Integrated metabolomics and network pharmacology to reveal the lipid-lowering mechanisms of Qizha Shuangye granules in hyperlipidemic rats.

BACKGROUND: Qizha Shuangye granules (QSG) comprise six traditional Chinese herbal medicines (TCHMs), which have a long history of treating hyperlipidemia (HLP) in China. This study aimed to evaluate the potential lipid-lowering effects of QSG in an HLP rat model and investigate possible mechanisms. The HLP rat model was induced by a high-fat diet. Lipid-related indicators in serum were detected. Serum and liver metabolites were investigated using a liquid chromatography-mass spectrometry-based metabolomics approach. A herb-compound-target-metabolite (H-C-T-M) network was further constructed to reveal the possible molecular mechanism of QSG to alleviate HLP. RESULTS: The administration of QSG inhibited the HLP-induced changes in total cholesterol, triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and non-esterified fatty acid (NEFA) levels. Additionally, QSG significantly attenuated the liver histopathological changes induced by HLP. Metabolomic analysis showed the serum and liver metabolic disorders presented in HLP rats. QSG can reverse the abnormal metabolism caused by HLP. Through network pharmacology analysis, key proteins such as androgen receptor, 3-hydroxy-3-methylglutaryl-CoA reductase, and peroxisome proliferator-activated receptor-α were screened out, and they were speculated to be possible therapeutic targets for QSG to treat HLP. CONCLUSION: The present study integrated metabolomics and network pharmacology analysis to reveal the efficacy and possible mechanism of QSG in treating HLP, which provides a new reference for the research and development of QSG as a functional food. © 2023 Society of Chemical Industry.

Study on the mechanism of stir-fried Fructus Tribuli in enhancing the essential hypertension treatment by an integrated "spectrum-effect relationship-network pharmacology-metabolomics" strategy.

Essential hypertension (EH) is a leading cause of cardiovascular morbidity and mortality. Fructus Tribuli (FT), as a traditional medicine, has been frequently used for thousands of years. The crude Fructus Tribuli (CFT), decoction pieces being processed to remove impurities, have been listed as an important medicine for the treatment of hypertension in the elderly. According to the theory of traditional Chinese medicine, the CFT can enhance the EH treatment after being stir-fried into stir-fried Fructus Tribuli (SFT). At present, whether the SFT can enhance the EH treatment and its potential pharmacodynamic substances and mechanism are unknown. In this study, an integrated "spectrum-effect relationship-network pharmacology-metabolomics" strategy was used. Using male spontaneously hypertensive rats as an experimental model, we compared the therapeutic effects of CFT and SFT on EH. Subsequently, to define the pharmacodynamic material basis of SFT in enhancing the EH treatment, the steroidal saponins (main active components of FT) were selected for spectrum-effect relationship analysis. Furthermore, we applied the joint pathway analysis of network pharmacology and metabolomics to explore the underlying mechanism of SFT in enhancing the EH treatment. Results showed that SFT was better than CFT in the EH treatment. The steroidal saponins transformed by stir-frying were the potential pharmacodynamic substances that SFT could enhance the EH treatment. And the mechanism of action might be associated with regulating glycerophospholipid metabolism and arachidonic acid metabolism, especially arachidonic acid metabolism. This study provided a scientific basis for the clinical use of SFT as an important medicine for the EH treatment.

Network pharmacology-based study on the mechanism of ShenKang injection in diabetic kidney disease through Keap1/Nrf2/Ho-1 signaling pathway.

OBJECTIVE: To study the effect of ShenKang Injection (SKI) on the kidneys of DKD rats and its effect on oxidative stress mediated by the Keap1/Nrf2/Ho-1 signaling pathway through network pharmacology and in vivo and in vitro experiments. METHODS: SKI drug targets were screened by TCMSP, DKD targets were screened by GenGards, OMIM, Drugbank, TTD, and Disgenet databases, and the two intersected for PPI network analysis and target prediction was performed by GO and KEGG. A total of 40 SD rats were randomly divided into 10 in the control group and 30 in the model group. After the model group was fed 8 W with high-sugar and high-fat diets, a DKD model was constructed by one-time intraperitoneal injection of streptozotocin (35 mg/kg). According to the weight, the model animals were randomly divided into three groups: 8 for model validation group, 8 for Irbesartan (25 mg/kg daily) group, and 8 for SKI group (5 ml/kg). Gavaged deionized water was given to the control group and the model validation group equally. The general conditions of the rats were observed, their body weights measured and their urine volumes recorded for 24 h. After the intervention of 16 W, serum was collected to detect Urea, Scr, blood lipids, and oxidative stress and lipid peroxidation indicators; Transmission electron microscopy, HE and Mallory staining were used to observe the pathological morphology of renal tissue. Immunohistochemistry and RT-PCR were used to detect the expression of Keap1, Nrf2, Ho-1, Gpx4 proteins and mRNA in rat kidney tissues. HK-2 cells were cultured in vitro and divided into: the control group, AGEs (200 µg/ml) group and AGEs + SKI group. The cell activity of the groups was detected using CCK-8 after 48 h of cell culture, and ROS were detected using fluorescent probes. Gpx4 expression was detected by immunofluorescence, while Keap1, Nrf2, Ho-1, and Gpx4 were detected by Western Blot. RESULTS: Network pharmacological analysis predicted that SKI may delay DKD kidney injury by affecting redox-related signaling pathways and mitigating AGEs-induced oxidative stress. In the animal experiment, compared with the model validation group, the general state of rats in the SKI group was improved, and 24-hour urine protein levels were significantly reduced, and the Scr in the serum was reduced. A decreasing trend was seen in Urea, and TC, TG, and LDL levels significantly decreased and the levels of ROS, LPO and MDA were significantly lowered. Pathological staining showed that renal interstitial fibrosis was significantly improved, and electron microscopy showed that foot process effacement was alleviated. Immunohistochemistry and RT-PCR showed decreased expression of Keap1 protein and mRNA in kidney tissues of the SKI group. Additionally, Nrf2, Ho-1, and Gpx4 proteins and mRNA were expressed significantly. In the cell experiment, after 48 h treatment with AGEs, ROS in HK-2 cells increased significantly and cell activity decreased significantly, while cell activity in AGEs + SKI group increased significantly and ROS decreased. The expression of Keap1 protein in HK-2 cells in the AGEs + SKI group decreased, while the expression of Nrf2, Ho-1 and Gpx4 proteins increased significantly. CONCLUSION: SKI can protect kidney function in DKD rats, delay DKD progression, inhibit AGEs-induced oxidative stress damage in HK-2 cells, and the mechanism of SKI to improve DKD may be achieved by activating the Keap1/Nrf2/Ho-1 signal transduction pathway.

Network pharmacology-based approach uncovers the pharmacodynamic components and mechanism of Fructus Tribuli for improving endothelial dysfunction in hypertension.

ETHNOPHARMACOLOGICAL RELEVANCE: Fructus Tribuli (FT), a traditional Chinese medicinal herbal, has been used for the clinical treatment of cardiovascular diseases for many years and affects vascular endothelial dysfunction (ED) in patients with hypertension. AIM OF THE STUDY: This study aimed to demonstrate the pharmacodynamic basis and mechanisms of FT for the treatment of ED. MATERIALS AND METHODS: The present study used ultra-high-performance liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) to analyze and identify the chemical components of FT. The active components in blood were determined after the oral administration of FT by comparative analysis to blank plasma. Then, based on the active components in vivo, network pharmacology was performed to predict the potential targets of FT in treating ED. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were also performed, and component-target-pathway networks were constructed. Interactions between the major active components and main targets were verified by molecular docking. Moreover, spontaneously hypertensive rats (SHRs) were divided into the normal, model, valsartan, low-dose FT, medium-dose FT, and high-dose FT experimental groups. In pharmacodynamic verification studies, treatment effects on blood pressure, serum markers (nitric oxide [NO], endothelin-1 [ET-1,], and angiotensin Ⅱ [Ang Ⅱ)]) of ED, and endothelial morphology of the thoracic aorta were evaluated and compared between groups. Finally, the PI3K/AKT/eNOS pathway was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot of the thoracic aorta of rats in each group to detect the mRNA expression of PI3K, AKT, and eNOS and the protein expression of PI3K, AKT, p-AKT, eNOS, and p-eNOS. RESULTS: A total of 51 chemical components were identified in FT, and 49 active components were identified in rat plasma. Thirteen major active components, 22 main targets, and the PI3K/AKT signaling pathway were screened by network pharmacology. The animal experiment results showed that FT reduced systolic blood pressure and ET-1 and Ang Ⅱ levels and increased NO levels in SHRs to varying degrees. The therapeutic effects were positively correlated with the oral dose of FT. Hematoxylin-eosin (HE) staining confirmed that FT could alleviate the pathological damage of the vascular endothelium. qRT-PCR and Western blot analysis confirmed that up-regulated expression of the PI3K/AKT/eNOS signaling pathway could improve ED. CONCLUSIONS: In this study, the material basis of FT was comprehensively identified, and the protective effect on ED was confirmed. FT had a treatment effect on ED through multi-component, multi-target, and multi-pathways. It also played a role by up-regulating the PI3K/AKT/eNOS signaling pathway.

Polysaccharide of Atractylodes macrocephala Koidz(PAMK) protects against DEHP-induced apoptosis in grass carp hepatocytes.

Di(2-ethylhexyl) phthalate (DEHP) is a new environmental pollutant, which is widely used in plastic additives. DEHP and its metabolites pollute surface water and threaten the survival of fish. In order to investigate the mechanism of DEHP-induced apoptosis on grass carp hepatocytes, we treated grass carp hepatocytes with DEHP, and selected Atractylodes macrocephala Koidz (PAMK) to study its inhibitory effect on DEHP. The results showed that after DEHP exposure, apoptosis related proteins expression were increased significantly, leading to hepatocytes apoptosis. Moreover, AO/EB staining and Hoechst staining also showed that the number of apoptotic cells increased after DEHP exposure. It should be noted that PAMK simultaneous treatment could alleviate apoptosis induced by DEHP. The innovation of this study is that the application of Chinese herbal medicine (PAMK) to antagonize the damage of DEHP in fish was investigated for the first time. This study indicated that traditional Chinese medicine can also be used in fish production to reduce the accumulation of food-derived drugs.

Paiteling induces apoptosis of cervical cancer cells by down-regulation of the E6/E7-Pi3k/Akt pathway: A network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Human papillomavirus (HPV) infection is considered to be the main pathogen causing intraepithelial neoplasia. Paiteling (PTL) has been used to treat intraepithelial neoplasia caused by human papillomavirus (HPV) infection for more than 20 years in China, but its specific mechanism of action is not very clear, and further research is still needed. OBJECTIVE: This study designed a comprehensive strategy to study the pharmacological mechanism of paiteling in regulating cervical cancer cell apoptosis by integrating LC-MS/MS, network pharmacology and pharmacological experiments. METHODS: We used liquid chromatography-tandem mass spectrometry to detect the active substances in PTL and performed protein-protein interaction analysis on the intersection of the targets of these key compounds and the targets of intraepithelial neoplasia. Additionally, by using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes (KEGG), the potential pathway of PTL against HPV-induced intraepithelial neoplasia was predicted. Finally, we used HeLa and Ect1/E6E7 cells for experimental verification. RESULTS: The protein-protein interaction network predicted that AKT1, TP53, MYC, STAT3, MTOR, and MAPK were pivotal targets for PTL to inhibit epithelial neoplasia. KEGG enrichment analysis showed that the Pi3k/Akt pathway and HPV infection had scientific significance. Compared to the control group, after PTL diluent stimulated HeLa and Ect1/E6E7 cells for 24 h, cell viability, migration, and invasion capabilities were significantly reduced, and cell apoptosis was significantly increased, conforming to a dose-effect relationship and time-effect relationship. PCR, cellular immunohistochemistry, and western blot experiments showed that PTL reduced the expression of E6, Pi3k, E7, Akt, Bcl-xl, while increasing the expression of Bad in HeLa and Ect1/E6E7 cells. CONCLUSION: PTL can induce cervical cancer cell apoptosis by inhibiting the E6/E7-Pi3k/Akt signaling pathway. It may provide an effective alternative strategy of traditional Chinese medicine for the treatment of epithelial neoplasia caused by HPV infection.

Probiotics ameliorate polyethylene microplastics-induced liver injury by inhibition of oxidative stress in Nile tilapia (Oreochromis niloticus).

Microplastic particles (MPs) are environmental pollutants that can cause varying levels of aquatic toxicity. Probiotics have been shown to reduce the negative effects of toxic substances. However, the protective effect of probiotics against the adverse effects of MPs has yet to be reported. The current study sought to determine the effects of the commercial probiotic AquaStar® Growout on polystyrene (PS)-MPs-mediated hepatic oxidative stress in Nile tilapia (Oreochromis niloticus). Fishes were assigned into four groups: the first group was the control, the second group was exposed to 1 mg/L of 0.5 µm PS-MPs, and the third and fourth groups were exposed to 1 mg/L of 0.5 µm PS-MPs and pre-fed with probiotics at levels of 3 g/kg and 6 g/kg diet, respectively. At the end of the experiment, probiotics administration reversed liver damage caused by the PS-MPs, reducing serum levels of malondialdehyde, aspartate aminotransferase, and alanine aminotransferase, and increasing the total antioxidant capacity. Furthermore, probiotics alleviated PS-MPs-induced oxidative stress by restoring antioxidant enzyme activities (superoxide dismutase, catalase, glutathione S-transferase, and glutathione peroxidase) and reducing oxidized glutathione and enhancing the redox state. Besides, probiotics supplementation decreased the transcriptional level of C-reactive protein and tumor necrosis factor-α following PS-MPs exposure. Furthermore, probiotics counteracted PS-MPs-associated reactive oxygen species production and mitogen-activated protein kinases (MAPKs) phosphorylation status. These findings suggested that probiotics could decrease liver damage caused by PS-MPs through their antioxidant properties and modulation of MAPK signaling pathways.

Terrestrosin D, a spirostanol saponin from Tribulus terrestris L. with potential hepatorenal toxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Fructus Tribuli (FT) has been commonly used as a traditional medicine for thousands of years. With the diverse uses of FT, more attention has been paid to its hepatorenal toxicity. However, the compounds causing the hepatorenal toxicity of FT remain undetermined. Terrestrosin D (TED), a major spirostanol saponin isolated from FT, may exert hepatorenal toxicity. AIM OF THE STUDY: This study aimed to evaluate the potential hepatorenal toxicity of TED, and preliminarily explore the possible mechanism of TED-induced hepatorenal toxicity. MATERIALS AND METHODS: Cytotoxicity assays, a repeated-dose 28-day in-vivo study, a toxicokinetic study, and a tissue distribution study were used to evaluate the potential hepatorenal toxicity of TED. Furthermore, network pharmacology was applied to preliminarily explore the possible mechanism of TED-induced hepatorenal toxicity. RESULTS: Both the in vitro and in vivo studies showed that the spirostanol saponin TED had potential hepatorenal toxicity. Nonetheless, hepatorenal toxicity induced by oral treatment with TED at a dosage range of 5 - 15 mg/kg daily for 28 consecutive days to Sprague-Dawley (SD) rats was reversible after 14 days of TED withdrawal. The toxicokinetic study demonstrated that the systematic exposure of SD rats to TED had an accumulation phenomenon and a dose-dependent trend after a 28-day repeated-dose oral administration. The tissue distribution study revealed that TED had a targeted distribution in the liver and kidneys accompanied by a phenomenon of accumulation in SD rats. Network pharmacology combined with molecular docking methods was used to screen for the key targets (HSP90AA1, CNR1, and DRD2) and the key pathways of TED-induced hepatorenal toxicity. CONCLUSIONS: The spirostanol saponin TED, a major spirostanol saponin isolated from FT, had potential hepatorenal toxicity.

Immunomodulatory effects of flazin from Crassostrea sikamea on splenic lymphocytes of Sprague-Dawley rats.

Crassostrea sikamea (C.sikamea) is an important edible and medicinal seafood in China. In the present study, a compound named flazin was separated and identified from the ethyl acetate extract of C.sikamea (EAECs) for the first time. In addition, the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra zolium (MTS) assay revealed that EAECs and flazin inhibited the transformation of splenic lymphocytes in vitro. Moreover, flazin (20 µg·mL-1) altered the populations of splenic lymphocyte subtypes. Real-time quantitative PCR (RT-qPCR) analysis and enzyme-linked immunosorbent assay (ELISA) showed that flazin suppressed the mRNA expression and secretion of TNF-α and IL-2, and reversed Concanavalin A (ConA)-induced mRNA up-regulation and protein secretion of TNF-α and IL-2. Western blot results showed that flazin reversed ConA-induced increases in p-ERK1/2 and p-p38 in splenocytes. In conclusion, flazin exhibits effective immunomodulatory function and may be useful for treating immune-related disorders, which indicates the application potential of C.sikamea as a functional food or immunomodulator.

Pharmacokinetic comparisons of six steroid saponins in rat plasma following oral administration of crude and stir-fried Fructus Tribuli extracts by UHPLC-MS/MS.

Modern pharmacological studies have shown that Fructus Tribuli can improve sexual function and treat cardiovascular diseases. In this study, we focused on comparing the pharmacokinetics of crude Fructus Tribuli (CFT) and stir-fried Fructus Tribuli (SFT) to further clarify the changes in chemical composition in vivo. The quantitation of six analytes was performed in a triple quadrupole mass spectrometer using the multiple reaction monitoring mode. Separation was performed on a Halo® C18 column using 0.05% formic acid and 5 µmol/L sodium formate in water, and 0.05% formic acid and 5 µmol/L sodium formate in acetonitrile as the mobile phase. The selectivity, precision, accuracy, extraction recovery, matrix effect and stability of the method were fully validated. Compared with the crude group, the parameters Cmax and AUC0-t of terrestroside B and terrestrosin K increased significantly (P < 0.05), but the Cmax and AUC0-t of polianthoside D, terrestrinin D, tribuluside A and terrestrosin D were decreased, terrestrosin D being especially decreased (P < 0.05), after oral administration of SFT extract. These results showed that the developed method was suitable for pharmacokinetic analysis of the six steroid saponins of CFT and SFT in rat plasma, and can be used to facilitate future clinical studies.

Study on the Potential Mechanism of Fructus Tribuli in the Treatment of Hypertensive Vascular Remodeling Based on Network Pharmacology and Molecular Docking.

BACKGROUND: Hypertensive vascular remodeling (HVR) is the pathophysiological basis of hypertension, which is also an important cause of vascular disease and target organ damage. Treatment with Fructus Tribuli (FT), a traditional Chinese medicine, has a positive effect on HVR. However, the pharmacological mechanisms of FT are still unclear. Therefore, this study aimed to reveal the potential mechanisms involved in the effects of FT on HVR based on network pharmacology and molecular docking. MATERIALS AND METHODS: We selected the active compounds and targets of FT according to the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and the Swiss Target Prediction database, and the targets of HVR were collected from the Online Mendelian Inheritance in Man (OMIM), GeneCards, and DrugBank databases. The protein-protein interaction network (PPI) was established using the STRING database. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses and network analysis were performed to further explore the potential mechanisms. Finally, molecular docking methods were used to evaluate the affinity between the active compounds and the main target. RESULTS: Seventeen active compounds of FT and 164 potential targets for the treatment of HVR were identified. Component-target and PPI networks were constructed, and 12 main active components and 33 main targets were identified by analyzing the topological parameters. Additionally, GO analysis indicated that the potential targets were enriched in 483 biological processes, 52 cellular components, and 110 molecular functions. KEGG analysis revealed that the potential targets were correlated with 122 pathways, such as the HIF-1 signaling pathway, ErbB signaling pathway, and VEGF signaling pathway. Finally, molecular docking showed that the 12 main active components had a good affinity for the top five main targets. CONCLUSION: This study demonstrated the multiple compounds, targets, and pathway characteristics of FT in the treatment of HVR. The network pharmacology method provided a novel research approach to analyze potential mechanisms.

Increased thalamic volume and decreased thalamo-precuneus functional connectivity are associated with smoking relapse.

The thalamus, with the highest density of nicotinic acetylcholine receptor (nAChR) in the brain, plays a central role in thalamo-cortical circuits that are implicated in nicotine addiction. However, little is known about whether the thalamo-cortical circuits are potentially predictive of smoking relapse. In the current study, a total of 125 participants (84 treatment-seeking male smokers and 41 age-matched male nonsmokers) were recruited. Structural and functional magnetic resonance images (MRI) were acquired from all participants. After a 12-week smoking cessation treatment with varenicline, the smokers were then divided into relapsers (n = 54) and nonrelapsers (n = 30). Then, we compared thalamic volume and seed-based thalamo-cortical resting state functional connectivity (rsFC) prior to the cessation treatment among relapsers, nonrelapsers and nonsmokers to investigate the associations between thalamic structure/function and smoking relapse. Increased thalamic volume was detected in smokers relative to nonsmokers, and in relapsers relative to nonrelapsers, especially on the left side. Moreover, decreased left thalamo-precuneus rsFC was detected in relapsers relative to nonrelapsers. Additionally, a logistic regression analysis showed that the thalamic volume and thalamo-precuneus rsFC predicted smoking relapse with an accuracy of 75.7%. These novel findings indicate that increased thalamic volume and decreased thalamo-precuneus rsFC are associated with smoking relapse, and these thalamic measures may be used to predict treatment efficacy of nicotine addiction and serve as a potential biomarker for personalized medicine.

Analysis of variations in the contents of steroidal saponins in Fructus Tribuli during stir-frying treatment.

Just as natural saponins transform into aglycones, secondary glycosides and their derivatives using biotransformation technology, steroidal saponins may also undergo similar transformation after stir-frying. The purpose of this study was to elucidate the variations and the reasons for these variations in the contents of steroidal saponins in Fructus Tribuli (FT) during a stir-frying treatment. Stir-fried FT was processed in different time-temperature conditions. An UHPLC-MS/MS method was established and fully validated for quantitative analysis. In addition, the simulation processing products of tribuluside A, terrestroside B, terrestrosin K, terrestrosin D and 25R-tribulosin were determined by qualitative analysis using UHPLC-Q-TOF-MS. The established UHPLC-MS/MS method provides a rapid, flexible, and reliable method for the quality assessment of FT. The present study revealed that furostanol saponins with a C22-OH group could transform into corresponding furostanol saponins with a C-20-C-22 double bond (FSDB) via dehydroxylation. Additionally, FSDB could be successively converted into its secondary glycosides via a deglycosylation reaction. The transformation of spirostanol saponins into corresponding aglycones via deglycosylation led to a decrease in spirostanol saponins and an increase in aglycones. The results of this research provided scientific evidence of variation and structural transformation among steroidal saponins. These findings might be helpful for elucidating the processing mechanism of FT.

Solvent-Induced Protein Precipitation for Drug Target Discovery on the Proteomic Scale.

High-throughput drug discovery is highly dependent on the targets available to accelerate the process of candidates screening. Traditional chemical proteomics approaches for the screening of drug targets usually require the immobilization/modification of the drug molecules to pull down the interacting proteins. Recently, energetics-based proteomics methods provide an alternative way to study drug-protein interaction by using complex cell lysate directly without any modification of the drugs. In this study, we developed a novel energetics-based proteomics strategy, the solvent-induced protein precipitation (SIP) approach, to profile the interaction of drugs with their target proteins by using quantitative proteomics. The method is easy to use for any laboratory with the common chemical reagents of acetone, ethanol, and acetic acid. The SIP approach was able to identify the well-known protein targets of methotrexate, SNS-032, and a pan-kinase inhibitor of staurosporine in cell lysate. We further applied this approach to discover the off-targets of geldanamycin. Three known protein targets of the HSP90 family were successfully identified, and several potential off-targets including NADH dehydrogenase subunits NDUFV1 and NDUFAB1 were identified for the first time, and the NDUFV1 was validated by using Western blotting. In addition, this approach was capable of evaluating the affinity of the drug-target interaction. The data collectively proved that our approach provides a powerful platform for drug target discovery.

Convallatoxin induces HaCaT cell necroptosis and ameliorates skin lesions in psoriasis-like mouse models.

Psoriasis is considered an immune-mediated inflammatory skin disorder that affects the quality of life of nearly four percent of the world population. Considering the side effects of existing therapeutic drugs and the urgent need for new drug development, we screened more than 250 traditional Chinese medicine compounds to identify drugs that significantly reduced the viability of human HaCaT keratinocytes, a psoriasis-related model cell line. Convallatoxin (CNT) was found to be a highly effective inhibitor of HaCaT cell viability. Subsequent mechanistic studies revealed that CNT induced HaCaT cell death by necroptosis rather than by apoptosis. CNT destroyed the membrane integrity of HaCaT cells, as detected by nuclear propidium iodide (PI) staining and lactate dehydrogenase (LDH) release. Additionally, the intercellular levels of adenosine triphosphate (ATP) were lower in HaCaT cells treated with CNT than in control HaCaT cells, and typical necroptosis-associated characteristics were observed by electron microscopy in cells treated with CNT. Furthermore, compared with control HaCaT cells, CNT-treated HaCaT cells produced more reactive oxygen species (ROS), but this effect was inhibited by the antioxidants N-acetyl-cysteine (NAC), diphenyleneiodonium chloride (DPI), and apocynin and the necroptosis inhibitor Nec-1. In addition, antioxidant treatment attenuated necroptotic cell death, suggesting that CNT-induced HaCaT necroptosis is mediated by oxidative stress. More importantly, CNT ameliorated skin lesions and inflammation in imiquimod (IMQ)- and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced psoriasis-like mouse models. In conclusion, our results demonstrate that CNT is cytotoxic against HaCaT cells in vitro and exerts antipsoriatic activities in two mouse models of psoriasis in vivo, making CNT a potential promising candidate drug for future research.

An Improved Protocol for the Virtual Screening Discovery of Novel Histone Deacetylase Inhibitors.

Histone deacetylases (HDACs) are enzymes that play a key role in structural modification and gene expression. The overexpression of HDAC is associated with cancer, and thus inhibiting the enzyme could be an efficient cancer therapy. To discover new HDAC inhibitors (HDACis), we proposed an improved protocol combining a hierarchical pharmacophore search, molecular docking, and molecular dynamic simulations. The test results showed that the improved screening protocol effectively reduced the false-positive rates of drug-like chemicals. Based on the protocol, we obtained 16 hit compounds as potential HDACis from the Life Chemicals database. Enzyme inhibition experiments showed that two of the hit chemical compounds had HDAC-inhibitory effects. In vitro assays showed that Z165155756 could selectively inhibit the proliferation of cancer cells and specifically promoted apoptosis and induced G1/S phase arrest in A2780 cells. It may have potential therapeutic effects in ovarian cancer and is worthy of further investigation.

Functionality-Independent DNA Encoding of Complex Natural Products.

DNA encoded chemical libraries (DELs) link the powers of genetics and chemical synthesis via combinatorial optimization. Through combinatorial chemistry, DELs can grow to the unprecedented size of billions to trillions. To take full advantage of the DEL approach, linking the power of genetics directly to chemical structures would offer even greater diversity in a finite chemical world. Natural products have evolved an incredible structural diversity along with their biological evolution. Herein, we used traditional Chinese medicines (TCMs) as examples in a late-stage modification toolbox approach to annotate these complex organic compounds with amplifiable DNA barcodes, which could be easily incorporated into a DEL. The method of end-products labeling also generates a cluster of isomers with a single DNA tag at different sites. These isomers provide an additional spatial diversity for multiple accessible pockets of targeted proteins. Notably, a novel PARP1 inhibitor from TCM has been identified from the natural products enriched DEL (nDEL).

Alantolactone suppresses APOC3 expression and alters lipid homeostasis in L02 liver cells.

A high level of APOC3 expression is an independent risk factor for some lipid metabolism-related diseases, such as cardiovascular disease (CVD), nonalcoholic fatty liver disease (NAFLD) and atherosclerosis (AS). This suggests that down-regulating APOC3 expression is a potential way of regulating lipid levels. In this study, we used luciferase reporter screening to identify a natural compound, alantolactone (ALA), that can inhibit the promoter activity of APOC3. ALA decreased APOC3 expression at both mRNA and protein levels. Then we pretreated L02 liver cells with oxLDL to investigate the function of ALA in lipid homeostasis. Intriguingly, ALA attenuated oxLDL-induced foam cell formation by reducing total cholesterol (TC) and triglyceride (TG) contents. Furthermore, these results could be reversed by overexpressing APOC3 protein. ALA inhibited tyrosine phosphorylation (Tyr705pho) of STAT3 to down-regulate APOC3 expression. Intriguingly, overexpression of a wild-type STAT3 or a constitutively active form of STAT3 (STAT3-C) up-regulated APOC3 expression and partly reversed the effect of ALA in oxLDL-induced L02 cells. Overexpression of wild-type STAT3 also increased TC but not TG contents in L02 cells. However, overexpression of STAT3-C significantly increased TC and TG contents, and the effect of ALA was partly attenuated by STAT3-C, although this was not statistically significant. These results suggest that ALA attenuates lipid accumulation through down-regulation of APOC3 expression, at least in part by inhibiting STAT3 signaling.

[Studies on quality control methods of nuxvomica].

Two quality control methods of nuxvomica were established for mutual. By selecting the appropriate measuring wavelength or wavelength range, the contents of strychnine and brucine in nuxvomica were determined without any preliminary separation by a new rapid spectrophotometry and a multi-wavelength linear regression spectrophotometry and with a computer program. The linear range of strychnine measured was 8.0-30.0 microg x mL(-1) (r = 0.999 9); The linear range of brucine measured was 7.0-31.2 microg x mL(-1) (r = 0.999 4). The average recoveries and relative standard deviations of strychnine and brucine were 98.18%-99.82%, 0.56%-1.54% and 100.5%-100.6%, 0.57%-0.62%, respectively. The methods are simple, rapid and reproducible, the interference of two components with each other may be eliminated and the methods are appropriate for quality control of nuxvomica.