Hepatocellular carcinoma (HCC) shows recurrence and lung metastasis even after treatment. 23-hydroxybetulinic acid (23-HBA), a major active constituent of Pulsatilla chinensis, exhibits potent antitumor activities. We herein investigate the biological effect of 23-HBA on metastasis and immunosuppression in a mouse model of HCC. Microarray-based gene expression profiling was employed to identify the target genes of 23-HBA in the treatment of HCC. The effect of 23-HBA on the progression of HCC was evaluated by in-vitro cell function measurements along with in-vivo xenograft implantation, lung metastasis and CD11b+Gr1+ staining experiments. The potential mechanism involving target signaling pathway was investigated by western blot analysis. Bioinformatics analysis revealed that matrix metalloproteinase 2 (MMP2) was a key target gene mediated by 23-HBA in HCC, whereas Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis demonstrated that MMP2 mainly affects the development and metastasis of HCC. 23-HBA significantly reduced cell malignant functions in vitro while delaying the HCC growth and metastasis in vivo. In addition, the number of myeloid-derived suppressor cells was shown to be reduced following administration of 23-HBA in mice. Mechanistic analysis indicated that these effects of 23-HBA during HCC were involved with the mitogen-activated protein kinase (MAPK) signaling pathway inactivation and resulted in decreased phosphorylation of both mitogen-activated protein kinases 1/2 and extracellular signal-regulated kinase 1/2. Our study reveals that 23-HBA acts as a tumor suppressor agent and suppresses HCC tumorigenesis, metastasis and immunosuppression via blockade of the MAPK signaling pathway, suggesting that 23-HBA may serve as a promising drug target to treat HCC.

The COVID-19 outbreak has created turbulence and uncertainty into multiple aspects of life in countries around the world. In China, the pandemic continues to pose a great challenge to the nature of traditional in-class education in schools. Chinese education has faced the difficult decision of whether to resume in-person teaching in an unprecedented and time-pressured manner.To ensure the quality of teaching and learning during this time, this study aims to explore the effectiveness of an "online + in-person" hybrid teaching model with a new three-part approach to the hybrid teaching lab, where students prepare for the in-person lab using virtual simulated experiments and learning modules and debrief their learning afterwards online as well. This approach not only enhances the efficiency during the in-person lab but also strongly reinforces concepts and laboratory skills by providing a "practice run" before physically attending the lab. A total of 400 medical undergraduates from Dalian Medical University in China were recruited for this study. In an undergraduate molecular biology laboratory course, we observed 200 students in a hybrid teaching model. We evaluated the learning outcomes from the "online + inperson" hybrid teaching model with a questionnaire survey and assessed the quality of experiment execution, report writing, and group collaboration. Moreover, the 200 students from the hybrid group were evaluated during an annual science competition at the university and compared to 200 students from the competition cohort who had no experience with a hybrid learning model. The comparison data were analyzed using a student's t-test statistical analysis. The students in the hybrid learning group demonstrated a strong enthusiasm for the model, high amount of time utilizing the online system, and high scores on laboratory evaluation assignments. Approximately 98% of the hybrid learning students reported that they preferred mixed teaching to the traditional teaching mode, and all students scored above 96% on the online laboratory report.Teachers of the course observed that the hybrid group had a noticeably higher level of proficiency in lab skills compared to the previous students. At the Abbreviations: ICC, immunocytochemistry; q-PCR, quantitative real time polymerase chain reaction.Zheng Sun and Zihan Xu contributed equally to this work.

The aim of the present study was to determine the cell proliferation, apoptotic pathway analysis through protein, mRNA and cell cycle arrest mechanism in nerolidol induced osteosarcoma MG-63 cells. The osteosarcoma MG-63 cells were treated with various doses of nerolidol (15 and 20 μM/ml) for 24 h. Cell proliferation was examined using assist method of MTT assay, fixed the IC50 value of nerolidol 15 μM/ml. Reactive oxygen species (ROS) generation was analyzed by DCFH-DA dye, mitochondrial potential detected by Rh-123 dye, apoptotic morphological changes identified by AO/EtBr, PI, DAPI staining, and cell adhesion were detected by using fluorescence microscope. Cell proliferation, and apoptotic molecular protein and mRNA expressions such as ERK, P38, p-PI3K, p-JNK, Bcl-2, JNK, p-P38, cyclin-D1, and Bax were analyzed in osteosarcoma MG-63 cells. Nerolidol significantly suppressed the osteosarcoma cells progression in a dose dependent manner (p < .05) evident in the oxidative stress induction and apoptotic morphological changes.Nerolidol also regulated the protein PI3K/AKT mechanistically via induction of apoptosis Nerolidol suppresses osteosarcoma MG-63 cells by PI3K/AKT by cell cycle arrest at early phase of G0/G1. To sum up, nerolidol suppressed the growth of bone cancer cells and can be finally targeted as a potent drug for analyzing its chemotherapeutic effects in future.