ABSTRACT
Background: Ubiquitination is medicated by three classes of enzymes and has been proven to involve in multiple cancer biological processes. Moreover, dysregulation of ubiquitination has received a growing body of attention in osteosarcoma (OS) tumorigenesis and treatment. Therefore, our study aimed to identify a ubiquitin-related gene signature for predicting prognosis and immune landscape and constructing OS molecular subtypes. Methods: Therapeutically Applicable Research to Generate Effective Treatments (TARGET) was regarded as the training set through univariate Cox regression, Lasso Cox regression, and multivariate Cox regression. The GSE21257 and GSE39055 served as the validation set to verify the predictive value of the signature. CIBERSORT was performed to show immune infiltration and the immune microenvironment. The NMF algorithm was used to construct OS molecular subtypes. Results: In this study, we developed a ubiquitin-related gene signature including seven genes (UBE2L3, CORO6, DCAF8, DNAI1, FBXL5, UHRF2, and WDR53), and the gene signature had a good performance in predicting prognosis for OS patients (AUC values at 1/3/5 years were 0.957, 0.890, and 0.919). Multivariate Cox regression indicated that the risk score model and prognosis stage were also independent prognostic prediction factors. Moreover, analyses of immune cells and immune-related functions showed a significant difference in different risk score groups and the three clusters. The drug sensitivity suggested that IC50 of proteasome inhibitor (MG-132) showed a notable significance between the risk score groups (p < 0.05). Through the NMF algorithm, we obtained the three clusters, and cluster 3 showed better survival outcomes. The expression of ubiquitin-related genes (CORO6, UBE2L3, FBXL5, DNAI1, and DCAF8) showed an obvious significance in normal and osteosarcoma tissues. Conclusion: We developed a novel ubiquitin-related gene signature which showed better predictive prognostic ability for OS and provided additional information on chemotherapy and immunotherapy. The OS molecular subtypes would also give a useful guide for individualized therapy.
ABSTRACT
BACKGROUND: The mitochondrial apoptotic pathway is an important pathway in cell apoptosis. Previous studies have found that Bushen Zhuangdu Fang can improve intervertebral disc degeneration by inhibiting the mitochondrial apoptotic pathway in animal experiments. However, its mechanism of action is to be clarified. OBJECTIVE: To observe the effect of serum containing Bushen Zhuangdu Fang on mitochondrial apoptotic pathway key proteins of human nucleus pulposus cells, and to explore the mechanism by which this drug-containing serum improves intervertebral disc degeneration. METHODS: Thirty-seven male Sprague-Dawley rats were randomly divided into blank group, low-dose Chinese medicine group (0.506 g/kg per day), medium-dose Chinese medicine group (1.012 g/kg per day) and high-dose Chinese medicine group (2.024 g/kg per day). After 2 weeks of continuous administration, drug-containing serum was prepared. Human nucleus pulposus cells were randomly divided into normal group, cell model group, low-dose drug-containing serum group, medium-dose drug-containing serum group, and high-dose drug-containing serum group. The cell model group was treated with 200 μmol/L H2O2 for 6 hours, and the normal group received no treatment. The three drug-containing serum groups were treated with corresponding treatments for 48 hours. The pathological changes of nucleus pulposus cells were observed by transmission electron microscopy. Apoptotic rate of nucleus pulposus cells was detected by flow cytometry and mitochondrial membrane potential was detected by flow cytometry. Apaf1, Bcl-2, Bax and Cytc expressions were detected by real-time quantitative PCR and western blot assay. RESULTS AND CONCLUSION: Compared with the normal group, the apoptotic rate of nucleus pulposus cells with obvious apoptotic morphology was significantly increased (P < 0.05), the expression of Apaf1, Cytc, and Bax were significantly increased at mRNA and protein levels (P < 0.05), and the mitochondrial membrane potential and expression of Bcl-2 mRNA and protein were significantly decreased in the cell model group (P < 0.05). After treatment with drug-containing serum, the apoptotic rate of nucleus pulposus cells decreased significantly (P < 0.05), the expression of Apaf1, Cytc, Bax and their proteins decreased significantly (P < 0.05), and the mitochondrial membrane potential, Bcl-2 and their proteins increased significantly (P < 0.05). Therefore, the serum containing Bushen Zhuangdu Fang can effectively inhibit apoptosis of nucleus pulposus cells in a dose-dependent manner. The drug-containing serum may alleviate intervertebral disc degeneration by reducing the expression of Apaf1, Cytc and Bax and increasing the expression of Bcl-2 at protein and gene levels, and inhibiting the mitochondrial apoptotic pathway.
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Objective:To explore the mechanism of mitochondrial apoptotic pathway in rat degenerative intervertebral disc cells in improving intervertebral disc degeneration under the action of Bushen Zhuangdu recipe. Method:The 100 SD male rats were randomly divided into blank group, model group, low, medium and high dose Bushen Zhuangdu recipe group (0.38,0.77,1.53 g·kg-1).Histopathological changes of rat intervertebral discs were observed by hematoxylin-eosin(HE) staining after 4 weeks of continuous administration of Chinese medicine. The apoptotic rate of nucleus pulposus cells in degenerative intervertebral discs was detected by TUNEL(terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling), and the levels of active Cysteinyl aspartate-specific proteinase-3(active Caspase-3), B cell leukemia-2(Bcl-2), cytochrome C (cytC) and Bcl-2-associated X protein(Bax) protein in intervertebral discs were detected by Western blot. Result:Compared with blank group, the histopathological score of intervertebral disc in the model group was significantly increased, the apoptosis rate of nucleus pulposus was significantly increased (PPPPPPConclusion:Bushen Zhuangdu recipe may improve the degeneration of intervertebral disc by reducing the expression of active Caspase-3, cytC and Bax, increasing the expression of Bcl-2 and inhibiting the apoptotic pathway of mitochondria in a dose-dependent manner.
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<p><b>OBJECTIVE</b>To investigate the chemical constituents from the leaves of Vaccinium bracteatum.</p><p><b>METHOD</b>Many column chromatographic techniques were used for the isolation and separation of chemical constituents. Their structures were elucidated on the basis of spectral analysis and chemical evidences.</p><p><b>RESULT</b>Twelve compounds were isolated from the plant, and they were identified as chrysoeriol (1), scopoletin (2), trans-p-hydroxycinnamic acid (3), trans-p-hydroxycinnamic acid ethyl ester (4), cafeic acid ethyl ester (5), beta-sitosterol (6), iuteolin (7), quercetin (8), esculetin (9), cafeic acid (10), isolariciresinol-9-O-beta-D-xyloside (11), 10-O-trans-p-coumaroylsandoside (12).</p><p><b>CONCLUSION</b>Compounds 4, 5, 11, 12 were isolated from the genus Vaccinium for the first time, and compounds 1, 2, 9, 10 were isolated from this plant for the first time.</p>
