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Objective: Rapid restoration of corneal epithelium integrity after injury is particularly important for preserving corneal transparency and vision. Mesenchymal stem cells (MSCs) can be taken into account as the promising regenerative therapeutics for improvement of wound healing processes based on the variety of the effective components. The extracellular vesicles form MSCs, especially exosomes, has been considered as important paracrine mediators though transferring microRNAs into recipient cell. This study investigated the mechanism of human umbilical cord MSC-derived exosomes (HUMSC-exosomes) on corneal epithelial wound healing.Methods: Exosomes extracted from HUMSCs were identified by transmission electron microscopy, nanoparticle tracking analysis, and Western blot. Corneal fluorescein staining and histological staining were evaluated in a corneal mechanical wound model. Changes in HCECs proliferation after HUMSC-exosomes or miR-21 mimics treatment were evaluated by CCK-8 and EdU assays, while migration was assessed by in vitro scratch wound assay. Full-length transcriptome sequencing was performed to identify the differentially expressed genes associated with HUMSC-exosomes treatment, followed by validation via real-time PCR and Western blot.Results: The exosomes derived from HUMSCs can significantly promote corneal epithelial cells proliferation, migration in vitro and corneal epithelial wound healing in vivo. Similar effects were obtained after miR-21 transfection, while the beneficial effects of HUMSC-exosomes were partially negated by miR-21 knockdown. Results also show that the benefits are associated with decreased PTEN level and activated the PI3K/Akt signaling pathway in HCECs.Conclusion: HUMSC-exosomes could enhance the recovery of corneal epithelial wounds though restraining PTEN by transferring miR-21, and may represent a promising novel therapeutic agent for corneal wound repair.

BackgroundRapid restoration of corneal epithelium integrity after injury is particularly important for preserving corneal transparency and vision. Mesenchymal stem cells (MSCs) can be taken into account as the promising regenerative therapeutics for improvement of wound healing processes based on the variety of the effective components. The extracellular vesicles form MSCs, especially exosomes, has been considered as important paracrine mediators though transferring microRNAs into recipient cell. This study investigated the mechanism of human umbilical cord MSC-derived exosomes (HUMSC-exosomes) on corneal epithelial wound healing.MethodsExosomes extracted from HUMSCs were identified by transmission electron microscopy, nanoparticle tracking analysis, and Western blot. Corneal fluorescein staining and histological staining were evaluated in a corneal mechanical wound model. Changes in HCECs proliferation after HUMSC-exosomes or miR-21 mimic treatment were evaluated by CCK-8 and EdU assays, while migration was assessed by in vitro scratch wound assay. Full-length transcriptome sequencing was performed to identify the differentially expressed genes associated with HUMSC-exosomes treatment, followed by validation via real-time PCR and Western blot.ResultsThe exosomes derived from HUMSCs can significantly promote corneal epithelial cells proliferation, migration in vitro and accelerate corneal epithelial wound healing in vivo. Similar effects were obtained after miR-21 transfection, while the beneficial effects of HUMSC-exosomes were partially negated by miR-21 knockdown. Results also show that the benefits are associated with decreased PTEN level and activated the PI3K/Akt signaling pathway in HCECs.Conclusions HUMSC-exosomes could accelerate the recovery of corneal epithelial wounds though restraining PTEN by transferring miR-21, and may represent a promising novel therapeutic agent for corneal wound repair.

Background Acute lymphoblastic leukemia (ALL) is an aggressive hematopoietic malignancy that is most commonly observed in children. Alantolactone (ALT) has been reported to exhibit anti-tumor activity in different types of cancer. The aim of the present study was to investigate the anti-tumor activity and molecular mechanism of ALT in ALL. Methods ALL cell lines were treated with 1, 5 and 10 μM ALT, and cell viability was assessed using an MTT assay and RNA sequencing. Flow cytometry, JC-1 staining and immunofluorescence staining assays were used to measure cell apoptosis and autophagy. Additionally, western blot analysis was used to detect expression of apoptosis and autophagy related proteins. Finally, the effects of ALT on tumor growth were assessed in a BV173 xenograft nude mouse model. Results ALT inhibited the proliferation of ALL cells in a dose-dependent manner. Additionally, it was demonstrated that ALT inhibited cell proliferation, colony formation, autophagy, induced apoptosis and reduced tumor growth in vivo through upregulating the expression of adaptor related protein complex 2 subunit mu 1 (AP2M1). Moreover, the autophagy activator rapamycin, attenuated the pro-apoptotic effects of ALT on BV173 and NALM6 cell lines. Overexpression of AP2M1 decreased the expression of Beclin1 and the LC3-II/LC3-1 ratio, and increased p62 expression. Knockdown of Beclin1 increased the levels of bax, cleaved caspase 3 and cytochrome C, and decreased bcl-2 expression. Conclusions The present study demonstrated that ALT exerts anti-tumor activity through inducing apoptosis and inhibiting autophagy by upregulating AP2M1 in ALL, highlighting a potential therapeutic strategy for treatment of ALL.

Background: Acute lymphoblastic leukemia (ALL) is an aggressive hematopoietic malignancy and most commonly seen in children. Alantolactone (ATL) has been reported to have anti-tumor activities in different types of cancer. This study aimed to evaluate the anti-tumor activity and molecular mechanisms of ATL in ALL. Methods: The ALL cells were treated with 1, 5 and 10μM of of ALT, and then subjected to MTT assay and RNA sequencing. Flow cytometry, JC-1 staining and immunofluorescence staining assay were employed to measure cell apoptosis and autophagy. Meanwhile, western blot analysis was used to detect apoptosis and autophagy-related proteins. Finally, the effect of ALT on tumor growth was measured in BV173 xenograft nude mouse model. Results: In this study, we demonstrated that ALT could inhibit the proliferation of ALL cells by inducing apoptosis and inhibiting autophagy. Administration of rapamycin activated autophagy while reversing the effect of ALT on apoptosis. Mechanically, ALT could induce apoptosis and inhibit autophagy by promoting AM2P1 expression. Further, AM2P1 was figured to inhibit beclin1 phosphorylation so that the apartment between beclin1 and bcl-2 was alleviated to participate in the regulation of autophagy and apoptosis in ALL cell. Conclusions: This study disclosed that Alantolactone can inhibit cell autophagy and promote apoptosis through targeting AP2M1 in acute lymphotic leukemia, indicating a potential therapeutic strategy for ALLtreatment.

Translationally controlled tumor protein (TCTP) is a highly conserved, growth‑associated and small molecule protein, which is highly expressed in various types of tumor cell. TCTP can promote the growth and suppress apoptosis of tumor cels. However, few studies have reported the effects of TCTP in gliomas. In the present study, a glioma cell line was established, which was stably transfected with TCTP short hairpin ribonucleic acid (shRNA), to investigate the impact of downregulated expression of TCTP on the proliferation, apoptosis and invasion of glioma cells. Western blot and reverse transcription-quantitative polymerase chain reaction analyses demonstrated that TCTP shRNA effectively reduced the expression of TCTP in the U251 glioma cell line. MTT and colony formation assays revealed that downregulated expression of TCTP significantly inhibited glioma cell proliferation. Cell cycle analysis using flow cytometry revealed that the cells in the pRNA‑H1.1‑TCTP group were arrested in the G0/G1 phase of the cell cycle. Western blot analysis detected downregulated expression levels of cyclins, including Cyclin D1, Cyclin E and Cyclin B. Annexin V‑fluorescein isothiocyanate/propidium iodide and Hoechst staining demonstrated that the apoptotic rate of the cells in the pRNA‑H1.1‑TCTP group was significantly higher than that of the cells in the pRNA‑H1.1‑control group, with upregulated expression levels of B-cell-associated X protein and cleaved‑caspase‑3 and downregulated expression of B-cell lmyphoma-2 in the apoptotic process. Wound healing and Transwell assays revealed that downregulated expression of TCTP significantly inhibited the migration and invasiveness of the glioma cells; and the expression levels and activities of matrix metalloproteinase (MMP)‑2 and MMP‑9 were also significantly affected. In conclusion, the present study demonstrated that downregulated expression of TCTP significantly inhibited proliferation and invasion, and induced apoptosis in the glioma cells. These results suggested that TCTP may be important in glioma development and metastasis. Therefore, TCTP is expected to become an effective target for glioma gene therapy.

Background Thalidomide (Thal) treatment of patients with multiple myeloma (MM) is associated with vascular thrombosis, but the underlying mechanism is unknown. Objectives To evaluate the hypothesis that Thal, dexamethasone (Dex) and MM serum perturb the hemostatic balance on human umbilical vein endothelial cells (HUVECs). Methods Drugs with or without the serum of MM patients or healthy controls were incubated with HUVECs. Analyses of phosphatidylserine (PS), tissue factor (TF), endothelial protein C receptor (EPCR) and thrombomodulin (TM) were performed using flow cytometry. The production of thrombin and activated protein C (APC) were measured by chromogenic assay. The roles of IL-6 and TNFα in regulating these indicators were also investigated. Results We found that Thal or Dex alone could not increase TF and PS expression in HUVECs. However, when pretreated with MM serum, their expression was significantly increased by either Thal or Dex. Concurrent changes were also detected in thrombin generation. In contrast, Thal and Dex had a direct inhibitory effect on the expression of EPCR and TM, and this inhibitory effect was especially significant when MM serum was added. The generation of APC paralleled the expression of EPCR and TM. All of the above outcomes were reversed to a variable extent by anti-IL-6R and anti-TNFα antibodies. Conclusions These findings suggest Thal may act as a procoagulant by altering the balance between procoagulant and anticoagulant proteins on the surface of HUVECs, thereby contributing to thrombogenesis. MM serum plays a synergistic role in this process.

Although gemcitabine is currently the best chemotherapeutic agent available for the treatment of advanced pancreatic cancer, eventual failure of response is a significant clinical problem. Therefore, novel therapeutic approaches against this disease are highly needed. The aim of this study was to evaluate whether shikonin, a naphthoquinone derivative, has potential in the treatment of pancreatic cancer when used either alone or in combination with gemcitabine. Our in vitro results showed that shikonin inhibited the proliferation of three different human pancreatic cancer cell lines and potentiated the cytotoxic effect of gemcitabine, which correlated with the down-regulation of constitutive as well as gemcitabine-induced activation of NF-κB and NF-κB-regulated gene products. Most importantly, using a xenograft model of human pancreatic cancer, we found shikonin alone significantly suppressed tumor growth and argumented the antitumor activity of gemcitabine. These effects also correlated with the down-regulation of NF-κB activity and its target genes, decreased proliferation (PCNA and Ki-67), decreased microvessel density (CD31), and increased apoptosis (TUNEL) in tumor remnants. Collectively, our results suggest that shikonin can suppress the growth of human pancreatic tumors and potentiate the antitumor effects of gemcitabine through the suppression of NF-κB and NF-κB-regulated gene products.

The methylation-mediated silencing of tumor suppressors, including key apoptosis-related genes plays an important role in the pathogenesis and therapeutic resistance in human cancer. In this study, we aimed to elucidate the role and mechanisms of resistance to apoptosis with caspase-8 gene downregulation in human malignant glioma. Reverse transcription-polymerase chain reaction (RT-PCR) and methylation-specific PCR (MSP) were used to examine caspase-8 expressoin at the mRNA level and gene methylation status in normal brain tissue, glioma tissue and cancer cell lines. Caspase-8 protein kinase activity was measured by caspase-8 colorimetric assays; cell apoptosis was examined by Annexin V/propidium iodide (PI) staining; the rates of tumor cell apoptosis were detected by flow cytometry. Our results revealed that caspase-8 gene silencing may result from the methylation of its gene promoter in human glioma tissues. The expression of caspase-8 at the mRNA level was significantly associated with the grade of human glioma. In certain human cancer cell lines, the expression at the mRNA level, protein kinase activity and tumor cell anti-apoptotic activity and resistance were related to the methylation status of the caspase-8 gene promoter. Thus, the caspase-8 gene methylation status may be used as an indicator for the early diagnosis of human malignant glioma. Combination therapy with demethylation reagents may overcome therapeutic resistance in the same malignancy.

Norepinephrine (NE) is an important neurotransmitter in the brain, and regulates antinociception. However, the mechanism of action of NE on pain-related neurons in the hippocampal CA3 region is not clear. This study examines the effects of NE, phentolamine on the electrical activities of pain-excited neurons (PENs) and pain-inhibited neurons (PINs) in the hippocampal CA3 region of rats. Trains of electric impulses applied to the right sciatic nerve were used as noxious stimulation. The electrical activities of PENs or PINs in the hippocampal CA3 region were recorded by using a glass microelectrode. Our results revealed that, in the hippocampal CA3 region, the intra-CA3 region microinjection of NE decreased the pain-evoked discharged frequency and prolonged the discharged latency of PEN, and increased the pain-evoked discharged frequency and shortened discharged inhibitory duration (ID) of PIN, exhibiting the specific analgesic effect of NE. While intra-CA3 region microinjection of phentolamine produced the opposite response. It implies that phentolamine can block the effect of endogenous NE to cause the enhanced response of PEN and PIN to noxious stimulation. On the basis of above findings we can deduce that NE, phentolamine and alpha-adrenoceptor are involved in the modulation of nociceptive information transmission in the hippocampal CA3 region.

In order to investigate the effects of testosterone-replacement therapy on peripheral blood stem cells and angiogenesis after acute myocardial infarction, a castrated rat acute myocardial infarction model was established by ligation of the left anterior descending coronary followed by treatment with testosterone. CD34(+) cells in myocardium and in peripheral blood after 1 and 3 days were measured by immunohistochemistry and flow cytometry, respectively. In the early phase of acute myocardial infarction, the expression levels of hypoxia-inducible factor 1a (HIF-1a), stromal cell-derived factor 1a (SDF-1a) and vascular endothelium growth factor (VEGF) in ischemic myocardium were determined by real time RT-PCR and immunohistochemistry, respectively. Infarct size, cardiomyocyte apoptosis, capillary density and cardiac function were assessed after 28 days. These results showed that the number of CD34(+) cells in the peripheral blood and in myocardium was significantly decreased in castrated rats, and the early expression levels of HIF-1a, SDF-1a and VEGF in the myocardium were also decreased. Furthermore, reduced capillary density, worsened cardiac function, increased infarct size and cardiomyocyte apoptosis at 28 days post-infarction were found in castrated rats. But these adverse effects could be reversed by testosterone-replacement therapy. These findings suggested that testosterone can increase the mobilization and homing of CD34(+) cells into the ischemic myocardium and further promote neoangiogenesis after myocardial infarction. The pro-angiogenesis effect of testosterone-replacement therapy is associated with the enhanced expression of HIF-1a, SDF-1a and VEGF in myocardium after myocardial infarction.