First Report of Achromobacter Xylosoxidans Causing Stem Soft Rot on Amorphophallus Konjac in China.

Amorphophallus konjac is one of the important commercial crops cultivated in south China and has long been used as a food source and a traditional medicine, because it is the only species with glucomannan and other trace elements (Ban et al. 2009; Melinda et al. 2010). In June of 2021, an outbreak of stem soft rot disease was observed on A. konjac plants in more than 2,000 square meters of agricultural planting fields in the Fuyuan country (25°34'50″N, 104°04'21″E), Qujing City, Yunnan Province, China. The disease incidence ranged from 30 to 35% in severely infested fields. The diseased plants displayed the first symptoms were damp brown spots. As the brown spots expanded, dark brown water stains appeared at the basal part of the stem and the bulbs were rotting with a foul smell, gradually extending to the underground parts. Progressively, the whole plants wilted and collapsed, and even the plants ultimately died. To identify the pathogen, symptomatic stems were cut into pieces, surface sterilized with 75% (v/v) ethanol, and placed on LB (tryptone/yeast extract/NaCl) medium for 24 to 48 hours at 28 ± 2°C. Six single-colony isolates were obtained from the diseased stems. The colonies on LB present a raised milky white opaque colonies moisture on the surface, round and convex in shape, with neat edges. Scanning electron microscopy showed that the cells were short rods (0.3∼0.5) × (1.9∼2.1)µm in size without any flagellum and were often arranged in pairs or clusters at certain angles. The 16S rDNA sequence of the randomly selected strain MY-G1 with primers 27F/1492R (Ying et al. 2012) and the housekeeping genes nusA, eno, lepA and nuoL (Spilker et al. 2012) were amplified and sequenced. The 16S rDNA sequence of the 1326 bp product was deposited in GenBank (accession no. ON786717) and showed a 99.77% similarity to A. xylosoxidans strain E2 (accession no. MK849863.1). The nusA (OP680477), eno (OP680479), lepA (OP680481) and nuoL (OP680482) sequences showed 94.71%, 97.24%, 94.64% and 95.95% similarity to A. xylosoxidans strain DN002 (accession no. CP045222.1), respectively. The phylogenetic trees built based on 16S rRNA and the nusA-eno-lepA-nuoL multilocus analysis showed the isolate MY-G1 to cluster with A. xylosoxidans. Based on morphological and molecular analysis, the isolated MY-G1 was identified as A. xylosoxidans, which indicates that MY-G1 is a new strain of A. xylosoxidans. Pathogenicity tests were confirmed on the stem and petiole of one-year-old A. konjac. The wounds were made by puncturing with a MY-G1 bacteria suspension containing 108 CFU/ml (15ul/inoculation site). As a negative control, control seedlings were injected with the same amount of sterilized distilled water. Control and inoculated seedlings (each six) were kept in greenhouses and watered as needed in controlled conditions: 28°C, 75% relative humidity. Inoculated seedlings presented similar symptoms of stem soft rot, inner medulla disintegration, and wilt of leaves on developed within 3 to 9 days. The bacterial pathogen was re-isolated from inoculated seedlings and identified by morphological and molecular methods to fulfill Koch's postulates test. According to previous research, A. xylosoxidans can cross-kingdom infect animals and plants (Aisenberg et al.,2004; Ye et al.,2018). To the best of our knowledge, this is the first report of A. xylosoxidans causing stem soft rot of A. konjac in China, expanding the known pathogen for the soft rot of A. konjac, and also the host range of A. xylosoxidans.

Therapeutic Effects of Berberine on Liver Fibrosis are associated With Lipid Metabolism and Intestinal Flora.

Liver cirrhosis is a form of liver fibrosis resulting from chronic hepatitis caused by various liver diseases, such as viral hepatitis, alcoholic liver damage, nonalcoholic steatohepatitis, autoimmune liver disease, and by parasitic diseases such as schistosomiasis. Liver fibrosis is the common pathological base and precursors of cirrhosis. Inflammation and disorders of lipid metabolism are key drivers in liver fibrosis. Studies have determined that parts of the arachidonic acid pathway, such as its metabolic enzymes and biologically active products, are hallmarks of inflammation, and that aberrant peroxisome proliferator-activated receptor gamma (PPARγ)-mediated regulation causes disorders of lipid metabolism. However, despite the ongoing research focus on delineating the mechanisms of liver fibrosis that underpin various chronic liver diseases, effective clinical treatments have yet to be developed. Berberine (BBR) is an isoquinoline alkaloid with multiple biological activities, such as anti-inflammatory, anti-bacterial, anti-cancer, and anti-hyperlipidemic activities. Many studies have also found that BBR acts via multiple pathways to alleviate liver fibrosis. Furthermore, the absorption of BBR is increased by nitroreductase-containing intestinal flora, and is strengthened via crosstalk with bile acid metabolism. This improves the oral bioavailability of BBR, thereby enhancing its clinical utility. The production of butyrate by intestinal anaerobic bacteria is dramatically increased by BBR, thereby amplifying butyrate-mediated alleviation of liver fibrosis. In this review, we discuss the effects of BBR on liver fibrosis and lipid metabolism, particularly the metabolism of arachidonic acid, and highlight the potential mechanisms by which BBR relieves liver fibrosis through lipid metabolism related and intestinal flora related pathways. We hope that this review will provide insights on the BBR-based treatment of liver cirrhosis and related research in this area, and we encourage further studies that increase the ability of BBR to enhance liver health.

[Electroacupuncture pretreatment alleviates apoptosis of renal tubular epithelia cells by down-regulating expression of TRPC6 in hyperglycemic mice].

OBJECTIVE: To observe the effect of electroacupuncture (EA) pretreatment on apoptosis of renal tubular epithelial cells in mice with hyperglycemia, so as to explore its mechanisms underlying protecting the kidney from hyperglycemia-induced injury. METHODS: Eighty male C57BL/6 mice were equally and randomly divided into control, model, EA and sham EA groups. The hyperglycemia model was established by intraperitoneal injection of streptozocin (STZ, 50 mg·kg-1·d-1) for 5 consecutive days. Before modeling, EA (2 Hz/15 Hz, 0.3-0.5 mA) was applied to bilateral "Zusanli" (ST36) and "Shenshu" (BL23) for 30 min, once daily for 7 days, while mice in the sham EA group were treated with the same acupoints but without electrical stimulation. The blood glucose values were measured after fasting for 6 hours after 3 days of modeling. The degree of renal tissue injury was observed by microscope after H.E. staining, and the apoptosis level of renal tubular epithelial cells observed by TUNEL staining. The expression levels of transient receptor potential channel 6 (TRPC6) and related apoptotic proteins Caspase-3, Bax and Bcl-2 in the renal tissue were detected by Western blot and immunohistochemistry, separately. RESULTS: Compared with the control group, the blood glucose content and the expression levels of TRPC6, Caspase-3 and Bax proteins, as well as the level of the renal apoptotic cells were significantly increased (P<0.001, P<0.000 1), while the expression level of Bcl-2 protein and the ratio of Bcl-2/Bax were remarkably decreased in the model group (P<0.000 1). In comparison with the model and sham EA groups, the blood glucose content, percentage of apoptotic cells and the expression levels of TRPC6, Caspase-3 and Bax were significantly decreased (P<0.01, P<0.000 1, P<0.05, P<0.001), and the expression level of Bcl-2 and the ratio of Bcl-2/Bax were apparently increased in the EA group (P<0.01, P<0.05, P<0.001). HE statin showed abnormal dilation of the capillary lumen and disappearance of the proximal tubules in the model group, which was relatively milder in the EA group. CONCLUSION: EA pretreatment can lower blood glucose level and reduce renal apoptosis in hyperglycemia mice, which may be related to its effects in down-regulating the expression of TRPC6 and Caspase-3 and up-regulating the ratio of Bcl-2/Bax.

Electroacupuncture Pretreatment Attenuates Cerebral Ischemia-Reperfusion Injury in Rats Through Transient Receptor Potential Vanilloid 1-Mediated Anti-apoptosis via Inhibiting NF-κB Signaling Pathway.

Our previous study showed that electroacupuncture (EA) pretreatment elicited protective effect on cerebral ischemia-reperfusion injury (CIRI) in rats, at least partly, which was associated with transient receptor potential vanilloid 1 (TRPV1)-regulated anti-oxidant stress and anti-inflammation. In this study, we further investigated the possible contribution of TRPV1-mediated anti-apoptosis in EA pretreatment-evoked neuroprotection in CIRI. After EA pretreatment at Baihui (GV20), bilateral Shenshu (BL23) and Sanyinjiao (SP6) acupoints, transient focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion for 6 h in rats. Then, infarct volume, nerve cell injury, neuronal apoptosis, NF-κB signaling activation, and expression of TRPV1 were evaluated by TTC staining, Hematoxylin-Eosin staining, transmission electron microscopy, immunochemistry, immunofluorescence, and Western blot, respectively. The presented data showed that EA pretreatment significantly reduced infarct volume, relieved nerve cell injury, decreased the expression of pro-apoptotic proteins Bax and cleaved caspase-3, increased the level of anti-apoptotic protein Bcl-2, inhibited NF-κB (p65) transcriptional activity, and curbed TRPV1 expression in MCAO rats. By contrast, enhancement of TRPV1 expression accompanying capsaicin application, the specific TRPV1 agonists, markedly accelerated nerve cell damage, aggravated neuronal apoptosis, prompted nuclear translocation of NF-κB (p65), resulting in the reversion of EA pretreatment-evoked neuroprotective effect in MCAO rats. Thus, we conclude that EA pretreatment-induced downregulation of neuronal TRPV1 expression plays an anti-apoptosis role through inhibiting NF-κB signaling pathway, thereby protecting MCAO rats from cerebral ischemia-reperfusion injury.

Phragmunis a suppresses glioblastoma through the regulation of MCL1-FBXW7 by blocking ELK1-SRF complex-dependent transcription.

Glioblastoma (GBM) is a highly aggressive brain tumor. During screening work, we found a new compound named phragmunis A (PGA), which is derived from the fruitbody of Trogia venenata, exhibits a potential cytotoxic effect on patient-derived recurrent GBM cells and temozolomide (TMZ)-resistant cell lines. The present study was designed to investigate the potential molecular mechanism of the anti-glioma effects of PGA in vitro and in vivo. Studies investigating the mechanism revealed that PGA diminished the binding efficiency of ETS family of transcription factor (ELK1) and Serum response factor (SRF), and suppressed ELK1-SRF complex-dependent transcription, which decreased the transcriptional levels of downstream genes Early growth response protein 1 (EGR1)-Polycomb ring finger (BMI1), thus inducing the imbalanced regulation between Myeloid cell leukaemia-1 (MCL1) and F-Box and WD repeat domain containing 7 (FBXW7). Finally, orthotopic xenograft models were established to confirm the anti-glioma effect of PGA on tumour growth. We showed, for the first time, that the cytotoxic effects of PGA occurred by inducing MCL1 inhibition and FBXW7 activation by blocking ELK1-SRF complex-dependent transcription. The blockage of ELK1-mediated transcription resulted in the suppression of EGR1-BMI1, which led to the upregulation of FBXW7 expression and downregulation of MCL1. These findings suggested that PGA could be a therapeutic drug candidate for the treatment of recurrent GBM by targeting the ELK1-SRF complex.

[Electroacupuncture preconditioning alleviates kidney injury in hyperglycemic mice].

OBJECTIVE: To explore the protective effect and molecular mechanism of electroacupuncture (EA) preconditioning on renal injury induced by streptozotocin (STZ) in hyperglycemic mice. METHODS: Eighty male C57BL/6 mice were randomly divided into 4 groups: control, model, EA and sham EA groups, n=20 in each group. The hyperglycemic model was established by intraperitoneal injection of 0.1% STZ solution (50 mg/kg) for 5 days. EA preconditioning or sham EA was applied at "Zusanli" (ST36) and "Shenshu" (BL23), once daily for 7 successive days in the EA or sham EA group. Three days after mode-ling, the blood glucose was measured after fasting for 6 hours. The degree of renal injury was observed by HE staining and PAS staining; the expressions of transient receptor potential cation channel 6 (TRPC6) and Nephrin protein in glomerulus were observed by immunohistochemistry; the expressions of TRPC6 and Nephrin protein in renal cortex were detected by Western blot. RESULTS: Compared with the control group, the blood glucose level was significantly increased (P<0.01), the glomerular cross-sectional area was apparently increased (P<0.000 1), the glomerular capillaries dilated, the matrix proliferated, the brush edge of renal tubules disappeared, the proportion of abnormal renal tubules increased (P<0.000 1), and the expressions of TRPC6 and Nephrin in glomeruli and renal tissue were significantly increased (P<0.01) in the model group. Following EA treatment and compared with the model and sham EA groups, the blood glucose was significantly down-regulated (P<0.01), the renal tissue injury was apparently alleviate, the cross-sectional area of glomerulus was reduced (P<0.05), the brush edge of renal tubules changes were obviously improved, the proportion of abnormal renal tubules decreased (P<0.000 1), and the expressions of TRPC6 and Nephrin in glomerulus and renal tissue decreased (P<0.05) in the EA group. CONCLUSION: EA preconditioning can alleviate renal injury in hyperglycemic mice, which is closely related to its effects in reducing renal TRPC6 and Nephrin expressions and inhibiting podocyte activation.

Climatic factors determine the yield and quality of Honghe flue-cured tobacco.

Flue-cured tobacco (Nicotiana tabacum L.) is a major cash crop in Yunnan, China, and the yield, chemical components, and their proportions decide the quality of tobacco leaves. To understand the effects of environmental factors (soil and climatic factors) on the yield and quality of flue-cured tobacco and determine the main regulating factors, we selected three flue-cured tobacco cultivars [K326, Yunyan87 (Yun87), and Honghuadajinyuan (Hongda)] grown in the Honghe Tobacco Zone. Indices related to yield and economic traits, chemical component properties, soil physical and chemical properties, and climatic factors at different planting sites, were evaluated. We used variance analysis, correlation analysis, and redundancy analysis (RDA) in this study. The results showed that the yield and chemical component properties of flue-cured tobacco, except for the number of left leaves and plant total sugar (PTS) content, were significantly correlated with climatic factors. Particularly, the yield increased in drier and sunnier weather. In terms of the carbon supply capacity, PTS, petroleum ether (PPE), and starch contents (PS) were higher under high-altitude and high-latitude climatic conditions, whereas for the nitrogen supply capacity, plant nitrogen (PTN) and nicotine (PN) contents improved under low-altitude and low-latitude climatic conditions. PTS, reducing sugar (PRS), potassium (PTK), chlorine (PCL), and PPE contents were negatively related to soil clay content, soil pH, and soil organic matter, whereas PRS and PTK contents were positively correlated with alkali-hydrolyzed nitrogen (AN). According to RDA, the soil clay, AN, available phosphorus (AP), and soil chlorine content (SCL) strongly affected the quality of flue-cured tobacco. The quality of the K326 and Yun87 cultivars was mostly influenced by moisture, whereas the quality of the Hongda cultivar was mostly affected by temperature. In conclusion, compared with soil properties, climatic factors more significantly affect the yield and quality of Honghe flue-cured tobacco leaves.

First Report of Botryosphaeria dothidea Causing Stem Canker and Dieback of Gleditsia sinensis in China.

Gleditsia Sinensis Lam is a kind of legume perennial woody plant, which is a traditional Chinese medicine with high economic and ecological value. Its planting area is about 0.1 million ha. In July 2018, symptoms of stem blight were observed on G. sinensis in An shun (26.072311°N, 106.097433°E), Guizhou province (southwestern China). Symptoms included stem canker and dieback, twig blight and extensive vascular discoloration, with incidence rate of 45 to 70%. Samples from plants with symptoms were washed with running tap water, surface sterilized with 2% sodium hypochlorite and then 75% ethanol, rinsed in sterile distilled water, plated on potato dextrose agar (PDA) and incubated at 28°C. Fungal isolates developed copious white aerial mycelium that became dark grey after four to five days, and formed black pycnidia after 23 days. Single hyphal tip cultures of putative 27 isolates were stored in the culture collection (CMW) of the Urban Modern Agriculture Engineering Research Center at the Kunming University. Conidia forming on pycnidia were one-celled, hyaline, aseptate, and fusiform, with dimensions of 20.3-25.9 µm x 4.2-6.5 µm (average 22.5 x 5.5 µm) (sixty conidia were measured). DNA sequence analysis of the ribosomal internal transcribed spacer regions ITS1-5.8S-ITS4, ß-tubulin, and the translation elongation factor 1-alpha (EF1-α) were performed. BLAST searches at GenBank showed the highest nucleotide sequence identity with Botryosphaeria dothidea reference sequence (ITS: >99.82%, KR708996; EF1-α: 99.62%, KP183214; ß-tubulin: > 99.67%, KU306116). Representative sequences of isolates from these regions were deposited in GenBank (ITS: Accession No. MT449017; EF1-α: Accession No. MT454342; ß-tubulin: Accession No. MT454343). Morphological and molecular results confirmed this species as B. dothidea (Aguirre et al. 2018). Pathogenicity tests were conducted by stem inoculation of two-year-old G. sinensis seedlings. Mycelial plugs (2-3 mm in diameter) from actively growing colonies of B. dothidea (PDA) were applied to same-size bark wounds on the middle point of the stems. Inoculated wounds were wrapped with Parafilm. Control seedlings received sterile PDA plugs. Inoculated and control seedlings (9 each), and kept in the greenhouse (28℃, 80% humidity); After 10 days, all of the inoculated plants showed dark vascular stem tissue, and the controls remained healthy. After 30 days, all of the inoculated but none of the control G. sinensis seedlings showed leaf wilting and tissue discoloration. B. dothidea was re-isolated from symptomatic tissues, thus fulfilling Koch's postulates. No symptoms were visible in the control seedlings, and no B. dothidea was re-isolated from the control seedlings tissues. B. dothidea is a member of Botryosphaeriaceae, commonly associated with cankers and dieback of woody plants. B. dothidea has been reported as a pathogen causing stem dieback and branch canker on Malosma laurina (Aguirre et al. 2018), Helwingia chinensis (Yu et al. 2012), and blueberry (Choi 2011; Yu et al. 2012). To our knowledge, this is the first report of B. dothidea on G. sinensis in China.

Electroacupuncture inhibits mast cell degranulation via cannabinoid CB2 receptors in a rat model of allergic contact dermatitis.

OBJECTIVE: Cannabinoid CB2 receptors (CB2Rs) are mainly present on immune cells including mast cells, which participate in 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis (ACD). In this study, we aimed to investigate whether inhibition of mast cell degranulation was involved in the anti-ACD effect of electroacupuncture (EA) at ST36 via CB2R. METHODS: Sprague-Dawley rats were sensitised and challenged with DNFB following EA stimulation for 1 week. Ear swelling, serum IgE levels, local cytokine production and mast cell infiltration were evaluated. Additionally, rat peritoneal mast cells (RPMCs) were isolated and cultured for detection of CB2R expression, mitogen-activated protein kinase (MAPK) signalling activation and mast cell degranulation (including ß-hexosaminidase and histamine release) in the presence or absence of CB2R antagonists. RESULTS: EA treatment inhibited ear swelling, suppressed IgE and cytokine production, decreased the number of mast cells and curbed mast cell degranulation, which was associated with the inhibition of p38 phosphorylation in DNFB-induced ACD. Importantly, EA enhanced the expression of CB2R mRNA and protein in the RPMCs. CB2R antagonist AM630 but not CB1R antagonist AM251 effectively reversed the suppressive effect of EA on p38 activation, mast cell infiltration and degranulation. CONCLUSION: These findings provide more evidence to support the hypothesis that EA promotes CB2R expression in mast cells, which is followed by inhibition of the p38 MAPK pathway, potentially resulting in the anti-ACD effect of EA. This suggests that EA at ST36 may be an effective candidate therapy for treating inflammatory skin diseases such as ACD.

Electroacupuncture Pretreatment Elicits Neuroprotection Against Cerebral Ischemia-Reperfusion Injury in Rats Associated with Transient Receptor Potential Vanilloid 1-Mediated Anti-Oxidant Stress and Anti-Inflammation.

Electroacupuncture (EA) pretreatment, electrical stimulation using metal needle at specific acupoints in advance, possesses the potential to prevent cerebral ischemia-reperfusion injury (CIRI). Transient receptor potential vanilloid 1 (TRPV-1) has been indicated to take part in cerebral protection of EA; however, the detailed mechanisms remain unclear. The aim of this study was to investigate whether neuroprotection of EA pretreatment against CIRI is associated with TRPV-1 and explore the underlying mechanisms. Middle cerebral artery occlusion (MCAO) was performed to induce CIRI after EA pretreatment at Baihui (GV20), bilateral Shenshu (BL23), and Sanyinjiao (SP6) acupoints in rats. Neurological deficit scores, infarct volumes, oxidative stress damage, inflammatory cytokine production, MAPK signaling activation, and the expression of TRPV-1 were assessed. EA pretreatment lowered neurological deficit scores, reduced infarct volumes, impeded oxidative stress injury, inhibited inflammatory cytokine production, curbed P38 phosphorylation, and suppressed TRPV-1 expression in MCAO rats. Attributing to inhibition of TRPV-1 expression, AMG-517 (TRPV-1 antagonist) showed the synergistic effect with EA pretreatment on the neuroprotection against ischemia-reperfusion injury. However, TRPV-1 agonists capsaicin significantly abrogated the neuroprotective effects of EA pretreatment in MCAO rats accompanying enhancement of TRPV-1 expression. These findings indicated EA pretreatment exerted neuroprotection in rats with cerebral ischemia-reperfusion injury, which at least partially were associated with TRPV1-mediated anti-oxidant stress and anti-inflammation via inhibiting P38 MAPK activation.

Microvascular Invasion as a Predictor of Response to Treatment with Sorafenib and Transarterial Chemoembolization for Recurrent Intermediate-Stage Hepatocellular Carcinoma.

Background The evidence of combining sorafenib with transarterial chemoembolization (TACE) for intermediate-stage recurrent hepatocellular carcinoma (HCC) is limited. Patient responses to this treatment varied because of the heterogeneous nature of intermediate-stage recurrent HCC, making it important to identify patients who are most likely to benefit from this combination therapy. Purpose To compare sorafenib administered in combination with TACE versus TACE alone in the treatment of recurrent intermediate-stage HCC after initial hepatectomy and to determine the relationship of microvascular invasion (MVI) to survival. Materials and Methods In this retrospective multicenter study, 3652 consecutive patients were found to have intrahepatic recurrences after initial hepatectomy of primary HCC from January 2010 to December 2016. Of these, 260 patients with intermediate-stage recurrent HCC underwent combination treatment with sorafenib and TACE or TACE alone. Overall survival (OS) and progression-free survival (PFS) were compared between these two treatments according to MVI status by using log-rank tests. Results A total of 128 patients were administered combination therapy (mean age, 55 years ± 7.6 [standard deviation]; 107 men) and 132 patients were administered TACE alone (mean age, 56 years ± 8.3; 110 men). The 5-year OS and PFS were higher in the combination group than in the TACE group (OS: 38.9% vs 20.5%, respectively, P = .01; PFS, 37.5% vs 18.7%, respectively, P = .003). For patients with MVI-positive lesions, the median OS and PFS after combination treatment (n = 55) were longer than those after TACE alone (n = 72; OS: 17.2 months vs 12.1 months, respectively, P = .02; PFS: 17.0 months vs 11.0 months, respectively, P = .02). Multivariable analysis showed that tumor number, MVI status, and treatment allocation were significant predictors of OS and PFS, whereas tumor size was a prognostic factor for PFS. Conclusion Patients with recurrent intermediate-stage hepatocellular carcinoma and lesions positive for microvascular invasion (MVI) had longer survival times by using a combined treatment of sorafenib with transarterial chemoembolization (TACE) compared with TACE alone; patients with MVI-negative lesions did not show survival benefit from combined therapy. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Malloy in this issue.

Electro-acupuncture Pretreatment at Zusanli (ST36) Acupoint Attenuates Lipopolysaccharide-Induced Inflammation in Rats by Inhibiting Ca2+ Influx Associated with Cannabinoid CB2 Receptors.

In this study, we aimed to investigate the effect of electro-acupuncture (EA) pretreatment at zusanli (ST36) acupoint on lipopolysaccharide (LPS)-induced endotoxemic rat model and explore the underlying molecular mechanisms. Rats were treated with EA at ST36 for 7 days before being subjected to LPS. Two hours post-LPS, samples such as serum, local acupoint tissues, and spleens were collected and processed for investigations including cytokine production, cytosolic calcium (Ca2+) concentration, Ca2+ influx, cannabinoid CB2 receptor (CB2R) expression, and TLR4/NF-κB signaling. Our results showed EA pretreatment significantly attenuated LPS-induced inflammatory cytokine production, such as TNF-α, IL-1ß, and IL-6. EA also enhanced CB2R expression, inhibited Ca2+ influx, and inactivated TLR4/NF-κB signaling, subsequently resulting in a substantial reduction of Ca2+ concentration. Importantly, CB2R antagonist AM630 effectively abrogated the suppressive effect of EA at ST36 on the endotoxemic rats, suggesting CB2R was involved in the anti-inflammatory effect of EA. EA pretreatment could enhance CB2R expression, inhibit Ca2+ influx, and inactivate TLR4/NF-κB signaling, which contributes to the alleviation of LPS-induced inflammation in rats.

Involvement of the Negative Feedback of IL-33 Signaling in the Anti-Inflammatory Effect of Electro-acupuncture on Allergic Contact Dermatitis via Targeting MicroRNA-155 in Mast Cells.

In this study, we aimed to investigate the effect of electro-acupuncture (EA) at the Zusanli acupoint (ST36) on interleukin (IL)-33-mediated mast cell activation. Firstly, 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis (ACD) in rats was developed with or without EA treatment. Then, rat peritoneal mast cells (RPMCs) were obtained and cultured in the presence of IL-33. EA treatment relieved ear swelling and reduced mast cell infiltration in the local inflammation area with DNFB challenge, accompanying the decrement of IL-33 production. RPMCs isolated from ACD rats with EA treatment showed significant downregulation of IL-6, TNF-α, IL-13, and MCP-1 production following IL-33 stimulation. However, there was no obvious difference in surface ST2 receptor expression among different groups. In addition, EA selectively altered IL-33 signaling, suppressing p38 phosphorylation as well as NF-κB- and AP-1-mediated transcription but not Akt phosphorylation. Importantly, EA lowered microRNA (miR)-155 expression in the RPMCs, which presented a positive correlation with IL-33-induced IL-6 production. Furthermore, overexpression of miR-155 in the RPMCs was established following miR-155 mimic transfection. RPMCs with the overexpressed miR-155 displayed an obvious increment of inflammatory cytokine and abrogated the inhibitive effect of EA on NF-κB- and AP-1-regulated transcription in response to IL-33 compared with those without transfected-miR-155. These findings demonstrate EA treatment inhibits NF-κB and AP-1 activation as well as promotes the negative feedback regulation of IL-33 signaling via targeting miR-155 in mast cells, which contribute to the anti-inflammatory effect of EA on DNFB-induced ACD in rats.

High-Tech Acupuncture for Prevention of Lifestyle Diseases-A Sino-Austrian Cooperation Research Protocol on Heart Rate Variability.

BACKGROUND: Acupuncture can not only be used for well-known diseases, but also for so-called modern lifestyle-related diseases. Using innovative methods like e.g. new analyses of heart rate variability (HRV), it is also possible to investigate diseases like burnout syndrome, ie., qi deficiency in Chinese medicine (CM). OBJECTIVE: The main object of this research protocol is to perform research on the relationship of burnout syndrome and heart rate (HR) and HRV. METHODS: A total of 175 patients with burnout syndrome (qi deficiency syndrome) in five groups and 35 healthy volunteers will be investigated. Based on random numbers generated by computer and concealed in opaque envelops, the patients will be assigned to four acupuncture groups using Zusanli (ST 36) acupuncture stimulation, Guanyuan (CV4) acupuncture stimulation, both points, and both points with Streitberger device respectively, and a moxibustion group using both points mentioned above, with 35 cases in each group. Altogether four different experiments are planned. Experiment 1 includes 70 subjects and is a comparison between a burnout group and a control group (healthy volunteers). The evaluation parameters are different scores and indices of HR and HRV. Experiment 2 includes 140 subjects and compares the efficacy of different acupuncture points. In experiment 3 (105 subjects), acupuncture and moxibustion should be compared to healthy volunteers. Experiment 4 (70 subjects) investigates the long-term therapeutic effects of acupuncture and moxibustion on the scores of qi deficiency and HR/HRV in qi deficiency patients. In both the acupuncture and moxibustion groups, a total of 10 treatments will be performed. CONCLUSIONS: The joint research aims at the scientific evaluation of CM, mainly in the field of HRV. This parameter could be a very good indicator of the state of health and can be inflfluenced by different acupuncture methods, as shown in the past.

[Comparison of Protective Effect of Electroacupuncture on Myocardial Ischemia Injury Between Different Acupoint Formulas in Rats].

OBJECTIVE: To observe the different effects of electroacupucnture of "Neiguan" (PC 6) grouping with other acupoints on myocardial ischemia (MI) injury, so as to provide evidence for clinical acupoint formulas. METHODS: Forty Wistar male rats were randomly divided into normal, model, EA 1[bilateral "Neiguan" (PC 6), bilateral "Zusanli" (ST 36),"Guanyuan" (CV 4)], and EA 2[bilateral "Neiguan" (PC 6), bilateral "Shenmen" (HT 7), "Danzhong" (CV 17)] groups, 10 rats in each group. The MI model was established by subcutaneous injection with ISO solution(5 mL/kg), once per day for 7 days. Both EA 1 and EA 2 groups were subjected to EA (2 Hz/100 Hz, 1 mA) for 10 min before subcutaneous injection, once daily for 21 days. Electrocardiogram (ECG) was recorded after treatment. Serum cTnT was detected by biochemistry. SOD, GSH-Px and MDA in myocardial tissues of left ventricular were measured by ELISA, and the ultrastructural changes of myocardial cells in the left ventricular were observed by electron microscope. RESULTS: Compared with the normal group, the amplitude of S-T segment and HR of ECG, cTnT and MDA contents were significantly increased in the model group (P<0.01) accompanied with seriously destroyed ultrastructure in the myocardial tissue, while SOD and GSH-Px levels were lower than those in the normal group (P<0.01). However, EA treatments reversed all the abnormal changes in the model group (P<0.05). There was no significant difference between EA 1 and EA 2 groups in the amplitude of S-T segment and HR (P>0.05); cTnT and MDA levels in the EA 1 group were lower than those in the EA 2 group (P<0.05), while SOD and GSH-Px levels were higher than those in the EA 2 group (P<0.05), accompanied with lighter degree of ultrastructure damage in the myocardial tissue in the EA 1 group. CONCLUSIONS: EA can prevent myocardial ischemic injury, and the effect of EA 1 treatment was better than that of EA 2.

Electro-Acupuncture at Zusanli Acupoint (ST36) Suppresses Inflammation in Allergic Contact Dermatitis Via Triggering Local IL-10 Production and Inhibiting p38 MAPK Activation.

Acupuncture has shown beneficial effect in the treatment of multiple dermatologic conditions including dermatitis, pruritus, urticaria, and hyperhidrosis; however, the detailed mechanisms are still kept unclear. This study aimed to investigate if electro-acupuncture (EA) treatment prevents 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis (ACD) in rats and explore its underlying mechanisms. ACD was induced by sensitizing and challenging with DNFB topically. Rats were treated daily following bilateral subcutaneous stimulation of EA at Zusanli acupoint (ST36) for 1 week. Ear swelling and serum IgE levels were measured. The ear biopsies were obtained for histology. Inflammatory cytokines on the dermatological ear and local acupoint tissue were assayed. Spleen lymphocytes and the homogenized supernatant of local acupuncture area were used to co-culture for flow cytology and immune analysis, respectively. EA treatment at ST36 notably inhibited ear swelling and inflammatory cell infiltration on DNFB-induced ACD. EA also decreased serum IgE concentrations and alleviated the production of inflammatory cytokines in dermatological ear. Additionally, EA treatment attenuated the percentage of CD4+IFN-γ+ and CD4+IL-4+ T cells associated with ACD. Interestingly, secretion of interleukin (IL)-10 in the local acupoint tissue following EA stimulation was increased and showed suppressive function when co-cultured with the spleen lymphocytes from DNFB group. Lastly, EA treatment demonstrably suppressed p38 MAPK activation in DNFB-treated rats. Our findings suggest that EA treatment at ST36 may ameliorate inflammation associated with DNFB-induced ACD via triggering local IL-10 production and inhibiting p38 MAPK activation, which provide an alternative and promising therapy for ACD.

Enhancement of immune cytokines and splenic CD4+ T cells by electroacupuncture at ST36 acupoint of SD rats.

Electroacupuncture at the ST36 acupoint can enhance the body's immune function. However, the mechanism for this enhancement has not been fully described. Our study was designed to investigate the effect of electroacupuncture on the immune function of Sprague-Dawley (SD) rats. The rats were randomly divided into three groups: a control group, a non-acupoint group (abdominal muscle acupuntured) and a ST36 acupoint group. Our results showed that successive electroacupuncture at the ST36 acupoint for 3 d significantly enhanced the interferon-γ (IFN-γ) level in the serum of SD rats. The results also showed that the serum and extracts from spleen cells of the ST36 acupoint group contained higher levels of interleukin (IL)-2 and IL-17 compared to those of the other two groups. Immunohistochemical analysis showed that electroacupuncture applied to the ST36 acupoint enhanced the expression level of CD4 in spleen cells. Furthermore, it was observed that CD4 co-localized with transient receptor potential vanilloid (TRPV) channels at the membrane of splenic CD4+ T cells and the expression level of CD4 was related to TRPV channels in the electroacupuncture treatment. These observations indicated that electroacupuncture stimulation at the ST36 acupoint enhanced the level of immune cytokines and splenic CD4+ T cells through TRPV channels in this system.

Electro-acupuncture at Acupoint ST36 Ameliorates Inflammation and Regulates Th1/Th2 Balance in Delayed-Type Hypersensitivity.

Increasing evidence indicates anti-allergic and anti-inflammatory effects of electro-acupuncture (EA) therapy. However, its underlying mechanism on delayed-type hypersensitivity (DTH), a classic allergic inflammatory disease, still remains unclear. In this study, we aimed to explore the immunomodulatory mechanism of EA intervention in a mouse model of ovalbumin (OVA)-induced DTH. Mice were randomly divided into four groups: Control, OVA-DTH, DTH + EA, DTH + Sham. "Zusanli" acupoint (ST36) was used for DTH + EA, whereas a non-acupoint (localized 5 mm below the "Zusanli" acupoint) was selected for DTH + Sham. Footpad thickness was checked, and the infiltration of inflammatory cells was estimated by hematoxylin and eosin staining. Levels of IgG and IgE in serum of different groups and inflammatory cytokines in the supernatants from homogenized footpads, including IFN-γ, TNF-α, IL-4, and IL-5, were determined by ELISA. Cell proliferation of spleen lymphocytes was assayed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT). The frequency of CD4+IFN-γ+ and CD4+IL-4+ T cells was analyzed with flow cytometry. In addition, the mRNA and protein expression of T-bet and GATA-3 were evaluated by real-time PCR and Western blotting, respectively. Our data showed EA treatment at acupoint ST36 relieved the pathological progression of DTH responses via reduction in footpad swelling, infiltration of inflammatory cells, levels of IgG and IgE as well as decreased production of IFN-γ and TNF-α in homogenized footpad tissue. Moreover, detailed studies were performed revealing that EA attenuated the percentage of CD4+IFN-γ+ T cells and prevented Th cells differentiation into Th1 cells, and this results from inhibiting secretion of IFN-γ and suppressing expression of T-bet, an IFN-γ transcription factor. The results indicated that EA treatment improved Th1-mediated allergic skin inflammation via restoring Th1/Th2 balance by curbing Th1 differentiation. These findings suggested that EA at acupoint ST36 might be a useful and promising therapeutic for allergic inflammatory as well as Th1-mediated inflammation response.

Cortex Mori Radicis Extract induces neurite outgrowth in PC12 cells activating ERK signaling pathway via inhibiting Ca(2+) influx.

Cortex Mori Radicis is a traditional Chinese herbal medicine which has a long history of use for the treatment of headaches, cough, edema and diabetes. However, its function and mode of action within nervous system remain largely unclear. In the present study, we have attempted to determine the effects of Cortex Mori Radicis Extract (CMRE) on neuronal differentiation. Here, we reported that CMRE induces the neurite outgrowth in pheochromocytoma PC12 cells and primary cortical neuron. Following the generation of neurite outgrowth, extracellular Ca(2+) influx was inhibited and intracellular Ca(2+) decreased. In addition, CMRE induced the extracellular signal-regulated kinase 1/2 (ERK1/2) activation and also stimulated the Rap1-GTP expression, which is closely linked to neuritogenesis. Moreover, the neurite outgrowth induced by CMRE was antagonized to a marked degree by suppressing activation of p-ERK1/2 with the specific ERK1/2 inhibitor (PD98059), suggesting the involvement of Rap1-GTP and ERK1/2 in CMRE-induced neurite outgrowth. Taken together, these results demonstrate that CMRE induces neurite outgrowth of PC12 cells through Rap1-ERK signaling pathway via inhibiting Ca(2+) influx, and provide a novel insight into the manner in which CMRE participates in neuritogenesis.

[Effects of electroacupuncture at "Neiguan" (PC 6) on p38 MAPK signaling pathway in rats with cardiac hypertrophy].

OBJECTIVE: To discuss the mechanisms of electroacupuncture at "Neiguan" (PC 6) on p38 MAPK signaling pathway in rats with cardiac hypertrophy. METHODS: Forty SD rats were randomly divided into four groups: a normal group, a model group, a model plus p38 MAPK inhibitor group, a model plus electroacupuncture group, ten rats in each group. The model rats were established by subcutaneous injection 3 mg/(kg x d) of Isoprenaline Hydrochloride; model plus p38 MAPK inhibitor group were injected 0.3 mg/(kg x d) of specific inhibitor SB 203580; model plus electroacupuncture group was treated by electroacupuncture at "Neiguan"(PC 6) with continuous-wave, 2 Hz and 1 mA for 20 minutes, once a day for 14 days. There was no treatment in other two groups. The contents of TNF-alpha and IL-1beta in heart tissue were detected by radioimmunoassay and the p38 MAPK, p-p38 MAPK by western blot. RESULTS: Compared with normal group, the contents of TNF-alpha, IL-1beta, p38 MAPK, p-p38 MAPK were significantly increased in model group (all P < 0.01). The contents of TNFalpha, IL-1beta, p38 MAPK, p-p38 MAPK were significantly decreased in model plus p38 MAPK inhibitor group and model plus electroacupuncture group, compared with model group, all P < 0.05; compared with normal group, P < 0.05, P < 0.01; but no significant difference between model plus p38 MAPK inhibitor group and model plus electroacupuncture group (P > 0.05). CONCLUSION: Electroacupuncture at "Neiguan" (PC 6) can prevent the phosphorylation of p38 MAPK of myocardial hypertrophy, and the mechanism maybe adjust p38 MAPK signaling pathway by inhibiting the expression of TNF-alpha and IL-1beta.