Abstract
Background
Routine coagulation tests are not widely accepted diagnostic criteria of trauma-induced hypercoagulopathy (TIH) due to insensitivity. Lymphatic vessels drain approximately 10% of the interstitial fluid into the lymphatic system and form lymph.
Subjective
The purpose of this study was to identify the potential lymph biomarkers for TIH.
Methods
Eighteen male Sprague-Dawley rats were randomly assigned to the sham (non-fractured rats with sham surgery and vehicle treatment), the VEH (fractured rats with vehicle treatment) and the CLO (fractured rats with clopidogrel treatment) group. Thoracic duct lymph was obtained to perform proteomics and untargeted metabolomics.
Results
A total of 1207 proteins and 16,695 metabolites were identified. The top 5 GO terms of lymph proteomics indicated that oxidative stress and innate immunity were closely associated with TIH and antithrombotic therapy. The top 5 GO terms of lymph metabolomics showed that homocystine and lysophosphatidylcholine were the differential expressed metabolites (DEMs) between the sham and VEH groups, while cholic acid, docosahexaenoic acid, N1-Methyl-2-pyridone-5-carboxamide, isoleucine and testosterone are the DEMs between the VEH and CLO group.
Conclusions
This study presents the first proteomic and metabolomic profiling of lymph after TIH and antithrombotic therapy, and predicts the possible lymph biomarkers for TIH.
Introduction
Trauma-induced coagulopathy (TIC) is a dynamic and complex coagulation dysfunction, characterized by hypocoagulability in the early hours, resulting in hemorrhagic shock, and hypercoagulability following the hypocoagulable state, resulting in venous thromboembolism and multiple organ failure [1]. As a result, TIC is regarded as an independent risk factor for poor prognosis among trauma patients [2,3,4,5]. Most studies on TIC mainly focus on hypocoagulability, however, 22.2–85.1% of trauma patients within days of injury develop trauma-induced hypercoagulopathy (TIH). TIH raises the risk of thrombotic events and mortality by 2–4 times [3, 6, 7]. Hence, discovering the potential biomarkers of TIH is essential for medical practice [4].
Routine coagulation tests, such as prothrombin time (PT), activated partial prothrombin time (APTT), D-dimers (DD) and fibrinogen (or fibrin) degradation products (FDPs), are suggested as the diagnostic indicators of TIH. However, these are not widely accepted diagnostic criteria of TIH due to the insensitivity [4]. Lymphatic vessels, as the second circulatory pathway, drain approximately 10% of the interstitial fluid into the lymphatic system and form lymph [8]. At the end of lymphatic draining pathways, most lymph is returned to the circulatory system through the thoracic duct. In contrast to blood vessels, lymphatic vessels play a vital role in macromolecule transport, lipid metabolism and waste clearance [9,10,11,12,13]. Previous research also demonstrates that lymph is not a plasma ultrafiltrate and presents some unique substances [14]. Therefore, to compensate for the shortcomings of serum and plasma, lymph might be a potential and valuable source of novel biomarkers for TIH.
Our previous investigation found that lymphatic drainage insufficiency was caused by a significant amount of lymphatic platelet thrombosis (LPT) blocking lymphatic vessels on the first day post -traumatic fracture [14]. Approximately 155 proteins were identified as uniquely existing in the lymph, including extracellular matrix-related proteins, actin cytoskeleton reorganization markers, and pancreatic proteins [14]. In our study, O88767 (Parkinson disease protein 7 homolog, Park7), P05197 (Elongation factor 2, Eef2), P07824 (Arginase-1, Arg1), P11232 (Thioredoxin, Txn), P30152 (Neutrophil gelatinase-associated lipocalin, Lcn2), P31044 (Phosphatidylethanolamine-binding protein 1, Pebp1), Q01129 (Decorin, Dcn), Q63716 (Peroxiredoxin-1, Prdx1), P07943 (Aldo-keto reductase family 1 member B1, Akr1b1) are the up-regulated proteins enriched in top 5 GO terms of BP (Fig. 1B). Monika’s proteomic data [14] suggests that wounded patients’ lymph, rather than plasma, may include Park7, Pebp1, and Prdx1. PARK7 is abundantly expressed in the brain, skeletal muscle, and adrenal gland (BioProject: PRJNA280600) and regulates mitochondrial dysfunction and oxidative stress [24, 25]. PEBP1, a tiny scaffold protein, inhibits protein kinase cascades and promotes ferroptosis cell death by binding with 15-lipoxygenases (15-LO) to produce hydroperoxy-PE [26]. Prdx1 is an enzyme with several functions, including oxidative defense, aging, inflammation, redox signaling, cell cycle, and carcinogenesis [27]. This finding revealed that Park7, Pebp1, and Prdx1 in collected lymph were possible diagnostic markers of injured individuals.
TIH is caused by a complex interaction of numerous processes, involving vascular endothelial injury, platelet hyperactivity, excessive release of procoagulants, hyperfibrinogenemia, anticoagulant pathways impairment, and fibrinolysis shutdown [4]. Immunoregulatory platelet dysfunction is the main pathological mechanism of TIH, despite the platelet count is at a normal level [1, 28]. On the one hand, injury-induced platelet activation promotes platelets to bind with leukocytes, forms platelet-leukocyte aggregates and activates innate immune response [29, 30]. On the other hand, activated neutrophils and macrophages release extracellular traps to simulate platelet aggregation and thrombin formation [1]. Therefore, we assumed that thrombolysis therapy reduced not just TIH but also excessive immune response. Figure 2C shows that the lymph of the CLO group had much lower levels of pro-inflammatory and immune-associated proteins, as confirmed by the top 5 GO terms of BP. Except for Q5WRG2 (Angiogenin, Ang) and Q6P6T1 (Complement C1s subcomponent, C1s) were previously reported to be presented in both plasma and lymph after trauma [14], we discovered following new protein molecules of lymph, including P50115 (Protein S100-A8, S100a8), P52925 (High mobility group protein B2, Hmgb2), F7FP65 (Retinoic acid receptor responder protein 2, Rarres2), D3ZWD6 (Complement C8 alpha chain, C8a), P01836 (Ig kappa chain C region, A allele), P00697 (Lysozyme C-1, Lyz1), G3V9C7 (Histone H2B, Hist1h2bk), M0R485 (Peptidoglycan recognition protein 2, Pglyrp2), P55314 (Complement component C8 beta chain, C8b), Q91YB6 (Complement inhibitory factor H, Cfh), D3ZPI8 (Complement C8 gamma chain, C8g). Although these down-regulated proteins are linked to inflammatory and immunological responses, it’s unclear if they’re just found in lymph fluid. Hence, more study is needed to identify particular lymph biomarkers following TIH or TIH plus thrombotic treatment.
Insight of lymph metabolome
The metabolomic researches of trauma-induced hemorrhagic shock are continuously reported [31,32,33,34,35], while the metabolomic changes of TIH are still little known. We depicted the lymph metabolomics of TIH and the potential pharmaceutical effect of antithrombotic therapy.
Compared to the sham group, homocystine, the up-regulated metabolite in the lymph of the VEH group, is enriched in cysteine and methionine metabolism (Fig. 3B). Homocystine is synthesized via transmethylation of methionine and enzymatic reaction [36]. Homocystine is regarded as an independent risk factor for thrombotic disorders and cardiovascular disease [37,38,39]. In addition, cysteine and methionine metabolism is also significantly increased in the plasma metabolome of injured animals and patients [31,32,33,34,35]. This result indicates that homocystine of lymph and plasma might be a potential biomarker of TIH.
Lysophosphatidylcholine is the main active component of oxidized low-density lipoprotein and can expand inflammation and exacerbate diseases by inducing the migration of lymphocytes and macrophages to produce pro-inflammatory cytokines [40,41,42,43]. Koji et al. detected lysophosphatidylcholine significantly increased in the mesenteric lymph on the model of trauma-induced hemorrhagic shock [43]. To our surprise, compared to the sham group, lysophosphatidylcholine is the down-regulated metabolite in the lymph of the VEH group and enriched in choline metabolism in cancer and glycerophospholipid metabolism (Fig. 3C). This result is opposite to that of previous research probably due to different animal models. Additionally, compared to the VEH group, lysophosphatidylcholine was found to significantly increase in the lymph of the CLO group (Fig. 4B). We inferred that (1) the sham, VEH, and CLO groups induced lysophosphatidylcholine generation due to operation; (2) In VEH group, lysophosphatidylcholine was accumulated in the injured sites and failed to be transported into thoracic duct due to the blockage of lymphatic vessels by lymphatic platelet thrombosis; (3) In CLO group, unblocked lymphatic vessels drained increased lysophosphatidylcholine of injured sites into thoracic duct.
Cholic acid is the main component of bile acids in the human body and has versatile roles in maintaining bile acid homeostasis, alleviating metabolic inflammation, and protecting neural injury [44]. Compared to the VEH group, cholic acid, enriched in bile secretion and primary bile acid biosynthesis, is significantly up-regulated in the CLO group. This result suggests that lymphatic platelet thrombolysis not only alleviates TIH but also improves systematical pathology.
Docosahexaenoic acid is an acknowledged neuroprotective and anti-inflammatory agent with multiple functions of alleviating endoplasmic reticulum and oxidative stress and regulating autophagy [45,46,47,48]. N1-Methyl-2-pyridone-5-carboxamide is one of the major metabolites of nicotinamide and is elevated in renal failure, vascular inflammation, chronic ulcerative colitis, and asthma [49,50,51,52]. Compared to the VEH group, docosahexaenoic acid, and N1-Methyl-2-pyridone-5-carboxamide are decreased in the CLO group (Fig. 4C). This is probably because antithrombotic therapy improves lymphatic transport of docosahexaenoic acid and N1-Methyl-2-pyridone-5-carboxamide, thus inhibiting their accumulation at fracture sides and decreasing oxidative response and nicotinamide metabolism.
Isoleucine is a kind of branchedchain and an essential amino acid for humans and animals [53]. Isoleucine plays diverse roles in physiological functions and metabolic pathways, including maintaining the growth and development of animals, enhancing immunity, regulating glucose transportation, and stimulating protein synthesis [53,54,55,56]. Testosterone is synthesized and secreted by testicular Leydig cells and the adrenal cortex and is regulated by the hypothalamic-pituitary-gonadal axis [57]. Testosterone plays irreplaceable roles in the growth and development of the human body, maintaining musculoskeletal homeostasis and regulating post-traumatic stress disorder [58,59,60]. Although little literature reports their direct relationships with trauma, the information above seems to imply that higher levels of isoleucine and testosterone are beneficial for patients with trauma. However, the data of lymph metabolomics indicated that the levels of isoleucine and testosterone in the CLO group were significantly lower than VEH group. Therefore, general analysis suggests that (1) thrombolysis therapy is good for relieving TIH, circulatory system disorder, and inflammatory and oxidative response; (2) but might lead to side effects, such as metabolic and endocrine disorders. In a word, these DEMs above are potential and sensitive biomarkers of TIH patients with antithrombotic therapy.
Limitations and outlook
TIH is a dynamic, sequential pathophysiological process. Our work focused on a single time point of TIH to evaluate the constitutive alterations of lymph using integrated proteome and metabolome. (2) To distinguish the different and unique biomarkers between blood-derived and lymph-derived samples, a time course co-analysis of plasma and lymph muti-omics in trauma patients is mandatory. (3) Because obtaining lymph is challenging, the previous collection of lymph in trauma patients is under the help of anesthesiologists and intensive care physicians. Multidisciplinary collaboration in clinical trials of trauma patients is required to investigate the relationship between the possible biomarkers and the prognosis of TIH in order to validate the sensitivity and specificity of screening lymph biomarkers in this study. (4) Given that men have a higher risk of traumatic fractures and TIH [61,62,63,64], we only employed young male rats in this experiment. To increase the study’s relevance and effect, both genders are suggested to be incorporated into the study design.
Data availability
1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD047692.2. The metabolomic data have been deposited to the MetaboLights (https://www.ebi.ac.uk/metabolights/) with the dataset identifier MTBLS9145.
References
Moore EE, Moore HB, Kornblith LZ, Neal MD, Hoffman M, Mutch NJ, Schöchl H, Hunt BJ, Sauaia A. Trauma-induced coagulopathy. Nat Rev Dis Primers. 2021;7(1):30.
Xu SX, Wang L, Zhou GJ, Zhang M, Gan JX. Risk factors and clinical significance of trauma-induced coagulopathy in ICU patients with severe trauma. Eur J Emerg Med. 2013;20(4):286–90.
Moore HB, Moore EE, Liras IN, et al. Targeting resuscitation to normalization of coagulating status: Hyper and hypocoagulability after severe injury are both associated with increased mortality[ J]. Am J Surg. 2017;214(6):1041–5.
Song JC, Yang LK, Zhao W, Zhu F, Wang G, Chen YP, Li WQ, Chinese People’s Liberation Army Professional Committee of Critical Care Medicine and Chinese Society of Thrombosis. Hemostasis and Critical Care, Chinese Medicine Education Association. Chinese expert consensus on diagnosis and treatment of trauma-induced hypercoagulopathy. Mil Med Res. 2021;8(1):25.
Spahn DR, Bouillon B, Cerny V, Duranteau J, Filipescu D, Hunt BJ, et al. The European guideline on management of major bleeding and coagulopathy following trauma: fifth edition. Crit Care. 2019;23(1):98.
Branco BC, Inaba K, Ives C, et al. Thromboelastogram evaluation of the impact of hypercoagulability in trauma patients[ J]. Shock. 2014;41(3):200–7.
Brill JB, Badiee J, Zander AL, et al. The rate of deep vein thrombosis doubles in trauma patients with hypercoagulable thromboelastography[ J]. J Trauma Acute Care Surg. 2017;83(3):413–9.
Olszewski WL. The lymphatic system in body homeostasis: physiological conditions. Lymphat Res Biol. 2003;1(1):11–21. discussion 21 – 4.
Petrova TV, Koh GY. Biological functions of lymphatic vessels. Science. 2020;369(6500):eaax4063.
Petrova TV, Koh GY. Organ-specific lymphatic vasculature: from development to pathophysiology. J Exp Med. 2018;215(1):35–49.
Oliver G, Kipnis J, Randolph GJ, Harvey NL. The lymphatic vasculature in the 21st Century: Novel Functional roles in Homeostasis and Disease. Cell. 2020;182(2):270–96.
Alitalo K. The lymphatic vasculature in disease. Nat Med. 2011;17(11):1371–80.
Wang Y, Oliver G. Current views on the function of the lymphatic vasculature in health and disease. Genes Dev. 2010;24(19):2115–26.
Dzieciatkowska M, D’Alessandro A, Moore EE, Wohlauer M, Banerjee A, Silliman CC, Hansen KC. Lymph is not a plasma ultrafiltrate: a proteomic analysis of injured patients. Shock. 2014;42(6):485–98.
Wang YJ, Zheng Y, Cong L, Wang P, Zhao L, **ng L, Liu J, Xu H, Li N, Zhao Y, Shi Q, Liang Q. Lymphatic platelet thrombosis limits bone repair by precluding lymphatic transporting DAMPs. Res Sq [Preprint]. 2023 Nov 14:rs.3.rs-3474507.
Boyd M, Risovic V, Jull P, Choo E, Wasan KM. A stepwise surgical procedure to investigate the lymphatic transport of lipid-based oral drug formulations: Cannulation of the mesenteric and thoracic lymph ducts within the rat. J Pharmacol Toxicol Methods. 2004 Mar-Apr;49(2):115–20.
Di Nisio M, van Es N, Büller HR. Deep vein thrombosis and pulmonary embolism. Lancet. 2016;388(10063):3060–73.
Goldhaber SZ, Bounameaux H. Pulmonary embolism and deep vein thrombosis. Lancet. 2012;379(9828):1835–46.
Bonnarens F, Einhorn TA. Production of a standard closed fracture in laboratory animal bone. J Orthop Research: Official Publication Orthop Res Soc. 1984;2:97–101.
Huang L, Harvie G, Feitelson JS, Gramatikoff K, Herold DA, Allen DL, Amunngama R, Hagler RA, Pisano MR, Zhang WW, Fang X. Immunoaffinity separation of plasma proteins by IgY microbeads: meeting the needs of proteomic sample preparation and analysis. Proteomics 5: 3314 – 3328, 2005.
Ma J, Chen T, Wu S, Yang C, Bai M, Shu K, Li K, Zhang G, ** Z, He F, Hermjakob H, Zhu Y. iProX: an integrated proteome resource. Nucleic Acids Res. 2019;47(D1):D1211–7.
Chen T, Ma J, Liu Y, Chen Z, **ao N, Lu Y, Fu Y, Yang C, Li M, Wu S, Wang X, Li D, He F, Hermjakob H, Zhu Y. iProX in 2021: connecting proteomics data sharing with big data. Nucleic Acids Res. 2022;50(D1):D1522–7.
Meng Z, Veenstra TD. Proteomic analysis of serum, plasma, and lymph for the identification of biomarkers. Proteom Clin Appl. 2007;1(8):747–57.
Lind-Holm Mogensen F, Scafidi A, Poli A, Michelucci A. PARK7/DJ-1 in microglia: implications in Parkinson’s disease and relevance as a therapeutic target. J Neuroinflammation. 2023;20(1):95.
Rønning SB, Andersen PV, Pedersen ME, Hollung K. Primary bovine skeletal muscle cells enters apoptosis rapidly via the intrinsic pathway when available oxygen is removed. PLoS ONE. 2017;12(8):e0182928.
Wenzel SE, Tyurina YY, Zhao J, St Croix CM, Dar HH, Mao G, Tyurin VA, Anthonymuthu TS, Kapralov AA, Amoscato AA, Mikulska-Ruminska K, Shrivastava IH, Kenny EM, Yang Q, Rosenbaum JC, Sparvero LJ, Emlet DR, Wen X, Minami Y, Qu F, Watkins SC, Holman TR, VanDemark AP, Kellum JA, Bahar I, Bayır H, Kagan VE. PEBP1 wardens ferroptosis by enabling lipoxygenase generation of lipid death signals. Cell. 2017;171(3):628–e64126.
Wu M, Deng C, Lo TH, Chan KY, Li X, Wong CM. Peroxiredoxin, Senescence, and Cancer. Cells. 2022;11(11):1772.
Vogel S, Bodenstein R, Chen Q, Feil S, Feil R, Rheinlaender J, Schäffer TE, Bohn E, Frick JS, Borst O, Münzer P, Walker B, Markel J, Csanyi G, Pagano PJ, Loughran P, Jessup ME, Watkins SC, Bullock GC, Sperry JL, Zuckerbraun BS, Billiar TR, Lotze MT, Gawaz M, Neal MD. Platelet-derived HMGB1 is a critical mediator of thrombosis. J Clin Invest. 2015;125(12):4638–54.
Vulliamy P, Kornblith LZ, Kutcher ME, Cohen MJ, Brohi K, Neal MD. Alterations in platelet behavior after major trauma: adaptive or maladaptive? Platelets. 2021;32(3):295–304.
Tweardy DJ, Khoshnevis MR, Yu B, Mastrangelo MA, Hardison EG, López JA. Essential role for platelets in organ injury and inflammation in resuscitated hemorrhagic shock. Shock. 2006;26(4):386–90.
Kinross JM, Alkhamesi N, Barton RH, Silk DB, Yap IK, Darzi AW, Holmes E, Nicholson JK. Global metabolic phenoty** in an experimental laparotomy model of surgical trauma. J Proteome Res. 2011;10(1):277–87.
D’Alessandro A, Moore HB, Moore EE, Wither M, Nemkov T, Gonzalez E, Slaughter A, Fragoso M, Hansen KC, Silliman CC, Banerjee A. Early hemorrhage triggers metabolic responses that build up during prolonged shock. Am J Physiol Regul Integr Comp Physiol. 2015;308(12):R1034–44.
Peltz ED, D’Alessandro A, Moore EE, Chin T, Silliman CC, Sauaia A, Hansen KC, Banerjee A. Pathologic metabolism: an exploratory study of the plasma metabolome of critical injury. J Trauma Acute Care Surg. 2015;78(4):742–51.
D’alessandro A, Nemkov T, Moore HB, Moore EE, Wither M, Nydam T, Slaughter A, Silliman CC, Banerjee A, Hansen KC. Metabolomics of trauma-associated death: shared and fluid-specific features of human plasma vs lymph. Blood Transfus. 2016;14(2):185–94.
Cohen MJ, Erickson CB, Lacroix IS, Debot M, Dzieciatkowska M, Schaid TR, Hallas MW, Thielen ON, Cralley AL, Banerjee A, Moore EE, Silliman CC, D’Alessandro A, Hansen KC. Trans-Omics analysis of post injury thrombo-inflammation identifies endotypes and trajectories in trauma patients. bioRxiv [Preprint]. 2023 Sep 9:2023.08.16.553446.
Froese DS, Fowler B, Baumgartner MR. Vitamin B12, folate, and the methionine remethylation cycle-biochemistry, pathways, and regulation. J Inherit Metab Dis. 2019;42(4):673–85.
Calderón-Larrañaga A, Saadeh M, Hooshmand B, Refsum H, Smith AD, Marengoni A, Vetrano DL. Association of Homocysteine, Methionine, and MTHFR 677C > T polymorphism with rate of Cardiovascular Multimorbidity Development in older adults in Sweden. JAMA Netw Open. 2020;3(5):e205316.
Yang M, Smith BC. Cysteine and methionine oxidation in thrombotic disorders. Curr Opin Chem Biol. 2023;76:102350.
Tøndel BG, Morelli VM, Hansen JB, Braekkan SK. Risk factors and predictors for venous thromboembolism in people with ischemic stroke: a systematic review. J Thromb Haemost. 2022;20(10):2173–86.
Liu P, Zhu W, Chen C, Yan B, Zhu L, Chen X, Peng C. The mechanisms of lysophosphatidylcholine in the development of diseases. Life Sci. 2020;247:117443.
Foster R, Jung J, Farooq A, McClung C, Ripsch MS, Fitzgerald MP, White FA. Sciatic nerve injury induces functional pro-nociceptive chemokine receptors in bladder-associated primary afferent neurons in the rat. Neuroscience. 2011;183:230–7.
Ryborg AK, Deleuran B, Søgaard H, Kragballe K. Intracutaneous injection of lysophosphatidylcholine induces skin inflammation and accumulation of leukocytes. Acta Derm Venereol. 2000 Jul-Aug;80(4):242–6.
Morishita K, Aiboshi J, Kobayashi T, Mikami S, Yokoyama Y, Ogawa K, Yokota H, Otomo Y. Lipidomics analysis of mesenteric lymph after trauma and hemorrhagic shock. J Trauma Acute Care Surg. 2012;72(6):1541–7.
Chiang JYL, Ferrell JM. Bile acid receptors FXR and TGR5 signaling in fatty liver diseases and therapy. Am J Physiol Gastrointest Liver Physiol. 2020;318(3):G554–73.
Yin Y, Sun G, Li E, Kiselyov K, Sun D. ER stress and impaired autophagy flux in neuronal degeneration and brain injury. Ageing Res Rev. 2017;34:3–14.
Marinelli S, Vacca V, De Angelis F, Pieroni L, Orsini T, Parisi C, Soligo M, Protto V, Manni L, Guerrieri R, Pavone F. Innovative mouse model mimicking human-like features of spinal cord injury: efficacy of docosahexaenoic acid on acute and chronic phases. Sci Rep. 2019;9(1):8883.
Descorbeth M, Figueroa K, Serrano-Illán M, De León M. Protective effect of docosahexaenoic acid on lipotoxicity-mediated cell death in Schwann cells: implication of PI3K/AKT and mTORC2 pathways. Brain Behav. 2018;8(11):e01123.
Oliver JM, Jones MT, Kirk KM, Gable DA, Repshas JT, Johnson TA, Andréasson U, Norgren N, Blennow K, Zetterberg H. Effect of Docosahexaenoic Acid on a biomarker of Head Trauma in American Football. Med Sci Sports Exerc. 2016;48(6):974–82.
Kelly RS, Sordillo JE, Lasky-Su J, Dahlin A, Perng W, Rifas-Shiman SL, Weiss ST, Gold DR, Litonjua AA, Hivert MF, Oken E, Wu AC. Plasma metabolite profiles in children with current asthma. Clin Exp Allergy. 2018;48(10):1297–304.
Ferrell M, Wang Z, Anderson JT, Li XS, Witkowski M, DiDonato JA, Hilser JR, Hartiala JA, Haghikia A, Cajka T, Fiehn O, Sangwan N, Demuth I, König M, Steinhagen-Thiessen E, Landmesser U, Tang WHW, Allayee H, Hazen SL. A terminal metabolite of niacin promotes vascular inflammation and contributes to cardiovascular disease risk. Nat Med. 2024;30(2):424–34.
Zhou R, Huang Y, Tian C, Yang Y, Zhang Z, He K. Coptis chinensis and Berberine Ameliorate Chronic Ulcerative Colitis: an Integrated Microbiome-Metabolomics Study. Am J Chin Med. 2023;51(8):2195–220.
Rutkowski B, Slominska E, Szolkiewicz M, Smolenski RT, Striley C, Rutkowski P, Swierczynski J. N-methyl-2-pyridone-5-carboxamide: a novel uremic toxin? Kidney Int Suppl. 2003;(84):S19–21.
Mao X, Gu C, Ren M, Chen D, Yu B, He J, Yu J, Zheng P, Luo J, Luo Y, Wang J, Tian G, Yang Q. l-Isoleucine administration alleviates Rotavirus infection and Immune Response in the weaned piglet model. Front Immunol. 2018;9:1654.
Fehlbaum P, Rao M, Zasloff M, Anderson GM. An essential amino acid induces epithelial beta -defensin expression. Proc Natl Acad Sci U S A. 2000;97(23):12723–8.
Zhao J, Feng L, Liu Y, Jiang W, Wu P, Jiang J, Zhang Y, Zhou X. Effect of dietary isoleucine on the immunity, antioxidant status, tight junctions and microflora in the intestine of juvenile Jian carp (Cyprinus carpio var. Jian). Fish Shellfish Immunol. 2014;41(2):663–73.
Zhang S, Zeng X, Ren M, Mao X, Qiao S. Novel metabolic and physiological functions of branched chain amino acids: a review. J Anim Sci Biotechnol. 2017;8:10.
Aghazadeh Y, Zirkin BR, Papadopoulos V. Pharmacological regulation of the cholesterol transport machinery in steroidogenic cells of the testis. Vitam Horm. 2015;98:189–227.
Feklicheva I, Boks MP, de Kloet ER, Chipeeva N, Maslennikova E, Pashkov A, Korobova S, Komelkova M, Kuznetsova Y, Platkovski P, Mamonova M, Sidorenko O, Vasilenko T, Tseilikman O, Tseilikman V. Biomarkers in PTSD-susceptible and resistant veterans with war experience of more than ten years ago: FOCUS ON cortisol, thyroid hormones, testosterone and GABA. J Psychiatr Res. 2022;148:258–63.
Kelly DM, Jones TH. Testosterone: a metabolic hormone in health and disease. J Endocrinol. 2013;217(3):R25–45.
Bandeira L, Silva BC, Bilezikian JP. Male osteoporosis. Arch Endocrinol Metab. 2022;66(5):739–47.
Van Gent JM, Calvo RY, Zander AL, Olson EJ, Sise CB, Sise MJ, Shackford SR. Risk factors for deep vein thrombosis and pulmonary embolism after traumatic injury: a competing risks analysis. J Trauma Acute Care Surg. 2017;83(6):1154–60.
Brinker MR, O’Connor DP. The incidence of fractures and dislocations referred for orthopaedic services in a capitated population. J Bone Joint Surg Am. 2004;86(2):290–7.
Wang H, Zhang Y, **ang Q, Wang X, Li C, **ong H, Zhou Y. Epidemiology of traumatic spinal fractures: experience from medical university-affiliated hospitals in Chongqing, China, 2001–2010. J Neurosurg Spine. 2012;17(5):459–68.
Mills LA, Simpson AH. The relative incidence of fracture non-union in the Scottish population (5.17 million): a 5-year epidemiological study. BMJ Open. 2013;3(2):e002276.
Acknowledgements
We thank to Professor Lian** **ng, from University of Rochester Medical Center, for guidance on lymphatic biology, and Professor Junling Liu, from Shanghai Jiao Tong University School of Medicine, for platelet biology. We thank the Shanghai Luming Biological Technology Co., LTD (Shanghai, China) for providing integrated proteomic and metabolomic services.
Funding
This work was sponsored by research grants from National Natural Science Foundation (82174407 to YJZ), The Inheritance and Innovation Team Project of National Traditional Chinese Medicine (ZYYCXTD-C-202202 to WYJ), State Administration of Traditional Chinese Medicine Qi Huang Scholar to WYJ and Shanghai Top Priority Research Center construction project (No. 2022ZZ01009 for Yong-jun Wang).
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YJW, YKZ conceived and designed the study; YKZ, PYW, LC performed the experiments and analyzed the data; YKZ drafted the manuscript; YJW, YJZ, QS revised the manuscript. All authors have approved the final version of the manuscript and have agreed to be accountable for all aspects of the work.
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12959_2024_634_MOESM2_ESM.tif
Supplementary Material 2. Supplemental Fig. 1 Scheme of the methodology. 18 male Sprague Dawley rats were randomly assigned to three groups, respectively sham group (6 non-fractured rats with sham surgery and vehicle-treated ), vehicle group (6 fractured rats with vehicle-treated), and clopidogrel group (6 fractured rats with clopidogrel-treated). Thoracic duct lymph on 24 h post-surgery was collected and centrifuged, the supernatant of lymph was detected by integrated proteomics and metabolomics to comprehensively describe the lymph profile of TIH. DEPs: differentially expressed proteins, DEMs: differentially expressed metabolites.
12959_2024_634_MOESM3_ESM.tif
Supplementary Material 3. Supplemental Fig. 2 Overview of proteomic data of lymph. (A) The number of proteins corresponding to different molecular weight distributions. (B) PCA of identified proteins in Sham, VEH, and CLO groups. (C) Hierarchical clustering dendrogram of sample Euclidean distance in Sham, VEH, and CLO groups. (D) The number of up-regulated and down-regulated DEPs in different groups. (E) Venn plot of the DEPs of lymph in different groups.
12959_2024_634_MOESM4_ESM.tif
Supplementary Material 4. Supplemental Fig. 3 Overview of metabolomic data of lymph. (A) Metabolites intensity distribution of QC samples. (B) The proportion of the identified metabolites in each chemical classification. (C) PCA plot of all samples of lymph. (D) OPLS-DA analysis of lymph. (E) Permutation analysis of lymph. To validate OPLS-DA mode, a cross-validation plot was analyzed by UPLC-Q-TOF/MS-based metabonomic data with 7-fold cross-validation and 200 times response permutation testing. (F) The number of up-regulated and down-regulated DEMs in different groups. (G) Venn plot of the DEMs of lymph in different groups.
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Zheng, Y., Wang, P., Cong, L. et al. Integrated proteomic and metabolomic profiling of lymph after trauma-induced hypercoagulopathy and antithrombotic therapy. Thrombosis J 22, 59 (2024). https://doi.org/10.1186/s12959-024-00634-3
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DOI: https://doi.org/10.1186/s12959-024-00634-3