin resistance (IR) implicated in type 2 diabetes (T2D) has become an epidemic health issue worldwide. In this study, we investigated whether SFF can improve insulin sensitivity in high-fat diet (HFD)-fed mice. Our present data showed that SFF significantly reduced fasting blood glucose and IR index along with improved glucose tolerance. Impaired phosphorylation of Akt was also restored by SFF. Furthermore, SFF decreased the levels of MDA and 4-HNE-modified protein and increased GSH/GSSG ratio as well as elevated antioxidant enzymes and activated Nrf2 signaling. SFF also increased the abundance and diversity of gut microbiota in the obese mice, as well as improved intestinal integrity and inflammation. Our findings suggested that SFF ameliorated HFD-induced IR through activating the Nrf2 pathway, remodeling gut microbiota, and reducing intestinal inflammation, thus providing a novel perspective into the treatment strategy on metabolic disease.

Joint replenishment of zinc and folic acid enhances the anti-depressive effect of paroxetine via increasing serum calcium and copper and decreasing serum arsenic.

Insufficient zinc and folic acid levels are associated with depression and poor response to antidepressants. This study aimed to investigate the influences of combined zinc and folic acid replenishment on the anti-depressive effect of paroxetine. Male rats were randomly divided into five groups: control (C), model (M), paroxetine (MP), zinc + folic acid (MZnF), and zinc + folic acid + paroxetine (MZnFP) groups. Rats were exposed to mild unpredictable stress for 3 weeks as a depression model. The combinations of drug and supplements were applied via daily gavage for 4 weeks. The open field test was conducted to observe behavioral changes. A chemiluminescence method was used to detect folacin, and inductively coupled plasma mass spectrometry was used to detect serum elements. Supplementation of zinc and folic acid significantly improved behavior responses to paroxetine, including movement speed, total distance, and central zone frequency. In addition, higher calcium and copper levels and a lower arsenic level were found in the serum of the MZnFP group. Thus, supplementation of zinc and folacin can enhance the anti-depressive effect of paroxetine, and the mechanism is potentially related to the improved levels of calcium and copper and a reduced level of arsenic.

[Electroacupuncture relieved neuropathic pain by suppressing L-Arg/NO/cGMP pathway in the lumbar spinal cord in rats with spared nerve injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on changes of expression of L-Arg transporter 2 (CAT-2) mRNA and nitric oxide synthase (iNOS) mRNA and protein and contents of NO and cGMP of L4-L6 segments of spinal cord in rats with spared nerve injury (SNI), so as to reveal its mechanism underlying reducing neuropathic pain. METHODS: A total of 120 male SD rats were randomly divided into sham operation, model, EA and NOS inhibitor (N omega-Nitro-L-arginine methyl ester hydrochloride, L-NAME) groups, with 30 rats in each group. The neuropathic pain model was established by ligating and cutting the tibial nerve and the common peroneal nerve. EA (2 Hz, 1-3 mA) was applied to "Weizhong" (BL40) and "Huantiao" (GB30)on the damaged hindlimb for 30 min, once daily from day 11 to 17 after SNI. Rats of the L-NAME group received i.p. of L-NAME (60 mg·kg-1·d-1) for 7 consecutive days. The mechanical pain threshold (PT) was determined before and 10 and 16 d after SNI, respectively. The expression le-vels of CAT-2 mRNA and iNOS mRNA, and iNOS protein in the L4-L6 segments of the spinal cord were detected by using reverse transcription - polymerase chain reaction (RT-PCR) and Western blot, respectively, and the contents of NO and cGMP of L4-L6 assayed using nitrate/nitrite reductase method and radioimmunoassay, respectively. RESULTS: After modeling, the PT was significantly decreased on day 10 and 16 after SNI in comparison with the sham operation group and their own baseline data of pre-operation in each group (P<0.01), and remarkably increased in the EA and L-NAME groups relevant to the model group on day 16 (P<0.01, P<0.05). Compared with the sham operation group, the expression levels of CAT-2 mRNA and iNOS mRNA and protein, as well as the contents of NO2－/NO3－and cGMP were signi-ficantly up-regulated in the model group (P<0.05, P<0.01). Following EA intervention, the levels of CAT-2 mRNA and iNOS mRNA and iNOS protein, and NO2－/NO3－and cGMP contents were all reversed in both EA and L-NAME groups (P<0.05, P<0.01). The effect of EA was significantly superior to that of L-NAME in raising the PT on day 16 after SNI (P<0.05), but obviously inferior to that of L-NAME in down-regulating the expression of CAT-2 mRNA and iNOS mRNA and protein (P<0.05). No significant differences were found between the EA and L-NAME groups in down-regulating NO2－/NO3－ andcGMP contents (P>0.05). CONCLUSION: EA intervention can effectively relieve neuropathic pain in SNI rats, which may be closely related to its function in suppressing L-Arg/NO/cGMP pathway in the lumbar spinal cord.

Antitumor bioactivity of porphyran extracted from Pyropia yezoensis Chonsoo2 on human cancer cell lines.

BACKGROUND: Pyropia yezoensis, rich in porphyran, is a medicine-edible red alga. In the present study, the physicochemical characteristics, conformational states and antitumor activities of a novel porphyran extracted from the high-yield algal strain Pyropia yezoensis Chonsoo2 and its two degraded derivatives by gamma irradiation were investigated. RESULTS: Pyropia yezoensis porphyran is a water-soluble, triple-helical sulfated hetero-galactopyranose, named PYP. PYP was degraded by gamma irradiation at 20 kGy and 50 kGy, giving two low molecular weight derivatives comprising PYP-20 and PYP-50, respectively. PYP with a higher molecular weight has a solution conformation different from PYP-20 and PYP-50. Three porphyrans had no toxicity in normal human liver cells (HL-7702) and showed antitumor effects on Hep3B, HeLa and MDA-MB-231. They had better antitumor against HeLa cells, exhibiting a similar inhibition ratio compared to 5-fluorouracil, with PYP especially exhibiting a higher inhibition ratio than 5-fluorouracil. With respect to HeLa cells, the different antitumor activities might be related to porphyran molecular weight and solution conformation. Furthermore, the HeLa cell cycle was blocked in the G2/M phase after PYP treatment, leading to cell proliferation inhibition. The induction of cell cycle arrest was related to the changes in the expression of p21, p53, Cyclin B1 and cyclin-dependent kinase 1. CONCLUSION: Pyropia yezoensis porphyran, as applied to medicine and functional food, could potentially be used as a non-toxic natural adjuvant in cancer therapy. © 2019 Society of Chemical Industry.

[Effect of Electroacupuncture of "Weizhong" (BL 40) and "Huantiao" (GB 30) on cAMP-PKA-CREB Signaling of Spinal Cord in Rats with Neuropathic Pain].

OBJECTIVE: We have demonstrated that electroacupuncture (EA) of "Weizhong" (BL 40) and "Huantiao" (GB 30) could evidently relieve mechanical hyperalgesia in spared nerve injury (SNI) rats. The present study was designed to observe the effect of EA on levels of cAMP, and protein kinase A (PKA) and cAMP response element binding protein (CREB) in the lumbar spinal cord of the same pain model rats, so as to explore its mechanisms underlying improvement of neuropathic pain. METHODS: One hundred male SD rats were randomly divided into 5 groups: sham operation, model, EA, AP-5 and L-NAME groups (n＝20 in each group). The sham operation group underwent a simple separation of the sciatic nerve but without ligation and abscission. The neuropathic pain model was established by abscission of the right tibial and common peroneal nerve. EA (2 Hz, 1－3 mA) was applied to right BL 40 and GB 30 for 30 min, once a day for 7 days, starting from day 11 after surgery. For rats of the AP-5 and L-NAME groups, AP-5 (a competitive antagonist for NMDA receptor, 0.7 mg•kg－1•d－1) and L-NAME (a non-selective antagonist for nitric oxide synthase, NOS, 60 mg•kg－1•d－1) were respectively administrated by intraperitoneal injection, once daily for 7 days. The mechanical pain threshold was measured, the cAMP content of spinal cord (L 4－L 6) was determined by radioimmunoassay, and the expression of PKA, p-PKA and CREB proteins of spinal cord (L 4－L 6) was detected by Western blot. RESULTS: The content of cAMP and the expression levels of PKA, p-PKA and CREB were significantly higher in the model group than in the sham operation group (P<0.01), and considerably lower in the EA group than in the model group (P<0.01, P<0.05). Compared to the model group, the expression levels of PKA and p-PKA were significantly decreased in the AP-5 and L-NAME groups (P<0.01, P<0.05). Compared to the EA group, the content of cAMP and the expression levels of CREB were significantly higher (P<0.05), and the expression of p-PKA was significantly lower in the AP-5 and L-NAME groups (P<0.05)．. CONCLUSION: EA intervention-induced down-regulation of cAMP, PKA and CREB levels in the lumbar spinal cord may contribute to its analgesic effect in neuropathic pain rats, suggesting an involvement of reduction of cAMP/PKA/CREB signaling of spinal cord in EA analgesia.

[Effect of Electroacupuncture on Intracellular Calcium Ion Concentration and Expression of Calcium/Calmodulin Dependent Protein Kinase â ¡ in Lumbar Spinal Cord in Rats with Neuropathic Pain].

OBJECTIVE: To observe the changes of intracellular calcium ([Ca2＋]i) concentration and expression of calcium/calmodulin dependent protein kinaseⅡ (CaMKⅡ) in spinal dorsal horn neurons of spared nerve injury (SNI) rats, so as to explore its mechanisms underlying improvement of neuropathic pain. METHODS: One hundred and ten SD rats were randomly divided into 5 groups: sham control, model, EA, AP-5 and L-NAME groups. The sham group underwent only a simple separation of the sciatic nerve but without ligation and abscission. The neuropathic pain model was established by abscission of the right tibial and common peroneal nerve. EA (2 Hz, 1－3 mA) was applied to right "Weizhong" (BL 40) and "Huantiao" (GB 30) for 30 min, once a day for 7 days, starting from day 11 after surgery. For rats of the AP-5 and L-NAME groups, AP-5 (a competitive antagonist for NMDA receptor, 0.7 mg·kg－1·d－1) and L-NAME (a non-selective antagonist for nitric oxide synthase [NOS], 60 mg·kg－1·d－1) were respectively administrated by intraperitoneal injection, once daily for 7 days. The mechanical pain threshold was measured, and the calcium fluorescence intensity (shown by Fluo-3/AM calcium fluorescence indicator) of the superficial layer of the lumbar spinal cord (L 4－L 6) was measured by immunohistochemical staining and the expression of spinal cord (L 4－L 6) CaMK Ⅱ protein was detected by Western blot (WB). RESULTS: After modeling, the mechanical pain threshold was significantly decreased on day 10 and 16 after operation in comparison with the sham operation group and baseline data of pre-operation in each group (P<0.01), and remarkably increased in the EA, AP-5 and L-NAME groups relevant to the model group on day 16 (P<0.01, P<0.05), while the effect of EA was significantly superior to that of AP-5 and L-NAME groups (P<0.05), suggesting a reduction of EA analgesia after administration of AP-5 and L-NAME. The concentration of intracellular [Ca2＋]i was significantly higher in the model group than in the sham group, and considerably lower in the EA, AP-5 and L-NAME groups than in the model group (P<0.01, P<0.05). Moreover, the expression level of CaMKⅡ shown by WB and immunohistochemical staining was significantly higher in the model group than in the sham group (P<0.05) and obviously lower in the EA group (not the AP-5 and L-NAME groups) than in the model group on day 16 after the intervention (P<0.05). It suggests an involvement of glutamate NMDA receptor and NMDAR-NOS/NO signaling in the analgesic effect and CaMKⅡ expression down-regulation of EA. CONCLUSIONS: EA can ease pain in rats with neuropathic pain, which is closely related to its effect in reducing the calcium concentration and the expression of CaMKⅡ in the lumbar spinal cord, possibly mediated by glutamate NMDA receptor and NMDAR-NOS/NO signaling.

[Effect of Electroacupuncture Intervention on CaMK II-CREB Pathway in Spinal Cord in Rats with Spared Nerve Injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of "Weizhong" (BL 40)-"Huantiao" (GB 30) on expression of phosphorylated calcium/calmodulin dependent protein kinase II (p-CaMK II) and cAMP response element binding protein (p-CREB) in the spinal cord in rats with spared nerve injury (SNI), so as to explore its mechanism underlying easing neuropathic pain. METHODS: Sixty SD rats were randomly divided into five groups: control (sham-operation) , model, EA, AP-5 (a NMDA receptor antagonist) and L-NAME (a non-selective nitric oxide synthase, NOS inhibitor) (n = 12 in each group). The neuropathic pain model was established by sectioning the right tibal nerve and common peroneal nerve. EA intervention (2 Hz, 1 mA, increasing 1 mA/10 min) was applied to "Weizhong" (BL 40) and "Huantiao" (GB 30) on the injured side for 30 min, once a day for 7 days. Rats of the AP-5 and L-NAME groups were treated by intragastric administration of AP-5 (0.7 mg · kg(-1) · d(-1)) and L-NAME (60 mg · kg(-1) · d(-1)) respectively from the 11 th day after operation, once daily for 7 days. The mechanical pain thresholds were measured before the SNI procedure (baseline) and at the 10th and 16th day after the procedure. The expression of p-CaMK II protein and p-CREB protein and gene of the spinal cord (L4-L6 segments) was determined by Western blot and fluorescence quantitative-polymerase chain reaction (PCR), separately. RESULTS: In comparison to the control group, the mechanical pain threshold was significantly decreased in the model group (P < 0.01). After EA intervention, the mechanical pain thresholds of the EA, AP-5 and L-NAME groups were obviously increased (P < 0.01, P < 0.05) on day 16 post SNI procedure. The expression levels of p-CaMK II and p-CREB proteins and CREB mRNA in the spinal cord were significantly higher in the model group than in the control group (P < 0.05). Compared with the model group, the expression levels of spinal p-CaMK II and p-CREB proteins and CREB mRNA were obviously down-regulated in the EA group (P < 0.05), but not in the AP-5 group and the L-NAME group (P > 0.055. CONCLUSION: EA intervention of BL 40-GB 30 may alleviate pain in neuropathic pain rats, which may be related to its effects in down-regulating spinal CaMK II-CREB pathway function.

[Effect of electroacupuncture intervention on N-methyl-D-aspartic acid receptor expression in spinal cord in rats with chronic constrictive injury of the sciatic nerve].

UNLABELLED: OBJECTIVE; To observe the changes of N-methyl-D-aspartic acid (NMDA) receptor expression of spinal cord after electroacupuncture (EA) intervention in rats with chronic constrictive injury (CCI) of the sciatic nerve so as to reveal the mechanism underlying improvement of neuropathic pain. METHODS: Sixty male SD rats were randomly divided into sham operation (sham) group, CCI model group and EA group (n = 20). CCI model was established by ligature of the right sciatic nerve with a piece of surgical chromic suture. For rats of the sham group, the right sciatic nerve was just isolated without ligature. The rats of the EA group were given with EA stimulation of "Weizhong" (BL 40) and "Huantiao" (GB 30) on the injured side at a frequency of 2 Hz, electric current of 1-3 mA for 30 min (increasing 1 mA every 10 min). The treatment was conducted once a day from the 11th day to the 20th day after modeling. NMDA receptor 2 B subunit (NR 2 B) protein and mRNA expression levels in the spinal cord were determined by immunohistochemistry, Western blot and reverse transcription (RT)-polymerase chain reaction (PCR), respectively, and spinal NMDA receptor subunit 1 (NR 1) protein and mRNA expression levels were measured by Western blot and RT-PCR, respectively. RESULTS: In comparison with the sham group, NR 1 protein and mRNA expression levels of the model group in the spinal cord were considerably upregulated after CCI (P < 0.01, P < 0.05). In comparison with the model group NR 1 protein and mRNA expression levels of the EA group in the spinal cord were evidently down-regulated (P < 0.05). No significant changes of NR 2 B protein and mRNA expression after CCI and EA stimulation were found after immunohistochemistry, Western blot and RT-PCR measurements (P > 0.05). CONCLUSION: EA intervention is effective in alleviating neuropathic pain in CCI rats, which may be closely related to its effects in lowering functional activity of NR 1 protein and mRNA in the spinal cord.

[Effects of electroacupuncture at acupoints "changbingfang" on serum IL-4 content and colonic NF-kappaB p65 expression in rats with ulcerative colitis].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Zhongwan"(CV 12), "Tianshu"(ST 20) and "Shangjuxu"(ST 37) (an acupoint prescription "Changbingfang" for treatment of intestinal disorders) on the expression of colonic nuclear factor kappaB p65 (NF-kappaB p65) protein expression and serum interleukin-4 (IL-4) content in rats, so as to explore its mechanism underlying improvement of ulcerative colitis (UC). METHODS: Thirty SD rats were randomly and equally divided into normal control, model and EA groups (10 rats/group). The UC model was made by enema with 8% acetic acid. EA stimulation (6 Hz/30 Hz) was delivered to "Zhongwan" (CV 12), "Tianshu" (ST 20) and "Shangjuxu" (ST 37) for 15 min separately, 0 h, 24 h and 48 h post-enema. The blood and colon tissues were taken for detection of serum IL-4 content with ELISA, and for assaying colonic NF-kappaB p65 protein expression with immunohistochemistry and Western blot, respectively. RESULTS: Compared with the normal control group, colonic NF-kappaB p65 protein expression level shown by immunohistochemistry and Western blot and serum IL-4 content were significantly higher in the model group (P < 0.01, P < 0.05). After EA stimulation, colonic NF-kappaB p65 protein expression levels shown by both immunohistochemistry and Western blot were down-regulated significantly (P < 0.01, P < 0.05) and serum IL-4 content was up-regulated further in comparison with the model group (P < 0.01). CONCLUSION: EA of acupoint recipe "Changbingfang" can down-regulate the proinflammatory NF-kappaB p65 protein expression and up-regulate serum anti-inflammatory cytokine IL-4 level in rats, which may contribute to its effect in improving ulcerative colitis.

[Effect of electroacupuncture on expression of spinal neuronal nitric oxide synthase protein and mRNA in rats with neuropathic pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on expression of neuronal nitric oxide synthase (nNOS) protein and mRNA in the spinal cord in rats with chronic constrictive injury (CCI) of the sciatic nerve so as to explore its mechanisms underlying pain relief. METHODS: Thirty-six male SD rats were randomly divided into sham operation (control) group (n = 12), CCI model group (n = 12) and EA group (n = 12). EA was applied to bilateral "Weizhong" (BL 40) and "Huan-tiao" (GB 30) on the injured side for 30 min, once a day for 7 days (from the 11th day on after CCI). The mechanical and thermal pain thresholds were measured before and after CCI. Spinal nNOS protein and mRNA expression levels were detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR), respectively. RESULTS: In comparison with the control group, both mechanical and thermal pain thresholds were significantly lower in the model group (P < 0.01). Compared with the model group, the two types of pain threshold were increased considerably on the 16th day in the EA group (P < 0.01). In comparison with the control group, the spinal nNOS protein and mRNA expression levels were increased significantly in the model group (P < 0.05, P < 0.01). In comparison with the model group, both nNOS protein and mRNA expression levels in the spinal cord were down-regulated obviously in the EA group (P < 0.05). CONCLUSION: Electroacupuncture can alleviate neuropathic pain in CCI rats, which is closely related to its effect in down-regulating the expression of nNOS protein and mRNA in the spinal cord.

[Selection of basic acupoints combined with "enteral-disorder-formuia" for electroacupuncture treatment of intestinal mucosal lesion in rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of differently combined acupoints of "Zhongwan" (CV 12), "Tianshu" (ST 25) and "Shangjuxu" (ST 37, for treatment of enteral disorders) on acetic acid-induced intestinal mucosal lesion in rats so as to select the best combinations of acupoints. METHODS: A total of 126 SD rats were divided into nine groups: normal control (normal), model, CV 12, ST 25, ST 37, CV 12 + ST 37, ST 25 + ST 37, CV 12 + ST 25 and CV 12 + ST 25 + ST 37. Intestinal mucosal lesion model was established by using enema (8% acetic acid 1 mL). EA was applied to the above-mentioned acupoints for 15 min, once daily for 3 days. General conditions, colonic mucosal damage index (CMDI) and pathological damage integral (PDI) were detected respectively. The colonic mucosal membrane tissue was stained with H. E. stain for observing its morphological changes under light microscope, and the ultrastructural changes were observed using electron transmission microscope. RESULTS: Compared with the control group, CMDI was decreased significantly (P < 0.05) and PDI of the colonic mucosal membrane increased significantly in the model group (P < 0.05). Compared with the model group, CMDI was decreased markedly and PDI increased significantly (P < 0.05) in the CV 12, ST 25, ST 37, CV 12 + ST 37, ST 25 + ST 37, CV 12 + ST 25 and CV 12 + ST 25 + ST 37 groups (P < 0.05). Comparison among the 7 EA treatment groups showed that both CMDI and PDI of the CV 12 + ST 25 + ST 37 group were significantly superior to those of CV 12, ST 25, ST 37, CV 12 + ST 37, ST 25 + ST 37 and CV 12 + ST 25 groups (P < 0.05). Under light microscope, the colonic mucosal injury was remarkable, while those of the 7 EA treatment groups were relatively milder. Ultramicrostructural findings showed that the colonic mucosal lesion including shortening or sparseness of the intestinal epithelial villi, swelling of mitochondria with arrangement disorder, breaking or fusion of the cristae, and enlargement of the rough endocytoplasmic reticulum was found in the model group, while the situations were lighter in the 7 EA groups particularly in the CV 12 + ST 25 + ST 37 group. CONCLUSION: EA of CV 12, ST 25 and ST 37 acupoint alone or combination can relieve acetic acid-induced intestinal mucosal lesion in rats, and the effect of joint application of these 3 acupoints is obviously better, suggesting their clinical applicability in clinical treatment of intestinal disorders as a basic formula.

[Effect of electroacupuncture on the levels of amino acid neurotransmitters in the spinal cord in rats with chronic constrictive injury].

OBJECTIVE: To observe the changes of amino acid neurotransmitter contents in the lumbar spinal cord after electroacupuncture (EA) in rats with neuropathic pain so as to study its spinal mechanism underlying pain relief. METHODS: Forty SD rats were randomly divided into control, sham surgery, model and EA groups (n=10). Chronic constrictive injury (CCI) (neuropathic pain) model was duplicated by ligature of the right sciatic nerve with a piece of catgut. EA (1-3 mA, 2 Hz) was applied to "Huantiao" (GB 30) and "Weizhong" (BL 40) on the injured side for 30 minutes, once a day for 7 days. The mechanical and thermal pain thresholds were measured before and after CCI, and after EA intervention. Concentrations of glutamate (Glu), aspartate (Asp), glutamine (Gln), gamma-aminobutyric acid (GABA), Glycine (Gly) and taurine (Tau) in the lumbar spinal cord (L4-6) were detected by O-phthaladehyde derivatization + high performance liquid chromatography (HPLC). RESULTS: Compared with the control group, both the mechanical and thermal pain thresholds of the model group were decreased significantly on day 10 and day 16 after CCI (P < 0.01). Compared with the model group, the mechanical and thermal pain thresholds in the EA group on day 16 after CCI were upregulated strikingly (P < 0.01). In comparison with the control group, Glu, Asp, Gin and GABA levels in the lumbar spinal cord were significantly higher in the model group (P < 0.05, P < 0.01), while Gly and Tau levels in the lumbar spinal cord were markedly lower in the model group (P < 0.05). In comparison with the model group, spinal Glu, Asp, and Gin contents were downregulated significantly in the EA group (P < 0.01), while Gly, GABA and Tau levels were upregulated obviously (P < 0.01, P < 0.05). No significant differences were found between the control and sham surgery groups in the mechanical and thermal pain thresholds, and in the spinal Glu, Asp, GIn, GABA, Gly and Tau levels (P > 0.05). CONCLUSION: EA of GB 30 and BL 40 may alleviate neuropathic pain in CCI rats, which is closely with its effects in reducing the release of excitatory amino acids and promoting the release of inhibitory amino acid neurotransmitters.

[Effect of electroacupuncture on expression of ionotropic glutamate receptor subunits and their genes in lumbar segments of spinal cord in rats with neuropathic pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of ionotropic glutamate receptor (iGluR) subunits and their mRNAs in the lumbar segments of spinal cord in rats with neuropathic pain, so as to explore its underlying mechanism in relieving spinal hyperalgesia. METHODS: Thirty SD rats were randomly divided into control, model, and EA groups, with 10 rats in each. The spared nerve injury (SNI) model was established by ligature of the sural nerve after cutting off the common peroneal nerve and anterior tibial nerve. EA (2 Hz, 1 mA) was applied to "Huantiao" (GB 30) and "Weizhong" (BL 40) for 30 min, once daily for 7 days. Mechanical pain threshold was detected before and after modeling and before and after EA treatment. The expression levels of N-methyl-d-aspartic acid (NMDA) receptor subunits NR1 and NR 2 B,and AMPA receptor subunit GluR 1 of iGluR and their genes were assayed by Western blot and reverse transcription polymerase chain reaction (RT-PCR) separately. RESULTS: In comparison with control group, the mechanical pain thresholds were decreased significantly on day 2, 7 and day 14 following modeling in the model group (P < 0.05, P < 0.01). While compared with the model group, the pain threshold was increased considerably on day 14 in the EA group (P < 0.01). Compared with the control group, the expression levels of lumbar spinal cord NR 2 B and NR 2 B mRNA in the model group were increased significantly (P < 0.05), and those of lumbar spinal cord NR 1 and NR 1 mRNA, GluR 1 and GluR 1 mRNA in the model group increased slightly (P > 0.05). In comparison with the model group, the expression levels of lumbar spinal cord NR 2 B and NR 2 B mRNA in the EA group were downregulated remarkably (P < 0.05), and those of lumbar spinal cord NR 1 and NR 1 mRNA, GluR 1 and GluR 1 mRNA in the EA group down-regulated slightly (P > 0.05). CONCLUSION: EA can significantly suppress pain reaction in rats with neuropathic pain probably through down-regulating the expression of lumbar spinal cord NR 2 B protein and NR 2 B mRNA.

[Effects of electroacupuncture of "Neiguan" (PC 6), "Zhongwan" (CV 12) and "Zusanli" (ST 36) on electrical activities of neurons subnucleus reticularis dorsalis in rats].

OBJECTIVE: To compare the effects of electroacupuncture (EA) stimulation of "Gastric Problem Formula" ["Neiguan" (PC 6), "Zhongwan" (CV 12) and "Zusanli" (ST 36)] on electrical activities of neurons of medullary subnucleus reticularis dorsalis (SRD) of the rat. METHODS: Experiments were performed on SD rats anesthetized by 10% urethane (1.0-1.5 g/kg). Under artificial ventilation and immobilization (2% gallamine triethiodide, 5 ml/kg), extracelluar discharges were recorded by glass micropipettes inserted into SRD of the medulla (Caudal to the Obex: - 1.0 - -2.0 mm, L/R= 0.5-1.5 mm, H: 0.6- 1.5 mm below the medullary surface). Following getting stable recordings, the acupuncture needle inserted in "Neiguan" (PC 6) was manipulated moderately (twisting range: 120 degrees, frequency = 80 times/min) for 30 s. The same manipulation was performed at "Zhongwan" (CV 12) and "Zusanli" (ST 36) separately. RESULTS: After moderate acupuncture stimulation, in the recorded 13 SRD neurons, the discharges were increased from (1.67 +/- 0.35) spikes/s to (17.55 +/- 1.40) spikes/s for "Neiguan" (PC 6), from (2.35 +/- 0.43) spikes/s to (16.96 +/- 1.83) spikes/s for "Zhongwan" (CV 12), and from (1.83 +/- 0.37) spikes/s to (17.39 +/- 1.58) spikes/s for "Zusanli" (ST 36). No apparent differences were found among 3 acupoints in their responses to manual manipulation. CONCLUSION: Moderate acupuncture stimulation of "Neiguan" (PC 6), "Zhongwan" (CV 12) and "Zusanli" (ST 36) can effectively activate spontaneous discharges of SRD neurons in the rat.

[Effects of electroacupuncture on long-term potentiation of synaptic transmission in spinal dorsal horn in rats with neuropathic pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Huantiao" (GB 30) and "Weizhong" (BL 40) on the mechanical pain threshold and the long-term potentiation (LTP) of the spinal dorsal horn in rats with neuropathic pain. METHODS: Thirty SD rats were randomized into control (sham-operation), model and electroacupuncture (EA) groups, with 10 cases in each. The neuropathic pain model was established by ligature and transaction of the popliteal nerve and common peroneal nerve. EA (2 Hz, 1 mA) was applied to "Huantiao" (GB 30) and "Weizhong" (BL 40) on the affected side for 30 min, once daily for 7 days. The mechanical pain threshold of the paw was detected before and after modeling and EA. LTP of C-fiber stimulation (2 mA, 0.5 ms, 100 Hz, interval 10 s, 4 strains) evoked field potentials in spinal dorsal horn (L4/L5) was recorded in rats under anesthesia. RESULTS: Compared with control group, the pain threshold of model group reduced significantly from the 1st day on after ligature and transaction of the nerve (P<0.01), while 14 days after EA, it elevated evidently in comparison with model group (P<0.01), suggesting an increase of the mechanical pain threshold after EA. Comparison of the variance rates of C-fiber induced LTP of the field potential among the 3 groups showed that the variance rate of EA group was significantly lower than that of model group (P<0.01). Self-comparison of EA group showed that after EA, the variance rate of LTP reduced significantly (P<0.01). CONCLUSION: EA of "Huantiao" (GB 30) and "Weizhong" (BL 40) can significantly relieve neuropathic pain in rats with nerve injury, and inhibit C-fiber high-frequency-stimulation induced LTP due to central sensitization induced abnormality synaptic transmission.

[Selection of basic acupoints for composing "gastric-disorder-formula" for electroacupuncture prevention of acute gastric mucosal lesion in rats].

OBJECTIVE: To select the best combination of the commonly-used acupoints [Zhongwan (CV 12), Neiguan (PC 6), and Zusanli (ST 36)] for treatment of gastric disorders. METHODS: Forty-eight Wistar rats were randomly divided into 8 groups: model, ST36, CV12, PC6, ST36 + PC6, ST36 + CV12, CV12 + PC6 and ST36 + CV12 + PC6 by factorial design method. Acute gastric mucosal lesion model was established by intragastric perfusion of anhydrous alcohol. EA(1- 3 mA, 30 Hz) was applied to the above-mentioned acupoints for 20 min respectively. And after EA, gastric mucosal lesion index (LI), pathological lesion integral score (PLIS) were calculated and mucosal ultramicrostructural changes observed by optical microscope and transmission electron microscope respectively. RESULTS: Compared with model group, the gastric mucosal LI and PLIS of ST36, CV12, PC6, ST36 + CV12, ST36 + PC6, CV12 + PC6, ST36 + CV12 + PC6 groups all decreased significantly (P < 0.05). Comparison among 7 EA groups showed that both LI and PLIS of ST36 + CV12 + PC6 group were significantly lower than those of ST36, V12, PC6, ST36 + CV12, ST36 + PC6 and CV12 + PC6 groups (P < 0.05). No significant differences were found between every two groups of ST36, CV12, PC6, ST36+ CV12, ST36 + PC6 and CV12 + PC6 groups (P > 0.05) in both LI and PLIS. Ultrastructural results showed that the injury of gastric mucosal parietal cells and delomorphous cells including mitochondrial swelling, crista arrange disorder and breaking, enlargement of the rough endoplasmic reticulum, ribosome exfoliation, etc. in model group was serious, but lightened significantly in EA groups particularly in ST36 + CV12 + PC6 group. CONCLUSION: ST36, PC6 and CV12 combination can effectively reduce anhydrous alcohol-induced gastric mucosal lesion in rats, and may be used as a basic formula

[Effects of electroacupuncture at points "Weibingfang" on EGF and EGFR mRNA expressions in rats with lesion of gastric mucosa].

OBJECTIVE: To probe the mechanism of electroacupuncture at points "Weibingfang" in treatment of acute gastric mucosal lesion. METHODS: Forty Wistar rats of sanitary degree were randomly divided into 4 groups, normal group, model group, sham-model group and Weibingfang group, 10 rats in each group. The acute gastric mucosal lesion model was made by intragastric perfusion of anhydrous alcohol. The Weibingfang group were treated by electroacupuncture at "Neiguan" (PC 6), "Zhongwan" (CV 12) and "Zusanli" (ST 36) with sparse-dense wave, frequency of 10-30 Hz, current intensity of 2 mA, for 20 min. One hour after the treatment, the blood from the abdominal aorta and the gastric mucosa tissue were taken, and serum epidermal growth factor (EGF) level and epidermic growth factor receptor mRNA (EGFR mRNA) expression in the gastric mucosa were detected with enzyme linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) respectively. RESULTS: There were significant differences in blood EGF level (41.62 +/- 12.58) ng/L and EGFR mRNA expression (0.78 +/- 0.03) in the model group were significantly different from [(60.37 +/- 12.01) ng/L and 0.55 +/- 0.04] in the normal group and [(61.21 +/- 13.46) ng/L and 0.53 +/- 0.05] in the sham-model group (P < 0.05); after electroacupuncture, blood EGF level (70.59 +/- 10.14) ng/L increased and the EGFR mRNA expression (1.18 +/- 0.02) in the gastric mucosa was up-regulated with a significant differences as compared with those in the model group (P < 0.05). CONCLUSION: Point "Weibingfang" can promote proliferation, differentiation and migration of the gastric mucosal epidermic cells to repair the gastric mucosal lesion.