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Systemic sclerosis (SSc) is a chronic autoimmune disease that can involve multiple organs throughout the body. It is characterized by extensive vascular disease and fibrosis of the skin and internal organs, but its mechanism is still unclear. Studies have confirmed that the Wnt pathway is involved in SSc fibrosis, but its pathological role in vascular lesions has not been reported. In this study, the bleomycin (BLM)-induced SSc mouse model was used to investigate the role of Wnt pathway in SSc cutaneous vascular lesions. Eighteen BALB/C mice were randomly divided into control, model and treatment groups. The control group of mice was injected with PBS 100 μL/d. The model group was injected with BLM 100 μL/d at a concentration of 1 mg/mL. The treatment group of mice received injection of BLM 100 μL as the model group of mice and retroperitoneal injection of iCRT3 (Wnt and beta-catenin inhibitors) 5 mg/kg/d. Mice were sacrificed on day 28. The thickness of dorsal dermis and epidermis in the model group induced by BLM was significantly higher than that in the control group (P <0. 05). The skin appendages such as sebaceous glands and hair follicles in the model group were significantly reduced. At the same time, the thickness of fat layer became thinner and surrounded by fibrous tissue, and the collagen deposition in the model group was higher than that in the control group. It was identified at the histological level by immunohistochemical staining that α-SMA expression in model group and treatment group α-SMA is highly expressed in skin tissues, and the positive expression of α-SMA around blood vessels was significantly higher than that in the control group. In addition, the expression of IL-6 and IL-17 in serum of model group was significantly higher than that in control group (P<0. 05), and the expression of IL-6 and IL-17 in serum of treatment group was significantly lower than that in model group (P<0. 05). Furthermore, q-PCR analysis showed that the mRNA levels of β-catenin in the skin microvessels of the mice were higher in the model and the treatment groups as compared with the control group. The protein levels of Wnt5A, β-catenin, α-SMA and col1A1 were analyzed by Western blotting and results showed that the levels of fibrosis-related proteins, α-SMA and col1A1, were increased in the model group injected with BLM as compared with the control group (P<0. 05), whereas iCRT3 treatment attenuated the upregulation of α-SMA and col1A1 induced by BLM in the treatment group (P<0. 05). The levels of Wnt pathway-related proteins β-catenin and Wnt5A were significantly increased in the model group as compared with the control (P<0. 05). This study suggests that BLM can successfully induce the skin phenotype of mice with systemic sclerosis. Abnormal activation of Wnt signaling pathway is involved in BLM-induced skin microangiopathy in mice with scleroderma, and the specific Wnt pathway inhibitor iCRT3 can reduce BLM-induced scleroderma. The expression of α-SMA and col1A1 proteins in mouse skin microvessels can improve the microvascular lesions of mouse skin. Inhibition of Wnt pathway may directly or indirectly down-regulate the abnormal expression of cytokines IL-6 and IL-17, and interfere with BLM-induced progression of vascular lesions in mice.
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Aim To study the effect of tetrandrine derivative HL-49 on the conformation and biological ac-tivity of Bloom helicase ( BLM ) , and to explore its antitumor mechanism.Methods The effect of HL-49 on the conformation of BLM helicase was studied by ultra- violet spectroscopy.The effects of HL-49 on DNA binding activity, DNA chain dissociation activity and ATPase activity of HL-49 on BLM DNA helicase were analyzed by fluorescence polarization and malachite green-ammonium phosphomolybdate colorimetric method.Results HL-49, a tetrandrine derivative, indirectly inhibited the ATPase activity of BLM DNA heli- case and DNA unwinding activity by reversible binding with DNA.The results of fluorescence polarization experiments showed that HL-49 could not affect the bind ing activity of BLM DNA helicase to DNA (dsDNA/ss- DNA) , but could bind to DNA in a concentration-de- pendent manner (P < 0.01).With the increase of HL- 49 concentration, the DNA unwinding ability of BLM DNA helicase decreased, and the Kobs value decreased gradually.The results of malachite green-ammonium phosphomolybdate colorimetry showed that HL-49 could significantly inhibit the ATPase activity of BLM DNA helicase.Conclusions HL49 can inhibit the ATPase activity and DNA unwinding activity of BLM DNA helicase by the reversible binding with DNA.
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Aim To study the variations of endogenous differential metabolites and metabolic pathways in PF rat model induced by bleomycin based on GC-MS.Methods The rat model for pulmonary fibrosis was induced by intratracheal instillation of bleomycin.Model rats were randomly divided into 7 days group of PF model(M7), 14 days group of model(M14), 21 days group of model(M21)and 28 days group of model(M28), with 6-8 rats in each group.On the 8th, 15th, 22nd and 29th days of the modeling, the rat model was evaluated by histopathology and hydroxyproline(HYP)detection.The non-targeted metabolomics was studied by GC-MS in order to find the related metabolites in the serum of PF rats and the metabolic pathways were analysed by MetaboAnalyst.Results Compared with control group, the phenomenon of rat lung lesions gradually appeared in each model group(M7, M14, M21, M28)with the modeling process.9, 14, 35, and 13 statistically significant metabolites were screened and identified respectively in M7, M14, M21 and M28.Among them, glycerol and phosphate were the early significant changes of pulmonary fibrosis.3-(3-hydroxyphenyl)propionic acid, alanine, galactonic acid, and serine changed significantly in the later stages of pulmonary fibrosis, and linoleic acid was the common main different metabolite.The main pathways affecting metabolism involved lipid metabolism, amino acid metabolism.Conclusion Abnormal amino acid metabolism and lipid metabolism are the main metabolic characteristics during the formation and development of PF by BLM.
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; Aim To explore the anti-tumor mechanism of dihydromyricetin (DMY), a kind of flavonoid compound with anti-inflammatory and anti-tumor effects, via studying the effect of DMY on biological activities of Bloom helicase. Methods The effect of DMY on the biological activities of BLM helicase was studied by ultraviolet spectrum (UV), circular dichroism (CD), fluorescence polarization and free phosphorus detection. Results The results of CD and UV showed that DMY could bind to a site of the BLM helicase. In the concentration of DMY in 0 ~ 25 μmol . L 1 range, DMY showed a positive correlation with the interference ability of BLM helicase secondary structure with the increase of concentration, while in the concentration of DMY in 25 ~ 75 μmol . L 1 range, DMY showed a negative correlation. Fluorescence polarization and free phosphorus detection experiments showed that DMY could bind to BLM helicase, thus inhibiting the helicase activity of BLM helicase. Conclusions DMY can competitively bind to the DNA binding site of BLM helicase and change the spatial structure of BLM helicase, inhibiting the binding of BLM helicase to DNA and the biological activity of BLM helicase accordingly.
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Aim To investigate the effects of bisbenzyl-isoquinoline alkaloid tetrandrine derivative HL-27 on the biological properties of the BLM642-1290 helicase. Methods Fluorescence polarization technique was used to investigate the effects of bisbenzylisoquinoline alkaloid tetrandrine derivative HL-27 on the DNA bind-ing activity and unwinding activity of the BLM642-1290 helicase. Malachite green-phosphate ammonium molyb-date colorimetry was used to investigate the effects of HL-27 on the ATPase activity of the BLM642-1290 heli-case. Ultraviolet spectral scanning was used to investi-gate the effects of HL-27 on the conformation of the BLM642-1290 helicase. Results When the concentra-tion of HL-27 reached 33.34 μmol·L-1, the inhibi-tion ratio of dsDNA and ssDNA binding activity of the BLM642-1290 helicase was 41.35% and 59.54% , re-spectively. When the concentration of HL-27 reached 50 μmol·L-1, the inhibition ratio of DNA unwinding activity of the BLM642-1290 helicase was 78.68% . When the concentration of HL-27 reached 100 μmol· L-1, the inhibition ratio of ATPase activity of the BLM642-1290 helicase was 43.8% . Conclusion The DNA binding activity, ATPase activity and unwinding activity of the BLM642-1290 helicase can be inhibited by bisbenzylisoquinoline alkaloid tetrandrine derivative HL-27.
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Objective:To explore the mechanism of the embolism and sclerotherapy of fibrin glue combined with bleomycin (FG/BLM) for the treatment of cervicofacial vascular malformations by color doppler ultrasound.Methods:10 patients with venous malformation(VM) and 10 patients with arterio-venous malformation(AVM) were included.All patients underwent embolism and sclerotherapy of FG/BLM guided by ultrasound.Color doppler ultrasound was used to record the real-time two-dimensional ultrasonography and color doppler image.The flow and distribution of FG/BLM after injection into the lesions were observed.Results:Two-dimensional ultrasonography showed clumps or flake strong echo after immediate injection of FG/BLM into the cavity of VMs,then floated in the abnormal venous lumen and diffused throughout the cavity.At the later stage the lesions were filled by a large number of flocculent and netted low echo,and patchy strong echo.The volume of VMs cavity expanded dramaticlly,and the blood flow signal was significantly decreased.After injection of FG/BLM into the lumen of AVMs,clumps or flake strong echo were observed,then most of the snowflake strong echo rapidly filled or scattered along with blood stream to the distal part of the vessels.The color doppler showed significantly decrease of blood flow signal.Conclusion:FG/BLM injection can embolize and block the draining vein of VM,and play a role on the storage of sclerozing agent.FG/BLM injection can embolize both the dilated blood vessels and capillary network of AVM.
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In the last years many populations of anurans have declined and extinctions have been recorded. They were related to environmental pollution, changes of land use and emerging diseases. The main objective of this study was to determine copper sensitivity of the anuran of the Amazon Rhinella granulosa and Scinax ruber tadpoles at stage 25 and Scinax ruber eggs exposed for 96 h to copper concentrations ranging from 15 µg Cu L-1 to 94 µg Cu L-1. LC50 at 96 h of Rhinella granulosa Gosner 25, Scinax ruber Gosner 25 and Scinax ruber eggs in black water of the Amazon were 23.48, 36.37 and 50.02 µg Cu L-1, respectively. The Biotic Ligand Model was used to predict the LC50 values for these species and it can be considered a promising tool for these tropical species and water conditions. Copper toxicity depends on water physical-chemical composition and on the larval stage of the tadpoles. The Gosner stage 19-21 (related to the appearance of external gills) is the most vulnerable and the egg stage is the most resistant. In case of contamination by copper, the natural streams must have special attention, since copper is more bioavailable.
Nos últimos anos foram registrados muitas extinções e declínios de populações de anuros. Eles estavam relacionados com a poluição do ambiente, a mudanças no uso da terra e ao surgimento de doenças. O principal objetivo deste estudo foi determinar a sensibilidade dos anuros amazônicos ao cobre. Os girinos de Scinax ruber e Rhinella granulosa no estadio 25 e os ovos de Scinax ruber foram expostos por 96 horas a concentrações de cobre entre 15 µg Cu L-1 a 94 µg Cu L-1. A CL50 -96 h dos girinos de Rhinella granulosa, dos girinos de Scinax ruber e dos ovos de Scinax ruber em águas pretas da Amazônia foram 23,48; 36,37 e 50,02 µg Cu L-1, respectivamente. O modelo do ligante biótico foi usado para prever os valores de CL50 para essas duas espécies e pode ser considerado uma ferramenta promissora para essas espécies tropicais e para essas condições de água. A Toxicidade de cobre depende da composição físico-química da água e do estagio larval dos girinos. O estadio 19-21 de Gosner (relacionados ao aparecimento das brânquias externas) são os mais vulnerável e o estagio de ovo é o mais resistente. Em caso de contaminação por cobre, os igarapés naturais devem ter uma atenção especial, uma vez que o cobre é mais biodisponível nesse ambiente.
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This study was aimed to confirm animal model and evaluating indicators of the chronic obstructive pulmonary disease combined with pulmonary interstitial fibrosis (PIF-COPD). PIF-COPD rats were induced by intratracheal administration of Bleomycin(BLM, 6 mg·kg-1) and cigarette smoke stimulation for 47 days. After that, the lung function and index were measured. Changes of pathomorphism of lung were observed with hematoxylin-eosin staining. Collagen protein I and III were determined by Sirius red staining. The protein antibody microarray chip was employed for serum cytokine expression pattern measurement. The results showed that in the lung function testing, the central airway resistance, tissue damping, dynamic elasticity of model rats increased significantly and the dynamic compliance reduced significantly. The lung index of model rats increased significantly (compared with the control group, P < 0.01). The pathological tests showed that lung tissues of model rats showed emphysema and fibration with inflammatory infiltration, and content of collagen type I and III increasing significantly. The contents of MMP-8, TIMP-1, IL-10, IL-13, PDGF-AA, Beta-NGF of model rats serum increased obviously compared with the control group and RAGE reduced compared with the normal control. It was concluded that rats which were induced by intratracheal administration of BLM (6 mg·kg-1) and cigarette smoke stimulation for 47 days can be used as animal model establishment of PIF-COPD. The respiratory function and serum levels of MMP-8, TIMP-1, IL-13, PDGF and NGF can be used as evaluating indicators of PIF-COPD model.
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<p><b>OBJECTIVE</b>To study the effect of fleroxacin (FLRX) on biological properties of Bloom (BLM) helicase catalytic core (BLM642-1290 helicase) in vitro and the molecular mechanism of interaction between the two molecules.</p><p><b>METHODS</b>DNA-binding and unwinding activities of BLM642-1290 helicase were assayed by fluorescence polarization and gel retardation assay under conditions that the helicase was subjected to different concentrations of FLRX. Effect of FLRX on helicase ATPase activity was analyzed by phosphorus-free assay based on a colorimetric estimation of ATP hydrolysis-produced inorganic phosphate. Molecular mechanism of interaction between the two molecules was assayed by ultraviolet and fluorescence spectra.</p><p><b>RESULTS</b>The DNA unwinding and ATPase activities of BLM642-1290 helicase were inhibited whereas the DNA-binding activity was promoted in vitro. A BLM-FLRX complex was formed through one binding site, electrostatic and hydrophobic interaction force. Moreover, the intrinsic fluorescence of the helicase was quenched by FLRX as a result of non-radioactive energy transfer. The biological activity of helicase was affected by FLRX, which may be through an allosteric mechanism and stabilization of enzyme conformation in low helicase activity state, disruption of the coupling of ATP hydrolysis to unwinding, and blocking helicase translocation on DNA strands.</p><p><b>CONCLUSION</b>FLRX may affect the biological activities and conformation of BLM642-1290 helicase, and DNA helicase may be used as a promising drug target for some diseases.</p>
Subject(s)
DNA , Metabolism , Fleroxacin , Pharmacology , Nucleic Acid Synthesis Inhibitors , Pharmacology , RecQ Helicases , Metabolism , Spectrometry, Fluorescence , Spectrophotometry, UltravioletABSTRACT
Objective; To explore the possible effect of hyperthermia combined with Prog on a human tongue cancer cell line Tca8113 and drug-resistance cell line Tca8113/BLM in vitro. Methods;The effect of hyperthermia combined with Prog on Tca8113 and Tca8113/ BLM cells were determined by MTT. The concentration of ADM, the expression of P-gp and MRP were detected by flow cytometry (FCM). Results;Compared with Prog or hyperthermia, the hyperthermia at 41 X. for 1 h combined with Prog showed obvious inhibitory effect in Tca8113 and Tca8113/BLM cells(P<0.01). The concentration of ADM in Tca8113 and Tca8113/BLM cells increased obvi-ously(P<0.01). Expression levels of P-gp and MRP in Tca8113 and Tca8113/BLM cells decreased significantly. Conclusion:The hyperthermia combined with Prog can increase the intracellular concentration of ADM in Tca8113 and Tca8113 /BLM cells, which reverse the drug-resistance of Tca8113/BLM cells to BLM and down-regulate expressions of P-gp and MRP.
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Patients with Bloom syndrome (BS) show an immunodeficiency, an enhanced sister chromatid exchanges (SCEs), a strong genetic instability and an increased predisposition to all. In order to investigate the differential expression of BLM protein in hematopoietic tumor cell strains and study the effects of BLM gene on ultraviolet (UV)- or hydroxyurea (HU)-induced apoptosis, Western blot was used to detect the expression of BLM protein in normal human bone marrow mononuclear cells and 4 kinds of hematopoietic tumor cell strains. The 4 kinds of hematopoietic tumor cells were exposed to UV light with a germicidal UV lamp or treated with 2 mmol/L hydroxyurea and the apoptotic rate was detected by using AnnexinV-FITC. The results showed that these tumor cells ex- pressed BLM protein higher than the normal human bone marrow mononuclear cells (P＜0.01). In the 4 hematopoietic tumor cells, BLM protein was all specially cleaved in response to UV- or HU-induced apoptosis. The increase of BLM protein expression may play an important role in the evelopment of these tumors, and BLM proteolysis is likely to be a general feature of the apoptotic esponse.
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Objective:To establish a multidrug resistant human salivary gland adenoid cystic carcinoma cell line.Methods:Human salivary gland adenoid cystic carcinoma cell line ACC-2 was treated by 24-hour-exposure to high dose of Bleomycin(BLM)(20 ?g/ml).Drug sensitivity was evaluated by MTT assay.Cell counting was employed to make the growth curve and to calculate the cell doubling time.Flow cytometry(FCM) was used to study the cell cycle distribution and apoptosis.The colony formation ability was also observed.Results:Multidrug resistant cell line of human salivary gland adenoid cystic carcinoma was established and named ACC-2/BLM.After 10 times repeated exposure to BLM,the resistance index(RI) to BLM,5-Fluorouracil(5-FU),Cisplatin(CDDP),Cyclophosphamide(CTX),Vincristine(VCR) were 7.299,1.03,2.15,1.114 and 5.96 respectively.Compared with ACC-2,the proliferation potential of ACC-2/BLM cells was decreased.The ACC-2 apoptosis cells were much more than ACC-2/BLM cells after 9-day-treatment by BLM at 60 ?g/ml.Conclusion:ACC-2/BLM cell line has multidrug resistant characteristics.
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Objective To explore the reverse effect and mechanism of progesterone(Prog) on a human tongue cancer drug-resistant cell line Tca8113/BLM in vitro. Methods The reverse effect of Prog on Tca8113/BLM was determined by MTT. The effects of Prog on the intracellular ADM congregation, cell cycle distribution and the expression of P-gp in the cell membrane were detected by flow cytometry (FCM). Results The value of IC50 of Tca8113 and Tca8113/BLM cells without Prog was 9 56 and 120 1,respectively. After treated with Prog(2umol/L),The value of IC50 of Tca8113 and Tca8113/BLM cells was 9 56 and 12 1,respectively. The potent fold of BLM in Tca8113/BLM cells was 9 92, the concentration of intracellular ADM in Tca8113/BLM cells significantly increased(P