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Traditional microtubule inhibitors fail to significantly enhance the effect of colorectal cancer;hence,new and efficient strategies are necessary.In this study,a supramolecular nanoreactor(DOC@TA-Fe3+)based on tannic acid(TA),iron ion(Fe3+),and docetaxel(DOC)with microtubule inhibition,reactive oxygen species(ROS)generation,and glutathione peroxidase 4(GPX4)inhibition,is prepared for ferroptosis/apoptosis treatment.After internalization by CT26 cells,the DOC@TA-Fe3+nanoreactor escapes from the lysosomes to release payloads.The subsequent Fe3+/Fe2+conversion mediated by TA reducibility can trigger the Fenton reaction to enhance the ROS concentration.Additionally,Fe3+can consume gluta-thione to repress the activity of GPX4 to induce ferroptosis.Meanwhile,the released DOC controls microtubule dynamics to activate the apoptosis pathway.The superior in vivo antitumor efficacy of DOC@TA-Fe3+nanoreactor in terms of tumor growth inhibition and improved survival is verified in CT26 tumor-bearing mouse model.Therefore,the nanoreactor can act as an effective apoptosis and ferroptosis inducer for application in colorectal cancer therapy.
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ObjectiveTo investigate the effect of microRNA-223-3p （miR-223-3p） on hepatic stellate cell （HSC） activation and its mechanism. MethodsHuman HSC LX2 cells were selected for the study， and LX2 cells were stimulated by TGF-β to establish a model of HSC activation； quantitative real-time PCR was used to measure the change in the expression level of miR-223-3p during HSC activation. After LX2 cells were transfected with miR-223-3p mimic， quantitative real-time PCR， Western blot， and immunofluorescence assay were used to clarify the regulatory effect of miR-223-3p on HSC activation， and dual-luciferase reporter assay was used to verify the association between miR-223-3p and the target gene MAP1B. After LX2 cells were transfected with MAP1B siRNA， Western blot was used to clarify the influence of inhibiting MAP1B expression on HSC activation； after LX2 cells were transfected with miR-223-3p， quantitative real-time PCR and Western blot were used to verify the regulatory effect of miR-223-3p on MAP1B. The independent-samples t test was used for comparison of continuous data between two groups. ResultsHSC in the activated state had a significant reduction in the expression level of miR-223-3p compared with those in the resting state （t=9.12， P<0.001）. Overexpression of miR-223-3p inhibited the mRNA and protein expression levels of the markers for HSC activation alpha-smooth muscle actin and collagen type Ⅰ （mRNA expression： t=8.35 and 12.23， both P<0.01； protein expression： t=16.24 and 20.90， both P<0.001）. The dual-luciferase reporter assay confirmed that MAP1B was a potential target gene of miR-223-3p. Compared with the control group， LX2 cells with miR-223-3p overexpression had significant reductions in the mRNA and protein expression levels of MAP1B （mRNA expression： t=5.95， P<0.01； protein expression： t=11.12， P<0.001）. ConclusionThis study shows that miR-223-3p can inhibit HSC activation by targeting MAP1B.
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A well-coordinated process is required to construct a complicated structure like the cell wall, which consists of several elements that must be joined appropriately from various sources inside the cell. In order to successfully moderate dynamic responses to developmental and environmental signals, further complexity is necessary. The plasma membrane is continually and actively transporting sugars, enzymes, and other cell wall elements throughout diffused development. Actin filaments and microtubules make up the cytoskeletal pathways used to transport cell wall elements in vesicles during cell division. In addition to these elements, other proteins, vesicles and lipids are transported from and to the cell plate while cytokinesis occurs. Adding additional cell wall material or building a new cell wall requires a rearrangement of the cytoskeleton, which we examine in this review first. We next look at the commonalities between these two processes. Our next topic is the transport of cell wall-building polysaccharides and enzymes via motor proteins and other interactions with the cytoskeleton. Final thoughts on cytokinesis-generated cell walls include a look at some of their unique properties.
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Objective:To investigate the early evaluation potential of serum levels of apolipoprotein B/apolipoprotein A1 (Apo B/A1), microtubule-associated protein 1-light chain 3 (MAP1-LC3) and intercellular adhesion molecule-1 (ICAM-1) in acute pancreatitis (AP) patients.Methods:A total of 413 AP patients who were treated at the Second Affiliated Hospital of Anhui Medical University between January 2019 and August 2020 were enrolled. Serum samples were collected from AP patients within 24 h of admission. Patients were divided into the non-severe acute pancreatitis (Non-SAP, n=315) and severe acute pancreatitis (SAP, n=98) groups according to the severity of the disease. Sixty healthy controls were recruited. The differences of serum Apo B/A1, MAP1-LC3 and ICAM-1 among the three groups were compared by one-way analysis of variance, and the correlation between Apo B/A1, MAP1-LC3 and ICAM-1 and the severity of AP was analyzed by Pearson correlation analysis. Sensitivity and specificity in assessing AP severity were predicted by receiver operating characteristic curve (ROC). Results:The early levels of Apo B/A1, MAP1-LC3 and ICAM-1 were all significantly higher for AP patients than for healthy controls ( P<0.05), and the levels of Apo B/A1, MAP1-LC3 and ICAM-1 in SAP patients were significantly higher than those in non-SAP patients[Apo B/A1: 2.21±1.40 vs. (0.96±0.34); MAP1-LC3: 0.92±0.29 vs. (0.48±0.24) ng/mL and ICAM-1: (235.57±54.50 ) vs. (120.28±61.69)ng/mL; P<0.05]. Pearson correlation analysis showed that levels of Apo B/A1, MAP1-LC3 and ICAM-1 were positively correlated with the first Ranson score after admission ( P<0.05), and ICAM-1 showed the highest degree of correlation with AP severity ( r=0.519). Areas under the receiver operating characteristic curve (AUROC) were 0.769 for Apo B/A1, 0.811 for MAP1-LC3, 0.828 for ICAM-1, and 0.938 for combined detection. Conclusions:Serum levels of Apo B/A1, MAP1-LC3 and ICAM-1 within 24 h after admission are significantly correlated with the severity of AP, which has clinical significance for early prediction of the severity of AP.
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Objective:To explore the effect of Aspergillus fumigatus ( A. fumigatus) on the autophagic flux in murine bone marrow-derived macrophages (BMDM) . Methods:Murine BMDM were in vitro cultured with heat-killed A. fumigatus for 0, 0.5, 4, and 12 hours. Then, cellular proteins were extracted, and Western blot analysis was performed to detect the conversion of the key autophagy protein microtubule-associated protein 1 light chain 3 (LC3) -Ⅰ to LC3-Ⅱ, and to determine the protein expression of phosphorylated mammalian target of rapamycin (p-mTOR) Ser2481. Additionally, murine BMDM were in vitro cultured with A. fumigatus alone or in combination with different lysosomal inhibitors, including the cysteine cathepsin inhibitor E-64d + pepstatin, bafilomycin-A1 (BAF-A1) , ammonium chloride (NH 4Cl) , and chloroquine, for 4 or 12 hours. Then, Western blot analysis was performed to investigate the effect of A. fumigatus on newly formed LC3-Ⅱ and basal autophagic flux, and confocal laser scanning fluorescence microscopy to analyze the colocalization of A. fumigatus with LC3 and Rubicon (a RUN domain Beclin-1-interacting and cysteine-rich-domain-containing protein) . Experimental results at different treatment time points were analyzed by using unpaired t test, and results of experiments evaluating the effect of two factors ( A. fumigatus spores and autophagosome inhibitors) were analyzed by 2 × 2 factorial analysis. Results:After in vitro co-culture with A. fumigatus for 0.5, 4, 12 hours, Western blot analysis showed that the conversion of LC3-Ⅰ to LC3-Ⅱ increased over time in murine BMDM compared with the control (0 hour) group ( t = 6.58, 3.28, 3.02, respectively, all P < 0.05) , but the protein expression level of p-mTOR (Ser2481) did not significantly differ at different treatment time points ( t = 0.441, 0.477, 0.382, P = 0.682, 0.660, 0.722, respectively) . After 4- and 12-hour in vitro treatment, the accumulation levels of LC3-Ⅱ in BMDM significantly increased in the A. fumigatus + chloroquine group compared with the chloroquine-alone group ( t = 2.13, 2.78, respectively, both P < 0.05) , in the A. fumigatus + NH 4Cl group compared with the NH 4Cl-alone group ( t = 2.92, 2.92, respectively, both P < 0.05) , in the A. fumigatus + BAF-A1 group compared with the BAF-A1-alone group ( t = 2.13, 2.13, respectively, both P < 0.05) , and in the A. fumigatus + E-64d + pepstatin group compared with the E-64d + pepstatin group ( t = 2.13, 2.92, respectively, both P < 0.05) . After 8-hour treatment with calcofluor white-labeled A. fumigatus spores, confocal laser scanning fluorescence microscopy showed that LC3 and Rubicon mainly surrounded A. fumigatus, suggesting their colocalization with A. fumigatus. Conclusion:A. fumigatus can in vitro increase the basal autophagic flux in murine BMDM.
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Objective:To observe the effects of high copper diet on neurobehavioral functions and synaptic associated protein expression in hippocampus of rats.Methods:Thirty male SD rats were randomly divided into control group and high copper diet group with 15 rats in each group according to the random number table method. The rats in control group were fed with ordinary diet and ordinary water, while the rats in high-copper diet group were fed with high-copper diet containing 1 g/kg copper sulfate and 0.185% copper sulfate deionized water for 12 weeks. The content of copper in serum and hippocampus of rats were detected by inductively coupled plasma-atomic emission spectrometry(ICP-AES) and ICP-mass spectrometry(ICP-MS). The neurobehavioral indicators were detected by stereotypic behavior test, open field test and Morris water maze test. The expression levels of microtubule associated protein 2(MAP2) and growth associated protein 43 (GAP43) in hippocampus were detected by Western blot.SPSS 22.0 software was used for statistical analysis, and two independent sample t-test was used for comparison between the two groups. Results:Compared with the control group, the content of serum copper((1.67±0.69)mg/L, (1.98±0.24)mg/L, t=17.53, P<0.05) and hippocampal free copper((3.52±1.24)mg/g, (4.78±0.57)mg/g, t=10.34, P<0.05) in the high copper diet group increased significantly, and the stereotypic behavior score increased significantly ((0.29±0.08), (2.97±0.72), t=14.33, P<0.01), the number of space crossing in the open field experiment ((153.40±24.73)points, (92.46±19.46)points, t=7.50, P<0.01) and the times of standing((19.34±1.98)times, (10.57±2.71)times, t=10.12, P<0.01) were significantly decreased. The average latency in Morris water maze navigation test was significantly prolonged ((3.14±1.67)s, (8.29±2.26)s, t=7.10, P<0.01), the number of crossing the original platform position in the space exploration test decreased significantly ((7.89±2.48)times, (2.98±1.73) times, t=3.23, P<0.01). Compared with control group, protein levels of GAP43((1.03±0.05), (0.48±0.02), t=39.56, P<0.05)and MAP2((0.93±0.05), (0.30±0.08), t=25.86, P<0.05) of high copper diet group were significantly decreased. Conclusion:High copper diet causes abnormality in a variety of neurobehavioral function indexes in rats, and a decrease in expression of MAP2 and GAP43 at the synaptic interface of hippocampal neurons may be involved in the process of learning and memory impairment in the neurobehavioral functions.
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Huachansu is a traditional Chinese medicine widely used in the clinic for cancer therapy, while the underlying mechanism is not fully clarified. This study was to investigate the targets and mechanisms of cinobufagin (CBG), an active component of Huachansu, in terms of blocking mitosis of cancer cells. Propidium iodide (PI) DNA staining was used to analyze the effect of CBG on cell cycle. The effect of CBG on mitosis of cancer cells was examined by α-tubulin and pericentrin staining after synchronization by a double thymidine block. Tubulin turbidity, tubulin polymerization and α-tubulin immunofluorescence assays were used to evaluate the effect of CBG on microtubule polymerization. CRISPR/Cas9 gene-editing technology was used to knockout microtubule-severing protein Katanin regulatory subunit B1 (KATNB1) in HCT116 cells, and the inhibitory effect of CBG on wild-type cells and knockout cells was measured by CCK-8. The engagement of CBG with KATNB1 was measured by CETSA and DARTS assays. The effect of CBG on KATNB1 protein and mRNA level was examined by Western blot and real-time PCR, respectively. Our data showed that CBG arrested HCT116 cell cycle at the G2/M phase, disrupted mitosis and induced centriole overduplication. CBG significantly inhibited tubulin polymerization in vitro and in vivo. The cytotoxicity of CBG inhibition on HCT116 was significantly attenuated upon KATNB1 depletion. Moreover, CBG bound to KATNB1 and decreased its protein level, while mutated KATNB1 weakened this effect. In conclusion, CBG inhibited microtubule polymerization via targeting KATNB1, thereby disrupting mitosis in cancer cells.
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Aurora kinase A (AURKA),a family member of aurora kinases,is involved in mitotic entry,maturation and separation of centrosome,assembly and stabilization of bipolar spindle,and condensation and separation of chromosome.Studies have demonstrated that AURKA plays a similar role in meiosis,while the specific mechanism and the similarities and differences in its role between meiosis and mitosis remain unclear.Therefore,we reviewed the studies about the localization and activation of AURKA in oocyte meiosis,and compared the role of AURKA in regulating spindle formation,activating spindle assembly checkpoint,and correcting the kinetochore-microtubule attachment between the meiosis of oocytes and the mitosis of somatic cells.This review will lay a theoretical foundation for revealing the mechanism of AURKA in the regulation of cell division and for the clinical research related to cancer and reproduction.
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Humans , Aurora Kinase A/genetics , Cell Cycle Proteins/genetics , Chromosome Segregation , Meiosis , OocytesABSTRACT
ObjectiveTo investigate the effects of Anmeidan （AMD） on neuronal structure and neuronal marker protein expression in the hippocampal CA1 region of sleep-deprived （SD） rats. MethodRats were randomly divided into control group， model group， an AMD group （9.09 g·kg-1·d-1）， and melatonin group （0.27 g·kg-1·d-1）. Rats in the control group and the model group received equal volumes of physiologicol saline. The SD model was induced by the self-made sleep deprivation box for four weeks. Ethovision XT system detected and analyzed the spontaneous behaviors of rats. The histomorphology of neurons in the hippocampal CA1 region was observed by hematoxylin-eosin （HE） staining and Nissl staining， and the changes in Nissl bodies were observed by Nissl staining. The ultrastructure of hippocampal cells was observed by transmission electron microscopy （TEM）. Immunohistochemistry was used to detect the expression of glial fibrillary acidic protein （GFAP）， microtubule-associated protein 2 （MAP2）， nestin， and neuronal nuclei （NeuN） in the CA1 region. ResultCompared with the control group， the model group showed longer distance， increased average activity speed， cumulative duration， average body fill， and higher activity frequency （P<0.01）. Besides， the neurons in the CA1 region were reduced in number with disorganized arrangement， wrinkled nuclei， deeply stained cytoplasm， reduced Nissl bodies， swollen and deformed mitochondria， shortened cristae， and swollen Golgi vesicles. Furthermore， the mean integral absorbance （IA） value of GFAP increased and those of MAP2， nestin， and NeuN decreased （P<0.01）. Compared with the model group， the AMD group showed shortened distance traveled， lower average activity speed， shorter cumulative duration， decreased average body fill， and reduced activity frequency （P<0.05， P<0.01）. Moreover， the neurons in the CA1 region were relieved from damage with increased cell number， clear nuclei and cytoplasm， increased Nissl bodies， and relieved mitochondrial damage. The IA value of GFAP decreased and those of MAP2， nestin， and NeuN increased （P<0.05， P<0.01）. ConclusionAMD can improve structural damage of neurons in the hippocampal CA1 region of sleep-deprived rats， which may be achieved by decreasing GFAP expression and increasing MAP2， nestin， and NeuN expression.
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ObjectiveTo observe the preventive and control effects of Danggui Niantongtang against adjuvant arthritis differentiated into wind-damp-heat impediment in rats and its influences on the expression of autophagy-related proteins microtubule-associated protein 1 light chain 3 （LC3）， homolog of yeast Atg6 （Beclin1） and p62. MethodThe six-week-old male SD rats were randomly divided into the normal group， wind-damp-heat impediment model group， low-， medium-， and high-dose Danggui Niantongtang （5.67， 11.34， 22.68 g·kg-1） groups， and methotrexate （MTX， 1.35 mg·kg-1） group， with 10 rats in each group. A rat model of adjuvant arthritis was established by subcutaneous injection of inactivated Mycobacterium tuberculosis into the tail root， followed by exposure to the manual climatic box for 16 d for inducing the wind-damp-heat impediment. The drugs were administered intragastrically on the day of immunization for 28 d. The general conditions of rats were observed and the swelling degree of toes and arthritis index （AI） were detected. The pathological changes in the synovial tissues of the knee joints were observed by hematoxylin-eosin （HE） staining. The mRNA expression levels of LC3， Beclin1， and p62 in the synovial tissues were measured by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， followed by the assay of their protein expression by Western blot and immunohistochemistry. ResultCompared with the normal group， the wind-damp-heat impediment model group exhibited significantly increased swelling degree of toes （P<0.01）， increased AI （P<0.01）， proliferated synovial cells （P<0.01）， up-regulated LC3 and Beclin1 protein and mRNA expression （P<0.01）， and down-regulated p62 protein and mRNA expression （P<0.01） after 16， 20， 24， 28-d medication. Compared with the wind-damp-heat impediment model group， each medication group displayed alleviated toe swelling and synovial hyperplasia to different degrees， decreased mRNA and protein expression levels of LC3 and Beclin1 （P<0.01）， and increased p62 mRNA and protein expression （P<0.05，P<0.01）， with the best outcomes observed in the medium-dose Danggui Niantongtang group. ConclusionDanggui Niantongtang effectively relieves adjuvant arthritis due to wind-damp-heat impediment in rats， which may be related to its regulation of the expression of autophagy-related proteins LC3， Beclin1， and p62 and the inhibition of autophagy.
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Objective:To observe the effect of Qiyu Sanlong decoction （QYSL） on the expressions of key molecules in signal axis of mammalian rapamycin target protein （mTOR）/yeast Atg6 homologous （Beclin1）/ microtubule-associated protein1 light chain3 （LC3） in A549 cells. Method:With A549 cells as the research object， the effect of QYSL medicated serum on cell viability of A549 cells were detected by cell counting kit-8 （CCK-8） method. The effect of QYSL decoction on A549 cell apoptosis， autophagosome formation and the expression of autophagy markers were detected by Terminal-deoxynucleoitidyl transferase mediated nick end labeling （TUNEL） method， transmission electron microscope （TEM）， Real-time polymerase chain reaction （Real-time PCR） and Western blot. Result:QYSL medicated serum could inhibit the viability of A549 cells in a concentration-dependent manner. Compared with the blank serum group， the number of apoptotic A549 cells in the QYSL medicated serum group was significantly increased （P<0.01）， and the formation of autophagosome was significantly increased. Compared with the blank serum group， the mRNA and protein expressions of mTOR in A549 cells in the QYSL serum group were significantly decreased （P<0.01）， while mRNA and protein expressions of Beclin-1， autophagy related genes 5 （ATG5）， autophagy related genes 13 （ATG13） were significantly increased （P<0.01）. Conclusion:QYSL decoction can induce autophagy in A549 cells， and its specific mechanism may be related to the down-regulation of mTOR expression， the up-regulation of Beclin1， ATG5， ATG13 and LC3 expression， and the promotion of LC3Ⅰ conversion to LC3Ⅱ.
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OBJECTIVES@#This study aimed to explore the effect of sex determining region Y-box 9 (SOX9) on the microtubule formation and epithelial-mesenchymal transition (EMT) of human oral squamous cell carcinoma (OSCC) CAL27 and the underlying mechanism.@*METHODS@#SOX9-shRNA1 and SOX9-shRNA2 were designed and synthesized and then transfected into CAL27 cells. The expression of SOX9 was detected by quantitative real-time polymerase chain reaction. Microtubule formation assay was used to detect the change in the number of microtubule nodules after interfering with SOX9. Immunofluorescence was used to detect the Vimentin content. Western blot was used to detect the protein expression of EMT marker molecules and Wnt/β-catenin pathway-related proteins, such as E-cadherin, N-cadherin, Fibronectin, Wnt, β-catenin, T-cell factor-4 (TCF-4).@*RESULTS@#The expression level of SOX9 significantly decreased after transfection with SOX9-shRNA1 and SOX9-shRNA2 in CAL27 cells (@*CONCLUSIONS@#Interference with SOX9 decreased Vimentin content and inhibited the microtubule formation and protein expression of EMT marker molecules, as well as the expression of proteins related to the Wnt/β-catenin pathway. Thus, SOX9 can induce microtubule formation and EMT in CAL27, which was related to the inhibition of the Wnt/β-catenin pathway activation.
Subject(s)
Humans , Carcinoma, Squamous Cell , Cell Line, Tumor , Epithelial-Mesenchymal Transition , Head and Neck Neoplasms , Microtubules/metabolism , Mouth Neoplasms , SOX9 Transcription Factor/metabolism , Squamous Cell Carcinoma of Head and Neck , Wnt Signaling Pathway , beta Catenin/metabolismABSTRACT
Objective:To investigate the efficacy of atenolol combined with perindopril in the treatment of chronic heart failure in older adult patients and its effects on ventricular function, serum connective tissue growth factor (CTGF), and nexin.Methods:120 older adult patients with chronic heart failure who received treatment in the Department of Cardiology, the First People's Hospital of Fuyang District from January 2016 to January 2018 were included in this study. They were randomly assigned to receive either basic treatment + atenolol treatment (control group, n = 60) or atenolol combined with perindopril treatment (observation group, n = 60). Clinical efficacy and clinical symptom, serum CTGF, nexin level and ventricular function pre- and post-treatment, as well as adverse reactions were compared between the control and observation groups. Results:After treatment, effective rate in the observation group was significantly higher than that in the control group (91.6% vs. 78.3%, χ2 = 4.183, P = 0.041). After treatment, CTGF and nexin levels in the control and observation groups were decreased compared with before treatment (both P < 0.05). After treatment, CTGF and nexin levels in the observation group were (4.42 ± 0.46) μg/L and (0.82 ± 0.03) μg/L, respectively, which were significantly lower than those in the control group [(4.82 ± 0.51) μg/L, (0.98 ± 0.04) μg/L, t = 18.153, 4.511, 19.335, 24.787, all P < 0.05]. After treatment, left ventricular end diastolic diameter and left ventricular end systolic diameter in the control and observation groups were deceased compared with before treatment (both P < 0.05). After treatment, left ventricular end diastolic diameter and left ventricular end systolic diameter in the observation group were (48.73 ± 4.41) mm and (41.13 ± 4.15) mm, respectively, which were significantly lower than those in the control group [(56.01 ± 4.67) mm, (47.45 ± 4.17) mm, t = 5.700, 8.799, 8.317, 8.351, all P < 0.05]. After treatment, left ventricular ejection fraction in the control and observation groups was significantly increased compared with before treatment. After treatment, left ventricular ejection fraction in the observation group was significantly higher than that in the control group [(44.86 ± 4.59) % vs. (39.05 ± 4.69) %, P < 0.05]. Conclusion:Atenolol combined with perindopril in the treatment of older adult patients with chronic heart failure can reduce clinical symptoms, improve ventricular function, decrease serum CTGF and nexin levels and is highly safe. Therefore, this method is worthy of clinical application.
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Objective: The objective of this study was to evaluate the uptake and specificity of [11C]MPC-6827, a MT targeted PET ligand in prostate, glioblastoma and breast cancer cells. Methods: [11C]MPC-6827 was synthesized by reacting corresponding desmethyl precursors with [11C]CH3I in a GE-FX2MeI/FX2M radiochemistry module. In vitro binding of [11C]MPC-6827 was performed in breast cancer MDA-MB-231, glioblastoma (GBM) patient-derived tumor (GBM-PDX), GBM U251 and prostate cancer 3 (PC3) cell lines at 37 °C in quadruplicate at 5, 15, 30, 60, and 90 minute incubation time. The nonspecific bindings were determined by incubation with unlabeled microtubule targeting agents MPC-6827, HD-800, colchicine, paclitaxel and docetaxel (5.0 mM). Results: [11C]MPC-6827 provided the highest binding in the breast cancer cell, MDA-MB-231, among all the cells studied, with 90% specific binding. [11C]MPC-6827 binds to glioblastoma PDX and U251 cells with ~50% and 40% specific binding, whereas, prostate cancer cell line, PC3 cells showed 40% specific binding. [11C]MPC-6827 also exhibits binding to the taxane and colchicine binding sites of MTs, in MDA-MB-231 cells. Conclusion: These data indicate that [11C]MPC-6827 can be a promising PET radiotracer for preclinical imaging of the brain and peripheral cancers.
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Humans , Male , Male , Actin Cytoskeleton , Axoneme , Cytoskeleton , Infertility, Male , Kartagener Syndrome , Microtubule-Associated Proteins , Microtubules , Organelles , Sperm Head , Sperm Motility , Spermatogenesis , Spermatozoa , TailABSTRACT
Objective:To study the effect of exercise preconditioning on the expression of autophagy-related protein Beclin 1 and microtubule-associated protein 1 light chain 3 (LC3), and apoptosis after myocardial ischemia-reperfusion injury in rats, to explore the mechanism of myocardial protection by exercise preconditioning. Methods:A total of 36 male Sprague-Dawley rats were randomly divided into sham operation group, model group and exercise preconditioning group, with twelve rats in each group. The exercise preconditioning group received electric treadmill training for four weeks before modeling. The model of myocardial ischemia-reperfusion was made by ligation of the left anterior descending coronary artery 30 minutes and reperfusion. One hour after reperfusion, the expression of Beclin 1 and LC3 protein in myocardium was detected by Western blotting, and apoptosis of myocardial cells was observed by TUNEL staining, then the apoptotic index was calculated. Results:Compared with the model group, the level of LC3-I protein in the myocardial ischemic area increased, the levels of Beclin 1 and LC3-II decreased, the ratio of LC3-II/LC3-I decreased (P < 0.05); and the apoptotic index decreased in the preconditioning group (P < 0.05). Conclusion:Exercise preconditioning could up-regulate LC3-I in ischemic area, down-regulate the expression of Beclin 1 and LC3-II, and inhibit cardiomyocyte apoptosis after ischemia-reperfusion.
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BACKGROUND: A high-intensity eccentric exercise may lead to skeletal muscle damage, endoplasmic reticulum stress, increasing misfolded proteins. Aggrephagy works as an effective way of controlling quality of proteins and plays an important role in damage repair. OBJECTIVE: To discover the activation degree of aggrephagy in the damage repair process after an overload eccentric exercise, and to discuss the role of aggrephagy in the exercise-induced muscle damage, which is supplementary to the protein degradation pathway in skeletal muscle damage. METHODS: Sixty-four male adult Sprague Dawley rats were randomly divided into a static control group (n=8) and seven exercise groups according to post-exercise time (0, 3, 6,12, 24,48 and 72 hours, eight rats in each). Damage models were established by downhill running (-16°, 16 m/min, 90 minutes). Subsequently, the histological changes of the rat soleus muscle was observed under an electron microscope and protein expressions of histone deacetylase 6 and microtubule-associated protein 1 light chain 3II/I related to aggrephagy were detected using western bot assay. RESULTS AND CONCLUSION: (1) Under the transmission electron microscope, the rat soleus muscle was disordered and widened and appeared to have transient sarcomere, the Z line was water wave-like, broken and disappeared, and the mitochondria were swollen and unevenly distributed with an unclear structure after high-intensity eccentric exercise. The model rats suffered severest muscle damage at 12 hours and recovered completely at 72 hours after high-intensity eccentric exercise. (2) After one-time eccentric exercise, the expression of histone deacetylase 6 had a transient increase. The histone deacetylase 6 expression reached the peak at 6 hours, then gradually reduced and recovered at 72 hours after exercise. The microtubule-associated protein 1 light chain 3II/I expression was significantly increased at 3 hours after exercise, reached the peak at 12 hours, then reduced significantly and recovered at 72 hours after exercise. All these findings indicate that aggrephagy transiently happens and exerts important roles in the clearance of misfolded proteins, maintenance of cellular environmental homeostasis and recovery of skeletal muscle damage.
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Objective::To study the effect of Qiyu Sanlong decoction on the growth of subcutaneous tumor in lung cancer mice and the expressions of key autophagy molecule, yeast Atg6 homologous (Beclin1), autophagy related genes5 (Atg5), and microtubule-associated protein1 light chain3 (LC3B). Method::Lewis lung carcinoma cells (LLC) were used to reproduce the lung cancer mice transplanted model. After the modeling, the mice were randomly divided into model group, Qiyu Sanlong decoction group, chemotherapy group and combination group, with 18 transplanted mice in each group. In model group, mice were fed with 0.9% saline 20 mL·kg-1 daily. In Qiyu Sanlong decoction group, mice were fed with Qiyu Sanlong decoction 80.48 g·kg-1 daily. The chemotherapy group was intraperitoneally injected with 0.4 mL cisplatin solution (DDP) at the 1st, 3rd and 5th day. The combination group was orally given the drugs at the concentration of 80.48 g·kg-1, and 0.4 mL DDP solution was intraperitoneally injected at the 1st, 3rd and 5th day. After 21 days of continuous treatment, tumor tissue was exfoliated and weighed, and the tumor inhibition rate was calculated. Hematoxylin-eosin (HE) staining was used to observe the histological changes of tumor. The expressions and localizations of Beclin1 and LC3B in tumor tissues were detected by immunohistochemical staining. Protein expressions of Beclin1, Atg5, LC3B-Ⅰand LC3B-Ⅱ were determined by Western blot, and the ratio of LC3B-Ⅱ/LC3B-Ⅰ was calculated. The transcription levels of Beclin1, Atg5 mRNA in tumor tissues were detected by Real-time PCR. Result::Qiyu Sanlong decoction had a mild inhibitory effect on transplanted tumor, with an inhibitory rate of 31.2%. Under microscope, patchy necrotic tumor cells were observed in the tumor tissues of Qiyu Sanlong decoction group. Immunohistochemical staining and Western blot analysis showed that Qiyu Sanlong decoction could up-regulate the expressions of Beclin1, Atg5 and LC3B protein (P<0.01), and promote the conversion from LC3B-Ⅰ into LC3-Ⅱ compared with the model group. Real-time PCR results showed that Qiyu Sanlong decoction could promote the transcription of Beclin1 mRNA and Atg5 mRNA compared with the model group (P<0.01). Conclusion::Qiyu Sanlong decoction has a mild inhibitory effect on lung tumors, and its mechanism may be related to up-regulating the expressions of autophagy key proteins Beclin1, Atg5 and LC3B, and promoting the conversion from LC3B-Ⅰ to LC3B-Ⅱ.
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Objective:To investigate the effect of Danggui Yinzi on allergic reaction in chronic urticaria (CU) mice model and the mechanism of autophagy intervention. Method:The SPF BALB/c mice were used to replicate the CU mice model by intraperitoneal injection of ovalbumin and aluminum hydroxide suspension. The animals were randomly allocated into six groups: a normal group (normal saline 20 mL·kg-1·d-1), a model group (normal saline 20 mL·kg-1·d-1), a loratadine group(0.001 3 g·kg-1·d-1), a Danggui Yinzi high，medium and low-dose group（39.3，19.6，9.8 g·kg-1·d-1）. The pathological changes of skin tissues were observed by hematoxylin-eosin (HE) staining. Morphological changes of autophagy in skin tissues epithelial cells were observed by transmission electron microscope. The mRNA levels of microtubule-associated protein 1 light chain 3B(LC3B) and ubiquitin-binding protein p62 mRNA in skin tissues were detected by real-time quantitative polymerase chain reaction (Real-time PCR). The expressions of LC3B and p62 in skin tissues were detected by immunohistochemistry (IHC). Result:Danggui Yinzi can significantly improve the pathological manifestations of dermal edema, collagen bundles separation, telangiectasia in CU mice， it can also improve autophagosomes formation and abnormal cell ultrastructure such as nuclear chromatin condensation, mitochondrial swelling, endoplasmic reticulum expansion, etc. Compared with the normal group, the protein expressions of LC3B in skin tissues of the model group was significantly increased (P<0.01), LC3B mRNA level was increased too, while p62 mRNA levels and its protein expressions were decreased-regulated (P<0.01). Compared with the model group, levels of LC3B mRNA and protein expressions of the Danggui Yinzi groups were significantly increased (P<0.05,P<0.01), while p62 mRNA levels and its protein expressions were significantly decreased-regulated (P<0.05,P<0.01). Conclusion:Danggui Yinzi can regulate the expression of LC3B, p62 mRNA and protein expressions, enhance the level of autophagy, and improve the pathological state of CU mice.
ABSTRACT
IMB5046 is a newly discovered nitrobenzoate functioning as a microtubule inhibitor. Here we report its synthesis and in vitro anti-angiogenic activity. IMB5046 was synthesized by conjugation of 2-morpholin-4-yl-5-nitrobenzoic acid with 4-(methylthio)benzyl alcohol via two-step reactions. The structure of the end product was verified using 1H NMR and HR-MS spectroscopy. The effect of these compounds on cell proliferation was determined using MTT assay, and their impact on cytoskeleton was investigated using fluorescence assay. Flow cytometry was performed to examine the effect of IMB5046 on cell cycle. Cell wound scratch assay and Transwell assay were performed to examine cell migration. Endothelial tube formation assay was used to evaluate the anti-angiogenic activity of IMB5046. The results indicated that IMB5046 induced endothelial cell contraction and microtubule depolymerization, and inhibited the proliferation of endothelial cells and tumor cells, while two raw materials showed no obvious effects. IMB5046 arrested cell cycle at G2/M phase, even at low-cytotoxic concentrations it significantly inhibited the motility of endothelial cells. IMB5046 inhibited the tube formation of endothelial cells according to the number of tubes and junctions. In conclusion, IMB5046 is a promising microtubule-targeting drug with anti-angiogenic activity.