Zuogui Wan alleviated maternal kidney-yin deficiency-induced thymic epithelial cell dysfunction in newborn rats through Wnt/ß-catenin signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Kidney-yin deficiency (KYD) during pregnancy is common and associated with possibility of thymus hypoplasia in neonates. Zuogui Wan (ZGW) is a classic traditional medicine to treat KYD. AIM OF STUDY: The Wnt/ß-catenin signaling pathway is essential for thymic epithelial cell (TEC) viability, function and for thymus integrity. We evaluated whether maternal diets with ZGW in KYD rats ameliorates epithelial cell dysfunction in the fetal thymus, and investigated its underlying mechanism in which the Wnt/ß-catenin signaling pathway is involved. MATERIALS AND METHODS: Rats were randomly assigned to four groups (n = 8). Two experimental groups received KYD induction with or without ZGW supplementation. The other 2 vehicle groups were sham operated and administrated with normal saline or ZGW. KYD was established using periodically chronic shaken stimulus and threaten stress. Success of the model induction was evaluated by the general observation, changing of the body weight and plasma thyroxine level. Then, pregnant of vehicle and KYD rats were fed with or without ZGW-supplemented diet throughout the F1 gestation. Postnatal thymi samples were obtained after delivery for histological examination. In vitro, TECs of the newborn rats whose mother suffered KYD were isolated, and cultured using the serum containing ZGW with or without the supplement of Wnt4/ß-catenin pathway inhibitor ICG-001. Cell viability was evaluated by CCK-8 assay. Meanwhile, the thymi tissues and TECs were collected for biochemical analysis. Levels of thymosin ß4 (TMSß4) and thymosin α1 (Tα1) were detected by ELISA assay. The mRNA and protein expression of Wnt4, ß-catenin, and Foxn1 were determined by RT-qPCR and Western blot respectively. RESULTS: In vivo, KYD resulted in significantly increased apoptosis of TECs and atrophy of the thymi, especially in the medullary zone. The morphological changes observed in KYD rats were ameliorated by ZGW treatment. Meanwhile, the decreased TMSß4, Tα1, Wnt4, ß-catenin, and Foxn1 levels in KYD rats were also significantly alleviated by ZGW administration. In vitro, elevated TMSß4 and Tα1 levels accompanied with upregulated Wnt4, ß-catenin, and Foxn1 expressions in the TECs were observed after ZGW intervention, however, which were significantly downregulated by ICG-001 supplement. CONCLUSIONS: Maternal kidney-yin deficiency could result in TEC dysfunction in newborn rats. ZGW was able to improve the growth and development of TEC, potentially by regulating the Wnt/ß-catenin pathway.

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.

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.

Effect of Mahuang Gancao Ganjiang Decoction on Fusion and Fission of Mitochondria and Apoptosis of Lymphocytes in Mice under Cold Stress.

Mahuang Gancao Ganjiang Decoction (MGGD) can effectively alleviate the symptoms of the patients suffering from exogenous cold stress. However, the curative mechanism has not been fully clarified. This study was designed to investigate the effect of MGGD on the apoptosis of lymphocytes induced by cold stress in mice. The model mice were randomly divided into four groups: the normal control group (no handling mice), cold stress group, MGGD + cold stress group, and MGGD group. Lymphocytes of the mice were isolated from the peripheral blood. Electron microscopy analysis revealed cold stress resulted in mitochondrial fragmentation. Accompanied with the change of morphology of mitochondria, ATP production and the activity of respiratory chain complex decreased in these cells. Western blot analysis showed that these cells expressed decreased fusion-related proteins Mitofusin 1 (Mfn1), Mitofusin 2 (Mfn2), and optic atrophy protein-1 (Opa-1) and increased fission-related proteins dynamin-related protein 1 (Drp1) and fission 1 (Fis-1); our results also show that decreased mitochondrial fusion induces cell apoptosis during cold stress. Meanwhile, we found MGGD can inhibit cell apoptosis induced by cold stress through regulating expression level of Mfn1, Mfn2, Drp1, Fis-1, and Opa-1. These findings are very significant for understanding how MGGD regulates cold-stress-induced cell apoptosis.

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.

Clinical experience with radio-, chemo- and hyperthermotherapy combined trimodality on locally advanced esophageal cancer.

Esophageal cancer is a highly malignant and lethal disease with a low 5-year survival rate. Therefore, an effective treatment modality is required. To investigate the treatment efficacy and toxicity of radio-, chemo- and hyper-thermotherapy combined trimodality on locally advanced esophageal cancer, the medical records of 78 patients with pathologically confirmed esophageal cancer treated with chemoradiotherapy plus hyperthemia at our institution were retrospectively investigated and the 3-year outcome was carefully assessed. All 78 patients received intensity-modulated radiation therapy at a total dose of 60-66 Gy, in a conventional schedule of 1.8-2.1 Gy/fraction, 5 fractions/week. They also received 4-6 courses of chemotherapy, consisting of 450 mg/m2 5-fluorouracil for 1-5 days and 25 mg/m2 cisplatin for 1-5 days, in addition to 6-12 sessions of hyperthermia, performed twice a week. Out of the 78 cases, complete remission of the primary tumor was observed in 31 (39.7%), partial remission in 44 (56.4%) and no change in 3 (3.9%) cases. The treatment response rate was 96.1%. The overall survival (OS) rate at 1, 2 and 3 years was 67.9, 41.0 and 33.3%, respectively. No significant difference in adverse effects was observed between this treatment regimen and other similar studies. Our preliminary results demonstrated that the chemo-, radio- and hyperthermotherapy combined trimodality exhibited excellent short-term clinical outcomes as regards tumor response rate and a sound long-term OS, with endurable adverse events. This trimodal treatment requires further investigation to establish its beneficial role in the treatment of patients with locally advanced esophageal cancer.