Natural exosome-like nanovesicles from edible tea flowers suppress metastatic breast cancer via ROS generation and microbiota modulation

Although several artificial nanotherapeutics have been approved for practical treatment of metastatic breast cancer, their inefficient therapeutic outcomes, serious adverse effects, and high cost of mass production remain crucial challenges. Herein, we developed an alternative strategy to specifically trigger apoptosis of breast tumors and inhibit their lung metastasis by using natural nanovehicles from tea flowers (TFENs). These nanovehicles had desirable particle sizes (131 nm), exosome-like morphology, and negative zeta potentials. Furthermore, TFENs were found to contain large amounts of polyphenols, flavonoids, functional proteins, and lipids. Cell experiments revealed that TFENs showed strong cytotoxicities against cancer cells due to the stimulation of reactive oxygen species (ROS) amplification. The increased intracellular ROS amounts could not only trigger mitochondrial damage, but also arrest cell cycle, resulting in the in vitro anti-proliferation, anti-migration, and anti-invasion activities against breast cancer cells. Further mice investigations demonstrated that TFENs after intravenous (i.v.) injection or oral administration could accumulate in breast tumors and lung metastatic sites, inhibit the growth and metastasis of breast cancer, and modulate gut microbiota. This study brings new insights to the green production of natural exosome-like nanoplatform for the inhibition of breast cancer and its lung metastasis via i.v. and oral routes.

Observation of dendrite osteocytes of mice at different developmental stages using Ploton silver staining and phalloidin staining / 南方医科大学学报

OBJECTIVE@#To assess the value of Ploton silver staining and phalloidin-iFlour 488 staining in observation of the morphology of osteocyte dendrites of mice at different developmental stages.@*METHODS@#The humerus and femurs were harvested from mice at 0 (P0), 5 (P5), 15 (P15), 21 (P21), 28 (P28), and 35 days (P35) after birth to prepare cryo-sections and paraffin sections. HE staining of P35 mouse femur sections served as a reference for observing osteocytes in the trabecular bone and cortical bone. The humeral sections at different developmental stages were stained with Ploton silver staining to observe the morphology of osteocytes and canaliculi, and the canalicular lengths in the cortical and trabecular bones of the humerus of the mice in each developmental stage were recorded. The cryo-sections of the humerus from P10 and P15 mice were stained with phalloidin iFlour-488 to observe the morphology of osteocytes and measurement of the length of osteocyte dendrites in the cortical bone.@*RESULTS@#In the trabecular bone of the humerus of P0-P15 mice, Ploton silver staining only visualized the outline of the osteocytes, and the morphology of the canaliculi was poorly defined. In P21 or older mice, Ploton silver staining revealed the morphology of the trabecular bone osteocytes and the canaliculi, which were neatly arranged and whose lengths increased significantly with age (P21 @*CONCLUSIONS@#Mouse osteocyte dendrites elongate progressively and their arrangement gradually becomes regular with age. Ploton silver staining can clearly visualize the morphology of the osteocytes and the canaliculi in adult mice but not in mice in early stages of development. Phalloidin iFlour-488 staining for labeling the cytoskeleton can be applied for mouse osteocytes at all developmental stages and allows morphological observation of mouse osteocytes in early developmental stages.

Effect of long-chain non-coding CDKN2B on miR-19 in chronic myeloid leukemia / 中国免疫学杂志

Objective:To investigate the effect of long-chain non-coding CDKN2B targeting miR-19 on the biological behavior of chronic myeloid leukemia cells and its mechanism.Methods: The expression of CDKN2B in different leukemia cells were detected by qPCR.Double luciferase reporter gene was used to detect the interaction between CDKN2B and miR-19.MTT proliferation assay and flow cytometry were used to detect the effect of CDKN2B on the proliferation and apoptosis of HL-60 cells.The changes of migration ability of leukemia HL-60 cells after overexpress of CDKN2B were detected by scratch test.The changes of invasion ability of leukemia HL-60 cells after silencing CDKN2B were detected by Transwell invasion assay.Scaling healing experiment and Transwell invasion assay were used to detect the effect of miR-19 on the migration and invasion of leukemia cells after silencing CDKN2B.The morphological changes of cytoskeleton microfilament microtubules after silencing CDKN2B were detected by phalloidin staining.Western blot was used to detect the expression of PI3K/AKT signaling pathway after silencing CDKN2B.Results: The expression level of CDKN2B was the lowest in leukemia cell HL-60.CDKN2B binds specifically to the 3′UTR of miR-19;overexpression of CDKN2B could inhibit the proliferation and enhance the apoptosis of leukemia HL-60 cells.Overexpression of CDKN2B can inhibit the invasion and migration of leukemia HL-60 cells.After overexpressed of CDKN2B,the cytoskeleton showed decreased pseudopodia and decreased exercise capacity.The expression of actin was down-regulated.The expression of PI3K/AKT pathway protein was down-regulated after overexpressed of CDKN2B.Conclusion: CDKN2B can target the regulation of miR-19 to regulate the biological behavior of leukemia cells.

Morphological Change of Mouse Inner Ear Hair Cells after Noise Exposure / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Mechanism of inner ear hair cell distortion after noise exposure has been well described. The present study was designed to determine the response to the auditory system of a genetically well-defined laboratory mouse in preparation for examining the effect of noise on mice with specific genetic mutations. So it is important to recognize the relationship between noise exposure duration and hair cell morphological changes. We try to reveal the hearing loss and inner ear hair cell morphological changes after applying the noise protocol. SUBJECTS AND METHOD: The mice were BALB/c hybrids and aged 8 weeks. Six mice served as non-noise-exposed controls and 8 mice were exposed for 3 hours per day to white band noise with a center frequency from 0.2 kHz to 70 kHz and a sound pressure level of 120 dB. And we divided the noise exposure group into 3 subgroups(1 day, 3 day, 5 day noise exposure group). We checked the photographs of FITC phalloidin stain and scanning electron microscopy of cochlea after noise exposure. RESULTS: The hearing level of mice decreased after noise exposure. We could see the stereocilia damage in cochlea after FITC phalloidin stain in cochlea and sterocilia loss was more severe in basal turn. In scanning electron microscopy, morphological changes of stereocilia were observed to be more severe in the cochlear basal turn than other area. Significant hair cell loss in the cochlear basal turn could be calculated using cochleocytogram. CONCLUSION: 120dB broad white band noise can damage the hair cell of cochlea in mice. These changes were especially severe in the cochlear basal turn. Noise exposure duration is the other important factor in damaging cochlear hair cells. Therefore, we can guess that harmful noise level and noise exposure duration are the main risk factors that injure the inner ear hair cell.

Morphological Changes of Cultured Rat Hepatocytes Induced by Phalloidin and Cytochalasin D / 대한해부학회지

The microfilaments of hepatocyte are distributed throughout the vicinity of cell membranes, especially numerous around the region of bile canaliculus, and provide the maintenance of cell shape, cellular wall tension, canalicular motility, the secretion for bile, etc. To evaluate the relationship between the microfilament and alteration of cell shape, we examined the morphological changes of cultured rat hepatocytes, following treatments with phalloidin or cytochalasin D with fluorescent and electron microscopes. 1. In the fluorescent micrographs, actin microfilament was distributed near the plasma membrane and bile canaliculus. 2. Both drugs, phalloidin or cytochalasin D, produce the cytoplasmic protrusions from the surface. Their shapes were pedunculated with narrow neck or bulged with broad base, respectively. 3. In the phalloidin treated group, cytoplasmic protrusion was seperated from the internal cytoplasm by microfila-ments networks at the narrow base. In contrast, in the cytochalasin D treated group, cytoplasm was bulged with broad base and kept in direct continuity with the canalicular ectoplasm. 4. Pericanalicular ectoplasm of phalloidin treated group was widened and accumulated with microfilaments. But, bile canaliculus of cytochalasin D treated group was markedly dilated and devoid of microvilli, and the ectoplasm was almost disappeared. Considering above results, dysfunction of microfilaments leads to the structural changes and inhibition of bile secretion of hepatocytes.

Ultrastructural Changes of the Cultured Hepatocytes in Microfilamentous Dysfunction induced by Drugs / 대한해부학회지

To examine the role of actin microfilaments which are located at beneath the plasma membrane, we observed the ultrastructural changes of rat hepatocyte induced by alteration of the microfilamentous integrity. The isolated hepatocytes from Sprague-Dawley were cultured in the L-15 medium containing phalloidin (agent that cause polymerization of actin) or cytochalasin D (agent that cause depolymerization of actin) for 30 min, 1 hour, 2 hours, 4 hours, 10 hours and 20 hours, respectively. The results observed with scanning and transmission electron microscope were as follows. 1. Following the alteration of actin microfilaments, bile canaliculi were dilated and devoid of microvilli. In phalloidin treated group, the thickening of microfilamentous ectoplasm was more marked than that of cytochalasin D treated group. Whereas, the dilation of bile canaliculi was more marked in cytochalasin D group. 2. Both drugs, phalloidin or cytochalasin D, produced the alteration of cell shape to form cytoplasmic protrusions at the cell surface. In the phalloidin treated group, protrusions were pedunculated, and the microfilament networks were accumulated at the narrow neck region. 3. In cytochalasin D treated group, no microfilament barrier was seen at the broad base of protrusion which exhibit direct continuity with the internal cytoplasm. 4. Single hepatocyte tend to recover their structural integrity as those in vivo. The new bile canaliculus was sealed off at the intercellular space by tight junctions, and intercellular contacts were established by the junctional complexes. The results demonstrated that excessive accumulation or depletion of microfilaments induced by phalloidin or cytochalasin D altered the cell shape different, respectively. The microfilaments of ectoplasm play an important role in the maintenance of the structural integrity of cultured hepatocytes.

Organotypic Culture of Organ of Corti with Floating Drop Method from Newborn Rat / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Organotypic culture of organ of Corti maintains the basic organization of the spiral lamina and can conserve several factors responsible for the neuronal growth of the nervous components. The explant culture technique has been widely used in organ culture system, however, the floating drop method using collagen gel was also developed as a simple and reliable method. In order to study the effect of growth factors on the regenerative and protective ability of cochlear hair cells, we first had to establish an in vitro model of the inner ear. MATERIALS AND METHODS: Organ of Corti was obtained from newborn rats and cultured with the floating drop method using collagen gel. Immunohistochemical staining was used to visualize the stereocilia and scanning electron microscopic study was also carried out. RESULTS: Explants were maintained up to 10 days without contamination. Morphologically, immunofluorescent staining with phalloidin showed well preserved outer and inner hair cells with stereocilia on the second day of culture. On the tenth day of culture, the staining result showed inner and outer hair cells, although the stereocilia were poorly stained. In scanning electron microscopic examination, an explant on the tenth day of culture showed preserved outer and inner hair cells and stereocilia, although damaged hair cells and stereocilia were also observed. CONCLUSION: The floating drop method was an appropriate method for maintaining the organ of Corti in vitro with the advantage being the easiness in its manual manipulation.

IMMUNOFLUORESCENCE OBSERVATION ON CYTOSKELETON AND CELL SURFACE FN BEFORE AND AFTER CELL TRANSFORMATION OF NIH3T3 CELL LINE / 解剖学报

The experiment was designed to compare the changes of cytoskeleton (including tubulin and actin) and cell surface fibronectin (FN) between NIH 3T3 cell line and transformed NIH 3T3 cell line by genome DNA of human lung adenocarcinoma cell line AGZY. We stained and observed these cells using immunohistochemical methods with antibody against bovine brain tubulin, phalloidin and self-made affinity column purified antibody against porcine plasma FN.Our results showed that the bundles of actin and tubulin are damaged seriously, demonofrating an unclear cytoskeleton structure and diffused fluorescence over the cells when they were transformed. The amount of membrane FN on transformed cell surface decreases significantly which is only 1/9 of NIH 3T3. The FN distribution altered markedly from thin threadlike network to spots and speckles.These results suggested that cell transformation was a complex event including the changes of cytoskeleton system and cell surface glycoproteins. In addition, it might also indicate that cause and effect relationship existed between these changes and the alteration of cell phenotype and loss of growth control.