Uncovering the mechanism of Qidan Dihuang Granule in the treatment of diabetic kidney disease combined network pharmacology, UHPLC-MS/MS with experimental validation.

Background and aim: Diabetic Kidney Disease (DKD) is a common microvascular complication of diabetes mellitus. Multi-center, randomized controlled trials have shown that Qidan Dihuang Granule (QDDHG) reduces the levels of albuminuria of DKD. However, the specific mechanisms of QDDHG on DKD are not clarified. Thus, this study utilized network pharmacology, UHPLC-MS/MS (Ultra-High Performance Liquid Chromatography - Mass Spectrometry) and animal experiments to reveal the mechanisms of QDDHG on DKD. Experimental procedure: Screening and retrieving active ingredients and corresponding targets of QDDHG on DKD through the TCMSP, ETCM, Disgenet, GeneCards, Omim and DrugBank databases. The PPI were performed with BioGrid, STRING, OmniPath, InWeb-IM. AutoDock Vina molecular docking module to estimate the validation from the compounds and target proteins. Free energy to estimate the binding affinity for identified compounds and target proteins. The ingredients of QDDHG were analyzed utilizing UHPLC-MS/MS. In vivo experiment with db/db mice were used to verify the targets and pathway predicted by network pharmacology. Results and conclusion: The results demonstrated that QDDHG has 18 active compounds and 13 target proteins of QDDHG exerted a crucial role in treatment of DKD. QDDHG affect the multiple biological processes included cellular response to lipid, response to oxidative stress, and various pathways, such as AGE-RAGE, PI3K-Akt, MAPK, TNF, EGFR, STAT3. The results of UHPLC-MS/MS showed that six ingredients predicted by network pharmacology were also verified in experiment. In vivo experiment verified the effects of QDDHG on protecting the renal function mainly through inhibited the expression of EGFR, STAT3 and pERK in the db/db mice.

Network-Based Method to Investigate the Promoted Cell Apoptosis Mechanisms of Oridonin in OSCC through the RNA-Transcriptome.

The morbidity of oral cancer is high in the world. Oridonin is a traditional Chinese medicine that can effectively inhibit oral squamous cell carcinoma (OSCC) growth, but its mechanism remains unclear. Our previous data showed that oridonin inhibited CAL-27 cell proliferation and promoted apoptosis. Herein, we explored the mechanism and target of oridonin in human OSCC through RNA sequencing and integration of multiple bioinformatics analysis strategies. Differences in gene expression can be analyzed with RNA sequencing. Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), gene set enrichment analysis (GSEA), Disease Ontology (DO), and other enrichment analyses were used to evaluate differentially expressed genes (DEGs). Protein-protein interaction (PPI) networks were built via the STRING database. It was found that tumor necrosis factor (TNF) signaling pathway, cytokine-cytokine receptor interaction, and nuclear factor-kappa B (NF-kappaB) signaling pathway were associated with the therapeutic effects of oridonin in OSCC. Three key genes (BIRC3, TNFSF10, and BCL6) were found to associate with cell apoptosis in OSCC cells treated with oridonin. Quantitative PCR assays verified the expression of apoptosis-related DEGs: TNFSF10, BIRC3, AIFM2, BCL6, BCL2L2, and Bax. Western blots were employed for verifying proteins expression associated with DEGs: cleaved caspase 3, Bax, Bcl-w, anti-cIAP2, and anti-TRAIL. In conclusion, our findings reveal the molecular pathways and targets by which oridonin can treat and induce cytotoxic effects in OSCC: by affecting the signaling including TNF, NF-κB, and cytokine-cytokine receptor interaction and by regulating the key gene BIRC3, TNFSF10, and BCL6. It should be noted that further clinical trial validation is very necessary. Combined with current research trends, our existing research may provide innovative research drugs for the treatment of OSCC.

Norcantharidin counteracts acquired everolimus resistance in renal cell carcinoma by dual inhibition of mammalian target of rapamycin complex 1 and complex 2 pathways in Vitro.

Everolimus, an oral mammalian target of rapamycin complex 1 (mTORC1) inhibitor, presents a therapeutic option in metastatic renal cell carcinoma (RCC) patients who were intolerant to, or previously failed, immune- and vascular endothelial growth factor-targeted therapies. However, the onset of drug resistance limits its clinical use. One possible mechanism underpinning the resistance is that inhibiting mTORC1 by everolimus results in mTORC2-dependent activation of v-Akt murine thymoma viral oncogene (AKT) and upregulation of hypoxia-inducible transcription factors (HIF). Norcantharidin (NCTD) is a demethylated derivative of cantharidin with antitumor properties which is an active ingredient of the traditional Chinese medicine Mylabris. In this study, everolimus-resistant RCC cells (786-O-R) obtained by chronic everolimus treatment revealed higher level of HIF2α and over-activated mTORC2 pathway and NCTD inhibits cell proliferation in both everolimus-resistant and -sensitive RCC cells by arresting cell cycle in G0/G1 phase and reducing cell cycle-related proteins of C-Myc and cyclin D. Furthermore, NCTD shows synergistic anticancer effects combined with everolimus in everolimus-resistant 786-O-R cells. Mechanically, NCTD repressed both mTORC1 and mTORC2 signaling pathways as well as downstream molecular signaling pathways, such as p-4EBP1, p-AKT, HIF1α and HIF2α. Our findings provide sound evidence that combination of NCTD and everolimus is a potential therapeutic strategy for treating RCC and overcoming everolimus resistance by dual inhibition of mTORC1 and mTORC2.

Expression changes of hypothalamic Ahi1 in mice brain: implication in sensing insulin signaling.

Growing evidence suggests that the brain, in particular the hypothalamus, directly senses hormones and nutrients to initiate feeding behavior and metabolic responses in the control of energy homeostasis. However, the molecular mechanisms underlying this important process have remained largely unknown. Our study provides the evidence for the role of Abelson helper integration site 1 (Ahi1) protein as a sensor of insulin signaling in the hypothalamus. We found that fasting increased the expression of hypothalamic Ahi1 which was accompanied by lower levels of circulating insulin compared with satiated mice, while re-feeding decreased the expression of hypothalamic Ahi1 which was accompanied by higher levels of circulating insulin. We also found the up-regulated expression of hypothalamic Ahi1 in high-fat induced obese mice, db/db mice, and streptozotocin induced diabetic mice. In addition, we demonstrated that insulin could decrease the expression of Ahi1 in neuroblastoma cell line N18TG2. Taken together, our results indicate that hypothalamic Ahi1 functions as a sensor of insulin signaling.