The Inhibition Mechanism Of Pinocembrin Against Citrus Green Mold In Proteomic

Flavonoids have diverse physiological activity and potential medicinal value, and C5-OH and C7-OH is the primary group of antimicrobial activity of their structure。 Previous studies have shown that pinocembrin significantly inhibited respiration of Penicillium and induce Penicillium italium into programmed cell death; respiratory investigation demonstrated that mitochondrial respiratory enzyme complexes I, IV and V of the electron transport chain was inhibited by pinocembrin, and the action site of pinocembrin might be in the mitochondrial respiratory chain enzymes, which resulted in cell energy metabolism diorder, and finally induced cell apoptosis, or necroptosis. In this study, we applied i TRAQ, molecular docking, MRM and q PCR to study the possible mechanism that why pinocembrin inhibit the pathogan. The main findings are as follows:1.After the treatment of pinocembrin, Penicillium oxidative phosphorylation pathway and TCA cycle produce a variety of differentially expressed proteins which are more than 1.2 times, It is the Enzyme complexes I, IV and V in the mitochondrial respiratory chain, and respiratory enzyme Succinate dehydrogenase,Malate dehydrogenase that have significant differential expressed proteins, leading to cell energy metabolism; the indicators in the process of death such as apoptosis-inducing factor protein, apoptosis of BAX and peptidylprolyl isomerase（CYPA） expression has changed significantly; in addition, the chaperone protein HSP70 with abnormal expression of more than 1.4 times which is stress induced protein, has multiple roles in maintaining protein structure.2. By computer aided design software MOE, respiratory chain proteins are homology modeled, then through the pines of molecular docking experiments, we can inquiry the docking possibility between enzyme complexes and Pinocembrin. Through homology modeling and molecular docking, we found that Unigene2720_CK₀A（Complexes V） has the best binding possibility with Pinocembrin.And proteins which have the binding possibility are concentrated in the mitochondrial respiratory chain protein complexes I and protein complexes V. 5-hydroxyl in A ring of Pinocembrin binding cavity and 88-Tyr residue of the receptor Unigene2720_CK form hydrogen bonding interaction; the C ring carbonyl of Pinocembrin and 82-Ala residue of the receptor Unigene2720_CK form hydrogenbonds;besides,78-Glu,77-Glu,73-Pro,75-Ser,79-Ala81-Leu,80-Asn,96-Glu,95-V al,97-GLy,98-Gln,25-Val residues involved ligand interaction with pinocembrin.3. By using the MRM technology, we can verify the differences in protein expression.The results showed that complexes I, II, IV, V are consistent with the trend of ITRAQ,but the performance is more remarkable; The q PCR results indicate that the m RNA expression level of complexes I, II, IV and V is not consistent with protein expression. Only the Unigene2707_CK₀A and Unigene14205_CK₀A are completely consistent at the m RNA expression and protein expression.4. By using z-VAD-fmk,Necrostatin-1 and Evans blue staining,we can study the style of the programmed cell death;z-VAD-fmk is a pan-caspase inhibitor（Pan sulfur cysteamine acid protease inhibitor）, and Necrostatin-1 is RIP1 specific inhibitor, from the experiment results we can see that the inhibition pan-caspase can not inhibit the process of Penicillium cell death, but the use of RIP1 inhibitor can reduce the pinocembrin induced death of Penicillium cell.So we speculate that pinocembrin induced the necroptosis, which is not depending on caspase in the process of cell death.