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Ischaemia-reperfusion (I/R) injury is one of the leading causes of acute kidney injury (AKI). Its pathologic mechanism is quite complex, involving oxidative stress, inflammatory response, autophagy, and apoptosis. Fibroblast growth factor 10 (FGF10) and 5-hydroxydecanoate (5-HD) play essential roles in kidney injury. Rats were divided into four groups: (i) sham group, sham-operated animals with an unconstructed renal artery; (ii) I/R group, kidneys were subjected to 50 min of ischaemia followed by reperfusion for 2 days; (iii) I/R + FGF10 group, animals treated with 0.5 mg/kg FGF10 (i.p.) 1 h before ischaemia; and (iv) 5-HD group, animals treated with 5 mg/kg 5-HD (i.m.) 30 min before FGF10 treatment. Renal injury, apoptosis damage, mitochondrial oxidative damage, mitochondrial membrane potential (MMP), and expression of the ATP-sensitive K+ (KATP) channel subunit Kir6.2 were evaluated. FGF10 treatment significantly alleviated I/R-induced elevation in the serum creatinine level and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling-positive tubular cells in the kidney. In addition, FGF10 dramatically ameliorated renal mitochondrial-related damage, including reducing mitochondrial-dependent apoptosis, alleviating oxidative stress, maintaining the mitochondrial membrane potential, and opening the mitochondrial KATP channels. The protective effect of FGF10 was significantly compromised by the ATP-dependent potassium channel blocker 5-HD. Our data suggest that FGF10 offers effective protection against I/R and improves animal survival by attenuating mitochondrial damage.

To provide evidence for the better clinical use of traditional Chinese medicine preparations (TCMPs), comparison of the pharmacological mechanisms between TCMPs with similar therapeutic effect is necessary. However, methodology for dealing with this issue is still scarce. Danhong injection (DHI) and Naoxintong capsule (NXT) are representative TCMPs for ischemic stroke (IS) treatment, which are also frequently used in combination. Here they were employed as research objects to demonstrate the feasibility of systems pharmacology approach in elucidation of the independent and combined effect of TCMPs. By incorporating chemical screening, target prediction, and network construction, a feasible systems pharmacology model has been established to systematically uncover the underlying action mechanisms of DHI, NXT, or their pair in IS treatment. Systematic analysis of the created TCMP-Compound-Target-Disease network revealed that DHI and NXT shared common targets such as PTGS2, F2, ADRB1, IL6, ALDH2, and CCL2, which were involved in the vasomotor system regulation, blood-brain barrier disruption, redox imbalance, neurotrophin activity, and brain inflammation. In comparative mechanism study, the merged DHI/NXT-IS PPI network and pathway enrichment analysis indicated that DHI and NXT exerted the therapeutic effects mainly through immune system and VEGF signaling pathways. Meanwhile, they had their own unique pathways, e.g., calcium signaling pathway for DHI and gap junction for NXT. While for their synergistic mechanism, DHI and NXT participated in chemokine signaling pathway, T cell receptor signaling pathway, VEGF signaling pathway, gap junction, and so on. Our study provided an optimized strategy for dissecting the different and combined effect of TCMPs with similar actions.

Background: Doxorubicin-Induced Cardiotoxicity (DIC) has greatly limited the clinical benefits of this frontline drug in oncotherapy. Drug combination with Natural Compounds (NCs) that possess potency against DIC is considered as a promising intervention strategy. However, the Mechanisms of Action (MoAs) underlying such drug interactions remain poorly understood. The aim of this study was to systematically pursuit of the molecular mechanisms of NCs against DIC. Methods: First, the gene expression signatures of DIC were characterized from transcriptomics datasets with doxorubicin-treated and untreated cardiomyocytes using differentially expressed gene identification, functional enrichment analysis, and protein-protein interaction network analysis. Secondly, reverse pharmacophore mapping-based network pharmacology was employed to illustrate the MoAs of 82 publicly reported NCs with anti-DIC potency. Cluster analysis based on their enriched pathways was performed to gain systematic insights into the anti-DIC mechanisms of the NCs. Finally, the typical compounds were validated using Gene Set Enrichment Analysis (GSEA) of the relevant gene expression profiles from a public gene expression database. Results: Based on their anti-DIC MoAs, the 82 NCs could be divided into four groups, which corresponded to ten MoA clusters. GSEA and literature evidence on these compounds were provided to validate the MoAs identified through this bioinformatics analysis. The results suggested that NCs exerted potency against DIC through both common and different MoAs. Conclusion: This strategy integrating different types of bioinformatics approaches is expected to create new insights for elucidating the MoAs of NCs against DIC.

Background: Hepatoma is a common malignancy in the world with high morbidity and mortality. The treatment of hepatoma is limited by its poor response to many chemotherapeutic agents. Although paclitaxel (PTX) is widely used in clinical chemotherapy, the low sensitivity to hepatoma restricts its application. Combination therapy is a promising approach to resolve this dilemma. Objective: To evaluate the interaction between paclitaxel, bufalin (BFL) and cinobufagin (CBF), and explore the optimum combination efficiently. Methods: HepG2 cells were treated with PTX, BFL and CBF individually or in combination. Their interactions were evaluated by two classical models (Chou-Talalay model and Bliss independence). Response surface methodology (RSM) was used to explore the optimum combination. Furthermore, the optimum drug combination was verified by the morphological experiment. Results: Synergistic effects were observed when cells were exposed to binary mixtures of PTX+CBF and BFL+CBF. Although the interaction of PTX and BFL was summative, a strong synergistic effect was observed when cells were exposed to ternary mixtures of PTX+BFL+CBF. The interaction results of RSM were consistent with classical models, but more efficient. Moreover, the optimum combination dose was given by RSM without the combinatorial explosion of exhaustive testing. Conclusion: The combination of BFL and CBF synergistically enhanced the potency of PTX against HepG2 cells. RSM could give an accurate evaluation for drug interactions and efficient prediction of optimum combination.

Chansu, which is prepared from the skin secretions of toad (Bufo bufo gargarizans Cantor), is widely used in traditional Chinese medicine (TCM). Being the principal bioactive constituents of Chansu, bufalin (BFL) and cinobufagin (CBF) have been shown to possess anticancer properties. TCM confers bioactivities through the synergistic effect between potential active ingredients, so as to interfere with the development of the disease, and ultimately achieve the therapeutic effect. We found that the anticancer effect was significantly potentiated by cotreatment with BFL and CBF compared to monotreatment, suggesting their synergistic interaction. To reveal their synergistic mechanisms, metabolomic and lipidomic profiling based on liquid chromatography-mass spectrometry (LC-MS) was utilized to delineate the responses in HepG2 cells after treatment with BFL and CBF individually or in combination. Metabolic pathways, including methionine metabolism, energy metabolism, lipid metabolism, and amino acid metabolism, were modulated and subsequently led to apoptosis and cell cycle arrest of HepG2 cells. In particular, the discrepant regulation of methionine metabolism between the monotreatment and cotreatment with BFL and CBF may account for their synergistic effect. Our study provided novel insights into the mechanistic links between cellular metabolism and the synergistic effect, which may ultimately lead to better treatments for hepatoma.

Yangxinshi Tablet (YXST) is a Chinese patent medicine commonly used to treat cardiovascular diseases. However, its detailed chemical basis and mechanisms of action have not been clarified. In this study, high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS) was applied for comprehensive analysis of the chemical constituents in YXST. A total of 127 compounds, including 19 phenolic acids, 12 alkaloids, 51 flavanoids, 32 triterpenoids, 2 lignans, 2 phenylethanoid glycosides, 2 anthraquinones, 1 coumarin, and 6 other compounds, were identified or tentatively deduced by comparing their retention times and MS spectra with those of authentic standards or literature data. To further prove the antioxidant activity of YXST, its free radical scavenging capacity was assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) spectrophotometric assay and the antioxidants in YXST were rapidly screened by DPPH-HPLC experiment. Especially, salvianolic acid A and salvianolic acid B showed excellent DPPH scavenging activities with the IC50 of 151.9 and 275.6μg/mL, respectively, which were stronger than that of l-ascorbic acid (positive control) with the IC50 of 297.1μg/mL. Additionally, these two most potent antioxidants were detectable in rat plasma after oral administration. In conclusion, this study reported important clues for the further pharmacological and clinical studies of YXST. Meanwhile, it provided a practical strategy for rapid screening and identifying of in vivo antioxidant in traditional Chinese medicine preparations.

Screening agonists of peroxisome proliferator-activated receptor-γ (PPARγ) from natural products is particularly motivated by the need for effective anti-diabetic agents. However, method for direct identification of PPARγ ligands from a complex sample is rarely reported. Here we propose a novel immobilized fusion protein affinity chromatography (IFPAC) to achieve rapid multicomponent screening. First, functional human PPARγ ligand binding domain (hPPARγLBD) was bacterially produced by fusion to glutathione S-transferase (GST). The unpurified GST-hPPARγLBD was directly applied to a 96-well filter plate prepacked with glutathione sepharose. Due to the strong affinity between GST and glutathione, the fusion protein could selectively attach to the glutathione matrix with an oriented immobilization, which was rapidly purified under non-denaturing conditions. Experimental results indicated that the prepared 96-affinity column array exhibited excellent selectivity and sensitivity for fishing novel interacting compounds. The proposed approach was applied in the high-throughput screening of PPARγ ligands from natural products, followed by rapid characterization of active compounds using HPLC-ESI-Q-TOF-MS/MS. Isochlorogenic acid A in Dendranthema indicum flowers was found to be a PPARγ ligand. Our findings suggested the IFPAC could be a powerful tool for drug discovery from natural products.

Danhong injection (DHI) is a widely used Chinese medicine injection (CMI) for the clinical treatment of cardiovascular and cerebrovascular diseases. In this study, a simple and efficient in vitro method based on ultrafiltration LC-MS and molecular modeling has been developed to study the human serum albumin (HSA) binding of the compounds in DHI. Seven major components including protocatechuic aldehyde, p-coumaric acid, salvianolic acid D, rosmarinic acid, salvianolic acid E, lithospermic acid and salvianolic acid B were identified as HSA ligands and their binding degrees in the proposed non-saturated model were 26.17, 37.69, 99.77, 91.78, 96.91, 99.42 and 98.10%, respectively. Considering the drug-HSA binding property of the compounds in DHI may change during drug combination therapy, competitive binding assay was carried out to evaluate the influence of aspirin on the DHI-HSA binding. Experimental results revealed that the salvianolic acids in DHI had stronger binding ability to HSA than sodium salicylate. To further verify the results above, molecular modeling and probe displacement assay were conducted to investigate the optimum binding site and binding affinity of the ligands on HSA. Our findings suggested that the established method could be a powerful tool to study the drug-HSA binding property of CMIs.

Screening brain-penetrable antioxidants from natural products is a promising way for neuroprotective drug discovery. However, there is no screening methodology enables simultaneous investigation of antioxidant activity and blood-brain barrier (BBB) permeability of compounds from complex samples. Here we propose a novel strategy by combining BBB specific parallel artificial membrane permeability assay with 1,1-diphenyl-2-picrylhydrazyl recognition (BBB-PAMPA-DPPH) to achieve rapid multicomponent screening. First, BBB specific artificial membrane was constructed to separate the compounds with high BBB permeability in herbal extracts. The antioxidant activity of the isolated compounds could be optically recognized through the bleaching of the purple-colored DPPH. By off-line combined HPLC-UV/Q-TOF-MS analysis, the exact BBB-penetrable compounds responsible for the antioxidant activity could be rapidly screened. With this approach, compound 2,6,4'-trihydroxy-4-methoxybenzophenon in Rhizoma Anemarrhena was found to be an antioxidant with very high BBB permeability, which could also be detected in rat plasma and brain tissue after oral administration. Our findings suggested the BBB-PAMPA-DPPH method could be a powerful tool for neuroprotective drug discovery from natural products.

Although Danhong injection (DHI) has been clearly shown to attenuate ischemic myocardial injury and improve heart function, there is no research regarding its role in doxorubicin (DOX)-induced cardiomyopathy. In this study, we aimed to investigate the reverse effect of DHI on DOX-induced cardiotoxicity in H9c2 cells. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay demonstrated that DHI had no cytotoxicity towards the relevant cell line unless the concentration was as high as 50 μL/mL. The satisfactory cardioprotective effect of DHI exerted at the concentration of 10 μL/mL, which agreed well with the result of real-time cell viability assay. Then non-targeted metabolomics based on LC–MS was employed to characterize metabolic alterations in DOX-induced cells with DHI treatment. Multivariate analysis, including PCA and PLS-DA, revealed 31 altered metabolites after DOX treatment that were primarily related to the disturbance of amino acids and nucleotides metabolism. While DHI could intervene in some disturbed metabolic pathways, such as the metabolism of arginine, glutathione (GSH), pantothenic acid, cytidine, inosine and 5′-methylthioadenosine. These results suggested that DHI exerted the therapeutic effect by improving energy metabolism and attenuating oxidative stress. The present study can lay a foundation for further research on the promising therapeutic effect of DHI in managing DOX-induced cardiotoxicity.