Based On Chemical Proteomics Research On The Mechanism Of Action Of Cornus Loganin In Protecting Liver Injury In Mice

As one of the precious traditional Chinese medicinal plants in China,Corni Fructus（CF）has the effect of tonifying the liver and kidney and relieving astringency.Loganin,one of the main components of CF,has a variety of pharmacological activities,for instance,the protection of nerve damage,hyperglycemia,protection of liver damage caused by diabetes.However,its protective effects on liver injury and its underlying mechanism remain to be clarified.In this study,the experiments in vivo and vitro were performed to determine the significant protective effects of loganin from CF on acute liver injury induced by acetaminophen（APAP）in mice models,and chemical proteomics was used to explore the mechanism of loganin in the treatment of liver injury induced by acetaminophen.The molecular probe was synthesized and co-incubated with living human hepatocellular carcinoma cell line HepG2 to "capture"the targets bound to loganin.The protein was enriched and purified with the biotin-streptomavidin system,and then identified by mass spectrometry based on gel affinity proteomics.After data analysis,target proteins were screened,and GO and KEGG analysis were performed on the targets.The main results and conclusions are as follows:（1）Effect of loganin in CF on cell liver injury.HepG2 cells were taken as the main object of study.HepG2 cells were stimulated with 800 μM hydrogen peroxide（H2O2）for 18 h to induce liver injury model.At the same time,the HepG2 cells were treated with 40 μM and 80 μM of loganin to observe the morphological changes of cells and detect the cell viability,cell apoptosis and oxidative stress levels.The results showed that the cells in the control group adhered firmly to the wall and grew in clumps.However,in the APAP model group,the cell morphology significantly changed,which was manifested as the shrinkage of cell membrane,the decrease of adherent cells and the increase of cell debris.The cell shrinkage was obviously reduced and adhered to the wall better in the loganin groups,and the growth state was better than that in the model group.Flow cytometry results showed that the apoptosis rate of H2O2 model group was significantly increased,and loganin could protect the liver cell injury caused by H2O2 by reducing apoptosis.ROS test results showed that severe oxidative stress reaction occurred in the H2O2 group,whereas loganin could improve the oxidative stress response in a dose-dependent manner.（2）Protective effect of loganin from CF on acute liver injury in mice.APAP induced liver injury in mice model is adopted to intragastric administration of different doses of loganin,observe and record the change of the mice’s weight before and after the experiment,the detection of hepatic function indexes（ALT and AST）,liver redox indicators（SOD,GSH and CAT）,liver tissue pathological changes and the level of liver cell apoptosis,detection of liver cell toxicity by use of immunofluorescence CYP2E1 expression situation,combined with immunohistochemical detection of apoptosis related proteins Bax expression changes.The results showed that Loganin could significantly reduce the levels of ALT and AST,increase the levels of SOD,GSH and CAT,significantly reduce the expression of hepatocyte apoptosis and cytotoxic substance CYP2E1,and inhibit the expression of pro-apoptotic factor Bax.（3）Explore the protective mechanism of loganin on APAP-induced acute liver injury based on chemical proteomics.The terminyl acetylene was synthesized on loganin,and then the probe and HepG2 cells were co-incubated.After the loganin binds the target proteins,the labeled protein was enriched and purified by the functional groups of the probe.Finally,the captured targets were identified by mass spectrometry and other techniques.The results showed that the 100 μM loganin probe and HepG2 cells were incubated for 3 h and showed a good labeling effect.In order to avoid producing non-specific marker proteins,the parent compound was added in advance as a competitive inhibitor.The results showed that the synthesized probe was consistent with the target protein of the parent compound.1098 proteins in the control group,496 proteins in the probe group,918 proteins in the competition group,403 proteins in the three overlapping groups and 34 specific proteins in the probe group,such as CREB5,MP2K2 and Atg9a,were detected by the probe-biotin-streptavidin system.A total of 127 pathways were enriched,including the PI3K-Akt,HIF-1,FOXO,AMPK,and Autophagy signaling pathways.What’s more,all of these signaling pathways are associated with inhibiting oxidative stress and anti-apoptosis.To sum up,loganin can protect liver injury in mice induced by APAP by anti-oxidative stress and reducing apoptosis.The results provide the basic data and theoretical basis for the foundation and the core function of the mechanism of action of loganin,which can be used as quality evaluation and control of cornel medicinal materials to provide new insights and methods,and lay the foundation for the clinical application and the development and utilization of CF and industrial chain development.