| Drug-induced liver injury is commonly one of the most reasons which novel drugs development failed and restrictly applied or retreated after marketing. Therefore, in the pre-marketing or post-marketing, it is vital to set up an effective, sensitive and specific in vitro model for DILI screening.Polygonum multiflorum as a tonified Chinese herbal medicine was widely used. The data of adverse effect of drugs showed that it had the risk of hepatotoxicity. Although some studies have been investigated throughout the world, the reasons of its toxic constituents are still unclear. Therefore, systematic research on toxic constituents and probably mechanism of P. multiflorum needs to be conducted.First, we conducted the comparision research on five liver cells model in vitro. Acetaminophen, carbamazepine, diclofenac sodium, isoniazid, sodium valproate, amiodarone hydrochloride, tetracycline hydrochloride, rifampicin, etoposide, labetalol hydrochloride, danazol triptolide, diosbulbin B and monocrotaline were chosen as DILI positive drugs. Bambuterol hydrochloride, buspirone hydrochloride and scopolamine butylbromide were referred as DILI negative drugs. We compared the L-02, HepG2, HepaRG and hiHeps cell lines and primary rat liver cells by CCK-8 assay, high-content screening assay and liver-related biochemical analysis. The results implied that the HepaRG cell line embraced the best ability of discriminating DILI, and the arrangement was HepaRG> L-02> HepG2= hiHeps> PRLs. HepaRG cell line showed more sensitivity on reactive oxygen species, mitochondrial damage and endoplasmic reticulum dysfunction with dose-dependent type compared to other liver cells. In the correlation analysis and significance testing, AST, LDH and MDH could characterize the effects of diclofenac sodium, buspirone hydrochloride and danazol on HepaRG cell line which implied as potential DILI-relevant biomarkers in the future.Then, we studied the toxic constituents derived from P. multiflorum which might cause liver injury. On one side, we systematically extracted and separated the rhizome of P. multiflorum, and acquired the 70% total ethanol parts and eight fractions. On the other side, we bought sixteen chemicals derived from P. multiflorum as screening targets. We studied the 70% total ethanol parts, eight fractions and sixteen chemical constituents by CCK-8 assay, UPLC-PAD quantification, and liver-related biochemical analysis. The results showed that Fr.4 was the toxic fraction of P. multiflorum which might induce liver injury. Gallic acid, resveratrol, emodin and rhein were the toxic constituents of P. multiflorum which exhibited the cytotoxic effects obviously. Combined with the features of L-02 cell injuries and chemical constituents’content analysis, we concluded that gallic acid could be one of the main toxic constituents of P. multiflorum. Besides, we found that GLDH and y-GT all reflected the hepatotoxicity of 70% total ethanol extracts and gallic acid.Last but not the least, we explored the preliminary mechanism of P. multiflorum induced liver injury. Cell cycle, cell apoptosis, high-content screening assay, two dimension electrophoresis and validation with molecular biology techniques were investigated thoroughly on HepaRG cell line treated with gallic acid at different doses at different times. The results were inferred that gallic acid didn’t infect the cell cycle significantly. Gallic acid significantly induced liver cells apoptosis in a dose-dependent manner. Gallic acid caused endoplasmic reticulum injury, reduced reactive oxygen species and calcium concentrations in the HepaRG cell line in a dose-dependent manner and time-dependent manner, and no significant lesions were observed in mitochondrial damage and neutral lipid metabolism disorders. When HepaRG cell line was treated with gallic acid at a dose of 0.5 mM at 24 h and 48 h, eleven differentiated expression proteins in all were identified together, in which six of them were in 24 h treatment and five of them were in 48 h treatment. Gene ontology and pathway analysis exhibited that the folding and processing of functional proteins in endoplasmic reticulum was the main course in 24 h treatment of gallic acid. In addition, the folding and processing of functional proteins in endoplasmic reticulum, the regulation and control of the precursor (including mRNA, pre-mRNA) of function proteins, inflammation and host defense were the main courses in 48 h treatment of gallic acid. Molecular biological techniques were applied and exhibited the consistency of protein expression and gene transcription of PPIA. Then, we postulated peptidyl-prolyl cis-trans isomerase A was the potential target of gallic acid which functionalized in the process of functional proteins in the endoplasmic reticulum.Conclusion:HepaRG cell line was proved to be a superior cell model (compared with other four liver cells) in DILI screening. AST, LDH and MDH were highly correlated with DILI drugs toxicity which could be a potent biomarker. Gallic acid was inferred to be one of the main toxic chemical constituents of P. multiflorum. We applied HepaRG cell line as a cell model to deeply explore the mechanism of liver injury by gallic acid. The outcome implied that gallic acid restrained the folding and processing of functional proteins which caused endoplasmic reticulum stress and generated liver cell apoptosis by repressing the biological course of transcription and expression of PPIA in a dose-dependent manner. |
PDF Full Text Request