Functional annotation map of natural compounds in traditional Chinese medicines library: TCMs with myocardial protection as a case

The chemical complexity of traditional Chinese medicines (TCMs) makes the active and functional annotation of natural compounds challenging. Herein, we developed the TCMs-Compounds Functional Annotation platform (TCMs-CFA) for large-scale predicting active compounds with potential mechanisms from TCM complex system, without isolating and activity testing every single compound one by one. The platform was established based on the integration of TCMs knowledge base, chemome profiling, and high-content imaging. It mainly included: (1) selection of herbal drugs of target based on TCMs knowledge base; (2) chemome profiling of TCMs extract library by LC‒MS; (3) cytological profiling of TCMs extract library by high-content cell-based imaging; (4) active compounds discovery by combining each mass signal and multi-parametric cell phenotypes; (5) construction of functional annotation map for predicting the potential mechanisms of lead compounds. In this stud TCMs with myocardial protection were applied as a case study, and validated for the feasibility and utility of the platform. Seven frequently used herbal drugs (Ginseng, etc.) were screened from 100,000 TCMs formulas for myocardial protection and subsequently prepared as a library of 700 extracts. By using TCMs-CFA platform, 81 lead compounds, including 10 novel bioactive ones, were quickly identified by correlating 8089 mass signals with 170,100 cytological parameters from an extract library. The TCMs-CFA platform described a new evidence-led tool for the rapid discovery process by data mining strategies, which is valuable for novel lead compounds from TCMs. All computations are done through Python and are publicly available on GitHub.

Past and future of trypanosomatids high-throughput phenotypic screening

Diseases caused by trypanosomatid parasites affect millions of people mainly living in developing countries. Novel drugs are highly needed since there are no vaccines and available treatment has several limitations, such as resistance, low efficacy, and high toxicity. The drug discovery process is often analogous to finding a needle in the haystack. In the last decades a so-called rational drug design paradigm, heavily dependent on computational approaches, has promised to deliver new drugs in a more cost-effective way. Paradoxically however, the mainstay of these computational methods is data-driven, meaning they need activity data for new compounds to be generated and available in databases. Therefore, high-throughput screening (HTS) of compounds still is a much-needed exercise in drug discovery to fuel other rational approaches. In trypanosomatids, due to the scarcity of validated molecular targets and biological complexity of these parasites, phenotypic screening has become an essential tool for the discovery of new bioactive compounds. In this article we discuss the perspectives of phenotypic HTS for trypanosomatid drug discovery with emphasis on the role of image-based, high-content methods. We also propose an ideal cascade of assays for the identification of new drug candidates for clinical development using leishmaniasis as an example.

Intermedin improves renal structure and function and microvascular injury in IgA nephropathy rats / 中国药理学通报

Aim To investigate the hepatotoxicity of rutecarpine(RUT)by using high-content screening technology.Methods HepG2 cells were exposed to different concentrations of RUT for different time, then cell viability was detected by MTT method.Cell count, nucleus injury, mitochondrial membrane potential(MMP), reactive oxygen species(ROS), internal flow of calcium, cell membrane integrity(DIR)were measured by high-content screening technology.The activation of MAPK, NF-κB and JAKs-STATs was assayed by high-content screening technology.The apoptosis was detected by flow cytometry.Results The viability was significantly reduced by 100 μmol·L-1 RUT(P<0.01)after HepG2 cell exposure to RUT for 24 h, the nuclear area decreased and the nuclear morphology was uneven, and after 48 h, the cell count was significantly reduced(P<0.01), the early apoptosis was detected(P<0.01).After HepG2 cell exposure to RUT for 6 h, the levels of ROS and internal flow of calcium significantly increased(P<0.01), and the cell membrane integrity was obviously damaged(P<0.01).After exposure to 100 μmol·L-1 RUT for 24 h, the phosphorylation of ERK, JNK, STAT3 and p38 significantly increased(P<0.01, P<0.05), but there was no significant change in total protein level.The expression of c-Jun and c-Fos was up-regulated at 3 h(P<0.01), and at 3h time point, the phosphorylation of NF-κB p65 significantly increased(P<0.01), but nuclear translocation was not significant.Conclusions Under certain conditions, RUT shows cytotoxicity on HepG2 cells, and its toxic mechanism is mainly related to injury caused by oxidative stress and inflammatory response.

Study on hepatoxic liver of rutecarpine based on high-content screening technology / 中国药理学通报

Aim To investigate the hepatotoxicity of rutecarpine(RUT)by using high-content screening technology.Methods HepG2 cells were exposed to different concentrations of RUT for different time, then cell viability was detected by MTT method.Cell count, nucleus injury, mitochondrial membrane potential(MMP), reactive oxygen species(ROS), internal flow of calcium, cell membrane integrity(DIR)were measured by high-content screening technology.The activation of MAPK, NF-κB and JAKs-STATs was assayed by high-content screening technology.The apoptosis was detected by flow cytometry.Results The viability was significantly reduced by 100 μmol·L-1 RUT(P<0.01)after HepG2 cell exposure to RUT for 24 h, the nuclear area decreased and the nuclear morphology was uneven, and after 48 h, the cell count was significantly reduced(P<0.01), the early apoptosis was detected(P<0.01).After HepG2 cell exposure to RUT for 6 h, the levels of ROS and internal flow of calcium significantly increased(P<0.01), and the cell membrane integrity was obviously damaged(P<0.01).After exposure to 100 μmol·L-1 RUT for 24 h, the phosphorylation of ERK, JNK, STAT3 and p38 significantly increased(P<0.01, P<0.05), but there was no significant change in total protein level.The expression of c-Jun and c-Fos was up-regulated at 3 h(P<0.01), and at 3h time point, the phosphorylation of NF-κB p65 significantly increased(P<0.01), but nuclear translocation was not significant.Conclusions Under certain conditions, RUT shows cytotoxicity on HepG2 cells, and its toxic mechanism is mainly related to injury caused by oxidative stress and inflammatory response.

Comparative study of three cell morphological analysis methods on in vitro micronucleus assay / 上海预防医学

Objective To investigate the feasibility of genotoxicity assessment for chemicals via flow cytometry (FCM) and high-content screening (HCS) based on high-throughput screening in vitro micronucleus assays. Methods In reference to the methodology of OECD TG487, the typical positive controls, cyclophosphamide (CP) and mitomycin C (MMC), were selected.And no serum MEM medium was treated as negative control.Dose range of CP was 5-20 mg/L and MMC was 0.25-1.0 mg/L.CHL cells were treated with three concentrations of each chemical for 4 h.High-throughput screening in vitro micronucleus assays based on FCM and HCS were established.The results of the frequency of micronuclei were compared to traditional cytokinesis blocking micronucleus assay in each group with or without metabolic activation. Results The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately 1.9%, 7.6%, 10.4% and 5.9%, 11.4%, 16.7%, which were obtained by conventional microscopic scoring.The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately 2.8%, 2.6%, 7.8% and 3.2%, 3.7%, 5.1%, which were obtained by flow cytometry screening.The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately2.8%, 6.2%, 9.1% and 7.9%, 10.1%, 10.2%, which were obtained by high-content screening.Compared with negative controls, the differences of the results were statistically significant(P < 0.05), and there was a dose-response relationship. Conclusion In this study, the results of high-throughput screening assays of FCM and HCS are in accordance to the results of traditional cytokinesis blocking micronucleus assay, indicating that high-throughput screening in vitro micronucleus assays could detect micronucleus formation automatically and improve the efficiency.Therefore, the method could provide data support for using high-throughput screening in vitro micronucleus assays into genotoxicity assessment of chemicals.

Comparative study of three cell morphological analysis methods on in vitro micronucleus assay / 上海预防医学

Objective To investigate the feasibility of genotoxicity assessment for chemicals via flow cytometry (FCM) and high-content screening (HCS) based on high-throughput screening in vitro micronucleus assays. Methods In reference to the methodology of OECD TG487, the typical positive controls, cyclophosphamide (CP) and mitomycin C (MMC), were selected.And no serum MEM medium was treated as negative control.Dose range of CP was 5-20 mg/L and MMC was 0.25-1.0 mg/L.CHL cells were treated with three concentrations of each chemical for 4 h.High-throughput screening in vitro micronucleus assays based on FCM and HCS were established.The results of the frequency of micronuclei were compared to traditional cytokinesis blocking micronucleus assay in each group with or without metabolic activation. Results The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately 1.9%, 7.6%, 10.4% and 5.9%, 11.4%, 16.7%, which were obtained by conventional microscopic scoring.The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately 2.8%, 2.6%, 7.8% and 3.2%, 3.7%, 5.1%, which were obtained by flow cytometry screening.The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately2.8%, 6.2%, 9.1% and 7.9%, 10.1%, 10.2%, which were obtained by high-content screening.Compared with negative controls, the differences of the results were statistically significant(P < 0.05), and there was a dose-response relationship. Conclusion In this study, the results of high-throughput screening assays of FCM and HCS are in accordance to the results of traditional cytokinesis blocking micronucleus assay, indicating that high-throughput screening in vitro micronucleus assays could detect micronucleus formation automatically and improve the efficiency.Therefore, the method could provide data support for using high-throughput screening in vitro micronucleus assays into genotoxicity assessment of chemicals.

Hepatotoxicity study of sulfur-fumigated Ophiopogonis Radix using high content screening / 中草药

Objective: An efficient method was established using high content screening (HCS) for the hepatotoxicity evaluation of sulfur-fumigated Ophiopogonis Radix. Methods: Cytotoxicity of positive control group, negative control group, Ophiopogonis Radix extracts group and sulfur-fumigated Ophiopogonis Radix extracts group were tested based on HepG2 human hepatoma cells. HCS was applied to detect the cell number, DNA content, level of glutathione (GSH), reactive oxgyen species (ROS), and mitochondrial membrane potential (MMP). Results: Compared with the cells of Ophiopogonis Radix extracts group, GSH of sulfur-fumigated Ophiopogonis Radix extracts decreased significantly at the concentration of 50 mg/mL; The MMP of sulfur-fumigated Ophiopogonis Radix extracts changed signally at the concentration of 12.5 mg/mL. Conclusion: Ophiopogonis Radix showed pontential cytotoxicity after sulfur-fumigated. The hepatotoxicity of sulfur-fumigated Ophiopogonis Radix may be related to mitochondria-mediated apoptosis according to the influence of its MMP from the results.

Enhancement Study of Macrophage Phagocytic Activity and the Quality Evaluation of Dioscorea opposita Thunb. Based on the Analysis of High Content Screening / 中国药学杂志

OBJECTIVE: To compare the differences of macrophages phagocytic activities between Dioscorea opposita Thunb. cv. Tiegun(abbreviated as TG) and Dioscorea opposita Thunb. but not Tiegun(abbreviated as NTG). METHODS: CCK-8 assay was used to test the cytotoxicity. On the basis of non-toxic dose, the high content screening(HCS) cell imaging analysis was applied to test the phagocytic ability of RAW264.7 cells engulfing GFP-E. coli in vitro, and the phagocytic rates between different kinds of samples at various concentrations were calculated. Furthermore, the biological potency was measured according to the qualitative response parallel method to better evaluate the difference of TG and NTG, by translating phagocytic rates into biological potency. RESULTS: At the range of 0.695 - 5.56 mg(crude drug) •mL-1, all 11 batches samples have no toxicity. The results of HCS displayed that every sample could promote cell phagocytic activity in varying degrees at a certain concentration. RAW264.7 cells could engulf the GFP-E. coli, and the images of HCS reflected the situation of phagocytosis clearly. In addition, the results of biopotency showed that the biopotency value of different samples was between 39.56 to 100, and the average biopotency value of TG was significantly higher than NTG (P < 0.05). CONCLUSION: In vitro experiments showed that all the samples could promote the phagocytic activity of RAW264.7 cells at different degrees. And the average biopotency value of TG was significantly higher than NTG, which is consistent with the saying that "TG has a better quality".

High-content screening in studies of toxicology and its application in drug-induced hepatotoxicity / 中国药理学与毒理学杂志

Drug-induced liver injury(DILI) is one of the major causes of termination of drug development.The establishment of a high-throughput test system to predict potential clinical hepatotoxicity is a valuable approach in the pharmaceutical industry.The high-content imaging-based in vitro assays allow simultaneous detection of cellular multiple parameters in the system.The real-time monitoring of multiple signaling pathways can shed light on many mechanisms of cell injury,with high sensitivity and specificity.Many types of liver cell models have been applied to high-content screening(HCS) so far.This paper introduceds the HCS technology and reviews the data of hepatotoxicity obtained from HCS technology in recent years.At the same time,we discuss the application of this technology in exploring the mechanism of hepatotoxicity and the potential of HCS technology in studying DILI and mechanisms.

Quantitative cardiotoxicity assessment of gambogic acid using multiple cellular phenotype analysis / 中国药理学与毒理学杂志

OBJECTIVE To evaluate the cardiotoxicity of a widen-spectrum antineoplastic drug, gambogic acid, through quantitative multiple cellular phenotypic characterization. METHODS H9c2 cell line was used as a model with doxorubicin (Dox) and amiodarone (Ami) as positive controls, hypaconi?tine as negative control and 0.1% DMSO as normal control. An optimized protocol was established to identify the morphology and function of cell nuclei. The effect of drugs on cell viability, nuclear area (Hoechst33342), mitochondria mass (MitoTracker Deep Red) and cytoplasmic calcium ion mobilization (Rhod2 AM)was studied. EC50 and Z′values were calculated to evaluate the degree of toxicology and to estimate the precision and false-positive rate, respectively. RESULTS Dose-response analysis indicated that EC50 of Dox on cell viability, nuclear area, mitochondrial mass was 0.72, 0.014 and 1.21μmol · L-1, respectively. On the other hand, EC50 of Ami on the parameters of cell viability, nuclear area and mitochon?drial mass was 14.83, 6.72 and 4.54μmol·L-1, respectively with Z′value above 0.5. Hypaconitine decreased the SER ridge of mitochondria. Gambogic acid caused significant mortality of H9c2 cells and induced nuclear shrinkage as Ami did. The EC50 values of cell viability and nuclear area were 0.24 and 1.16 μmol · L- 1. Meanwhile,gambogic acid disturbed the mitochondrial function as indicated by the increased mitochondrial area (EC50=0.44 μmol · L-1), abnormal SER Ridge(EC50=0.99 μmol · L-1) and decreased mitochondrial mass(EC50=1.21 μmol · L- 1). Cellular calcium mobilization was lower than normal (EC50=0.41 μmol · L-1). CONCLUSION The EC50 values of positive controls calculated from our assessment are similar those reported in literature. A multi-parameter and simultaneous evaluation enables a comprehensive analysis of the morphology of nuclei and mitochondria of cardiomyocytes and a preliminary assessment of the mechanisms of toxicity.

High content screening for promoting effect of Dioscorea opposita on macrophage phagocytosis / 中草药

Objective: To establish a high content screening (HCS) method by testing the phagocytic function of RAW 264.7, and to evaluate the effect of Dioscorea opposita on cell proliferation and phagocytosis in vitro. Methods: CCK-8 was used to measure the proliferation of RAW 264.7, and HCS was helpful to determine the ability of RAW 264.7 to engulf GFP-E.coli. Results: The best method was established by examining the time of administration, the amount of bacteria added, and the time of bacterial stimulation: administration time of 12 h, 50 times of bacteria, and stimulation time of 1.5 h. Then, the repeatability of this method was tested, and the RSD value was 2.31%. Compared with the group without drugs, at the concentration of 0.156-1.25 mg/mL, the water extract of D. opposita could promote the proliferation, and improve the average phagocytosis rate of every single RAW264.7 cell. And the samples had the strongest effect on proliferation and phagocytosis when the concentration was 1.25 mg/mL. Conclusion: An HCS method is established and firstly used in D. opposita. HCS has the advantage of accurate, intuitive, and high throughput. D. opposita can not only promote cell proliferation, but also improve the phagocytic ability of each single RAW264.7 in vitro. A new method is provided for further study on immune activity and on the mechanism of enhancing the immune activity that some tonic herbs may have, such as D. opposita.

Cytotoxic effect of geniposide and its metabolite genipin on HepG2 cells and mechanism / 中国药理学通报

Aim To compare the cytotoxicity of geni-poside (GS)and its metabolite genipin (GP)on hu-man hepatocelluar HepG2 cells and explore the sub-stance and mechanism of hepatotoxicity induced by Fructus Gardeniae.Methods The cytotoxic effect of GS and GP was analyzed by MTT method;the antioxi-dant enzyme activities of manganese superoxide dis-mutase (Mn-SOD),catalase (CAT)and levels of glu-tathione (GSH)were detected by respective kits;the change of intracellular reactive oxygen species (ROS ) was measured by 2′,7′-dichlorofluorescin diacetate (DCFH-DA)staining method;multiparameter cytotox-icity analysis (cell loss,nuclear size and morphologi-cal changes,DNA content,cell membrane permeabili-ty,mitochondrial membrane potential changes,cyto-chrome C release ) were measured simultaneously by high content screening (HCS)assays.Results No cytotoxicity was found in GS (20~1 000 μmol·L-1 ) groups (P>0.05 ),but GP was found to exert obvi-ous cytotoxic effect,and 50μmol·L-1 GP could obvi-ously inhibit HepG2 cell proliferation (P0. 0 5 ),GP could significantly decrease the activity of Mn-SOD,CAT and the level of GSH,and obviously increase the content of ROS (P0.05 ). Conclusion GP is the direct substance of hepatotox-icity induced by Fructus Gardeniae,and the mecha-nism might be associated with oxidative stress,mito-chondria injury and apoptosis.

Protection of 20 Chinese materia medica extracts to Aβ25-35-induced SH-SY5Y cell injury using high content screening / 中草药

Objective: To identify neuroprotective extracts with the protective effects on Aβ25-35-induced SH-SY5Y cell injury via high content screening (HCS). Methods: Hoechst 33342/PI double staining method was used to screen neuroprotective extracts from 60 Chinese materia medica (CMM) extracts. Further more, the effects of neuroprotective extracts on Aβ25-35-induced changes in the levels of Caspase-3/7 were detected. Results: The results showed that 17 extracts had obviously neuroprotective effects. Among the 17 extracts, 8 of them inhibited Aβ25-35-induced up-regulation of Caspase-3/7. Conclusion: HCS is an efficient method to screen neuroprotective extracts with the protective effects of Aβ25-35-induced SH-SY5Y cell injury. The neuroprotective extracts have potential medicinal value in Alzheimer's disease.

Optimization of effective component formula from active ingredients of Salvia Miltiorrhiza and Panax Ginseng through orthogonal design method to resist breast cancer / 中国药理学通报

Aim To optimize the most effective compo-nent formula from the active ingredients of Salvia Milti-orrhiza and Panax Ginseng through the orthogonal de-sign method to resist breast cancer, and to reveal its antitumor mechanism in MCF-7 cells. Methods The human breast cancer cells MCF-7 were employed as the research object and the normal breast epithelial cells MCF-10A were used as control,optimizing the most ef-fective component formula from the active ingredients of Salvia Miltiorrhiza and Panax Ginseng by using CCK-8 assay and orthogonal design method; real-time cell a-nalysis was used to monitor the best combination formu-la on cell proliferation, and high content screening was used to detect the best combination drug on cell apop-tosis. Results The best combination of the salvianolic acids, saponins of Panax Ginseng and ginseng polysac-charides that were screened out were 5 , 10 , 5 mg · L-1 . Compared with control group, the treatment group had effective response inhibiting the proliferation on MCF-7 cells, but those effects were weaker on MCF-10A cells through real-time cell analysis. Ho-echst, Annexin V, PI staining fluorescence showed no significant difference ( P >0. 05 ) on MCF-10 A cells compared with the control group,but there was signifi-cant difference ( P <0. 01 ) on MCF-7 cells by HCS. Conclusions The most effective component formula from the active ingredients of Salvia Miltiorrhiza and Panax Ginseng have a strong inhibition of proliferation and induction of apoptosis to resist breast cancer with selection, and there is no significant difference in MCF-10A cells.

High content screening analysis for systematic study on traditional Chinese medicine / 中草药

High content screening(HCS) analysis is an advanced technique in the era of systematic biology,which can comprehensively reflect the changes of cell with live,dynamic,and multiparametric characters.It is a good tool to study the complex ingredients and mechanism of traditional Chinese medicine(TCM).Application of the technique would contribute to the discovery of effective ingredients,elucidation of mechanism,and development of TCM theory.