Study On The Effects Of RCCS Simulated Microgravity On The Metabolomics Of Human HaCaT Cells

Objective The manned spaceflight is in the stage of rapid development,and there will be more and more astronauts going into space to carry out manned spaceflight missions.It is urgent to strengthen the medical supervision and medical insurance of astronauts.As an important and unavoidable factor in the space environment,microgravity induce a wide range of effects on organisms.The weightlessness has impacts on human keratinocyte cell according to the literature.Foreign scholars has found that weightlessness has impacts on human keratinocyte cell lines.Metabolomics is mainly used to explore molecular substances less than 150 kDa,quantitatively analyze related cellular metabolic produced in vivo and to find the related effects of metabolites with physiological and pathological changes.In this study,the rotary cell culture system(RCCS)was used to simulate the microgravity environment,and combined with UHPLC-LTQ-MS metabolomics detection to find the change of metabolism and metabolic pathways of human immortalized keratinocyte HaCaT cells under simulated microgravity environment,and aimed to provide a theoretical basis for the management of spaceflight related medical problems in the field of skin metabolism and wounds healing.Methods The rotary cell culture system(RCCS)was used to simulate the microgravity environment,and the 4th to 10 th generation HaCaT cells with good cell phase were cultured in vitro and divided randomly into simulated microgravity group(SMG)and normal gravity group(NG).Each group contained 6 samples,and the cells were collected after 1 d,2 d,and 3 d.The UHPLC-LTQ-MS techniques was used to detect and analyze the HaCaT cells samples.After obtaining the total ion chromatogram of the metabolite,the data was imported into the metabolomics processing software Progenesis QI for baseline filtration,peak identification,integration,retention time correction,peak alignment.And finally a data matrix was obtained which contained retention time,mass-to-charge ratio and peak intensity.Principal component analysis,partial least square-discriminant analysis and orthogonal partial least square-discriminant analysis were carried out then by using SIMCA-P 14.0 software.Principal component analysis and orthogonal partial least squares discriminant analysis were used for pattern recognition.Samples of variable importance projection(VIP)> 1 in the OPLS-DA model were selected for independent sample t-test to P <0.05 is the threshold to screen out potential biomarkers and analyze the metabolic pathways associated with it.Permutation testing was used to prevent over fitting.The results of the two methods were compared to find similarities and differences in biomarkers.Results(1)The results of LC/MS metabolism showed that there were 74 different metabolites in HaCaT cells under microgravity environment after 1 d culture,among which 16 were up-regulated and 58 down-regulated,and there were 89 different metabolites under microgravity environment after 2 d culture,among which 15 were up-regulated and 74 down-regulated.After 3 d culture,there were 100 different metabolites,of which 23 were up-regulated and 77 down-regulated.Forty nine metabolites expressed differently(VIP > 1 and P < 0.05)in 3 d were set as target metabolites.It was showed that the sphingosine,glutamate,and docosapentaenoic acid were down-regulated,and dehydrated sorbitol were up-regulated.(2)KEGG metabolite identification and pathway analysis were performed on the selected differential metabolites,and the results showed that the mainly metabolic activities involved were sphingolipids,propiophenone,protein synthesis,and amino acid.The related pathways were sphingolipids signal pathway,fork head box transcription factor O(FoxO)signaling pathway,and phospholipase D signaling pathway.Conclusion(1)The metabolomics technique based on UHPLC-LTQ-MS combined with multivariate statistical series has advantages of non-invasive,effective,rapid,and sensitive,and could provide a basis for the study of spaceflight related medical problems including stress injuries and repairment.(2)The metabolism of protein,glycolipid,citric acid cycle,phospholipid and amino acid of HaCaT cells were affected significantly under RCCS simulated weightlessness.(3)Sphingosine,docosapentaenoic acid,phosphatidylethanolamine and phosphatidyl serine are the potential biomarkers for prognosis of skin damage repair in weightless environment.