ABSTRACT
Objective: The PRAS40 is an essential inhibitory subunit of the mTORC1 complex, which regulates autophagy. It has been suggested that Erxian Decoction (EXD) could treat spinal cord injury (SCI) via the autophagy pathway. However, the mechanism of whether EXD acts through PRAS40 remains unclear. Methods: With the help of immobilized PRAS40, isothermal titration calorimetry (ITC) and molecular docking, the bioactive metabolites in the EXD were screened. To establish in vitro SCI models, PC12 cells were exposed to hydrogen peroxide (H2O2) and then treated with the identified EXD substances. Furthermore, Western blot assay was carried out to identify potential molecular mechanisms involved. For assessing the effect of metabolites in vivo, the SCI model rats were first pretreated with or without the metabolite and then subjected to the immunohistochemistry (IHC) staining, Basso, Beattie & Bresnahan (BBB) locomotor rating scale, and H&E staining. Results: The immobilized PRAS40 isolated indole, 4-nitrophenol, terephthalic acid, palmatine, sinapinaldehyde, and 3-chloroaniline as the potential ligands binding to PRAS40. Furthermore, the association constants of palmatine and indole as 2.84 × 106 M-1 and 3.82 × 105 M-1 were elucidated via ITC due to the drug-like properties of these two metabolites. Molecular docking results also further demonstrated the mechanism of palmatine binding to PRAS40. Western blot analysis of PC12 cells demonstrated that palmatine inhibited the expression of p-mTOR by binding to PRAS40, activating the autophagic flux by markedly increasing LC3. The injection of palmatine (10µM and 20 µM) indicated notably increased BBB scores in the SCI rat model. Additionally, a dose-dependent increase in LC3 was observed by IHC staining. Conclusion: This research proved that EXD comprises PRAS40 antagonists, and the identified metabolite, palmatine, could potentially treat SCI by activating the autophagic flux.
ABSTRACT
BACKGROUND: Emerging evidence shows that serum tumor biomarkers (TBs) and log odds of positive lymph node scheme (LODDS) are closely associated with the prognosis of colorectal cancer (CRC) patients. The aim of our study is to validate the predictive value of TBs and LODDS clinically and to develop a robust prognostic model to predict the overall survival (OS) of patients with CRC. METHODS: CRC patients who underwent radical resection and with no preoperative chemotherapy were enrolled in the study. The eligible population were randomized into training (70%) and test (30%) cohorts for the comprehensive evaluation of the prognostic model. Clinical implications of serum biomarkers and LODDS were identified by univariate and multivariate Cox proportion regression analysis. The predictive ability and discriminative performance were evaluated by Kaplan-Meier (K-M) curves and receiver operating characteristic (ROC) curves. Clinical applicability of the prognostic model was assessed by decision curve analysis (DCA), and the corresponding nomogram was constructed based on the above factors. RESULTS: A total of 1,202 eligible CRC patients were incorporated into our study. Multivariable COX analysis demonstrated that CA199 (HR = 1.304), CA125 (HR = 1.429), CEA (HR = 1.307), and LODDS (HR = 1.488) were independent risk factors for OS (all P < 0.0001). K-M curves showed that the high-risk group possessed a shorter OS than the low-risk counterparts. The area under curves (AUCs) of the model for 1-, 3- and 5-year OS were 86.04, 78.70, and 76.66% respectively for the train cohort (80.35, 77.59, and 74.26% for test cohort). Logistic DCA and survival DCA confirmed that the prognostic model displayed more clinical benefits than the conventional AJCC 8th TNM stage and CEA model. The nomograms were built accordingly, and the calibration plot for the probability of survival at 3- or 5-years after surgery showed an optimal agreement between prediction and actual observation. CONCLUSIONS: Preoperative serum TBs and LODDS have significant clinical implications for CRC patients. A novel prognostic model incorporating common TBs (CA199, CA125, and CEA) and LODDS displayed better predictive performance than both single factor and the TNM classification. A novel nomogram incorporating TBs and LODDS could individually predict OS in patients with CRC.
