Novel ADAM-17 inhibitor ZLDI-8 inhibits the proliferation and metastasis of chemo-resistant non-small-cell lung cancer by reversing Notch and epithelial mesenchymal transition in vitro and in vivo.

Acquired drug-resistant non-small cell lung cancer (NSCLC) has strong proliferation ability and is prone to epithelial-mesenchymal transition (EMT) and subsequent metastasis. Notch pathway mediates cell survival and EMT and is involved in the induction of multidrug resistance (MDR). ZLDI-8 is an inhibitor of Notch activating/cleaving enzyme ADAM-17 we found before. However, the effects of ZLDI-8 on resistant NSCLC was unclear. Here, we demonstrated for the first time that ZLDI-8 could induce apoptosis in lung cancer, especially in chemotherapy-resistant non-small cell lung cancer cells, and also inhibit migration, invasion and EMT phenotype of drug-resistant lung cancer. ZLDI-8 inhibits the Notch signaling pathway, thereby regulating the expression of survival/apoptosis and EMT-related proteins. Moreover, ZLDI-8 suppresses multidrug-resistant lung cancer xenograft growth in vivo and blocks metastasis in a tail vein injection mice model. Therefore, ZLDI-8 is expected to be an effective agent in the treatment of drug-resistant lung cancer.

Network pharmacology-based research uncovers cold resistance and thermogenesis mechanism of Cinnamomum cassia.

BACKGROUND: Cinnamomum cassia (L.) J.Presl (Cinnamon) was known as a kind of hot herb, improved circulation and warmed the body. However, the active components and mechanisms of dispelling cold remain unknown. METHODS: The effects of several Chinses herbs on thermogenesis were evaluated on body temperature and activation of brown adipose tissue. After confirming the effect, the components of cinnamon were identified using HPLC-Q-TOF/MS and screened with databases. The targets of components were obtained with TCMSP, SymMap, Swiss and STITCH databases. Thermogenesis genes were predicted with DisGeNET and GeneCards databases. The protein-protein interaction network was constructed with Cytoscape 3.7.1 software. GO enrichment analysis was accomplished with STRING databases. KEGG pathway analysis was established with Omicshare tools. The top 20 targets for four compounds were obtained according to the number of edges of PPI network. In addition, the network results were verified with experimental research for the effects of extracts and major compounds. RESULTS: Cinnamon extract significantly upregulated the body temperature during cold exposure.121 components were identified in HPLC-Q-TOF/MS. Among them, 60 compounds were included in the databases. 116 targets were obtained for the compounds, and 41 genes were related to thermogenesis. The network results revealed that 27 active ingredients and 39 target genes. Through the KEGG analysis, the top 3 pathways were PPAR signaling pathway, AMPK signaling pathway, thermogenesis pathway. The thermogenic protein PPARγ, UCP1 and PGC1-α was included in the critical targets of four major compounds. The three major compounds increased the lipid consumption and activated the brown adipocyte. They also upregulated the expression of UCP1, PGC1-α and pHSL, especially 2-methoxycinnamaldehyde was confirmed the effect for the first time. Furthermore, cinnamaldehyde and cinnamon extract activated the expression of TRPA1 on DRG cells. CONCLUSION: The mechanisms of cinnamon on cold resistance were investigated with network pharmacology and experiment validation. This work provided research direction to support the traditional applications of thermogenesis.

Cinnamomum cassia extract promotes thermogenesis during exposure to cold via activation of brown adipose tissue.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinnamomum cassia (L.) J.Presl (Lauraceae), a widely used traditional Chinese medicine, is well known to exert hot property. It is recorded as dispelling cold drug in ancient Chinese monographs, such as Synopsis of golden chamber published in Han dynasty. According to Chinese Pharmacopoeia (2015), Cinnamomum cassia (L.) J.Presl (Cinnamon) has the functions of dispersing cold, relieving pain, warming meridians and promoting blood circulation. AIM OF THE STUDY: The aim of this study is to evaluate the effect of Cinnamon extract (CE) on cold endurance and the mechanism of thermogenesis activity. MATERIALS AND METHODS: The improving effect of hypothermia were evaluated with body temperature by infrared camera and multi-thermo thermometer. In vivo, the thermogenic effect was observed with energy metabolism and substrate utilization. The activation of brown adipose tissue (BAT) was evaluated with the histomorphology and expression of thermogenic protein. In vitro, the uncoupling effect on mitochondrial was evaluated with Seahorse and fluorescent staining. The mechanism of thermogenesis was explored in brown adipocyte. RESULTS: The body temperature and energy expenditure were significantly increased by CE administration in cold environment. In morphology, lipid droplets were reduced and the number of mitochondrial was increased. CE significantly increased the non-shivering thermogenesis via upregulating the expression of thermogenic protein. In vitro, the uncoupling effect was obviously along with the decreased mitochondrial membrane potential and ATP production. It was confirmed that the thermogenesis effect was induced via lipolysis and energy metabolism. In addition, CE also alleviated myocardium injury in the morphology in cold environment. Moreover, the major constituent was identified as (1) coumarin, (2) cinnamic acid, (3) cinnamaldehyde and (4) 2-methoxy cinnamaldehyde. CONCLUSIONS: The mechanism of improving cold tolerance was related to lipolysis and activation of BAT. Meanwhile, we provided a kind of potential prevention methods for cold injury.

Novel ADAM-17 inhibitor ZLDI-8 inhibits the metastasis of hepatocellular carcinoma by reversing epithelial-mesenchymal transition in vitro and in vivo.

AIMS: Epithelial-mesenchymal transition (EMT) is one of the important regulators of metastasis in advanced hepatocellular carcinoma (HCC). Blocking the Notch signaling pathway and then reversing the EMT process is a hot spot in clinical tumor research. Here, we aimed to investigate the effect and underlying mechanisms of ADAM-17 (a key cleavage enzyme of Notch pathway) inhibitor ZLDI-8 we found before on the metastasis of hepatocellular carcinoma in vitro and in vivo. MAIN METHODS: The cell viability of HCC cells was evaluated by MTT and colony formation assays. Migration and invasion were assessed respectively with wound healing and transwell assays. The expression and location of proteins were detected by western blot and immunofluorescence, respectively. The effects of ZLDI-8 on metastasis of liver cancer in vivo were investigated in a tail vein injection model. KEY FINDINGS: In the present work, ZLDI-8 significantly inhibited proliferation, migration, invasion and EMT phenotype of highly aggressive MHCC97-H and LM3 cells. Moreover, ZLDI-8 could inhibit the migration and invasion of HepG2 and Bel7402 cells induced by TGF-ß1. ZLDI-8 suppressed the protein expression of interstitial markers and increased that of epithelial markers. Meanwhile, ZLDI-8 decreased the expression of proteins in the Notch signaling pathway. Finally, ZLDI-8 blocks metastasis in the lung metastasis model in vivo. SIGNIFICANCE: ZLDI-8 suppressed the metastasis of hepatocellular carcinoma, which was associated with reversing the EMT process and regulating Notch signaling pathway. The study laid the foundation for the discovery of drugs that reverse EMT to inhibit advanced HCC metastasis.

Jiang Gui Fang activated interscapular brown adipose tissue and induced epididymal white adipose tissue browning through the PPARγ/SIRT1-PGC1α pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Gui Zhi Tang, a well-known Chinese herbal formula recorded in the Eastern Han Dynasty, has been widely used to treat exogenous cold for thousands of years. Recent studies have shown that Gui Zhi Tang has the effect of regulating the body temperature. Because of its effect on heat production, protecting vital organs of the body and avoiding damage from the cold environment, Jiang Gui Fang (JG) was obtained from the Department of Traditional Chinese Medicine at the General Hospital of Northern Theatre Command where it has been used clinically for many years and has exhibited favourable efficacy. Based on research on Gui Zhi Tang, the principles of traditional Chinese medicine and survey of a large number of studies, this empirical formula was developed. The composition of JG included Dried ginger, Cassia twig, and Liquorice in Gui Zhi Tang, which play a major role in the treatment of exogenous cold, and combined these components with other Chinese medicines, such as Pueraria, Spatholobus, Acanthopanacis cortex, Evodiae fructus, and Codonopsis pilosula. AIM OF THE STUDY: To promote the core body temperature and prevent invasion of the major organs from the cold environment, we studied the effect of JG on the core body temperature of mice and then explored its regulation of interscapular brown adipose tissue (iBAT) and epididymal white adipose tissue (eWAT) and the possible mechanism. Finally, we determined the phytochemical composition of JG that plays a role in heat production. MATERIALS AND METHODS: In vivo study, we performed a 4-week treatment of JG in acute cold environment at -20 °C and chronic cold exposure at 4 °C. The core temperature, adipose tissue weight, serum parameters, and morphological observation of adipocytes, liver and kidney were measured. Then we investigated the expression levels of adipogenic factors, thermogenic factors and lipoprotein. In vitro, we determined the lipid droplet content, ATP content, and the maximum oxygen consumption of mitochondria. RESULTS: JG treatment promoted core temperature, inhibited eWAT weight, protected liver, and reduced glucose and lipids in Kunming (KM) mice. JG also increased the expression of BAT-associated thermogenic factors, including uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α). The levels of the lipogenic factor peroxisome proliferate-activator receptor gamma (PPARγ) and the lipolytic protein hormone-sensitive triglyceride lipase (HSL) in eWAT were elevated. The results of H&E and immunohistochemistry showed that JG significantly reduced the size of iBAT and eWAT and increased the content of UCP1. In vitro, JG reduced the content of lipid droplets and ATP in brown fat cells. The maximum oxygen consumption capacity of mitochondria and the expression levels of UCP1, PGC1α and silent mating type information regulation 2 homologue 1 (SIRT1) were enhanced after JG treatment. CONCLUSIONS: In vivo and in vitro studies, the results demonstrated that JG obviously increased the core temperature of mice by activating iBAT and inducing eWAT browning, which proved the mechanism is closely related to the PPARγ/SIRT1- PGC1α pathway. In this paper, we will provide a reference for further study of iBAT activation and eWAT browning.

Notopterygium incisum extract (NRE) rescues cognitive deficits in APP/PS1 Alzhneimer's disease mice by attenuating amyloid-beta, tau, and neuroinflammation pathology.

ETHNOPHARMACOLOGICAL RELEVANCE: Alzheimer's disease (AD) is a frequently occurring disease of the elderly, and "deficiency" is the root of AD. Most famous experts of traditional Chinese medicine believe that the disease is based on deficiency, and the deficiency of kidney essence is the basis. Notopterygium incisum (Qiang huo) is beneficial to bladder, liver, and kidneys. It is used to treat liver and kidney deficiency, language difficulties, and mental coma. Qiang huo yu feng tang has been used to treat liver and kidney deficiency, unclear language and mental paralysis in many traditional Chinese medicine books and records. In modern times, it has been used to treat AD and exhibited favourable efficacy. AIM OF THE STUDY: This study attempts to investigate the effects of furocoumarins from Notopterygium incisum (NRE) on the Aß cascade, tau pathology and inflammatory pathology of AD. MATERIALS AND METHODS: In this study, we reported a detailed protocol for stabilizing HEK APPswe293T cells with lentivirus for the first time. This cell line can secrete high concentration of Aß. In addition, we treated N2a cells with AKT/PKC specific inhibitors (wortmannin/GF-109203X) and established a tau pathological cell model (AKT/PKC N2a) by activating GSK3ß and triggering hyperphosphorylation of tau. The Aß levels and the expression of phosphorylated tau were detected by ELISA and Western blot. The cognitive ability of NRE on APP/PS1 mice was detected using a Morris water maze (MWM) assay and Aß contents were also evaluated. RESULTS: In HEK APPswe293T cells, NRE (10, 20, 40 µg/mL) significantly inhibited the secretion and production of Aß in dose dependent manner. In addition, NRE also suppressed the expression of phosphorylated tau in wortmannin/GF-109203X treated N2a cells. Furthermore, NRE ameliorated the cognitive impairment of APP/PS1 mice, and the contents of Aß, IL-1ß and TNF-α were significantly depressed in hippocampus and cortex. CONCLUSION: In conclusion, our results demonstrated that NRE has a potential anti-AD effect via the inhibition of the Aß cascade, tau pathology and neuroinflammation in vitro and in vivo.

Identification of New Potent Human Uncoupling Protein 1 (UCP1) Agonists Using Virtual Screening and in vitro Approaches.

Recent studies suggested that activation of Uncoupling Protein 1 (UCP1) has become an appealing therapeutic strategy against obesity and diabetes. In our research, the 3D structure of UCP1 was constructed through homology modelling, refined through molecular dynamics simulation, and evaluated by Ramachandran plot, the molecular docking of UCP1 activators brought about the proposal of an interaction mode inside the UCP1 active site. Remarkably, Reside Lys126 formed hydrogen bond; residues Pro121, Val125, Tyr146, Tyr149 and Arg150 formed hydrophobic interaction, which are key amino acids within UCP1 site. Then a pharmacophore model was generated consisting of three hydrophobic groups, a negative center and an additional hydrophobic group. Pharmacophore-based virutal screening of Specs database yield 5 hits. In vitro assay indicated ZINC 04660290 significantly increased the protein expression of UCP1 and decreased the fat droplet in a dose-dependent manner. Besides, pharmacokinetic properties were predicted for those five compounds through ADME/T prediction. All of these will guide us to design new UCP1 activators for the treatment of obesity and diabetes.

ZW290 Increases Cold Tolerance by Inducing Thermogenesis via the Upregulation of Uncoupling Protein 1 in Brown Adipose Tissue In Vitro and In Vivo.

To provide molecular evidence on the thermogenic mechanism of primary brown adipocytes, western blot analysis was used to detect brown adipose tissue (BAT)-specific gene expressions. BAT protects the mammals from hypothermia injury with a large amount of mitochondria and high expression of uncoupling Protein 1 (UCP1), which is the vital protein to determine the heat production in BAT. In our previous study, the compound ZW290 (the structure shown in Fig. 1) was obtained by molecular docking with a UCP1 inducer. In the present study, ZW290 not only significantly upregulated the expression of UCP1 protein (p < 0.01) and its related signaling pathway in the primary brown adipocytes, but also remarkably decreased the mitochondrial membrane potential and the concentration of adenosine triphosphate (ATP) (p < 0.01). Kunming (KM) mice were kept under acute cold exposure (-20°C) to evaluate the preventive and protective effects of ZW290 on cold injury, and revealed its regulating mechanism in vitro. The rectal and body temperatures of ZW290-treated mice were significantly higher than those of the control (or model) group both at room temperature and at -20°C (p < 0.001). Hematoxylin-eosin (HE) staining and immunohistochemistry indicated that ZW290 notably decreased the size of lipid droplets in BAT and increased the content of mitochondria and the expression of UCP1 in BAT and white adipose tissue (WAT). Furthermore, the survival rate showed that ZW290 could prolong the overall survival of mice. Therefore, we obtained the conclusion that ZW290 might transform energy into heat by inhibiting ATP synthesis and increasing the expression of UCP1. Additionally, ZW290 may enhance cold tolerance by increasing heat production through increasing the content of mitochondria and the expression of UCP1 in BAT and WAT.

In Silico Analyses for Key Genes and Molecular Genetic Mechanism in Epilepsy and Alzheimer's Disease.

BACKGROUND: Epilepsy and Alzheimer's disease are common neuropathies with a complex pathogenesis. Both of them have some correlations in etiology, pathogenesis, pathological changes, clinical manifestations and treatment. OBJECTIVE: This study investigated the key genes and molecular genetic mechanism in epilepsy and Alzheimer's disease by bioinformatics analysis. METHOD: Two gene expression profiles were used to screen differentially expressed genes by GEO2R tool. The Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Then the protein-protein interaction (PPI) network was constructed by Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape software which can be used to analyze modules with MCODE. RESULTS: A total of 199 differentially expressed genes (DEGs) in the two groups. According to GO_BP analysis and KEGG pathway enrichment by DAVID, we found DEGs referring to several pathways significantly down-regulated in endocytosis, such as endocytosis, synaptic vesicle cycle, lysosome, cAMP signaling pathway, circadian entrainment, LTP, glutamatergic synapse and GABAergic synapse pathway. The regulator genes of the upstream pathway of circadian rhythms were obviously downgraded. CONCLUSION: Our research demonstrated that the regulatory genes of the upstream pathway of circadian rhythms were obviously downgraded. These biological pathways and DEGs or hub genes may contribute to revealing the molecular relationship between Alzheimer's disease and epilepsy.