20(R)-Panaxatriol enhances METTL3-mediated m6A modification of STUB1 to inhibit autophagy and exert antitumor effects in Triple-Negative Breast Cancer cells.

BACKGROUND: Aberrant activation of autophagy in triple-negative breast cancer (TNBC) has led researchers to investigate potential therapeutic strategies targeting this process. The regulation of autophagy is significantly influenced by METTL3. Our previous research has shown that the Panax ginseng-derived compound, 20(R)-panaxatriol (PT), has potential as an anti-tumor agent. However, it remains unclear whether PT can modulate autophagy through METTL3 to exert its anti-tumor effects. OBJECTIVE: Our objective is to investigate whether PT can regulate autophagy in TNBC cells and elucidate the molecular mechanisms. STUDY DESIGN: For in vitro experiments, we employed SUM-159-PT and MDA-MB-231 cells. While in vivo experiments involved BALB/c nude mice and NOD/SCID mice. METHODS: In vitro, TNBC cells were treated with PT, and cell lines with varying expression levels of METTL3 were established. We assessed the impact on tumor cell activity and autophagy by analyzing autophagic flux, Western Blot (WB), and methylation levels. In vivo, subcutaneous transplantation models were established in BALB/c nude and NOD/SCID mice to observe the effect of PT on TNBC growth. HE staining and immunofluorescence were employed to analyze histopathological changes in tumor tissues. MeRIP-seq and dual-luciferase reporter gene assays were used to identify key downstream targets. Additionally, the silencing of STIP1 Homology And U-Box Containing Protein 1 (STUB1) explored PT's effects. The mechanism of PT's action on STUB1 via METTL3 was elucidated through mRNA stability assays, mRNA alternative splicing analysis, and nuclear-cytoplasmic mRNA separation. RESULTS: In both in vivo and in vitro experiments, it was discovered that PT significantly upregulates the expression of METTL3, leading to autophagy inhibition and therapeutic effects in TNBC. Simultaneously, through MeRIP-seq analysis and dual-luciferase reporter gene assays, we have demonstrated that PT modulates STUB1 via METTL3, influencing autophagy in TNBC cells. Furthermore, intriguingly, PT extends the half-life of STUB1 mRNA by enhancing its methylation modification, thereby enhancing its stability. CONCLUSION: In summary, our research reveals that PT increases STUB1 m6A modification through a METTL3-mediated mechanism in TNBC cells, inhibiting autophagy and further accentuating its anti-tumor properties. Our study provides novel mechanistic insights into TNBC pathogenesis and potential drug targets for TNBC.

Leonurine attenuates OVA-induced asthma via p38 MAPK/NF-κB signaling pathway.

Leonurine (Leo) is a natural alkaloid extracted from Herba leonuri, which has many biological activities. However, whether leonurine has a protective effect on asthma remains unknown. The purpose of this study was to investigate the protective effect of leonurine on asthma. We evaluated its therapeutic effect and related signal transduction in LPS-induced RAW264.7 cells and OVA-induced asthmatic mice. In addition, we used network pharmacology, molecular docking and molecular dynamics simulation to verify the experimental results. In LPS-induced RAW 264.7 cells, leonurine significantly reduced the production of TNF-α and IL-6, andinhibited the activation of p38 MAPK/NF-κB signaling pathway. In OVA-induced asthmatic mice, leonurine decreased the number of inflammatory cells in the bronchoalveolar lavage fluid (BALF), particularly neutrophils and eosinophils. Leonurine also reduced the contents of IL-4, IL-5, IL-13 in the BALF and OVA-IgE in the serum. Leonurine remarkly improved OVA-induced inflammatory cell infiltration and significantly inhibited mucus overproduction. In addition, leonurine inhibited the activation of p38 MAPK/NF-κB signaling pathway in the lung tissues of asthmatic mice. Network pharmacology suggested that p38 MAPKα was a potential target of leonurine in the treatment of asthma. Molecular docking and molecular dynamics simulations indicated that leonurine could stably bind to p38 MAPKα protein. In summary, leonurine attenuated asthma by regulating p38 MAPK/NF-κB signaling pathway.

Eupatilin inhibits keratinocyte proliferation and ameliorates imiquimod-induced psoriasis-like skin lesions in mice via the p38 MAPK/NF-κB signaling pathway.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease that is currently incurable and causes long-term distress to patients. Therefore, there is an urgent need to develop safe and effective psoriatic drugs. Eupatilin is a natural flavone, that has a variety of pharmacological effects. However, the anti-psoriatic effect of eupatilin and its underlying mechanism remain unclear. METHODS: HaCaT cells were treated with 20 µg/mL LPS for 24 h to establish the proliferation model of HaCaT cells. Cell viability was measured by MTT assay. Western blotting was used to detect the expression of p-p38 MAPK, p38 MAPK, p-NF-κB p65 and NF-κB p65 in HaCaT cells. Imiquimod (IMQ) was used to induce psoriasis-like mouse model. Psoriasis Area Severity Index (PASI) score was used to evaluate the degree of skin injury, H&E staining was used to observe the pathological damage of skin tissues, and the expression levels of TNF-α, IL-6, IL-23 and IL-17 in the serum were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: Eupatilin could inhibit the hyperproliferation of LPS-stimulated HaCaT cells through p38 MAPK/NF-κB signaling pathway in vitro. In psoriatic mice, eupatilin could significantly reduce skin erythema, scales and thickening scores, ameliorate skin histopathological lesions, and decrease the levels of TNF-α, IL-6, IL-23 and IL-17 in the serum. CONCLUSION: Eupatilin had a good anti-proliferative effect in LPS-stimulated HaCaT cells, and significantly alleviated IMQ-induced psoriasis-like lesions in mice. Eupatilin was a promising drug for the treatment of psoriasis.

Eupatilin Suppresses OVA-Induced Asthma by Inhibiting NF-κB and MAPK and Activating Nrf2 Signaling Pathways in Mice.

To investigate the effect of eupatilin in asthma treatment, we evaluated its therapeutic effect and related signal transduction in OVA-induced asthmatic mice and LPS-stimulated RAW264.7 cells. The BALF was tested for changes in lung inflammatory cells. Th2 cytokines in the BALF and OVA-IgE in the serum were measured by ELISA. H&E and PAS staining were used to evaluate histopathological changes in mouse lungs. The key proteins NF-κB, MAPK, and Nrf2 in lung tissues were quantitatively analyzed by Western blotting. Finally, we evaluated the effect of eupatilin on cytokines and related protein expression in LPS-stimulated RAW 264.7 cells in vitro. In OVA-induced asthmatic mice, eupatilin reduced the numbers of inflammatory cells, especially neutrophils and eosinophils. Eupatilin also decreased the levels of IL-5, IL-13 in the BALF and OVA-IgE in the serum. Furthermore, eupatilin inhibited the activation of NF-κB and MAPK pathways and increased the expression of Nrf2 in OVA-induced asthmatic mice. In vitro, eupatilin significantly reduced LPS-stimulated NO, IL-6, and ROS production. Additionally, the NF-κB, MAPK, and Nrf2 protein expression in LPS-stimulated RAW264.7 cells was consistent with that in OVA-induced asthmatic lung tissues. In summary, eupatilin attenuated OVA-induced asthma by regulating NF-κB, MAPK, and Nrf2 signaling pathways. These results suggest the utility of eupatilin as an anti-inflammatory drug for asthma treatment.

Nanofocusing of a metallized double periodic arranged nanocone array for surface-enhanced Raman spectroscopy.

A plasmonic double periodic arranged nanocone array (DPANA) integrated by nanotips and nanogaps exhibit strong capability of light compression, and thus lead to extremely enhanced electric near-field intensity. The DPANA is fabricated by the self-assembled mask integrated with the inductively couple plasma (ICP) etching technology. Finite-difference time-domain (FDTD) simulations suggest that the metallized DPANA can generate a strong hotspot at the sharp tip apex and the nanogap between adjacent sharp tips. The electric-field enhancement characteristic is firstly verified with the help of the second-order surface nonlinear optical response of the metallized DPANA. The surface-enhanced Raman spectroscopy (SERS) examination of the metallized DPANA exhibits high sensitivity due to clearly presenting the Raman spectra of Rhodamine-6G (R6G) with concentrations down to 10 pM and has excellent uniformity, time stability, and recyclability, simultaneously. Furthermore, the principle demonstration of SERS practical application is also performed for thiram. This as-prepared SERS substrate has great potential application for trace amount detection.

Design, synthesis and lipid-lowering activities of penipyridone derivatives.

On the basis of our earlier discovered natural product penipyridone G with potential lipid-lowering utility, 35 penipyridone derivatives were designed, synthesized and characterized. Based on the oleic acid-induced HepG2 cell lipid accumulation model, compounds 12c, 14, 15f, 15k, 15o, 15p and 16f showed potent lipid-lowering activities among the synthetic compounds at 10 µM. In particular, compounds 4, 15k, 15o showed significant activities on inhibiting lipid accumulation in insulin resistant HepG2 cells, and these three compounds were safe and non-toxic within the concentration range of 400 µM. In comparison, 15o possessed the best lipid-lowering activity. Compared with the vehicle group, the triglyceride inhibition rate of 15o was about 30.2%, and the total cholesterol inhibition rate was about 14.8% at 20 µM, which was equipotent to Simvastatin. Our research indicates that 15o may serve as a promising lead compound for the development of hypolipidemic drugs.

Photoprotective effect of Astragalus membranaceus polysaccharide on UVA-induced damage in HaCaT cells.

BACKGROUND: The skin provides a predominant barrier against chemical, physical and microbial incursion. The intemperate exposure to ultraviolet A (UVA) radiation can cause excessive cellular oxidative stress, leading to skin damage, proteins damage and mitochondrial dysfunction. There is sufficient evidences supporting the proposal that mitochondria is highly implicated in skin photo-damage. METHODS: In the present study, a polysaccharide isolated from Astragalus membranaceus was further purified to be an α-glucan, which was further investigated its beneficial influence on UVA-induced photo-damage in HaCaT cells. RESULTS: Our results showed that the purified Astragalus membranaceus polysaccharide (AP) can protect HaCaT cells from UVA-induced photo-damage through reducing UVA-induced intracellular ROS production and mitochondrial membrane potential, thereby altering ATP content. It was found that the UVA induced damage in HaCaT cells could be effectively restored by co-treatment with AP. CONCLUSIONS: AP exhibited promising potential for advanced application as multifunctional skin care products and drugs.

Plasmon-enhanced nonlinear nanofocusing of gold nanoprisms driven via an ultrafast azimuthal vector beam.

We present the plasmon-enhanced nonlinear nanofocusing of a gold (Au) nanoprism array substrate (ANAS) driven via an ultrafast azimuthal vector beam (AVB). Theoretical calculations show that the electric-field intensity of the ANAS vertically excited via the femtosecond AVB is higher than that of LPB excitation. In this experiment, the second-order surface nonlinear optical response of the ANAS is adopted to examine the nonlinear plasmonic nanofocusing of the ANAS, and it was observed that the second harmonic (SH) intensity of the ANAS excited via the femtosecond AVB is ∼3.8 times higher than that of LPB excitation, revealing that the ANAS under AVB excitation has a better nonlinear plasmonic nanofocusing characteristic than that under LPB excitation. Furthermore, the GaSe nanosheets are transferred on the ANAS to examine the nonlinear plasmonic nanofocusing of the ANAS. The SH intensity of the GaSe nanosheets deposited on the ANAS via the femtosecond AVB excitation has been enhanced ∼4.7 times than that of LPB excitation, indicating that the ANAS via AVB excitation has better nonlinear plasmonic nanofocusing than that of LPB excitation. This method may be used as a nonlinear nanofocusing light source to increase the light-matter nonlinear interaction.

Protective effect of d-tetramannuronic acid tetrasodium salt on UVA-induced photo-aging in HaCaT cells.

UVA radiation from the sun is the main external stimulus in the pathogenesis of skin photo-aging. This process is associated with cellular oxidative stress. Here we aim at showing the protective effect of d-Tetramannuronic Acid Tetrasodium Salt (M4), a natural product, against UVA (30J/cm2) irradiation-induced oxidative stress and photo-aging in HaCaT cells, and to reveal the molecular mechanism underlying the protective efficacy. M4 pretreatment significantly increased HaCaT cell viability and MMP, suppressing UVA-induced ROS generation. Moreover, M4 treatment prevented the UVA-induced photo-aging of HaCaT cells (the reduction of cell viability, mitochondria dysfunction, and SIRT1/pGC-1α deregulation). Notably, the anti-photo-aging potential of M4 was directly associated with the increased expression of MMP and SIRT1, which was followed by the up-regulation of pGC-1α, D-LOOP, and Mt-TFA, and the transcriptional activation of NRF1/NRF2. Therefore, M4 is useful for the protection of skin cells from UVA-induced photo-aging.

Protective Effect of L-Hexaguluroic Acid Hexasodium Salt on UVA-Induced Photo-Aging in HaCaT Cells.

This study aimed to show the α-L-Hexaguluroic acid hexasodium salt (G6) protective effect against UVA-induced photoaging of human keratinocyte cells. We found that G6 localized to the mitochondria and improved mitochondrial functions. G6 increased respiratory chain complex activities, which led to increased cellular ATP content and NAD+/NADH ratio. Thus, G6 alleviated the oxidative stress state in UVA-irradiated cells. Moreover, G6 can regulate the SIRT1/pGC-1α pathway, which enhanced the cells' viability and mitochondria energy metabolism. Notably, the anti-photoaging potential of G6 was directly associated with the increased level of MMP and SIRT1, which was followed by the upregulation of pGC-1α, D-LOOP, and Mt-TFA, and with the transcriptional activation of NRF1/NRF2. Taking all of the results together, we conclude that G6 could protect HaCaT cells from UVA-induced photo-aging via the regulation of mitochondria energy metabolism and its downstream signaling pathways.

Allogeneic cell therapy using umbilical cord MSCs on collagen scaffolds for patients with recurrent uterine adhesion: a phase I clinical trial.

BACKGROUND: Intrauterine adhesions (IUA) are the most common cause of uterine infertility and are caused by endometrium fibrotic regeneration following severe damage to the endometrium. Although current stem cell treatment options using different types of autologous stem cells have exhibited some beneficial outcomes in IUA patients, the reported drawbacks include variable therapeutic efficacies, invasiveness and treatment unavailability. Therefore, the development of new therapeutic stem cell treatments is critical to improving clinical outcomes. METHODS: Twenty-six patients who suffered from infertility caused by recurrent IUA were enrolled in this prospective, non-controlled, phase I clinical trial with a 30-month follow-up. During the procedure, 1 × 107 umbilical cord-derived mesenchymal stromal cells (UC-MSCs), loaded onto a collagen scaffold, were transplanted into the uterine cavity following an adhesion separation procedure. Medical history, physical examination, endometrial thickness, intrauterine adhesion score and the biological molecules related to endometrial proliferation and differentiation were assessed both before and 3 months after cell therapy. RESULTS: No treatment-related serious adverse events were found. Three months after the operation, the average maximum endometrial thickness in patients increased, and the intrauterine adhesion score decreased compared to those before the treatment. A histological study showed the upregulation of ERα (estrogen receptor α), vimentin, Ki67 and vWF (von Willebrand factor) expression levels and the downregulation of ΔNP63 expression level, which indicates an improvement in endometrial proliferation, differentiation and neovascularization following treatment. DNA short tandem repeat (STR) analysis showed that the regenerated endometrium contained patient DNA only. By the end of the 30-month follow-up period, ten of the 26 patients had become pregnant, and eight of them had delivered live babies with no obvious birth defects and without placental complications, one patient in the third trimester of pregnancy, and one had a spontaneous abortion at 7 weeks. CONCLUSIONS: Transplanting clinical-grade UC-MSCs loaded onto a degradable collagen scaffold into the uterine cavity of patients with recurrent IUA following adhesiolysis surgery is a safety and effective therapeutic method. TRIAL REGISTRATION: Clinicaltrials.gov . NCT02313415 , Registered December 6, 2014.

Generation of clinical-grade human induced pluripotent stem cells in Xeno-free conditions.

INTRODUCTION: Human induced pluripotent stem cells (hiPSCs) are considered as one of the most promising seed cell sources in regenerative medicine. Now hiPSC-based clinical trials are underway. To ensure clinical safety, cells used in clinical trials or therapies should be generated under GMP conditions, and with Xeno-free culture media to avoid possible side effects like immune rejection that induced by the Xeno reagents. However, up to now there are no reports for hiPSC lines developed completely under GMP conditions using Xeno-free reagents. METHODS: Clinical-grade human foreskin fibroblast (HFF) cells used as feeder cells and parental cells of the clinical-grade hiPSCs were isolated from human foreskin tissues and cultured in Xeno-free media. Clinical-grade hiPSCs were derived by integration-free Sendai virus-based reprogramming kit in Xeno-free pluriton™ reprogramming medium or X medium. Neural cells and cardiomyocytes differentiation were conducted following a series of spatial and temporal specific signals induction according to the corresponding lineage development signals. Biological safety evaluation of the clinical-grade HFF cells and hiPSCs were conducted following the guidance of the "Pharmacopoeia of the People's Republic of China, Edition 2010, Volume III". RESULTS: We have successfully derived several integration-free clinical-grade hiPSC lines under GMP-controlled conditions and with Xeno-free reagents culture media in line with the current guidance of international and national evaluation criteria. As for the source of hiPSCs and feeder cells, biological safety evaluation of the HFF cells have been strictly reviewed by the National Institutes for Food and Drug Control (NIFDC). The hiPSC lines are pluripotent and have passed the safety evaluation. Moreover, one of the randomly selected hiPSC lines was capable of differentiating into functional neural cells and cardiomyocytes in Xeno-free culture media. CONCLUSION: The clinical-grade hiPSC lines therefore could be valuable sources for future hiPSC-based clinical trials or therapies and for drug screening.

Generation of induced pluripotent stem cells with high efficiency from human umbilical cord blood mononuclear cells.

Human induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine. Generating iPSCs from immunologically immature newborn umbilical cord blood mononuclear cells (UCBMCs) is of great significance. Here we report generation of human iPSCs with great efficiency from UCBMCs using a dox-inducible lentiviral system carrying four Yamanaka factors. We generated these cells by optimizing the existing iPSC induction protocol. The UCBMC-derived iPSCs (UCB-iPSCs) have characteristics that are identical to pluripotent human embryonic stem cells (hESCs). This study highlights the use of UCBMCs to generate highly functional human iPSCs that could accelerate the development of cell-based regenerative therapy for patients suffering from various diseases.

Identification of a small molecule 1,4-bis-[4-(3-phenoxy-propoxy)-but-2-ynyl]-piperazine as a novel inhibitor of the transcription factor p53.

AIM: To identify novel small compound inhibitor of p53 protein. METHODS: Mouse embryonic fibroblasts (MEF) and mouse embryonic stem (ES) cells were tested. Cell proliferation rate was determined using a Cell Proliferation Kit. The mRNA and protein levels of p53-related genes were measured using real-time PCR and Western blotting, respectively. Global response in the p53 signaling network was analyzed using Illumina whole-genome expression BeadChips. RESULTS: Treatment of MEF cells with a small molecule 1,4-bis-[4-(3-phenoxy-propoxy)-but-2-ynyl]-piperazine (G5) at 10 µmol/L for 24 h markedly reduced the mRNA and protein levels of the p53 downstream genes MDM2 and p21. In G5-treated ES cells, a total of 372 differentially expressed genes were identified, and 18 among them were direct downstream genes of p53; 6 out of 9 p53-repressed genes were upregulated, and 5 out of 9 p53-activated genes were downregulated. In both MEF cells and ES cells, treatment of with G5 (10 µmol/L) up to 48 h neither affected the proliferation rate nor caused morphological alterations. CONCLUSION: G5 inhibits p53 activity and simultaneously preserves the normal growth and proliferation of cells, therefore is a new compound for studies of p53-mediated cell manipulation.