ABSTRACT
To explore the effects of ß-Sitosterol upon hepatocellular carcinoma cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition (EMT), and to investigate the underlying mechanism using network pharmacology. Human hepatocellular carcinoma cell lines (Huh-7 and HCCLM3) were expose to gradient concentrations of ß-Sitosterol (5 µg/mL, 10 µg/mL, and 20 µg/mL). Cell viability and proliferation were assessed using MTT, CCK-8, colony formation, and EdU assays.Flow cytometry was employed to evaluate cell cycle and apoptosis. Scratch and Transwell assays were performed, respectively, to detect cell migration and invasion. The levels of apoptosis-associated proteins (BAX, BCL2, and cleaved caspase3) as well as EMT-associated proteins (E-cadherin, N-cadherin, Snail, and Vimentin) were detected in Huh-7 and HCCLM3 cell lines using Western blot analysis. The drug target gene for ß-Sitosterol was screened via PubChem and subsequently evaluated for expression in the GSE112790 dataset. In addition, the expression level of glycogen synthase kinase 3 beta (GSK3B) within the Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database was analyzed, along with its correlation to the survival outcomes of patients with hepatocellular carcinoma. The diagnostic efficiency of GSK3B was assessed by analyzing the ROC curve. Subsequently, Huh-7 and HCCLM3 cell lines were transfected with the overexpression vector of GSK3B and then treated with ß-Sitosterol to further validate the association between GSK3B and ß-Sitosterol. GSK3B demonstrated a significantly elevated expression in patients with hepatocellular carcinoma, which could predict hepatocellular carcinoma patients' impaired prognosis based on GEO dataset and TCGA database. GSK3B inhibitor (CHIR-98014) notably inhibited cell proliferation and invasion, promoted cell apoptosis and cell cycle arrest at G0/G1 phase in hepatocellular carcinoma cells. ß-Sitosterol treatment further promoted the efffects of GSK3B inhibitor on hepatocellular carcinoma cells. GSK3B overexpression has been found to enhance the proliferative and invasive capabilities of hepatocellular carcinoma cells. Furthermore it has been observed that GSK3B overexpression, it has been obsear can partially reverse the inhibitory effect of ß-Sitosterol upon hepatocellular. ß-Sitosterol suppressed hepatocellular carcinoma cell proliferation and invasion, and enhanced apoptosis via inhibiting GSK3B expression.
Subject(s)
Apoptosis , Carcinoma, Hepatocellular , Cell Proliferation , Epithelial-Mesenchymal Transition , Glycogen Synthase Kinase 3 beta , Liver Neoplasms , Sitosterols , Humans , Sitosterols/pharmacology , Glycogen Synthase Kinase 3 beta/metabolism , Glycogen Synthase Kinase 3 beta/genetics , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/pathology , Liver Neoplasms/genetics , Liver Neoplasms/drug therapy , Liver Neoplasms/metabolism , Cell Proliferation/drug effects , Cell Proliferation/genetics , Apoptosis/drug effects , Apoptosis/genetics , Cell Line, Tumor , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Cell Movement/drug effects , Cell Movement/genetics , Gene Expression/genetics , Gene Expression/drug effects , Phenotype , Neoplasm Invasiveness/genetics , Cell Survival/drug effects , Cell Survival/genetics , Network Pharmacology , Gene Expression Regulation, Neoplastic/drug effectsABSTRACT
Temperature and photoperiod are proximate environmental factors that have an important influence on the morphological, physiological and behavioral adjustments animals performance du-ring seasonal acclimatization. In this study, the effects of temperature and photoperiod on phenotypic flexibility in body mass, energy budget and digestive tract morphology in the Chinese bulbul (Pycnonotus sinensis) were examined, and the relationship between energy budget and digestive tract morphology was analyzed. Twelve male and sixteen female Chinese bulbuls were randomly assigned into 4 experimental groups so that each group was comprised of three males and four females. The groups were: 1) a warm and long photoperiod (30 â, 16 light: 8 dark) group, 2) a warm and short photoperiod (30 â, 8 light: 16 dark) group, 3) a cold and long photoperiod (10 â, 16 light:8 dark) group, and 4) a cold and short photoperiod (10 â, 8 light: 16 dark) group. Each group was acclimated to its respective temperature and photoperiod for 4 weeks. Birds in the cold temperature and short photoperiod group underwent a significant increase in body mass, gross energy intake (GEI) and digestible energy intake (DEI) compared to the other three groups, and there was a significant interaction between temperature and photoperiod on gross energy intake and diges-tible energy intake. The mass of the stomach, small intestine, rectum, and total digestive tract, all increased significantly in cold temperature treatment groups compared to those acclimated to a relatively warm temperature. There was a significant, positive correlation between GEI and DEI resi-duals and those of the length and dry mass of the small intestine and total digestive tract. These results suggested that the Chinese bulbul met the increased energy demands of winter (colder tempe-ratures and reduced foraging time due to shorter day-length) by increasing its body mass, digestible energy intake and digestive tract size.
Subject(s)
Body Weight , Cold Temperature , Gastrointestinal Tract , Glycosides/physiology , Photoperiod , Acclimatization , Animals , Cyclopentanes , Energy Intake , Energy Metabolism , Male , SeasonsABSTRACT
Chinese bulbuls (Pycnonotus sinensis) are small passerine birds that inhabit areas of central, southern and eastern China. Previous observations suggest that free-living individuals of this species may change their food intake in response to seasonal changes in ambient temperature. In the present study, we randomly assigned Chinese bulbuls to either a 30â or 10â group, and measured their body mass (BM), body temperature, gross energy intake (GEI), digestible energy intake (DEI), and the length and mass of their digestive tracts over 28 days of acclimation at these temperatures. As predicted, birds in the 30â group had lower body mass, GEI and DEI relative to those in the 10â group. The length and mass of the digestive tract was also lower in the 30â group and trends in these parameters were positively correlated with BM, GEI and DEI. These results suggest that Chinese bulbuls reduced their absolute energy demands at relatively high temperatures by decreasing their body mass, GEI and DEI, and digestive tract size.
