BACKGROUND This study used network pharmacology method and cell model to assess the eﬀects of Radix Astragali (RA) on cholangiocarcinoma (CCA) and to predict core targets and molecular mechanisms.
MATERIAL AND METHODS We performed an in vitro study to assess the effect of RA on CCA using CCK8 assay, the Live-Cell Analysis System, and trypan blue staining. The components and targets of RA were analyzed using the Traditional Chinese Medicine Systems Pharmacology database, and genes associated with CCA were retrieved from the GeneCards and OMIM platforms. Protein-protein interactions were analyzed with the STRING platform.
The components-targets-disease network was built by Cytoscape. The TIMER database revealed the expression of core targets with diverse immune infiltration levels. GO and KEGG analyses were performed to identify molecular-biology processes and signaling pathways. The predictions were verified by Western blotting.
RESULTS Concentration-dependent antitumor activity was confirmed in the cholangiocarcinoma QBC939 cell line treated with RA. RA contained 16 active compounds, with quercetin and kaempferol as the core compounds.
The most important biotargets for RA in CCA were caspase 3, MAPK8, MYC, EGFR, and PARP. The TIMER database revealed that the expression of caspase3 and MYC was related with diverse immune infiltration levels of CCA. The results of Western blotting showed RA significantly influenced the expression of the 5 targets that network pharmacology predicted. CONCLUSIONS RA is an active medicinal material that can be developed into a safe and effective multi-targeted anticancer treatment for CCA.
Sorting the Wheat From the Chaff: Programmed Cell Death as a Marker of Stress Tolerance in Agriculturally Important Cereals
Conventional methods for screening for stress-tolerant cereal varieties rely on expensive, labour-intensive field testing and molecular biology techniques. Here, we use the root hair assay (RHA) as a rapid screening tool to identify stress-tolerant varieties at the early seedling stage.
Wheat and barley seedlings had stress applied, and the response quantified in terms of programmed cell death (PCD), viability and necrosis. Heat shock experiments of seven barley varieties showed that winter and spring barley varieties could be partitioned into their two distinct seasonal groups based on their PCD susceptibility, allowing quick data-driven evaluation of their thermotolerance at an early seedling stage.
In addition, evaluating the response of eight wheat varieties to heat and salt stress allowed identification of their PCD inflection points (35°C and 150 mM NaCl), where the largest differences in PCD levels arise. Using the PCD inflection points as a reference, we compared different stress effects and found that heat-susceptible wheat varieties displayed similar vulnerabilities to salt stress.
Stress-induced PCD levels also facilitated the assessment of the basal, induced and cross-stress tolerance of wheat varieties using single, combined and multiple individual stress exposures by applying concurrent heat and salt stress in a time-course experiment. Two stress-susceptible varieties were found to have low constitutive resistance as illustrated by their high PCD levels in response to single and combined stress exposure.
However, both varieties had a fast, adaptive response as PCD levels declined at the other time-points, showing that even with low constitutive resistance, the initial stress cue primes cross-stress tolerance adaptations for enhanced resistance even to a second, different stress type.
Here, we demonstrate the RHA’s suitability for high-throughput analysis (∼4 days from germination to data collection) of multiple cereal varieties and stress treatments. We also showed the versatility of using stress-induced PCD levels to investigate the role of constitutive and adaptive resistance by exploring the temporal progression of cross-stress tolerance.
Our results show that by identifying suboptimal PCD levels in vivo in a laboratory setting, we can preliminarily identify stress-susceptible cereal varieties and this information can guide further, more efficiently targeted, field-scale experimental testing.