[Effect of wheat-grain moxibustion on Wnt/ß-catenin signaling pathway in bone marrow cell in mice with bone marrow inhibition].

OBJECTIVE: To observe the effect of wheat-grain moxibustion at "Dazhui" (GV 14), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6) on Wnt/ß-catenin signaling pathway in bone marrow cell in mice with bone marrow inhibition, and to explore the possible mechanism of wheat-grain moxibustion in treating bone marrow inhibition. METHODS: Forty-five SPF male CD1(ICR) mice were randomly divided into a blank group, a model group and a wheat-grain moxibustion group, 15 mice in each group. The bone marrow inhibition model was established by intraperitoneal injection of 80 mg/kg of cyclophosphamide (CTX). The mice in the wheat-grain moxibustion group were treated with wheat-grain moxibustion at "Dazhui" (GV 14), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6), 3 moxa cones per acupoint, 30 s per moxa cone, once a day, for 7 consecutive days. The white blood cell count (WBC) was measured before modeling, before intervention and 3, 5 d and 7 d into intervention. After intervention, the general situation of mice was observed; the number of nucleated cells in bone marrow was detected; the serum levels of interleukin-3 (IL-3), interleukin-6 (IL-6) and granulocyte macrophage colony stimulating factor (GM-CSF) were measured by ELISA; the protein and mRNA expression of ß-catenin, cyclinD1 and C-Myc in bone marrow cells was measured by Western blot and real-time PCR method. RESULTS: Compared with the blank group, the mice in the model group showed sluggish reaction, unstable gait, decreased body weight, and the WBC, number of nucleated cells in bone marrow as well as serum levels of IL-3, IL-6, GM-CSF were decreased (P<0.01), and the protein and mRNA expression of ß-catenin, cyclinD1 and C-Myc was decreased (P<0.01). Compared with the model group, the mice in the wheat-grain moxibustion group showed better general condition, and WBC, the number of nucleated cells in bone marrow as well as serum levels of IL-3, IL-6, GM-CSF were increased (P<0.01, P<0.05), and the protein and mRNA expression of ß-catenin, cyclinD1 and C-Myc was increased (P<0.05). CONCLUSION: Wheat-grain moxibustion shows therapeutic effect on bone marrow inhibition, and its mechanism may be related to activating Wnt/ß-catenin signaling pathway in bone marrow cells, improving bone medullary hematopoiesis microenvironment and promoting bone marrow cell proliferation.

Effect of wheat-grain moxibustion on Wnt/β-catenin signaling pathway in bone marrow cell in mice with bone marrow inhibition / 中国针灸

OBJECTIVE@#To observe the effect of wheat-grain moxibustion at "Dazhui" (GV 14), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6) on Wnt/β-catenin signaling pathway in bone marrow cell in mice with bone marrow inhibition, and to explore the possible mechanism of wheat-grain moxibustion in treating bone marrow inhibition.@*METHODS@#Forty-five SPF male CD1(ICR) mice were randomly divided into a blank group, a model group and a wheat-grain moxibustion group, 15 mice in each group. The bone marrow inhibition model was established by intraperitoneal injection of 80 mg/kg of cyclophosphamide (CTX). The mice in the wheat-grain moxibustion group were treated with wheat-grain moxibustion at "Dazhui" (GV 14), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6), 3 moxa cones per acupoint, 30 s per moxa cone, once a day, for 7 consecutive days. The white blood cell count (WBC) was measured before modeling, before intervention and 3, 5 d and 7 d into intervention. After intervention, the general situation of mice was observed; the number of nucleated cells in bone marrow was detected; the serum levels of interleukin-3 (IL-3), interleukin-6 (IL-6) and granulocyte macrophage colony stimulating factor (GM-CSF) were measured by ELISA; the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc in bone marrow cells was measured by Western blot and real-time PCR method.@*RESULTS@#Compared with the blank group, the mice in the model group showed sluggish reaction, unstable gait, decreased body weight, and the WBC, number of nucleated cells in bone marrow as well as serum levels of IL-3, IL-6, GM-CSF were decreased (P<0.01), and the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc was decreased (P<0.01). Compared with the model group, the mice in the wheat-grain moxibustion group showed better general condition, and WBC, the number of nucleated cells in bone marrow as well as serum levels of IL-3, IL-6, GM-CSF were increased (P<0.01, P<0.05), and the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc was increased (P<0.05).@*CONCLUSION@#Wheat-grain moxibustion shows therapeutic effect on bone marrow inhibition, and its mechanism may be related to activating Wnt/β-catenin signaling pathway in bone marrow cells, improving bone medullary hematopoiesis microenvironment and promoting bone marrow cell proliferation.

Embelia Laeta aqueous extract suppresses acute inflammation via decreasing COX-2/iNOS expression and inhibiting NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Embelia laeta (L.) Mez., which is called Suanjifeng in Chinese ethnic Yao medicine, is traditionally for inflammation-related diseases, such as oral ulcer, sore throat, enteritis, and rheumatoid arthritis. However, the biological properties and the underlying mechanisms of Embelia laeta still need further studies. AIM OF THIS STUDY: The present study aims to investigate the anti-inflammatory effect and its underlying mechanisms of Embelia laeta. MATERIALS AND METHODS: In this study, except acute toxicity experiments, Kunming (KM) mice of either sex were enrolled to establish inflammatory model induced by xylene, acetic acid and carrageenan, respectively. Mice were randomly divided into different groups and pretreated by oral gavage with different doses of Embelia laeta aqueous extract (ELAE) (2.5, 5, 10 g/kg) and 10 mg/kg of Indo for 7 days. Ear edema, vascular permeability, abdominal writhing, and paw edema degree were detected in related experiments. Moreover, in the carrageenan-induced paw edema mice model, histological changes were detected by H&E staining. MDA, MPO and NO were detected by assay kit. Proinflammatory cytokines of IFN-γ, TNF-α, IL-1ß, IL-6 and PGE2 were detected by ELISA. Additionally, COX-2, iNOS and NF-κB pathway-related proteins were detected by Western blotting. RESULTS: Results showed that the ELAE evoked an obvious dose-dependent inhibition of ear edema induced by xylene, paw edema induced by carrageenan, as well as suppressing the increase of vascular permeability and writhing times elicited by acetic acid. Histopathological analysis indicated that ELAE could significantly decrease the cellular infiltration in paw tissue. ELAE showed antioxidant property through markedly decrease the MDA level and MPO activity in edema paw. In addition, ELAE decreased the proinflammatory cytokines IFN-γ, TNF-α, IL-1ß, IL-6, PGE2 and NO that induced by carrageenan. Western blotting results also showed that ELAE could obviously downregulate the COX-2 and iNOS expression. Further analysis revealed that ELAE also inhibited NF-κB from the cytoplasm to the nucleus and stabilize the conversion of IκBα. CONCLUSION: ELAE had powerful anti-inflammatory property, which might be had a close relationship with mediating proinflammatory cytokines production, decreasing the COX-2 and iNOS expression, and inhibiting the activation of NF-κB signaling pathway.

Anneslea fragrans Wall. ameliorates ulcerative colitis via inhibiting NF-κB and MAPK activation and mediating intestinal barrier integrity.

ETHNOPHARMACOLOGICAL RELEVANCE: Anneslea fragrans Wall. is traditionally used as a folk medicine in treating indigestion, fever, dysentery, diarrhea, and liver inflammation in China, Vietnam and Cambodia. However, its anti-inflammatory activity and mechanism under a safety therapeutic dose as well as the main chemical components have not yet been fully investigated. AIM OF THE STUDY: This study aimed to explore the therapeutic effect and possible molecular mechanisms of aqueous-methanol extract (AFE) of A. fragrans leaves on dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) mice and illustrate its potent anti-inflammatory chemical compounds. MATERIALS AND METHODS: The AFE was obtained and then analyzed by high performance liquid chromatography (HPLC). Phytochemical investigation on the AFE was carried out to isolate and characterize its major components. The acute toxicity test was performed to provide the safety information of AFE. Subsequently, the protective effect of AFE on DSS-induced UC was evaluated by physiological changes, histopathological and immunohistochemical analysis, and the expressions of antioxidant enzyme, pro-inflammatory cytokines and anti-inflammatory cytokines. The expressions of target proteins in nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) were determined by western blot analysis. The tight junction (TJ) proteins in colon tissue were performed by immunohistochemical technique for evaluating the intestinal barrier integrity. RESULTS: HPLC guided isolation of AFE resulted into two dihydrochalcones, which were elucidated as vacciniifolin (1) and confusoside (2). Acute toxicity evaluation revealed that median lethal dose (LD50) of AFE was greater than 5000 mg/kg. Furthermore, AFE significantly attenuated ulcerative colitis symptoms, suppressed myeloperoxidase activity, and increased the expression of superoxide dismutase and glutathione. AFE treatment could also reduce the levels of tumor necrosis factor-α, interleukin-1ß, and interleukin-6 and increase the levels of interleukin-4 and interleukin-10 in colon tissues and serum of DSS-induced UC mice. In addition, AFE significantly increased the expression of zonula occludens-1, occludin and claudin-1, and inhibited the phosphorylation of target protein of the NF-κB and MAPK signaling pathways in colon tissue. CONCLUSION: Dihydrochalcone glycosides are the major chemical constituents in AFE. AFE ameliorated DSS-induced UC in mice by inhibiting the inflammatory response via modulation of NF-κB and MAPK pathways and maintaining the intestinal barrier function, indicating that the plant A. fragrans could be used as a therapeutic candidate for ulcerative colitis.

Sensitivity and Resistance of MET Exon 14 Mutations in Lung Cancer to Eight MET Tyrosine Kinase Inhibitors In Vitro.

BACKGROUND: MNNG HOS transforming gene (MET) exon 14 mutations in lung cancer, including exon 14 skipping and point mutations, have been attracting the attention of thoracic oncologists as new therapeutic targets. Tumors with these mutations almost always acquire resistance, which also occurs in other oncogene-addicted lung cancers. However, the resistance mechanisms and treatment strategies are not fully understood. METHODS: We generated Ba/F3 cells expressing MET exon 14 mutations by retroviral gene transfer. The sensitivities of these cells to eight MET-tyrosine kinase inhibitors (TKIs) were determined using a colorimetric assay. In addition, using N-ethyl-N-nitrosourea mutagenesis, we generated resistant clones, searched for secondary MET mutations, and then examined the sensitivities of these resistant cells to different TKIs. RESULTS: Ba/F3 cells transfected with MET mutations grew in the absence of interleukin-3, indicating their oncogenic activity. These cells were sensitive to all MET-TKIs except tivantinib. We identified a variety of secondary mutations. D1228 and Y1230 were common sites for resistance mutations for type I TKIs, which bind the active form of MET, whereas L1195 and F1200 were common sites for type II TKIs, which bind the inactive form. In general, resistance mutations against type I were sensitive to type II, and vice versa. CONCLUSIONS: MET-TKIs inhibited the growth of cells with MET exon 14 mutations. We also identified mutation sites specific for TKI types as resistance mechanisms and complementary activities between type I and type II inhibitors against those mutations. These finding should provide relevant clinical implication for treating patients with lung cancer harboring MET exon 14 mutations.

Celastrol treatment protects against acute ischemic stroke-induced brain injury by promoting an IL-33/ST2 axis-mediated microglia/macrophage M2 polarization.

BACKGROUND: Acute ischemic stroke (AIS) is the most common type of cerebrovascular disease and is a leading cause of disability and death worldwide. Recently, a study suggested that transformation of microglia from the pro-inflammatory M1 state to the anti-inflammatory and tissue-reparative M2 phenotype may be an effective therapeutic strategy for ischemic stroke. Celastrol, a traditional oriental medicine, may have anti-inflammatory and neuroprotective effects. However, the underlying mechanisms remain unknown. METHODS: We first determined the expression levels of inflammatory factors in patients and rodent models associated with AIS; we then determined the anti-inflammatory effects of celastrol in AIS, both in vivo and in vitro, using animal models of middle cerebral artery occlusion (MCAO) and cell models of oxygen-glucose deprivation (OGD) treatment with or without celastrol, respectively. RESULTS: The results indicated that expression of both inflammatory (interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α) cytokines, as well as the anti-inflammatory cytokine, IL-33, and IL-10, were increased following AIS in patients and in animal models. Furthermore, in vitro experiments confirmed that celastrol treatment decreased inflammatory cytokine expression induced by OGD through an IL-33/ST2 axis-mediated M2 microglia/macrophage polarization. Finally, celastrol is protected against ischemic-induced nerve injury, both in vivo and in vitro. CONCLUSIONS: Taken together, these data suggest that celastrol post-treatment reduces ischemic stroke-induced brain damage, suggesting celastrol may represent a novel potent pharmacological therapy.

Beetroot (Beta vulgaris) rescues mice from γ-ray irradiation by accelerating hematopoiesis and curtailing immunosuppression.

CONTEXT: Beetroot [Beta vulgaris Linné (Chenopodiaceae)], a vegetable usually consumed as a food or a medicinal plant in Europe, has been reported to have antioxidant and anti-inflammatory properties. Since the lymphohematopoietic system is the most sensitive tissue to ionizing radiation, protecting it from radiation damage is one of the best ways to decrease detrimental effects from radiation exposure. OBJECTIVE: In this study, we evaluated the radio-protective effects of beetroot in hematopoietic stem cells (HSCs) and progenitor cells. MATERIALS AND METHODS: Beetroot extract was administered at a dose of 400 mg/mouse per os (p.o.) three times into C57BL/6 mice and, at day 10 after γ-ray irradiation, diverse molecular presentations were measured and compared against non-irradiated and irradiated mice with PBS treatments. Survival of beetroot-fed and unfed irradiated animal was also compared. RESULTS: Beetroot not only stimulated cell proliferation, but also minimized DNA damage of splenocytes. Beetroot also repopulated S-phase cells and increased Ki-67 or c-Kit positive cells in bone marrow. Moreover, beetroot-treated mice showed notable boosting of differentiation of HSCs into burst-forming units-erythroid along with increased production of IL-3. Also, beetroot-treated mice displayed enhancement in the level of hematocrit and hemoglobin as well as the number of red blood cell in peripheral blood. Beetroot diet improved survival rate of lethally exposed mice with a dose reduction factor (DRF) of 1.1. DISCUSSION AND CONCLUSION: These results suggest that beetroot has the potency to preserve bone marrow integrity and stimulate the differentiation of HSCs against ionizing radiation.

Protective effects of paeoniflorin and albiflorin on chemotherapy-induced myelosuppression in mice.

Paeonia lactiflora root (baishao in Chinese) is a commonly used herb in traditional Chinese medicines (TCM). Two isomers, paeoniflorin (PF) and albiflorin (AF), are isolated from P. lactiflora. The present study aimed to investigate the protective effects of PF and AF on myelosuppression induced by chemotherapy in mice and to explore the underlying mechanisms. The mouse myelosuppression model was established by intraperitoneal (i.p.) injection of cyclophosphamide (CP, 200 mg·kg(-1)). The blood cell counts were performed. The thymus index and spleen index were also determined and bone morrow histological examination was performed. The levels of tumor necrosis factor-α (TNF-α) in serum and colony-stimulating factor (G-CSF) in plasma were measured by Enzyme-Linked Immunosorbent Assays (ELISA) and the serum levels of interleukin-3 (IL-3), granulocyte-macrophagecolony-stimulatingfactor (GM-CSF), and interleukin-6 (IL-6) were measured by radioimmunoassay (RIA). The levels of mRNA expression protein of IL-3, GM-CSF and G-CSF in spleen and bone marrow cells were determined respectively. PF and AF significantly increased the white blood cell (WBC) counts and reversed the atrophy of thymus. They also increased the serum levels of GM-CSF and IL-3 and the plasma level of G-CSF and reduced the level of TNF-α in serum. PF enhanced the mRNA level of IL-3 and AF enhanced the mRNA levels of GM-CSF and G-CSF in the spleen. PF and AF both increased the protein levels of GM-CSF and G-CSF in bone marrow cells. In conclusion, our results demonstrated that PF and AF promoted the recovery of bone marrow hemopoietic function in the mouse myelosuppression model.

Effects and mechanisms of Bazhen decoction, Siwu decoction, and Sijunzi decoction on 5-fluorouracil-induced anemia in mice.

OBJECTIVE: To investigate the effects of Bazhen decoction (BZD), Siwu decoction (SWD) and Sijunzi decoction (SJZD) in mice with anemia induced by 5-fluorouracil (5-FU) and discussed the possible pharmacological hematopoietic mechanism to provide experimental evidence for the clinical use of the three classical prescriptions in the treatment of anemia. METHODS: Anemia was induced by intravenous injection of 5-FU and 80 female Kunming mice were randomly, assigned to oral administration of SWD, SJZD, or BZD daily for 10 days. Peripheral blood cells count and bone marrow cell cycle were monitored to evaluate anti-anemia effects. Serum cytokines, interferon-γ (IFN-γ), interleukin-3 (IL-3), erythropoietin (EPO), granulocyte-macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α) were assayed. EPO mRNA expression was assayed in kidney and liver tissue homogenates. RESULTS: BZD and SWD significantly increased the number of red blood cells, hemoglobin concentration, and hematocrit, promoted bone marrow cells to enter the cell cycle, proliferate and differentiate, significantly increased IL-3 secretion, and significantly inhibited IFN-γ secretion. BZD stimulated transcription of EPO mRNA in the kidney and liver and enhanced serum EPO expression. A therapeutic effect of SJZD was not observed. CONCLUSION: BZD and SWD treatment specifically enhanced hematopoietic function and mediated myelopoiesis by altering serum cytokines levels and accelerating entry of bone marrow cells into the cell cycle. Better curative effects were achieved via nourishing both Qi and blood (BZD) than by enriching the blood (SWD) or invigorating Qi (SWZD) alone.

Effect of curcumin on p38MAPK expression in DSS-induced murine ulcerative colitis.

The aim of this study was to determine the therapeutic effect of curcumin on dextran sulfate sodium-induced ulcerative colitis (UC) and to explore the related mechanism. Sixty mice were randomly divided into 6 groups. A group was the normal control group; B group was the model group; C group was the 1.5 mg/kg dexamethasone group based on the B group; and D, E and F groups were 15, 30, and 60 mg/kg curcumin groups, respectively, based on the B group. The mice were killed 7 days after treatment; the expression of TNF-α and MPO in colon tissue was determined with ELISA, and colon p-p38MAPK and p38MAPK mRNA expression was evaluated by immunohistochemistry and RT-PCR, respectively. In the C, D, E, and F groups, TNF-α and MPO levels significantly decreased (P < 0.05), and the expression of p-p38MAPK also significantly decreased (P < 0.01). The expression of p38MAPK mRNA in the C, D, E, and F groups decreased (P < 0.01), and there was a statistically significant difference between the E and F groups (P < 0.01). Curcumin had a therapeutic effect, which probably played a role in UC treatment by inhibiting the p38MAPK signaling pathway, thereby reducing the release of TNF-α.

Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion.

Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils.

Impact of mitomycin C on the mRNA expression signatures of immunological biomarkers in eosinophilic nasal polyposis.

BACKGROUND: The topical application of mitomycin C has been evaluated as a complementary therapy for eosinophilic nasal polyposis (ENP). However, the mechanism underlying the additional benefits of mitomycin C for the control of eosinophilic inflammation and prevention of posttherapeutic relapse remains to be elucidated. In this work, the aim was to characterize the gene expression profile by quantitative real-time polymerase chain reaction (qPCR) of proinflammatory and regulatory biomarkers that are typically associated with ENP and to assess the impact of the topical application of mitomycin C on the nasal mucosal tissue immunologic milieu after ENP surgery. METHODS: We have selected 20 patients with ENP that were recommended to undergo surgical intervention. Normal mucosal tissue was obtained from healthy nasal mucosa from six patients with absence of eosinophilic infiltration. To test the effect of mitomycin C, one side of the maxillary sinus mucosa was selected for topical application of this drug and the other received no further treatment and acted as the control. The genes interleukin-4 (IL-4), IL-5, IL-10, IL-13, chemokine (C-C motif) ligand 5 (CCL5), CCL24, colony-stimulating factor 2 (CSF2), transforming growth factor beta 1 (TGFB1), tumor necrosis factor alpha (TNF-alpha), and beta actin (ACTB) were selected for gene expression analysis by qPCR. RESULTS: The data showed higher expression of proinflammatory biomarkers and lower levels of regulatory TGFB1 transcripts in ENP mucosal tissue. Surgery with topical application of mitomycin C induced a prominent transcriptional down-regulation of the immunologic biomarkers, CCL24, TNF-alpha, CSF2, and IL-5, in ENP mucosal tissue. Additionally, this treatment restored the levels of chemokines and cytokines to those observed in the nasal mucosal tissue of control subjects, except for TGFB1, which remained below the reference pattern. Moreover, CSF2 was identified as a putative biomarker with significant predictive value for complementary prophylactic purposes after surgery in ENP patients. CONCLUSION: After the characterization of the expression signatures of immunologic biomarkers in ENP, we observed that the topical use of mitomycin C is important for the reestablishment of the immunologic microenvironment of a normal expression profile of biomarkers involved in ENP mucosal tissue.

In vitro anti-inflammatory effects of beta-carboline alkaloids, isolated from Picrasma quassioides, through inhibition of the iNOS pathway.

In our previous study, fourty-eight compounds have been isolated and identified from the roots of Picrasma quassioides, which have been widely used as a traditional Chinese medicine for clearing heat and for detoxification as described in the Chinese Pharmacopoeia. ß-Carboline alkaloids are commonly considered as main active constituents of P. quassioides, but the molecular mechanism remains yet unknown. In the present paper, one new ß-carboline alkaloid together with two known ß-carboline alkaloids have been investigated for their anti-inflammatory effect and mechanism of action in cultured macrophage RAW 264.7 cells. Griess assay was used to evaluate the inhibitory effect on the overproduction of nitric oxide. ELISA was used to determine the level of proinflammatory cytokines including TNF-α and IL-6. The inhibitory effect on the enzymatic activity of COX-2 and inducible nitric oxide synthase were tested by colorimetric and fluorimetric methods, respectively. Western blot was used to detect the expression of inducible nitric oxide synthase and COX-2. All three ß-carboline alkaloids suppressed LPS-stimulated nitric oxide production and proinflammatory cytokines secretion, including TNF-α and IL-6 in a dose-dependent manner. They also strongly inhibited the expression of inducible nitric oxide synthase and inducible nitric oxide synthase enzymatic activity, whereas the expression of COX-2 and COX-2 enzymatic activity were not affected. These results indicated that potent inhibition of nitric oxide, TNF-α, and IL-6, but not COX-2 expression and COX-2 activity, might constitute the anti-inflammatory mechanism of ß-carboline alkaloids. ß-Carboline alkaloids suppressed the overproduction of nitric oxide through downregulation of inducible nitric oxide synthase expression and inducible nitric oxide synthase enzymatic activity in an LPS-stimulated macrophage.

Panax ginseng modulates cytokines in bone marrow toxicity and myelopoiesis: ginsenoside Rg1 partially supports myelopoiesis.

In this study, we have demonstrated that Korean Panax ginseng (KG) significantly enhances myelopoiesis in vitro and reconstitutes bone marrow after 5-flurouracil-induced (5FU) myelosuppression in mice. KG promoted total white blood cell, lymphocyte, neutrophil and platelet counts and improved body weight, spleen weight, and thymus weight. The number of CFU-GM in bone marrow cells of mice and serum levels of IL-3 and GM-CSF were significantly improved after KG treatment. KG induced significant c-Kit, SCF and IL-1 mRNA expression in spleen. Moreover, treatment with KG led to marked improvements in 5FU-induced histopathological changes in bone marrow and spleen, and partial suppression of thymus damage. The levels of IL-3 and GM-CSF in cultured bone marrow cells after 24 h stimulation with KG were considerably increased. The mechanism underlying promotion of myelopoiesis by KG was assessed by monitoring gene expression at two time-points of 4 and 8 h. Treatment with Rg1 (0.5, 1 and 1.5 µmol) specifically enhanced c-Kit, IL-6 and TNF-α mRNA expression in cultured bone marrow cells. Our results collectively suggest that the anti-myelotoxicity activity and promotion of myelopoiesis by KG are mediated through cytokines. Moreover, the ginsenoside, Rg1, supports the role of KG in myelopoiesis to some extent.

Hematopoietic and myeloprotective activities of an acidic Angelica sinensis polysaccharide on human CD34+ stem cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica sinensis (AS) is a Chinese herbal medicine traditionally used in prescriptions for replenishing blood and treating abnormal menstruation and other women's diseases. AIM OF THE STUDY: This study aimed to separate and identify the major hematopoietic fraction from Angelica sinensis polysaccharides (ASPS), and to investigate the myeloprotective activity of the major bioactive fraction of ASPS as a possible supporting agent for cancer treatments. MATERIALS AND METHODS: The ASPS was fractionated with DEAE-Sepharose CL-6B column to obtain four fractions (F1, F2, F3 and F4). Each fraction was cultured with human peripheral blood mononuclear cells (MNCs) to collect conditioned medium (CM). The hematopoietic ability of various MNC-CM was then evaluated by the colony-forming assay on CD34(+) cells collected by the MACS method from human umbilical cord blood (UCB). In myeloprotective experiment, Adriblastina was used to act as the myelosuppressive agent. The monosaccharide composition of ASPS was analyzed by high-performance anion-exchange chromatography-pulse amperometric detector. RESULTS: The F2 fraction, which was found to have the highest hematopoietic activity, stimulated the human peripheral blood MNCs to secret GM-CSF and IL-3. F2 could also protect the hematopoietic function of CD34(+) cells from Adriblastina. F2 occupies 19% of ASPS and contains 0.53% protein. The monosaccharide composition of F2 was arabinose (51.82%), fructose (1.65%), galactose (29.96%), glucose (4.78%) and galacturonic acid (14.80%), with molecular weight 2.5-295 kDa. CONCLUSIONS: The bioactive fraction identified and fractionated from ASPS may be used as a health-promoting agent for anemia patients and cancer patients under chemoradiation treatment.

Herbal cocktail ka-mi-kae-kyuk-tang stimulates mouse bone marrow stem cell hematopoiesis and janus-activated kinase 2/signal transducer and activator of transcription 5 pathway.

Ka-mi-kae-kyuk-tang (KMKKT) is an Oriental herbal medicinal cocktail. Our collaborative team has shown that it has potent anti-angiogenic, anti-cancer and anti-metastatic activities in vivo without observable side effects. We have documented evidence for KMKKT to alleviate drug-induced hematotoxicity in vivo. In the present study, we investigated the mechanistic and signaling events through which KMKKT enhances hematopoiesis, using hematopoietic stem cells (HSCs) isolated from the bone marrow of 8-12 week-old C57BL/6 mice. Our results show that KMKKT significantly increased the expression of the hematopoietic cytokines interleukin (IL)-3, stem cell factor (SCF), granulocyte-macrophage-colony stimulating factor (GM-CSF), thrombopoietin (TPO) and erythropoietin (EPO) at the level of mRNA and secretion in HSCs. KMKKT also increased the expression of c-Kit, a cytokine receptor expressed in HSCs. In addition, KMKKT enhanced phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5), and increased the binding activity of STAT5 to gamma interferon activated sites (GAS) that mediate JAK2 downstream signaling. Furthermore, we found that KMKKT significantly enhanced the growth rate of colony-forming unit granulocyte erythrocyte monocyte macrophages (CFU-GEMM) and burst forming unit erythroid (BFU-E) of mouse HSCs (mHSCs) stimulated by IL-3/EPO. Overall, our results demonstrated that KMKKT alleviated drug-induced side effects through enhanced hematopoiesis, at least in part through cytokine-mediated JAK2/STAT5 signaling.

Megakaryocytopoiesis and thrombopoiesis in hematopoietic stem cells exposed to ionizing radiation.

Hematopoietic processes, especially megakaryocytopoiesis and thrombopoiesis, are highly sensitive to extracellular oxidative stresses such as ionizing radiation and chemotherapeutic agents. This study examined the terminal maturation of megakaryocytes and platelet production in hematopoietic stem/progenitor cells (HSPCs) exposed to ionizing radiation. Highly purified CD34(+) cells derived from human placental/umbilical cord blood were exposed to X rays (2 Gy, 150 kVp, 20 mA; 0.5-mm aluminum and 0.3-mm copper filters) at a dose rate of approximately 1 Gy/min and then cultured in a serum-free medium supplemented with thrombopoietin and interleukin-3. The number of cells generated from X-irradiated CD34(+) cells decreased with the time in culture. However, the fraction of CD34(+)Tie-2(+) and CD41(+)Tie-2(+) cells among the total cells generated from X-irradiated cells increased significantly in comparison to nonirradiated controls on day 7. In addition, the CD42a(+) particles, which appeared to be platelets, generated from the X-irradiated HSPCs appeared to be normal. Quantitative real-time reverse transcriptase-polymerase chain reaction analysis of the expression of various genes in cells harvested from the cultures showed that the early hematopoiesis-related genes FLI1, HOXB4 and Tie-2, the cytokine receptor genes KIT and IL3RA, and the oxidative stress-related genes HO1 and NQO1 were upregulated on day 7. These results suggest that normal terminal maturation of megakaryocytes and platelet production occur in residual HSPCs after exposure to ionizing radiation despite the adverse effect of radiation on proliferation and differentiation of HSPCs. Ionizing radiation may have the potential to promote both megakaryocytopoiesis and thrombopoiesis.

Development of a high-throughput cell-based reporter assay for screening of JAK3 inhibitors.

JAK3 is an ideal target for the treatment of immune-related diseases and the prevention of organ allograft rejection. Several JAK3 inhibitors have been identified by biochemical enzymatic assays, but the majority display significant off-target effects on JAK2. Therefore, there is a need to develop new experimental approaches to identify compounds that specifically inhibit JAK3. Here, we show that in 32D/IL-2Rß cells, STAT5 becomes phosphorylated by an IL-3/JAK2- or IL-2/JAK3-dependent pathway. Importantly, the selective JAK3 inhibitor CP-690,550 blocked the phosphorylation and the nuclear translocation of STAT5 following treatment of cells with IL-2 but not with IL-3. In an attempt to use the cells for large-scale chemical screens to identify JAK3 inhibitors, we established a cell line, 32D/IL-2Rß/6xSTAT5, stably expressing a STAT5 reporter gene. Treatment of this cell line with IL-2 or IL-3 dramatically increased the reporter activity in a high-throughput format. As expected, CP-690,550 selectively inhibited the activity of the 6xSTAT5 reporter following treatment with IL-2. By contrast, the pan-JAK inhibitor curcumin inhibited the activity of this reporter following treatment with either IL-2 or IL-3. Thus, this study indicates that the STAT5 reporter cell line can be used as an efficacious cellular model for chemical screens to identify selective JAK3 inhibitors.

Anti inflammatory potential of Decalepis hamiltonii (Wight and Arn) as evidenced by down regulation of pro inflammatory cytokines-TNF-alpha and IL-2.

ETHNOPHARMACOLOGICAL RELEVANCE: Ancient tribes in the Western Ghats of India use the roots of Decalepis hamiltonii Wight and Arn (Asclepiadaceae) for several medicinal purposes particularly inflammation. AIM: To investigate whether the pure compounds obtained from the Decalepis hamiltonii have anti inflammatory activity. MATERIALS AND METHODS: The bioactive lead molecules from the roots of Decalepis hamiltonii were extracted into dichloromethane/methanol and purified by silica gel column chromatography. Structural elucidation of the purified compounds was performed with (1)H and (13)C NMR and mass spectrometry. The in vitro anti inflammatory activity of the pure compounds was studied in mitogen induced peripheral blood mononuclear cells (PBMCs) employing [(3)H] thymidine uptake assay and their effect on cytokine expression by reverse transcriptase polymerase chain reaction (RT-PCR). The inhibition of nuclear factor kappaB (NF-kappaB) activity in the presence of pure compounds was determined in J774 A.1 cells. The cytotoxicity of the compounds was tested using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit. RESULTS AND DISCUSSION: Lupeol acetate (Compound 1) and (2S)-5,7,4'-trihydroxy flavanone 4'-O-beta-d-glucoside (Compound 2) isolated from Decalepis hamiltonii roots inhibited the proliferation of mitogen induced PBMCs with an IC(50) value of 8 and 0.5mug/ml respectively. MTT assay revealed the compounds to be non-cytotoxic. Though, both the compounds down regulated the synthesis of mRNA of the pro inflammatory cytokines, interleukin-2 (IL-2) and tumour necrosis factor-alpha (TNF-alpha), the anti inflammatory cytokine interleukin-10 (IL-10), was found to be up regulated. NF-kappaB activation in J774 A.1 cells were also inhibited by both the compounds. CONCLUSION: Lupeol acetate and (2S)-5,7,4'-trihydroxy flavanone 4'-O-beta-d-glucoside isolated from Decalepis hamiltonii roots showed anti inflammatory activities by down regulating TNF-alpha and IL-2 specific mRNA, besides up regulating the synthesis of mRNA of IL-10.

Beta-androstenes and resistance to viral and bacterial infections.

This report illustrates that the beta-androstenes are indeed able to upregulate the host immune response to a level that enables the host to resist lethal infection by viruses or bacteria. These agents consist of a subgroup of steroids, which also mediates a rapid recovery of hematopoietic precursor cells after whole-body lethal radiation injury. In vivo, the androstenes increase the levels of the Th1 cytokines such as IL-2, IL-3, and IFN. Similar to hydrocortisone, they suppress inflammation, but without immune suppression, and have a role in the maintenance of the Th1/Th2 balance and immune homeostasis.