Network-Pharmacology-Based Study on Active Phytochemicals and Molecular Mechanism of Cnidii Fructus in Treating Colorectal Cancer.

AIMS: Cnidii Fructus (CF) is known for its antibacterial, anti-inflammatory, and antitumor properties, as well as its activities against kidney deficiency and impotence. In this study, we aimed to explore the anti-CRC cancer effect and molecular mechanism of CF via network pharmacology and in vitro antitumor experiments. METHODS: Network pharmacology was used to investigate the anti-CRC mechanism of CF. First, a series of databases was used to screen the active phytochemical targets and anti-CRC core targets. Then, the GO and KEGG pathways were analyzed to predict possible mechanisms. Molecular docking analysis explore core targets-phytochemicals interactions. In vitro antitumor experiments were carried on verifying anti-CRC mechanism of CF. RESULTS: In this study, 20 active ingredient targets and 50 intersecting targets were analyzed by Cytoscape software 3.9.1 to obtain the core genes and phytochemicals. Then, the GO and KEGG pathways of 50 intersecting targets were analyzed to predict possible mechanisms. The results from GO and KEGG indicated that CF has significant antitumor efficacy, which involves many signaling pathways, such as PI3K/AKT and p53. The five core targets and five core phytochemicals were screened for molecular docking to show protein-ligand interactions. According to the results of molecular docking, the compound O-acetylcolumbianetin was selected for the anti- CRC functional verification in vitro. MTT assay showed that O-acetylcolumbianetin significantly inhibited the proliferation of colorectal HCT116 cells in a time- and quantity-dependent manner. O-acetylcolumbianetin can promote the expression of CASP3 protein, induce HCT116 cells apoptosis, thus exert anti-CRC effect. CONCLUSION: This study preliminarily verified the anti-CRC effect and molecular mechanism of CF and provided a reference for Traditional Chinese Medicine anti-tumor subsequent research.

Effects and mechanisms of Xiaochaihu Tang against liver fibrosis: An integration of network pharmacology, molecular docking and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Liver fibrosis is a potentially harmful chronic liver disease caused by various etiologies. There is currently no specific drug for liver fibrosis. Xiaochaihu Tang (XCHT) is a traditional formula combined of seven herbs, which was first recorded in the Treatise on Febrile Diseases in Han Dynasty of ancient China. It is widely used in clinic to hepatic protection, analgesic, antipyretic and anti-inflammatory treatment. And it has been recommended for treating chronic hepatitis and chronic cholecystitis in the latest guidelines for the diagnosis and treatment of liver fibrosis with integrated traditional and western medicine. However, the underlying regulatory mechanisms remain elusive. AIM OF THE STUDY: This study aims to explore the therapeutic effects of XCHT on liver fibrosis and its underlying molecular mechanisms from the perspective of network pharmacology and experimental research. MATERIALS AND METHODS: Carbon tetrachloride (CCl4) induced and bile duct ligation (BDL) induced liver fibrosis models in mice were established to evaluate the anti-fibrosis effects of XCHT in vivo. Potential anti-fibrosis targets of XCHT were screened via network establishment. The underlying mechanisms were uncovered through GO and pathway enrichment analysis. Then, the core targets were identified from protein-protein interaction network by means of the Cytohubba plug-in of Cytoscape. Furthermore, two effective monomer components of XCHT were recognized by molecular docking. Moreover, the predicted components and pathways were verified by in vitro experiments. RESULTS: When treated with XCHT, liver fibrosis was alleviated in both mice models, showing as the improvement of liver function, the protection of hepatocytes, the inhibition of HSC activation and the reduction of hepatic collagen accumulation. 540 monomer components, 300 therapeutic targets, 109 signaling pathways, 246 GO biological processes, 77 GO cellular components, 107 GO molecular functions items and core targets were identified by network analysis. Then, 6-gingerol and baicalein were identified as the core components of anti-fibrosis effects of XCHT via leptin or Nrf2 signaling pathway. Furthermore, the experiment in vitro also validated the results. CONCLUSIONS: Our study suggests XCHT could alleviate liver fibrosis through multi-targets and multi-pathways; 6-gingerol and baicalein are its core components which may play an important role via leptin or Nrf2 signaling pathway.

A single-nucleotide polymorphism in WRKY33 promoter is associated with the cold sensitivity in cultivated tomato.

Natural variations in cis-regulatory regions often affect crop phenotypes by altering gene expression. However, the mechanism of how promoter mutations affect gene expression and crop stress tolerance is still poorly understood. In this study, by analyzing RNA-sequencing (RNA-Seq) data and reverse transcription quantitative real-time PCR validation in the cultivated tomato and its wild relatives, we reveal that the transcripts of WRKY33 are almost unchanged in cold-sensitive cultivated tomato Solanum lycopersicum L. 'Ailsa Craig' but are significantly induced in cold-tolerant wild tomato relatives Solanum habrochaites LA1777 and Solanum pennellii LA0716 under cold stress. Overexpression of SlWRKY33 or ShWRKY33 positively regulates cold tolerance in tomato. Variant of the critical W-box in SlWRKY33 promoter results in the loss of self-transcription function of SlWRKY33 under cold stress. Analysis integrating RNA-Seq and chromatin immunoprecipitation sequencing data reveals that SlWRKY33 directly targets and induces multiple kinases, transcription factors, and molecular chaperone genes, such as CDPK11, MYBS3, and BAG6, thus enhancing cold tolerance. In addition, heat- and Botrytis-induced WRKY33s expression in both wild and cultivated tomatoes are independent of the critical W-box variation. Our findings suggest nucleotide polymorphism in cis-regulatory regions is crucial for different cold sensitivity between cultivated and wild tomato plants.

Protective Effect of Danshen Zexie Decoction Against Non-Alcoholic Fatty Liver Disease Through Inhibition of ROS/NLRP3/IL-1ß Pathway by Nrf2 Signaling Activation.

Lipid metabolism disorders are a prominent characteristic in the pathological development of non-alcoholic fatty liver disease (NAFLD). Danshen zexie decoction (DZD) is a Chinese herbal medicine that is based on zexie decoction and has an effect of regulating lipid mechanism. However, the anti-NAFLD effect and mechanism of DZD remain unclear. In this study, we observed the therapeutic effect of DZD on NAFLD rats and investigated its possible mechanisms. Sixty Sprague Dawley rats were randomly assigned to six groups: control group, model group, Yishanfu (polyene phosphatidylcholine) group, and low, medium and high-dose DZD groups. High-fat diet (HFD) was fed to the rats to establish an NAFLD model, and each treatment group was given corresponding drugs at the same time for eight consecutive weeks. The results revealed that the obvious lipid metabolism disorder and liver injury induced by HFD were alleviated by treatment with DZD, which was verified by decreased serum TC, TG, ALT, AST, liver TC, TG, and FFA, as well as the alleviation of hepatic steatosis. The production of ROS in rats was reduced after treatment with DZD. The SOD activity and GSH content were increased with DZD treatment, while the MDA level was decreased. The administration of DZD could decrease serum IL-1ß and IL-18 contents. Moreover, DZD upregulated the expressions of Nrf2, HO-1, GCLC, and GCLM, while it suppressed the expressions of NLRP3, caspase-1, GSDMD, and GSDMD-N. In conclusion, the data showed that DZD can reduce lipid accumulation, alleviate oxidative stress and inflammation, and inhibit pyroptosis in NAFLD rats, which might be ascribed to suppression of the ROS/NLRP3/IL-1ß signaling pathway by activation of Nrf2. Overall, these results indicated that DZD is expected to be a therapeutic drug for NAFLD.

Loss-of-function alleles of ZmPLD3 cause haploid induction in maize.

Doubled haploid technology has been widely applied to multiple plant species and is recognized as one of the most important technologies for improving crop breeding efficiency. Although mutations in MATRILINEAL/Zea mays PHOSPHOLIPASE A1/NOT LIKE DAD (MTL/ZmPLA1/NLD) and Zea mays DOMAIN OF UNKNOWN FUNCTION 679 MEMBRANE PROTEIN (ZmDMP) have been shown to generate haploids in maize, knowledge of the genetic basis of haploid induction (HI) remains incomplete. Therefore, cloning of new genes underlying HI is important for further elucidating its genetic architecture. Here, we found that loss-of-function mutations of Zea mays PHOSPHOLIPASE D3 (ZmPLD3), one of the members from the phospholipase D subfamily, could trigger maternal HI in maize. ZmPLD3 was identified through a reverse genetic strategy based on analysis of pollen-specifically expressed phospholipases, followed by validation through the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) system. Mutations of ZmPLD3 resulted in a haploid induction rate (HIR) similar to that of mtl/zmpla1/nld and showed synergistic effects rather than functional redundancy on tripling the HIR (from 1.19% to 4.13%) in the presence of mtl/zmpla1/nld. RNA-seq profiling of mature pollen indicated that a large number of pollen-specific differentially expressed genes were enriched in processes related to gametogenesis development, such as pollen tube development and cell communication, during the double-fertilization process. In addition, ZmPLD3 is highly conserved among cereals, highlighting the potential application of these in vivo haploid-inducer lines for other important crop plant species. Collectively, our discovery identifies a novel gene underlying in vivo maternal HI and provides possibility of breeding haploid inducers with further improved HIR.

Astragaloside IV ameliorates high glucose­induced renal tubular epithelial­mesenchymal transition by blocking mTORC1/p70S6K signaling in HK­2 cells.

Astragaloside IV (AST) is the major active saponin in Astragalus membranaceus and, reportedly, has a variety of pharmacological activities. However, the potential of AST to ameliorate high glucose­mediated renal tubular epithelial­mesenchymal transition (EMT) remains undetermined. The aim of the present research was to explore the effect and mechanism of AST in EMT of renal tubular epithelial cells, as an underlying mechanism of renal fibrosis and a vital feature involved in diabetic nephropathy. The effect of AST on the EMT of renal tubular epithelial cells (HK­2) stimulated by high glucose was investigated and it was attempted to elucidate the potential underlying mechanism. The expression of E­cadherin and α­smooth muscle actin were determined by western blotting and immunofluorescence assays. The expression of the mammalian target of rapamycin complex 1 (mTORC1)/ ribosomal protein S6 kinase ß­1 (p70S6K) signaling pathway and protein levels of four transcriptional factors (snail, slug, twist and zinc finger E­box­binding homeobox 1) were also determined by western blotting. Additionally, extracellular matrix components, including fibronectin (FN) and collagen type IV (Col IV) were detected by ELISA. The results suggested that the EMT of HK­2 cells and the mTORC1/p70S6K pathway were activated by high glucose. The expression of snail and twist in HK­2 cells was elevated by high glucose. Furthermore, extracellular matrix components, FN and Col IV, were increased in HK­2 cells cultured with high glucose. In turn, treatment with AST reduced EMT features in HK­2 cells, inhibited mTORC1/p70S6K pathway activation, downregulated expression of snail and twist, and reduced secretion of FN and Col IV. In summary, the findings suggested that AST ameliorates high glucose­mediated renal tubular EMT by blocking the mTORC1/p70S6K signaling pathway in HK­2 cells.

Melatonin alleviates low-sulfur stress by promoting sulfur homeostasis in tomato plants.

Despite involvement of melatonin (MT) in plant growth and stress tolerance, its role in sulfur (S) acquisition and assimilation remains unclear. Here we report that low-S conditions cause serious growth inhibition by reducing chlorophyll content, photosynthesis and biomass accumulation. S deficiency evoked oxidative stress leading to the cell structural alterations and DNA damage. In contrast, MT supplementation to the S-deprived plants resulted in a significant diminution in reactive oxygen species (ROS) accumulation, thereby mitigating S deficiency-induced damages to cellular macromolecules and ultrastructures. Moreover, MT promoted S uptake and assimilation by regulating the expression of genes encoding enzymes involved in S transport and metabolism. MT also protected cells from ROS-induced damage by regulating 2-cysteine peroxiredoxin and biosynthesis of S-compounds. These results provide strong evidence that MT can enhance plant tolerance to low-S-induced stress by improving S uptake, metabolism and redox homeostasis, and thus advocating beneficial effects of MT on increasing the sulfur utilization efficiency.