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Impaired autophagy and excessive apoptosis disrupt cellular homeostasis and contribute to neural tube defects (NTDs), which are a group of fatal and disabling birth defects caused by the failure of neural tube closure during early embryonic development. However, the regulatory mechanisms underlying NTDs and outcomes remain elusive. Here, we report the role of the transcription factor nuclear factor I-C (NFIC) in maintaining cellular homeostasis in NTDs. We demonstrated that abnormally elevated levels of NFIC in a mouse model of NTDs can interact with the miR-200b promoter, leading to the activation of the transcription of miR-200b, which plays a critical role in NTD formation, as reported in our previous study. Furthermore, miR-200b represses autophagy and triggers apoptosis by directly targeting the autophagy-related gene Ambra1 (Autophagy/Beclin1 regulator 1). Notably, miR-200b inhibitors mitigate the unexpected effects of NFIC on autophagy and apoptosis. Collectively, these results indicate that the NFIC-miR-200b-Ambra1 axis, which integrates transcription- and epigenome-regulated miRNAs and an autophagy regulator, disrupts cellular homeostasis during the closure of the neural tube, and may provide new insight into NTD pathogenesis., (© 2021. The Author(s).)

Streptococcus pneumoniae is a Gram-positive and human-restricted pathogen colonizing the nasopharynx with an absence of clinical symptoms as well as a major pathogen causing otitis media (OM), one of the most common childhood infections. Upon bacterial infection, neutrophils are rapidly activated and recruited to the infected site, acting as the frontline defender against emerging microbial pathogens via different ways. Evidence shows that interleukin 17A (IL-17A), a neutrophil-inducing factor, plays important roles in the immune responses in several diseases. However, its function in response to S. pneumoniae OM remains unclear. In this study, the function of IL-17A in response to S. pneumoniae OM was examined using an in vivo model. We developed a model of acute OM (AOM) in C57BL/6 mice and found that neutrophils were the dominant immune cells that infiltrated to the middle ear cavity (MEC) and contributed to bacterial clearance. Using IL-17A knockout (KO) mice, we found that IL-17A boosted neutrophil recruitment to the MEC and afterwards induced apoptosis, which was identified to be conducive to bacterial clearance. In addition, our observation suggested that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was involved in the recruitment and apoptosis of neutrophils mediated by IL-17A. These data support the conclusion that IL-17A contributes to the host immune response against S. pneumoniae by promoting neutrophil recruitment and apoptosis through the p38 MAPK signaling pathway.

AIM: To investigate the oncolytic effect of human umbilical cord mesenchymal stem cells (hUMSCs) delivering reovirus type 3 (Reo3) on chronic myeloid leukemia (CML) K562 cells. METHODS: The expression of junctional adhesion molecule-A (JAM-A), a receptor susceptible to Reo3, on the surface of hUMSCs and K562 cells was assessed by flow cytometry. Intracellular viral inclusion body distribution 72 h after Reo3 infection in hUMSCs was observed by electron microscopy. The hUMSCs were infected with various multiplicities of infection (MOI) of Reo3 (M01=0, 1, 2 and 3) for 24, 48, 72, 96 and 120 h, and the most suitable MOI was identified by CCK-8 assay. Subsequently, hUMSCs were infected with the optimal titer of Reo3 for the same durations, and supernatants were collected. The titer of Reo3 in the supernatant from each group was measured using mouse fibroblast L929 cells combined with median tissue culture infectious dose (TCID50) method, determining the optimal infection time. The K562 cells were divided into 4 groups: control group, hUMSCs group, Reo3 group, and hUMSCs-Reo3 group. Ratios of hUMSCs to K562 cells in hUMSCs group and hUMSCs-Reo3 group were set at low, medium and high (5^1, 10^1 and 20: 1). The changes of K562 cell viability after co-cultured with hUMSCs-Reo3 for 24, 48 and 72 h were analyzed by CCK-8 assay. The apoptosis of K562 cells was evaluated by flow cytometry. The half maximal effective concentration (EC50) of anti-Reo3 monoclonal antibody was determined using L929 cells. The oncolytic effect of hUMSCs-Reo3 on K562 cells with antibody present in vitro was verified. Western blot analysis was used to detect the protein levels of Bcl-2, Bax, survivin and cleaved caspase-3 in K562 cells after treatment. A BALB/c nude mouse subcutaneous tumor model was constructed with K562 cells (n=6) to analyze the in vivo anti-tumor effect of hUMSCs-Reo3. RESULTS: The expression levels of JAM-A on the surfaces of hUMSCs and K562 cells were found to be 11. 0% and 99. 0%, respectively. Electron microscopy revealed a significant presence of viral inclusion bodies within hUMSCs 72 h following infection with Reo3. Within 120 h, no statistically significant difference was observed in the viability of hUMSCs between Reo3 (MOI=1) group and uninfected group, establishing the optimal MOI. The TCID50 results indicated that the highest virus titer in the lysate of hUMSCs in Reo3 (MOI=1) group occurred 48 h after infection, determining 48 h as the optimal infection time. The K562 cells co-cultured with hUMSCs-Reo3 for 24, 48 and 72 h showed a dose- and time-dependent inhibition of cell viability. The EC50 of the anti-Reo3 monoclonal antibody was found to be 1: 34. Even in the presence of antibodies at various concentrations (1: 34, 1 • 300 and 1: 600), hUMSCs were capable of transporting Reo3 to inhibit K562 cell viability and induce apoptosis in vitro. Compared with control group, significant down-regulation of Bcl-2 and survivin expression levels in K562 cells was noted after 48 h of co-culture with hUMSCs-Reo3 (P<0. 05), while Bax and cleaved caspase-3 expression levels were significantly up-regulated (P< 0. 05 or P<0. 01). In the BALB/c nude mouse tumor-bearing model, determination of tumor volume changes, pathological examination of tumor tissue and major organs, and assessment of cathepsin B/L activity using a small animal live imaging system confirmed the oncolytic effect of hUMSCs-Reo3 on K562 cells in vivo without adverse effects on normal tissues. CONCLUSION: The hUMSCs are effective in transporting Reo3, and this delivery system is capable of releasing sufficient quantities of Reo3 in both in vivo and in vitro settings to inhibit the malignant proliferation of K562 cells and promote apoptosis, thereby exerting an oncolytic effect. [ABSTRACT FROM AUTHOR]

AIM: To explore the synergistic sensitization effect of human umbilical cord mesenchymal stem cell culture supernatant (hUMSC-CM) combined with temozolomide (TMZ) on various glioma cell lines, and to elucidate the underlying mechanisms. METHODS: The hUMSC-CM was harvested using two different serum deprivation techniques at 24 and 48 h, and was converted into freeze-dried powder, which was then given to rat malignant glioma cell line RG-2, human astrocytoma cell line U251 and human glioblastoma cell line LN-428 at 5 concentrations (0, 1, 3, 6 and 9 g/L). The effectiveness and sensitivity of hUMSC-CM for inhibiting growth of glioma cells at 24, 48 and 72 h were assessed using CCK-8 assay. Hematoxylin-eosin (HE) staining combined with CCK-8 assay was employed to evaluate the chemotherapy sensitivity of glioma cells after 48 h of treatment with TMZ at 6 concentrations (0, 25, 50, 100, 200 and 400 µmol/L). Two concentrations (3 and 9 g/L) of hUMSC-CM and 3 concentrations (50, 100 and 200 µmol/L) of TMZ were chosen for concurrent treatment of glioma cells to assess the proliferation and pathological alterations. TUNEL staining was utilized to detect apoptosis. Flow cytometry was utilized to analyze cell cycle modifications. The expression alterations of apoptosis-inducing proteins, cleaved caspase-3, cleaved caspase-8 and cleaved PARP1, as well as autophagy-inducing proteins beclin-1 and LC3, were examined using Western blot to investigate the synergistic sensitization mechanism of hUMSC-CM combined with TMZ in vitro. RESULTS: The susceptibility of glioma cell lines to hUMSC-CM and TMZ varied, with RG-2 showing the highest sensitivity, followed by U251, and then LN-428. The inhibitory effect of hUMSC-CM (3 and 9 g/L) and TMZ (50, 100 and 200 µmol/L) combined treatment on glioma cells was significantly greater than that that of single-agent treatments (P<0. 05), demonstrating a dose- and concentration-dependent enhancement. Notably, the combination of 9 g/L hUMSC-CM (C9) with 50 µmol/L TMZ (T50) effectively suppressed glioma cell growth. CCK-8 assay indicated a significant reduction of cell viability in C9+T50 group compared with either C9 or T50 alone (P<0. 05). HE staining and TUNEL staining revealed pronounced morphological changes and significant apoptotic features in glioma cells treated with C9+T50. Flow cytometric analysis confirmed that C9+T50 induced cell cycle arrest in glioma cells. Furthermore, compared with control group, the levels of cleaved caspase-3, cleaved caspase-8, cleaved PARP1, beclin-1, and LC3-II/LC3-I were significantly elevated in the C9+T50-treated glioma cells (P<0. 01). CONCLUSION: (1) The concomitant administration of hUMSC-CM and TMZ exerts a broad inhibitory effect on glioma cells, with a synergistic sensitization observed across different cell lines. (2) The enhancement of glioma cell sensitivity to TMZ by hUMSC-CM may be attributed to the modulation of caspase-8/caspase-3/PARP1 signaling pathway and the induction of both apoptosis and autophagy in glioma cells. [ABSTRACT FROM AUTHOR]

Saikosaponin D (SSD), a saponin derivative, is extracted from Bupleurum falcatum. It exhibits an inhibitory effect on a number of tumor cells and is relatively safe when used at therapeutic doses. However, its effects on glioblastoma multiforme (GBM) have not been fully explored. This study is aimed at investigating the cytotoxic effects of SSD in GBM cell lines. SSD induces apoptosis and autophagy by activating endoplasmic reticulum (ER) stress in GBM cells. GBM cell proliferation activity and morphology were observed using the Cell Counting Kit-8 assay and hematoxylin and eosin staining. Hoechst 33258 fluorescence staining and flow cytometry were performed to assess apoptosis. Western blotting and immunocytochemical staining were used to detect protein expression and distribution. SSD significantly inhibited the proliferation of RG-2, U87-MG, and U251 cells in a dose-dependent manner, and the proportion of apoptotic cells increased significantly. Additionally, the expressions of ER-, apoptosis-, and autophagy-related proteins were significantly upregulated and distributed in the cytoplasm and nucleus. Therefore, SSD may be considered a novel treatment option for GBM. This study demonstrated the anti-GBM effect of SSD from the perspectives of cell apoptosis and autophagy. [ABSTRACT FROM AUTHOR]

• Functional efficacies of porcine bone collagen peptides with different molecular weights (MWs) were compared. • Collagen peptides (<1 kDa) increased proliferation and cell cycle and inhibited apoptosis. • The underlying mechanism was revealed that low-MW peptides exerted their effects by activating PI3K/Akt. Collagen peptides are widely used in food and medicine industries. Given the significant role of collagen in bone structure, collagen peptides are considered promising for osteoporosis prevention and treatment. To elucidate their effect on osteoporosis, we extracted porcine collagen, digested it into peptides, and investigated their biological function using osteoblast MC3T3-E1 cells. The collagen structure was relatively stable and we prepared peptides with different molecular weights (MWs) via hydrolysis with two commercially-available enzymes (alcalase/trypsin). Low-MW (<1 kDa) peptides, mainly comprised of tripeptides, increased proliferation and promoted cell cycle progression by upregulating CDK-2, CDK-4, Cyclin B1, and Cyclin D1, and inhibited apoptosis by decreasing Bax/Bcl-2 and cleaved caspase-3 expression. Meanwhile, they upregulated p-Akt and downregulated PTEN, indicating that low-MW peptides inhibited apoptosis and promoted osteoblast proliferation and differentiation by activating PI3K/Akt pathway. Thus, we report the mechanism through which porcine bone-derived collagen peptides could be used to prevent and treat osteoporosis. [ABSTRACT FROM AUTHOR]