Comparative Proteomics On The Mechanisms Of Neuropeptide Y-induced Apoptosis In Human Hepatocytes

Neuropeptide Y (NPY) is a36amid acid peptide abundantly expressed in the central andperipheral nervous system during development and adulthood. NPY is structurally similarto avian pancreatic polypeptide(APP) and peptide YY(PYY). NPY and norepinephrine(NE)are togather released from the sympathic nerves while the sympathic system is activated.NPY exerts its actions through binding to the NPY receptors which are members of the Gprotein-coupled receptor superfamily. In the brain, NPY regulates learning, memory andfeeding processes. In the peripheral, NPY regulates the circulatory system, reproductivesystem and digestive system.The liver is a vital organ and it is important for the metabolism.The live of mostmammalian species is richly innervated by afferent and efferent sympathetic andparasympathetic fibers. In human adult liver, NPY-ergic nerves are the most abundantpeptidergic nerve fibres.These nerve fibres are in close contact with the hepatic arteries,portal veins and bile ducts. These nerve fibres were found not only in the portal tracts butalso throughout the entire hepatic lobule, often extending to the centrilobular veins. Theyformed a network in hepatic lobules in close relation to hepatocytes. It is reported thatNPY may co-operate with norepinephrine in regulating hepatic blood flow. However, therole of NPY in hepatocyte function is largely unexplored.A novel proteomic approach based on2D-nano-LC-MS/MS is a high-performancetechnology. Firstly, the target proteins were hydrolyzed and the mixed peptides formatted.These peptides were subjected to2D-nano-LC separation followed by MS/MS analysis.SEQUEST software were used to analysis data.The technology was used for large-scaleprotein study due to its superior throughput and sensitivity.Given the lack of the information on the effect of NPY on L-02cells and themechanisms, our subject took the following research:1. The viability of different concentrations of NPY on L-02cells was measured byMTT.2. The apoptosis markers such as Bax, Bcl-2, Bad and caspase-3were detected byWestern Blot to evaluate NPY-induced apoptosis.3. The proteomic approach based on2D-nano-LC-MS/MS was applied to uncoverprotein alterations after NPY treatment of L-02cells and discovered the signaling of NPYinduced L-02cells apoptosis.4. The bioinformatic softwares were used to analysis data. We explored the proteins associated with apoptosis by literature retrieval and uncovered the signaling. Western blotwas used to verify the altered intensity of proteins which observed in2D-LC-MS/MSbased proteomic analysis.5. The inhibitor of mTOR rapamycin was used to observe the role of mTOR in NPYinduced apoptosis.The main results were as follows:1. Cytotoxicity was observed in the concentration of1nM and high concentration of10nM and100nM where viability was significantly decreased.2. Apoptosis markers were found the deregulation of Bcl-2(64%down-regulationed),Bax(103%up-regulationed), Bad(253%up-regulationed) and17kDa cleavedcaspase-3(197%up-regulationed).The results suggested that NPY induced L-02cellsapoptosis.3. Through the proteomic approach, we identified317proteins with NPY/CON(spectral count radio) more than5(80proteins) or less than0.2(237proteins). Further, wefound that mTOR, S6K, cytochrome c, and Hsp70had interconnected relationships.mTORcan directly modulated S6K and then phosphorylated the proapoptotic molecule Bad.Hsp70can regulate the release of cytochrome c through JNK, and cytochrome c reduced therelease of caspase9and caspase3and resulted in apoptosis.4. Further validation by Western blot showed that altered intensity of mTOR, S6K,cytochrome c and Hsp70matched well with the differences observed in2D-nano-LC-MS/MS based proteomic analysis.5. Rapamycin blocked the effect of NPY induced L-02cells apoptosis.In conclusion, NPY induced the L-02cells apoptosis in a dose-dependent manner. Theexpression of twelve proteins associated with apoptosis was altered while10nM NPYtreated L-02cells. And among of them, four proteins had closely interconnections. Wespeculated that mTOR/S6K/BAD, hsp70/cytochrome c/caspase-3might be two importantapoptotic pathway involved in NPY induced apoptosis. Further, the inhibitor of mTOR wasused and we found that rapamycin could block the effect of NPY induced apoptosis. Theseresults showed that the intrinsic apoptosis pathway was involved the NPY-inducedhepatocytes apoptosis and the comparative proteomics based on shotgun approach is avaluable tool for signals analysis.