Effects Of Simulated Microgravity On The Expression Of P-gp And Its Related Proteins

With the great progress of China space program in recent years,increasing manned space flight practices have brought significant opportunities for the develop ment of space medicine.Microgravity is an inextricable disadvantage during the space flight that induces extensively physiological changes in human body,such as body fluid shift,blood volume changes.This might affect pharmacokinetics.P-gp(Permea bility glycoprotein)is a drug-efflux protein located on the cell membrane.It is widely expressed in the drug-related organs and the blood-brain barrier that can maintain the brain environment stabilityis.It is closely related to the absorption,distribution,metabolism and excretion of drugs.Rat-tail suspension model was used to explore the P-gp under different simulated microgravity cycles in the following three aspects.1)The effect of expression of P-gp and MDR1 gene in the brain of simulated microgravityThe results showed that the P-gp expression was significantly increaseda by36.4 %(p<0.05)in simulated microgravity rats compared with the normal group.The expression of P-gp was increased in the 7 day simulated microgravity rats compared with the ground group(17.9 %),but there was no significant difference.P-gp expression in the rat brain showed tendency to restore the normal level at 14 and 21 day.The increase of protein expression was only 8.0 % and 5.45 % compared with the normal group,without significant difference.The expression of MDR1 was increased at 3,7 and 14 day after simulated microgravity,compared with the normal group.The expression of MDR1 was significantly increased in 3 day group.The expression of MDR1 was restored to the normal level at 21 day after tailsuspension.Furthermore the same trend was abserved between protein and gene expression.Acute stress may be caused in rats in short-term simulation of microgravity.The overexpression of P-gp can maintain the nervous system stability.With the prolongation of simulated microgravity and stress disappeared,rats were acclimated to microgravity.Then P-gp expression almost tended to return to normal level.2)Immunoprecipitation combined mass spectrometry were used to screen the interaction proteins with P-gpThe differential proteomic analysis of brain co-immunoprecipitation(CO-IP)complexes from different simulated microgravity periods was performed by usingnon-standard quantitative IBAQ proteomics techniques.66 differential proteins were identified from all of four groups.The clustering analysis of 66 different proteins was performed by using different bioinformatics tools PANTHER,DAVID,STRING.The results showd that most of the proteins were phosphatase or phosphodiesterase.Phosphorylation plays an important role in the regulation of P-gp pathway.In the biological process analysis,66 P-gp-interacting differential proteins were mainly enriched in the processes of drug response,potassium / calcium transport,glycoside response,ATP hydrolysis-coupled transport,cAMP catalytic process,etc.The cellular components were mainly distributed in the cell membrane,cytosol,exosomes,mitochondria,protein complexes,extracellular vesicles,etc.It can be mainly bind the calcium ion kinase,ATP,ubiquitin protein ligase,glycoprotein,misfolding protein binding,with guanylate kinase,cyclic nucleotide phosphodiesterase,ATPase activity in the molecular function.KEGG pathway analysis revealed that these proteins were mainly enriched in cGMP-PKG signaling pathway,cAMP signal pathway,gap junction,estrogen signaling pathway,calcium signaling pathway and glutamatergic synapse pathway.14 typical P-gp interacting differential proteins were found using the bioinform atics tools.The function of these 14 proteins was analyzed to explore its possible P-gp regulatory mechanisms and related signaling pathway use string software.In conclusions,all of the results showed that the protein may interact with P-gp in these pathways:(1)ATP energy supply of P-gp active transportation(2)The synthesis,transportation and degradation of P-gp in cells(3)cAMP/PKA-mediated phospho rylation of P-gp(4)Expression of P-gp in COX-2 cascade induced by glutamate.3)The validation of P-gp interacting proteins with Western-blot methodP-gp is an energy-dependent drug efflux protein.Significant interaction between Atp1b1 and P-gp was found by proteomic data showed that Atp1b1 might provide energy for active transport of P-gp.Atp1b1 was also verified by Western blot,which was consistent with mass spectrometry results.Thest results supported reliability of mass spectrometry data.