Nephrotoxicity screening of main active components in Zuojin Pills / 中国药理学通报

Aim To study the nephrotoxicity effects of the main monomers in Zuojin Pills. Methods CCK-8 and high-content toxicity screening were used to preliminarily screen the main alkaloids in Zuojin Pills that may cause renal cell damage. Further, by confirmation of cell morphology, release rate of lactate dehydrogenase and cytochrome C, and expression of apoptosis-related proteins, the alkaloids causing cell damage were preliminarily identified, providing in vitro toxicological evidence for the compatibility of components of traditional Chinese medicine and compatibility attenuation. Results Preliminary screening using CCK-8 method and high-content technology showed that evodiamine (EVO) could significantly reduce cell number, increase cell membrane permeability, and reduce mitochondrial membrane potential. In addition, cell morphology, apoptosis and cytochrome C expression were consistent with the results of high-content screening. Western blot experiments indicate that EVO could induce apoptosis and cause renal cell damage. Conclusions EVO can obviously cause renal cell damage, and may induce apoptosis by affecting mitochondria, cytochrome C and cell membrane permeability.

High-content analysis of in vitro hepatocyte injury induced by various hepatotoxicants

In vitro prediction of hepatotoxicity can enhance the performance of non-clinical animal testing for identifying chemical hazards. In this study, we assessed high-content analysis (HCA) using multi-parameter cell-based assays as an in vitro hepatotoxicity testing model using various hepatotoxicants and human hepatocytes such as HepG2 cells and human primary hepatocytes (hPHs). Both hepatocyte types were exposed separately to multiple doses of ten hepatotoxicants associated with liver injury whose mechanisms of action have been described. HCA data were obtained using fluorescence probes for nuclear size (Hoechst), mitochondrial membrane potential (TMRM), cytosolic free calcium (Fluo-4AM), and lipid peroxidation (BODIPY). Cellular alterations were observed in response to all hepatotoxicants tested. The most sensitive parameter was TMRM, with high sensitivity at a low dose, next was BODIPY, followed by Fluo-4AM. HCA data from HepG2 cells and hPHs were generally concordant, although some inconsistencies were noted. Both hepatocyte types showed mild or severe mitochondrial impairment and lipid peroxidation in response to several hepatotoxicants. The results demonstrate that the application of HCA to in vitro hepatotoxicity testing enables more efficient hazard identification, and further, they suggest that certain parameters could serve as sensitive endpoints for predicting the hepatotoxic potential of chemical compounds.

Stable co-expression of human kappa opioid receptor and PKAcat-EGFP in CHO cells and functional identification / 中国药理学通报

Aim To establish a cell model which stably co-ex-press human kappa opioid receptor (hKOR) and enhanced green fluorescent protein ( EGFP) labeled catalytic domain of cAMP-dependent protein kinase A(PKAcat) fusion protein (PKAcat-EGFP) in Chinese hamster ovary(CHO) cells, laying the foun-dation for the high-throughput screening of hKOR drugs and drug molecular mechanisms in vitro. Methods Hygromycin B resist-ant hKOR recombinant plasmid [ pcDNA3.1/Hygro ( + ) -hKOR] was transfected into CHO cells stably expressing PKA-cat-EGFP by a lipofectin based method. Transfected cells were selected in culture medium containing hygromycin B. The posi-tive clones were selected by PKA redistribution assay. Z’ factor was used for evaluation and validation the reliability of the cell model. PKA redistribution assay and LANCE cAMP 384 Kit were used to test the function of the receptors in selected clone. Results CHO-PKAcat-EGFP/hKOR-13 cell model exhibited stable response in PKA redistribution assay and LANCE cAMP 384 Kit. Treated with 100 nmol·L-1U-50488 for 30 min, the average value of Z’ factor was 0.596, proving the reliability of the cell model. The hKOR expression in cell model remained stable after a few generations. Conclusion The CHO-PKAcat-EGFP/hKOR-13 cell model with stable co-expression of hKOR and PKAcat-EGFP has been successfully established.

Renal Cell Toxicity Screening of Drugs by High Content Analysis / 中国药学杂志

OBJECTIVE: To discuss the feasibility of renal toxicity screening of drugs using high content analysis technology. METHODS: Rosiglitazone was selected as the negative drug and valinomycin was selected as the positive drug. High intension HK-2 renal cell toxicity screening model was established through testing five indexes including cell number, DNA intensity, cell permeability, mitochondrial membrane potential and cytochrome C, using high content analysis technology. Aristolochic acid A and cisplatine that has obvious renal toxicity was used to validate the model. RESULTS: Valinomycin, cisplatin and aristolochic acid A has obvious renal toxicity;rosiglitazone don't have renal toxicity. CONCLUSION: High content analysis can be used to screen renal toxicity of drugs.

Effect of Paeonia lactiflora formula granule against the damage of LO2 cells induced by carbon tetrachloride / 解放军医学杂志

Objective To investigate the effect of Paeonia lactiflora formula granule (PLFG) against the damage of LO2 cells induced by carbon tetrachloride (CCl4) and explore its mechanism. Methods LO2 cells were divided into control group, CCl4 model group, PLFG (1, 5, 10mg/L) groups and Vit E (50mmol/L) group. LO2 cells of PLFG groups and Vit E group were pretreated by drugs for 24 hours, then except control group, other groups were treated by 10mmol/L CCl4 for 6 hours, which induced hepatic cell damage, the alanine aminotransferase (ALT) and aspartate transaminase (AST) content of the cell culture supernatant were measured, the survival rates of LO2 cells were analyzed by MTT method. LO2 cells were divided into control group, CCl4 model group, PLFG (10mg/L) group and Vit E (50mmol/L) group, cell loss, changes in nuclear size and morphology, DNA content, mitochondrial membrane potential (MMP), cell permeability changes, and cytochrome C release were measured simultaneously by high content analysis (HCA). Results Compared with CCl4 model group, PLFG and Vit E pretreatment could obviously decrease the level of ALT and AST (P<0.05 or P<0.01), especially in the 10mg/L PLFG group and Vit E group. PLFG (1, 5, 10mg/L) and Vit E could also significantly weaken the decrease of LO2 cell viability, which were induced by CCl4 treatment, the results showed in dose-dependent to some extents (P<0.05 or P<0.01). Meanwhile, treatment with 10mg/L PLFG and Vit E could significantly increase the level of MMP (P<0.01), prevent cytochrome C release (P<0.01), decrease the membrane permeability (P<0.01), increase nuclear size (P<0.01), decrease total nuclear intensity (P<0.01) and increase the cell count (P<0.05). Conclusion PLFG may act an obvious protective effect against the liver injury induced by CCl4, and it might be due to protecting the integrity of mitochondria membrane, decreasing cell membrane permeability and inhibiting the apoptosis of LO2 cells.

In vitro activity of a novel cannabinoid receptor l selective antagonist LMJ07 / 国际药学研究杂志

Objective: LMJ07 is a novel cannabinoid receptorl (CB1R) selective antagonist discovered by our lab. In the present study, its affinity and antagonistic activity against CB1R were evaluated at the molecular and cellular levels by receptor binding experiment, CB1R internalization experiment and by monitoring the change in cytoskeletal and intracellular signal induced by CB1R activation. Methods: With the CB1R selective antagonist rimonabant (SR141716A) as control, the affinity and selectivity of LMJ07 to CB1R and CB2R were assayed by radioligand binding assays, and the G protein-independent antagonistic activity against cannabinoid receptor (CBR) was assayed by enhanced green fluorescein protein (EGFP)-CBR internalization with hierarchiae cluscer anclysis (HCA) analysis. At the same time, we evaluated the changes in cytoskeletal and the intracellular cAMP levels in response to LMJ07 treatment in CHO-CB1 cells by Cellkey label-free assays and homogeneous time-resolved fluorescence (HTRF). Additionally, we also confirmed the CB1R antagonistic efficacy of LMJ07 by detecting the content of Ca2+ in primary cultured hippocampal neuronal cells which could express CB1R with continuous fluorescence detection technology. Results: LMJ07 is a selective CB1R antagonist with high affinity, which can selectively antagonize receptor endocytosis induced by CB1RactivationIt’s affinity and antagonistic efficacy to CB1R were equal to those of rimonabant. In CHO-CB1 cells, LMJ07 (0.01-10μmol/L)could dose-dependently inverse the change in cytoskeletal as well as the increase in intracellular cAMP induced by CBR agonist Win55212-2. In the primary cultured hippocampal neuronal cells, LMJ07 (10 nmol/L-1 μmol/L) could block the increase in [Ca2+] induced by CB1R agonist Win55212-2. Conclusion: LMJ07 is a new selective CB1R antagonist, which shows equal affinity and antagonistic activity against CB1R as the widely accepted CB1R antagonist rimonabant. In addition, the combination of high content analysis (HCS) and Cellkey label-free assay provides a better research tool for rapid and high throughput screening of novel CB1R antagonists.

Recognition of environmental estrogen-like compounds based on fluorescent imaging analysis of ERα-EGFP nuclear granules formation / 军事医学

Objective To establish a quick method to probe the environmental estrogen-like compounds via fluorescent imaging analysis in high content analysis (HCA) technology.Methods HCA assays were performed to quantitatively in-vestigate the effect of collected environmental pollutants , including bisphenol A , nonylphenol , 1,2-phenylenediamine , 4-aminophenol, resorcinol, 3-aminophenol, 1,4-phenylenediamine and 2,5-diaminotoluene on nuclear granules formation ( Foci-formation) of estrogen receptor α( ERα)-EGFR( enhanced green fluorescent protein ) fusion-protein, which was se-lected as a research model in this study .The results were confirmed by the ERαtranscriptional activity by luciferase as-says.Results Compounds 1,2-phenylenediamine, 4-aminophenol, resorcinol, 3-aminophenol, 1,4-phenylenediamine and 2,5-diaminotoluene sulfate could not induce the ERα-EGFR nuclear granule formation.17-β-Estradiol, bisphenol A, or nonylphenol enhanced ERα-EGFR nuclear granule formation in a dose-dependent manner .The EC50 value was (4.17 ± 0.41) nmol/L, (1.48 ±1.79) μmol/L,or (3.70 ±0.78) μmol/L, respectively.The minimum detectable concentration was 1 nmol/L (17-β-estradiol)and 300 nmol/L (bisphenol A, nonylphenol).In luciferase tests, 17-β-estradiol, bisphe-nol A, or nonylphenol increased ERαtranscriptional activity in a dose-dependent manner ,and the EC50 value was (4.46 ±0.56) nmol/L, (2.31 ±0.21) μmol/L, or (6.60 ±0.94) μmol/L, respectively.The minimum detectable concentration was 3 nmol/L (17-β-estradiol), 300 nmol/L (bisphenol A),and 1 μmol/L (nonylphenol).Conclusion As an efficient method for ERαagonist identification , HCA assays based on the cell image phenotypes analysis can be used in quick recog -nition of environmental compounds with ER agonist-like activity.In all experimental compounds , only bisphenol A and non-ylphenol have a clear ER agonist-like activity .

Anti-tumor activity and mechanism of new microtubuIe-targeting compound WX-127-07 / 中国药理学与毒理学杂志

OBJECTIVE To evaluate the anti-tumor activities of WX-127-07,a new microtubule-tar-geting agent invitroand probe its molecular mechanism. METHODS The well-known microtubule-targe-ting anti-tumor drugs taxol,vincristine and anti-gout drug colchicine were used as positive controls. The anti-proliferation activity was examined in five different cell lines after treatment with WX-127-07(0.3 -300 nmol·L-1 )for 72 h by SRB assay. The cell cycle arrest profile was assayed by flow cytometry. The multiparameters of cytotoxicity,cell morphology,apoptosis and different signaling pathways related to tumorigenesis and inflammation were analyzed using the high content analysis platform. Tubulin tryptic digestion and competition inhibition assay for colchicine or vinblastine site were used to confirm the bind-ing site in microtubules at a molecular level. RESULTS All the tested compounds obviously inhibited the growth of A549,HepG2,HeLa,HLF and HUVEC cells. The lC50 values of WX-127-07 were 4.47±0.05, 5.18±0.08,4.90±0.19,4.10±0.16 and(5.04±0.08)nmol·L-1 respectively,lower than those of colchicine〔the lC50 values were 21. 17 ± 1. 22,14. 19 ± 0. 53,43. 80 ± 1. 64,145. 89 ± 10. 97 and( 27. 67 ± 1.79)nmol·L-1 ,respectively〕and those of vincristine〔the lC50 values were 16.51±0.36,16.76±0.33, 27.80±2.75,43.80±1.48 and(9.15±0.78)nmol·L-1 ,respectively〕,but were similar to or lower than those of taxol〔the lC50 values were 10. 68 ± 0. 61,12. 86 ± 0. 25,4. 81 ± 0. 61,102. 07 ± 15. 17 and( 3. 04 ± 0.12)nmol·L-1 ,respectively〕. High content multi-parameter analysis revealed that WX-127-07 induced a concentration-dependent microtubular depolymerization(P=0.0075)with the same pattern as colchicine and vincristine,but at a lower concentration. Both WX-127-07 and positive drugs could induce cell cycle arrest in A549 cells,increase nuclear membrane permeability and early signs of apoptosis in HepG2 cells,but neither cancer related pathways nor inflammation related pathways were affected. Microtubular competition inhibition assay showed that WX-127-07 inhibited the binding of colchicine with tubulin(P =0.0259). Tryptic digestion of tubulin-WX-127-07 premixture showed a similar electrophoretic band to that of tubulin-colchicine premixture. CONCLUSION WX-127-07 is a novel microtubule-depolymerizing agent with anti-proliferation activity and acting on the colchicine binding site.