[Preliminary study on effect of Phellinus igniarius ethanol extract on serum uric acid metabolism and gut microbiome in rats].

The aim of this paper was to investigate the effect of ethanol extract of Phellinus igniarius in lowering uric acid and changing the gut microbiome in hyperuricemia rats. A total of 36 SD rats were randomly divided into normal control group, model control group, positive drug control group, and high-dose, middle-dose and low-dose P. igniarius ethanol extract groups, with 6 rats in each group. Hyperuricemia rats were established by D-fructose combined with oteracil potassium(OAPS). One week later, the positive control group was given allopurinol 50 mg·kg~(-1) intragastrically, and P. igniarius ethanol extract groups were treated with 30, 60 and 90 mg·kg~(-1) drugs for 14 consecutive days. Body weight, blood glucose and serum uric acid(SUA) were monitored every week. After the model rats were administered with the ethanol extracts of P. igniarius by gavage for two weeks, the activities of creatinine, BUN, xanthine oxidase(XOD) and adenosine deaminase(ADA) were detected. The right kidney was taken to analyze the histological and morphological changes and the degree of damage to main organs of the extract of P. igniarius. The 16 S rDNA gene sequence technique was used to analyze the guts microbiota composition in feces. The results indicated that ethanol extract of P. igniarius could significantly lower the SUA level(P<0.01), while inhibiting the activities of XOD and ADA(P<0.05, P<0.01). Histological examination showed that the allopurine group showed slight renal tubular dilation and inflammatory cell infiltration compared with the normal group, with no significant difference between the P. igniarius ethanol extract groups and the normal group. The 16 S sequencing results showed that the composition of gut microbiota has changed in each group. Therefore, ethanol extracts of P. igniarius may reduce the level of SUA in rats by inhibiting the activities of XOD and ADA, with a certain effect on the composition of gut microbiota.

Prodigiosin isolated from Serratia marcescens in the Periplaneta americana gut and its apoptosis­inducing activity in HeLa cells.

Serratia marcescens are considered to be abundant and optimal resources for obtaining prodigiosin, which can be isolated from soil, water, plants and air but rarely from insects. In the present study, a strain of Serratia marcescens named WA12­1­18 was isolated from the gut of Periplaneta americana, which was capable of producing high levels of pigment reaching 2.77 g/l via solid fermentation and was identified as prodigiosin by ultraviolet, high performance liquid chromatography (LC), Fourier­transform infrared spectroscopy, LC­mass spectroscopy and nuclear magnetic resonance. The apoptotic tumor cells treated with prodigiosin were examined by 4',6­diamidino­2­phenylindole (DAPI) staining assays and transmission electron microscopy. Flow cytometry (FCM) was utilized to measure the apoptotic rate with Annexin V staining and the expression levels of proteins involved in apoptosis, including B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X (Bax) and caspase­3 were determined by western blot analysis and reverse transcription­quantitative polymerase chain reaction (RT­qPCR). The experimental results revealed that prodigiosin could inhibit the proliferation of HeLa cells and the half­maximal inhibitory concentration values of prodigiosin in HeLa were 2.1, 1.2 and 0.5 µg/ml over 24, 48 and 72 h, respectively. Furthermore, DAPI staining assays and transmission electron microscopy clearly demonstrated that prodigiosin could induce HeLa cell apoptosis. FCM results revealed that the cell apoptotic rates were 19.7±1.4, 23.7±2.4 and 26.2±2.3% following the treatment with 0.5, 1.0 and 2.0 µg/ml prodigiosin for 48 h, respectively. Western blot analysis and RT­qPCR revealed that prodigiosin could activate apoptosis­associated molecules including Bcl­2, Bax and caspase­3. Therefore, the results of the present study demonstrated that the prodigiosin could induce apoptosis in HeLa cells, which may be associated with the upregulation of Bax and caspase­3, the concomitant downregulation of Bcl­2 levels and also triggering the extrinsic apoptotic signaling pathway.

Selective and effective targeting of chronic myeloid leukemia stem cells by topoisomerase II inhibitor etoposide in combination with imatinib mesylate in vitro.

Imatinib mesylate (IM) and other BCR-ABL tyrosine kinase inhibitors (TKIs) have improved chronic myeloid leukemia (CML) patient survival markedly but fail to eradicate quiescent CML leukemia stem cells (LSCs). Thus, strategies targeting LSCs are required to induce long-term remission and achieve cure. Here, we investigated the ability of topoisomerase II (Top II) inhibitor etoposide (Eto) to target CML LSCs. Treatment with Eto combined with IM markedly induced apoptosis in primitive CML CD34+ CD38- stem cells resistant to eradication by IM alone, but not in normal hematopoietic stem cells, CML and normal mature CD34- cells, and other leukemia and lymphoma cell lines. The interaction of IM and Eto significantly inhibited phosphorylation of PDK1, AKT, GSK3, S6, and ERK proteins; increased the expression of pro-apoptotic gene Bax; and decreased the expression of anti-apoptotic gene c-Myc in CML CD34+ cells. Top II inhibitors treatment represents an attractive approach for targeting LSCs in CML patients undergoing TKIs monotherapy.

[Metabolism Changes of Main Active Ingredients During Liquid Fermentation of Cordyceps militaris].

Objective: To study the accumulation and changes of main active ingredients during liquid fermentation of Cordyceps militaris. Methods: The militaris varity GIM5. 270 was selected to extended fermentation time to 20 days on the basic fermentation condition. Meanwhile, the accumulation and dynamic changes of biomass, polysaccharide, cordycepic acid, adenosine and cordycepin in the fermentation system were detected by the analytical method of contents per 24 hour. Results: The foundation culture medium composed of complex nitrogen sources could reach a higher biomass level than single nitrogen sources. In addition, with the development of time, the mycelial biomass increased, the contents of polysaccharide and cordycepic acid( D-mannitol) increased firstly and then decreased, the contents of adenosine decreased gradually and cordycepin( 3-deoxy adenosine) increased gradually. Conclusion: The whole system is observed autolyzed phenomenon caused by absorbing self-generated nutrients. In this study, the dynamic changes of the main active ingredients in the fermentation system are researched and the optimum collecting time is determined, which provides evidence for reaching a better yield of the active ingredients.

Silencing of miR-21 sensitizes CML CD34+ stem/progenitor cells to imatinib-induced apoptosis by blocking PI3K/AKT pathway.

BCR-ABL tyrosine kinase inhibitor imatinib fails to eradicate leukemia stem cells (LSCs), the underlying mechanisms maintaining CML LSCs remain poorly understood. Here, we showed that transient inhibition of miR-21 by antagomiR-21 markedly increased imatinib-induced apoptosis in CML, but not normal CD34+ stem/progenitor cells. Furthermore, PI3K inhibitors also significantly sensitized CML CD34+ cells to imatinib-induced apoptosis. MiR-21 or PI3K inhibitor in combination with imatinib treatment significantly decreased AKT phosphorylation and c-Myc expression than either agent did alone, but did not affect Bim and Bcl-6 expresssion. These findings indicate that miR-21 is required for maintaining the imatinib-resistant phenotype of CML CD34+ cells through PI3K/AKT signaling pathway, thus providing the basis for a promising therapeutic approach to eliminate CML LSCs.

Targeting miR-21 sensitizes Ph+ ALL Sup-b15 cells to imatinib-induced apoptosis through upregulation of PTEN.

Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) cells are insensitive to BCR-ABL tyrosine kinase inhibitor imatinib, the underlying mechanisms remain largely unknown. Here, we showed that imatinib treatment induced significant upregulation of miR-21 and downregulation of PTEN in Ph+ ALL cell line Sup-b15. Transient inhibition of miR-21 resulted in increased apoptosis, PTEN upregulation and AKT dephosphorylation, whereas ectopic overexpression of miR-21 further conferred imatinib resistance. Furthermore, knockdown of PTEN protected the cells from imatinib-induced apoptosis achieved by inhibition of miR-21. Additionally, PI3K inhibitors also notably enhanced the effects of imatinib on Sup-b15 cells and primary Ph+ ALL cells similar to miR-21 inhibitor. Therefore, miR-21 contributes to imatinib resistance in Ph+ ALL cells and antagonizing miR-21 demonstrates therapeutic potential by sensitizing the malignancy to imatinib therapy.

[Gleevec induces apoptosis in K562 cells through activating caspase-3].

The present study is to elucidate the mechanisms underlying Gleevec-induced apoptosis of chronic myeloid leukemia (CML) K562 cells in vitro. The apoptotic cell death and cell cycle distribution after Gleevec treatment and the effect of PDCD4 siRNA on Gleevec-induced apoptosis of K562 cells were analyzed by flow cytometry. The effect of Gleevec on p-Crkl, caspase-3, PARP and PDCD4 protein levels, and the knockdown efficacy of PDCD4 siRNA were detected by Western blotting. The results showed that Gleevec dramatically suppressed the phosphorylation level of Crkl in a dose-dependent manner and induced significant apoptosis and G0/G1 cell cycle arrest of K562 cells in time- and dose-dependent manners. In addition, Gleevec activated caspase-3 and its downstream substrates PARP, and the caspase pan inhibitor Z-VAD-FMK (50 micromol x L(-1)) markedly reduced Gleevec-induced apoptosis from 47.97% +/- 10.56% to 31.05% +/- 9.206% (P < 0.05). Moreover, Gleevec significantly increased the protein expression of programmed cell death 4 (PDCD4). PDCD4 knockdown by siRNA reduced Gleevec-induced apoptosis from 46.97% +/- 14.32% to 42.8% +/- 11.43%. In summary, Gleevec induced apoptosis in K562 cells via caspase-3 activation.

Cecropin suppresses human hepatocellular carcinoma BEL- 7402 cell growth and survival in vivo without side-toxicity.

Conventional chemotherapy against hepatocellular carcinoma typically causes various side effects. Our previous study showed that cecropin of Musca domestica can induce apoptosis in human hepatocellular carcinoma BEL-7402 cells in vitro. However, whether cecropin inhibits BEL-7402 cell in vivo and the question of possible side effects remained undentified. The present study confirmed tumor-inhibitory effects of cecropin in vivo, and furthermore strongly suggested that cecropin cytotoxicity in BEL-7402 cells in vivo may be mainly derived from its pro-apoptotic action. Specifically, we found that cecropin exerted no obvious side effects in tumor-bearing mice as it had no significant hematoxicity as well as visceral toxicity. Therefore, cecropin may be a potential candidate for further investigation as an antitumor agent against hepatocellular carcinoma.

Inhibitory effects of α-pinene on hepatoma carcinoma cell proliferation.

BACKGROUND: Pine needle oil from crude extract of pine needles has anti-tumor effects, but the effective component is not known. METHODS: In the present study, compounds from a steam distillation extract of pine needles were isolated and characterized. Alpha-pinene was identified as an active anti-proliferative compound on hepatoma carcinoma BEL-7402 cells using the MTT assay. RESULTS: Further experiments showed that α-pinene inhibited BEL-7402 cells by arresting cell growth in the G2/M phase of the cell cycle, downregulating Cdc25C mRNA and protein expression, and reducing cycle dependence on kinase 1(CDK1) activity. CONCLUSION: Taken together, these findings indicate that α-pinene may be useful as a potential anti-tumor drug.

In vitro study of the cytotoxicities of two mixed-ligand oxovanadium complexes on human hepatoma cells.

The cytotoxicities of two oxovanadium complexes, VOI [VO(satsc)(phen)] (satsc = salicylaldehyde thiosemicarbazone, phen = 1,10-phenanthroline) and VOII [VO(3,5-dibrsatsc)(phen)](3,5-dibrsatsc = 3,5-dibromosalicylaldehyde thiosemicarbazone), were studied by performing MTT assays on human hepatoma cell lines BEL-7402, HUH-7 and HepG2. The results showed that both the VOI and VOII complexes possess significant anti-proliferative effects. In addition, the anti-proliferative mechanism of the complexes was analyzed by cell cycle analysis and an apoptosis assay and by detecting the mitochondrial membrane potential (delta psi m). The experimental results showed that the complexes can cause a G0/G1 phase cell cycle arrest and can significantly decrease delta psi m, causing depolarization of the mitochondrial membrane. Notably, the two complexes induced apoptosis in BEL-7402 cells and displayed typical morphological apoptotic characteristics. The cytotoxicities of the VOII complex are significantly stronger than that of the VOI complex, suggesting that the cytotoxic effects of oxovanadium complexes may be associated with the electronic effects of the complexes.

Effect of lipopolysaccharide mediating early- and late- activated THP-1 macrophages on ECV304 endothelial cell dysfunction: dysregulation of secretion of VEGF and proliferation and migration of ECV304.

OBJECTIVE: To investigate the different effects of lipopolysaccharide (LPS) mediating early and late activated THP-1 macrophages (Mφ) on ECV304 endothelial cell dysfunction: dysregulation of secretion of VEGF and proliferation, and migration of ECV304. METHODS: The inflammatory Mφ was divided into early phase (2 h) group and late phase (24 h) group according the different exposure time to LPS. Then the inflammatory Mφ and ECV304 were co-cultured via transwell chambers in both non-contacting and contacting systems. The levels of VEGF were determined by ELISA, and the proliferation index and apoptosis of ECV304 were analyzed by FACSCalibur. The migration of ECV304 was tested by modified Boyden chamber assay. RESULTS: The level of VEGF and the proliferation of ECV304 cell were increased more apparently in early-phase Mφ-treated group. But the proportion of early apoptotic and late apoptotic/necrotic cells in late-phase Mφ-treated group were higher than that of the former. Migration rate of ECV304 was enhanced in early-phase Mφ-treated group. All those effects were more significant in contacting system comparing with no-contacting system. CONCLUSION: Early-activated macrophages (mediated by LPS) could increase the secretion of VEGF and promote the proliferation and migration of ECV304; while the late-activated macrophages could promote/enhance the apoptosis of ECV304 more significant in contacting system when (it was) compared with no-contacting system.

Effects of Musca domestica cecropin on the adhesion and migration of human hepatocellular carcinoma BEL-7402 cells.

This study was designed to explore the effects of Musca domestica antimicrobial peptides cecropin on the adhesion and migration of human hepatocellular carcinoma BEL-7402 cells. The adhesive and migratory capacities were determined by adhesion assay and transwell assay, respectively. The changes in microvilli of tumor cells were determined by scanning electron microscopy (SEM). Western blotting and quantitative polymerase chain reaction (qPCR) were carried out to determine the expression levels of proteins related to adhesion and migration, such as matrix metalloproteinase-2 (MMP2), tissue inhibitors of metalloproteinase-2 (TIMP2), and epithelial cadherin (E-cadherin). We found that Musca domestica cecropin inhibited the adhesion and migration of BEL-7402 cells, which also displayed curling microvilli, increased ball structures on cell surface, gradually broken connections between tumor cells, and even disappeared microvilli on some cells. The expression of MMP2 was significantly reduced after cecropin treatment, while the levels of TIMP2 and E-cadherin were significantly increased. These results suggest that Musca domestica cecropin inhibits the adhesion and migration of human hepatocellular carcinoma BEL-7402 cells by destroying the microvilli of tumor cells and changing the expression of MMP2, TIMP2 and E-cadherin.

Curcumin induces FasL-related apoptosis through p38 activation in human hepatocellular carcinoma Huh7 cells.

AIM: The aim of this study is to explore the underlying molecular mechanism of curcumin-induced apoptosis in human hepatocellular carcinoma (HCC) Huh7 cells. MAIN METHODS: Fas and FasL mRNA expression was analyzed by reverse transcription PCR. Western blot was applied to detect the protein expression of Bcl-2 family members, MAPK family members, c-Jun, c-Fos, ATF-2, caspase-3, PARP, TNF receptor family members and the respective ligands. Apoptotic cells were assayed with annexin V/PI double staining and flow cytometry. KEY FINDINGS: Curcumin treatment resulted in a fast and significant increase of Fas and Fas ligand (FasL) along with activation of caspase-3 and cleavage of PARP in Huh7 cells. Inhibition of caspase-3 activity by the specific inhibitor Z-DEVD-FMK rescued Huh7 cells from curcumin-induced apoptosis. Neutralization of FasL significantly protected the cells from curcumin-induced caspase-3 activation and apoptosis in a dose-dependent manner. Moreover, p38 was rapidly activated in response to curcumin, and inactivation of p38 by pharmacologic inhibitor SB203580 dramatically suppressed curcumin-induced FasL expression and apoptosis. SIGNIFICANCE: Our results demonstrated that curcumin induces apoptosis through p38-denpendent up-regulation of FasL in Huh7 cells.

[Comparison of the effects of traditional and modern processing methods on antibacterial and anti-inflammatory effects of Musca domestica].

OBJECTIVE: To investigate the different effects of traditional and modern processing methods onantibacterial and anti-inflammatory effects of Musca domestica. METHODS: Antibacterial and anti-inflammatory effects of traditional and modem processing products were carried out on Staphylococcus aureus, Escherichia coli and macrophage RAW264.7 which activated by LPS. RESULTS: The antibacterial and anti-inflammatory effects were more pronounced in modern processing product treatment group than those of traditional processing product treatment group. CONCLUSION: Modern processing technology can protect the substances in Musca domestica which have antibacterial and anti-inflammatory effects.

ß-defensin 2 as an adjuvant promotes anti-melanoma immune responses and inhibits the growth of implanted murine melanoma in vivo.

ß-defensin 2 is a small antimicrobial peptide of the innate immune system and has been thought to regulate anti-tumor immunity. However, little is known on whether ß-defensin 2 could modulate melanoma-specific NK and T cell responses. In this study, we first cloned the murine ß-defensin 2 gene by RT-PCR and generated the ß-defensin 2 stably expressing B16 cells (B16-mBD2). Subsequently, we evaluated whether vaccination with irradiated B16-mBD2 could modulate the growth of implanted B16 cells and determined the potential mechanisms underlying the action of B16-mBD2 vaccine in modulating the growth of B16 tumors in C57BL/6. We found that vaccination with irradiated B16-mBD2, but not with control B16-p or parental B16, inhibited the development and progression of B16 tumors, and prolonged the survival of tumor-bearing mice. However, vaccination with irradiated B16-mBD2 failed to inhibit the development of B16 tumors in the CD4(+)- or CD8(+)-depleted recipients. Furthermore, vaccination with irradiated B16-mBD2 stimulated strong NK activity and promoted potent B16-specific CTL responses, accompanied by augmenting IFN-γ and IL-12, but not IL-4, responses in the recipient mice. Moreover, vaccination with irradiated B16-mBD2 promoted the infiltration of CD8(+) and CD4(+) T, NK cells and macrophages in the tumor tissues. These data suggest ß-defensin 2 may act as a positive regulator, promoting anti-tumor NK and T cell responses in vivo. Therefore, ß-defensin 2 may be used for the development of immunotherapy for the intervention of melanoma.

[The target of Musca domestica cecropin on human hepatocellular carcinoma BEL-7402 cells].

Human hepatocellular carcinoma BEL-7402 cells were treated with 50 micromol/L Musca domestica cecropin for 12 h, and observed under scanning electron microscope. The effect of Musca domestica cecropin labeled with FITC (FITC-cecropin) on BEL-7402 cells was detected by laser scanning confocal microscopy. The scanning electron microscopy showed that most microvilli on the surface of BEL-7402 cells disappeared at 12 h after cecropin treatment. The laser scanning confocal microscopy revealed that most FITC-cecropin combined with BEL-7402 cell membrane, and partly in the cytoplasm.

Housefly maggots (Musca domestica) protein-enriched fraction/extracts (PE) inhibit lipopolysaccharide-induced atherosclerosis pro-inflammatory responses.

AIM: To investigate the effects of housefly maggot (Musca domestica) protein-enriched fraction/extracts (PE) on lipopolysaccharide (LPS)-induced atherosclerosis (AS) pro-inflammatory responses in mice and macrophages. METHODS: The mouse model of AS was established by feeding a cholesterol-enriched diet and inducing by LPS. Changes in the levels of blood lipids (total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL) and high-density lipoprotein cholesterol (HDL)) and pro-inflammatory cytokines (interferon-gamma (IFNγ), tumor necrosis factor alpha (TNFα) and interleukin-1alpha (IL-1α)) were determined. Histomorphometric analysis of the pathological condition of the artery was also carried out. The macrophages were stimulated by LPS in the presence or absence of PE, and then the levels of TNFα, IL-1α and monocyte chemotactic protein 1 (MCP-1) in cell culture supernatant were measured. RESULTS: Compared with the negative control group, the levels of three pro-inflammatory cytokines were significantly enhanced in the PE treatment group (p< 0.01). The concentrations of TC, TG and LDL were lower in the PE treatment group than in the negative control group (p< 0.01). HDL concentration in the PE treatment group was higher than in the negative control group (p< 0.01). Histomorphometric analysis showed that the thickness of the intima and media area, as well as the area ratio of the intima to media in the PE treatment group were lower than in the negative control group (p< 0.01). The expression of TNFα, IL-1α and MCP-1 in LPS-induced macrophages was inhibited by different concentrations of PE (p< 0.01). CONCLUSION: These results indicate that PE potently inhibited multiple pro-inflammatory responses in experimental atherosclerosis lesions in vivo, and possessed anti-pro-inflammatory properties in vitro.

Fructose-1,6-diphosphate attenuates acute lung injury induced by lipopolysaccharide in mice.

Fructose-1,6-diphosphate (FDP), a high-energy glycolytic pathway intermediate, is reported to have a salutary effect in endotoxic shock and sepsis, but its underlying mechanism of action in inflammation is incompletely understood. In this study, our aim was to examine the function of FDP on acute lung injury (ALI) induced by lipopolysaccharide (LPS). We found that in vitro pretreatment with FDP remarkably repressed the production of TNF-alpha and IL-6 in murine alveolar macrophages MH-S exposed to LPS. In the mouse model of LPS-induced inflammatory lung injury, intravenous precondition of a single 400 mg/kg dose of FDP resulted in a significant reduction in LPS-mediated extravasation of Evans blue dye albumin, bronchoalveolar lavage leucocyte content, and lung tissue myeloperoxidase activity (reflecting phagocyte infiltration). Furthermore, histopathologic examination indicated that alveolitis with inflammatory cells infiltration and alveolar hemorrhage in the alveolar space was less severe in the FDP-treated mice than in the mice treated by LPS alone at 24 h. Additionally, pretreatment with FDP markedly decreased the transcription of TNF-alpha, IL-6 and inducible NO synthase (iNOS), and suppressed the nuclear translocation of NF-kappaB in lung tissues in response to LPS challenge. These results thus suggested that FDP plays an anti-inflammatory role in LPS-mediated acute lung injury, possibly through abrogation of NF-kappaB activation.