Glaucocalyxin A attenuates allergic responses by inhibiting mast cell degranulation through HMGB1/TLR4/NF-κB signaling pathways / 药学学报

The study is to investigate the effect of glaucocalyxin A (GLA) on mast cell-mediated anaphylaxis. The animal welfare and experimental process of this experiment followed the regulations of the Animal Ethics Committee of Yanbian University. BALB/c mice were used in the animal experiment and randomly divided into five groups, control group, model group, and GLA low, medium, and high dose groups (10, 20, and 40 mg·kg-1). Mice were sensitized by intradermal injection of anti-dinitrophenyl-immunoglobulin E (DNP-IgE) into the ears and challenged with a mixture of DNP-human serum albumin (HSA) and 4% evans blue into the tail veins to prepare an animal skin passive cutaneous anaphylaxis (PCA) model, which was collected from both ears for measurement of dye staining and histology. Rat peritoneal mast cells (RPMCs) were used in the cell experiment and divided into control, IgE + antigen (Ag), and IgE + Ag + GLA groups to determine histamine release as well as calcium influx levels. High-affinity IgE receptor (FcεRI)-mediated signaling pathway proteins and HMGB1/TLR4/NF-κB (high mobility group box 1/toll like receptor 4/nuclear transcription factor kappa B) signaling proteins were detected by Western blot. The results of animal experiments suggest that GLA inhibits PCA, reduces evans blue dye exudation, and reduces ear inflammation and ear thickness in mice. The results of cellular experiments suggested that GLA could reduce histamine release and calcium influx, and inhibit tumor necrosis factor-α (TNF-α), interleukin (IL)-4, IL-13, and IL-1β production; Western blot results showed that GLA inhibited FcεRI-mediated phosphorylation levels of spleen tyrosine kinase (Syk), Lck/Yes novel tyrosine kinase (Lyn), tyrosine kinase Fyn (Fyn), growth-factor receptor-bound protein 2 (Gab2), and phospholipase C (PLC) γ1, while GLA inhibited HMGB1/TLR4 signaling pathway to limit NF-κB p65 nuclear metastasis. The results indicate that GLA inhibits mast cell degranulation and attenuates allergic inflammation through the HMGB1/TLR4/NF-κB signaling pathway.

The Mechanism of Cathepsins S Inhibitor Affected Ischemia-induced Neovascularization in Experimental Mice / 中国循环杂志

Objectives: To investigate the potential mechanism of cathepsins S inhibitor (CatS-I) affected ischemia-induced neovascularization in experimental mice. Methods: 8 week-old wild type (C57/BL6) mice were randomly divided into 2 groups: Control group, the mice received basic diet with intraperitoneal injection of 5% carboxymethylcellulose sodium and CatS-I group, the mice received basic diet with intraperitoneal injection of CatS-I (1mg/kg·d); all animals were treated for 17 days. n=20 in each group. Hind limb ischemia model was established at 3 days after injection in both groups. Blood flow was measured by laser Doppler blood flow analyzer, the ratio of ischemic area to non-ischemic area was calculated; protein expressions of peroxidase proliferation activated receptors-γ (PPAR-γ), p-Akt, p-eNOS and VEGF were examined by Western blot analysis at day 4 after the operation; frozen section of ischemic skeletal muscle was taken at 7 days after operation to measure capillary density by immunohistochemistry.Results: ① CatS-I inhibited blood flow recovery. Compared with Control group, CatS-I group had slower blood flow recovery in ischemic hind limb, P<0.05. ② CatS-I inhibited capillary formation. At 14 days after operation, capillary formation in non-ischemic skeletal muscles was similar between 2 groups, P>0.05; while in ischemic skeletal muscles, capillary density was lower in CatS-I group than Control group, P<0.01. ③ Compared with Control group, CatS-I group showed decreased protein expressions of PPAR-γ, p-Akt, p-Enos and VEGF, P<0.05. Conclusions: CatS-I regulated ischemia-induced neovascularization might be related to PPAR-γ activation and PI3K/Akt/eNOS signaling pathway in experimental mice.

miR-24-mediated knockdown of H2AX damages mitochondria and the insulin signaling pathway

Mitochondrial deficits or altered expressions of microRNAs are associated with the pathogenesis of various diseases, and microRNA-operated control of mitochondrial activity has been reported. Using a retrovirus-mediated short-hairpin RNA (shRNA) system, we observed that miR-24-mediated H2AX knockdown (H2AX-KD) impaired both mitochondria and the insulin signaling pathway. The overexpression of miR-24 decreased mitochondrial H2AX and disrupted mitochondrial function, as indicated by the ATP content, membrane potential and oxygen consumption. Similar mitochondrial damage was observed in shH2AX-mediated specific H2AX-KD cells. The H2AX-KD reduced the expression levels of mitochondrial transcription factor A (TFAM) and mitochondrial DNA-dependent transcripts. H2AX-KD mitochondria were swollen, and their cristae were destroyed. H2AX-KD also blocked the import of precursor proteins into mitochondria and the insulin-stimulated phosphorylation of IRS-1 (Y632) and Akt (S473 and T308). The rescue of H2AX, but not the nuclear form of ΔC24-H2AX, restored all features of miR-24- or shH2AX-mediated impairment of mitochondria. Hepatic miR-24 levels were significantly increased in db/db and ob/ob mice. A strong feedback loop may be present among miR-24, H2AX, mitochondria and the insulin signaling pathway. Our findings suggest that H2AX-targeting miR-24 may be a novel negative regulator of mitochondrial function and is implicated in the pathogenesis of insulin resistance.

Influenza M1 Virus-Like Particles Consisting of Toxoplasma gondii Rhoptry Protein 4

Toxoplasma gondii infections occur throughout the world, and efforts are needed to develop various vaccine candidates expressing recombinant protein antigens. In this study, influenza matrix protein (M1) virus-like particles (VLPs) consisting of T. gondii rhoptry antigen 4 (ROP4 protein) were generated using baculovirus (rBV) expression system. Recombinant ROP4 protein with influenza M1 were cloned and expressed in rBV. SF9 insect cells were coinfected with recombinant rBVs expressing T. gondii ROP4 and influenza M1. As the results, influenza M1 VLPs showed spherical shapes, and T. gondii ROP4 protein exhibited as spikes on VLP surface under transmission electron microscopy (TEM). The M1 VLPs resemble virions in morphology and size. We found that M1 VLPs reacted with antibody from T. gondii-infected mice by western blot and ELISA. This study demonstrated that T. gondii ROP4 protein can be expressed on the surface of influenza M1 VLPs and the M1 VLPs containing T. gondii ROP4 reacted with T. gondii-infected sera, indicating the possibility that M1 VLPs could be used as a coating antigen for diagnostic and/or vaccine candidate against T. gondii infection.

Significance of metronomic therapy against Helicobacter pylori for the prevention and treatment of emetogenic chemotherapy of gastric cancer / 中国肿瘤临床

Objective:To investigate the significance of metronomic therapy against Helicobacter pylori (HP) in the prevention of delayed emesis caused by chemotherapy of gastric cancer compared with the routine therapy. Methods:HP infection was confirmed by carbon 14 breath test in 69 patients. Combined chemotherapy was employed for the first time in the patients, who were divided into groups A and B. Metronomic therapy was administered to group A (n=33). Briefly, triplex medication against Helicobacter bacil i triplex was oral y ad-ministered:20 mg of omeprazole and 0.5 g of amoxicillin twice daily, with 200 mg of tinidazole once daily. Oral administration in group A was performed for 14 days from the start of chemotherapy. Simultaneously, 5-HT3 antagonists were applied. By contrast, group B (n=36) was treated with the oral triplex medication against Helicobacter bacilli:20 mg of omeprazole and 1 g of amoxicillin twice daily, with 400 mg of tinidazole once daily. Oral administration in group B was performed for 7 days from the beginning of chemotherapy with simultaneous application of 5-HT3 antagonists. Both groups were simultaneously treated with the 5-HT3 antagonist granisetron at 3 mg once daily during the administration of anti-HP therapy. HP infection was evaluated by immunohistochemistry before and after treatment. Results:The total effective rate for emesis in group A was 84.85%, which was significantly higher than that in group B (55.56%). Among the patients in group A, 15.15%demonstrated delayed emesis, compared with 44.44%of the patients in group B;the number of individuals was significantly lower in group A than in group B. The average number of chemotherapy cycles in group A was significantly higher than that in group B at 3.1 cycles;the difference between groups was statistically significant (P<0.05). In addition, the HP infection in group B was significantly lower than that in group A (P<0.05). Conclusion:Compared with one week of treatment with the conventional dose, two weeks of low-dose metronomic therapy against HP during chemotherapy can significantly reduce chemotherapy induced delayed emesis and can significantly reduce the degree of HP infection in patients with gastric cancer with HP infection.

Expression and clinical significance of MTDH and VEGF in triple-negative breast cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the expression and clinical significance of MTDH and VEGF in triple-negative breast cancer (TNBC).</p><p><b>METHODS</b>Tissue samples of 168 breast cancers (including 112 TNBC tissue and 56 non-TNBC tissue), 10 breast fibroadenomas and 15 normal breast tissues were collected. Postoperative specimens were examined by immunohistochemistry for MTDH and VEGF expression. The correlation between the expression of MTDH and VEGF and clinicopathological features was analyzed.</p><p><b>RESULTS</b>MTDH and VEGF were expressed in 57.1% and 49.4% of breast cancer patients, 64.3% and 56.3% in TNBC patients, respectively, significantly higher than that in the non-TNBC tissues, breast fibroadenomas and normal breast tissues (P<0.05 for all). Statistically significant correlation was found between the MTDH and VEGF expressions (r=0.356, P<0.001). Moreover, MTDH expression was correlated with tumor size, BMI index, lymph node metastasis, pathological stage, recurrence and metastasis, and the expression of p53 and Ki-67 proteins (P<0.05 for all). The VEGF protein expression was correlated with lymph node metastasis, pathological staging, recurrence and metastasis, and the expression of Ki-67 protein (P<0.05 for all). The patients with high expression of MTDH and VEGF showed a lower DFS and OS (P<0.05 for both).</p><p><b>CONCLUSIONS</b>MTDH and VEGF expression may be correlated with tumor angiogenesis and progression and has the potential to be valuable prognostic factors in patients with TNBC.</p>

Effect of ginsenoside(Rg3)combined with delaying tamoxifen resistance in breast cancer / 实用肿瘤学杂志

Objective To evaluate the effect and the best concentration of Rg 3 combined with delaying tamoxifen resistance in breast cancer .Methods The tamoxifen-resistant(TAM-R)cell line with TAM was es-tablished.TAM-R cell line and WT -MCF-7 cell line were cultured in TAM at different concentrations and then detected the growth of cells .WT-MCF-7 cell line was cultured medium in TAM which contained Rg 3 at different levels .The growing situation of cells was detected with MTT method .Results Compared with wild -type WT-MCF-7 cells,TAM-R showed higher curves when they were both at TAM treatment .Rg3 could be associated with delaying the emergency of tamoxifen resistance and the best concentration was 1 ×10 -8 mol/L and 5 ×10 -8 mol/L.Conclusion It might be useful to reduce the tamoxifen resistance incidence by clinically com-bined Rg3 with TAM treatment .

Role of Bcl-xL in the cathepsin D-associated apoptosis of K562 cells / 中国实验血液学杂志

The purpose of study was to explore the possible functions of Bcl-xL in the glucosamine sulfate-induced apoptosis of chronic myeloid leukemia K562 cells. Light microscopy and Wright-Giemsa staining were used to investigate the morphologic evidences for apoptosis of K562 cells induced by glucosamine sulfate (GS); immunofluorescence was used to observe the translocation of cathepsin D and cytochrome C during the apoptosis; Western blot was performed to detect the expression of Bcl-xL, Bid, Bax in K562 cells treated by GS. The results showed that many vacuoles were observed in the cytoplasma of the K562 cells treated by GS; fluorescent signals of cathepsin D and cytochrome were fransformed from granules to disperse form by using immunofluorescence; the expression of Bcl-xL was found down-regulated in K562 cells treated by GS, but not in the cells pre-treated with pepstatin A; the significant changes were not detected in expression of Bax and Bid protein before or after apoptosis. It is concluded that Bcl-xL protein may mediate relationship between cathepsin D and mitochondia pathway, Cathepsin D may play an important role in the GS inducing apoptosis of K562 cells through downregulation of Bcl-xL expression.

Reversion of multidrug resistance in HL-60/VCR cells by down-regulation of bcl-2 with bcl-2 siRNA / 中国实验血液学杂志

To evaluate the feasibility of gene therapy using bcl-2 as target in multiple drug resistance of leukemia, the small interfering RNA eukaryotic expression vector specific to human bcl-2 gene was constructed by gene recombination, then transfected into HL-60/VCR cells. Stable transfectants were obtained by G418 screening. The growth curve and drug sensitivity were detected by using MTT. The expression of Bax and ZNRD1 was analyzed by Western blot. The results showed that mU6pro-bcl-2 siRNA was successfully constructed and transfected into HL-60/VCR cells. The IC(50) of transfected cells to vincristine and adriamycin was significantly reduced as compared with that of the control. The expression of ZNRD1 in transfected cells was decreased as compared with that of the control, while Bax not. It is concluded that the bcl-2 siRNA restores the sensitivity of HL-60/VCR cells to conventional chemotherapeutic agents to a certain degree.