Endothelial Glycocalyx Degradation Is Associated With Early Organ Impairment in Multiple Trauma Patients

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Trauma remains a leading cause of morbidity and mortality worldwide and in China mainland 1,2 . Organ function impairments are common in trauma, especially in multiple traumas 3 . Endothelial glycocalyx (EG) is a layer of gel-like macromolecules widely distributed on the surface of vascular endothelium, mainly composed of proteoglycans (PGs) and glycosaminoglycans (GAGs). PGs, as the core proteins, are anchored on the surface of vascular endothelium, and the side chains of GAGs are covalently connected to it 4 . Highly sulfated GAGs side-chains are negatively charged and have electrical effects on plasma protein components such as albumin, brinogen, bronectin, thrombomodulin, antithrombin-, peroxidase, and cell adhesion molecules 5 . EG maintains vascular homeostasis by regulating vascular tension and permeability, inhibiting thrombosis in microvessels and regulating leukocyte adhesion on endothelial cell surface [6][7][8] .Currently, EG abnormal degradations were widely detected in sepsis, tumor and other critical illness 9,10 . However, data of EG degradation in multiple trauma patients, and the association with early organ function impairment are obscure. PGs include SDC1, phosphatidylinositol PG, basal membrane PG, among which SDC1 is the main component 11 . GAGs include HS, hyaluronic acid (HA) and dermatan sulfates, HS accounts for more than 50% of GAGs 12 .
We selected SDC1 and HS as the representation of EG degradation, and conducted this study to detect EG degradation in multiple trauma patients, to fathom the correlation between EG degradation and early organ function impairment, and to demonstrate the predictive value of SDC1 and HS.

Study Design
A prospective observational study was designed to enroll multiple trauma patients (trauma group) and healthy participants (control group) in a university a liated hospital between February 2020 and October 2020. Trauma group inclusion criteria were: two or more severe injuries caused by single reason in at least two areas of the body, admitted to hospital after trauma less than 24 hours. Exclusion criteria were: age < 16 years, or >75 years, malignant tumor, chronic hepatic or kidney diseases, undrained pneumothorax.
The study was approved by the Ethics Committee of the 2nd A liated Hospital of Nantong University (No.20190612), Jiangsu, China.
The written informed consent was obtained from individual, patient or patient's guardian.

Data Collection
Gender, age and body mass index (BMI) of both groups were documented. In trauma group, the reasons of injury were recorded, injury severity scores (ISS), sequential organ failure assessment scores (SOFA) were graded, occurrences of hypothermia (T < 35℃), shock, acidosis, mechanical ventilation (MV), and traumatic brain injury (TBI) on admission were recorded, incidences of acute kidney injury (AKI), trauma induced coagulopathy (TIC) within 48 hours, and 28-day mortality were documented.

Laboratory Methods
Blood samples of both groups were centrifuged (2500g for 15 minutes at room temperature) and stored at 80℃. Double antibody sandwich ELISA tests were conducted by Enzyme calibration equipment (Thermo scienti c Inc., Waltham, MA, USA), to detect SDC1 (EK1339, BOSTER Biological Tech., Wuhan, China), HS (E-EL-H2364c, Elabscience Biotechnology Co., Ltd., Wuhan, China), interleukin-6 (IL-6) (KE00139, Proteintech Group Inc., Rosemont, IL, USA), and tumor necrosis factor-α (TNF-α) (KE00068, Proteintech Group Inc., Rosemont, IL, USA), respectively. The tests were processed using unthawed samples within 6  performed for continuous variables comparisons between two groups. Categorical variables were compared by Fisher's exact test. The correlation between the two variables was analyzed by Spearman's correlation. The receiver operating characteristic (ROC) curves were graphed and area under the curve (AUC) were calculated to investigate the accuracy of indicators for predicting AKI, TIC within 48 hours, and 28-day mortality in trauma group. All statistical tests were two-tailed, and P < 0.05 was considered statistically signi cant.

Correlation between EG degradation and organ function indicators
Spearman's correlation analyses were conducted for EG degradation indicators (SDC1 and HS, respectively) with organ function indicators in trauma group, and found that serum SDC1 and HS were both positively correlated with PT, APTT, EVLW, Cr, NT-pro BNP, Mb, Lac, IL-6 and TNF-α, both negatively correlated with PaO 2 /FiO 2 ratio, Ca 2+ , AT-III, pH and ALB (Table 2). Besides, SDC1 was signi cantly positively correlated with HS (r=0.639, P<0.001).

Comparison within trauma group by different degree of degradation
Trauma group was divided into high degradation groups (SDC1 ≥ median) and low degradation groups (SDC1 < medians) by SDC1 median (69.39 ng/ml), comparing the differences between the two sets. The differences in general conditions and injury mechanisms were not statistically signi cant, and the high degradation group had higher ISS, SOFA scores, higher hypothermia, acidosis ratio, and higher in ammation indicators (IL-6, TNF-α), while respiratory (oxygen index, EVLW), liver (ALB), kidneys (BUN, Cr), bleeding and coagulation (Hb, PLT, Ca 2+ , PT, APTT, AT-III) and metabolic indicators (pH, HCO 3 − , Lac) were worse than the low degradation group (P < 0.05, respectively, Table 3).

Discussion
Under physiological conditions, the synthesis and degradation of EG are in dynamic balance 5,17 . At present, studies on abnormal EG degradation and impaired endothelial barrier have become an important direction of critical diseases 18,19 .EG abnormal degradation was found in sepsis 20 , tumor 21 , burns 22 and major surgery 23 patients. Our study demonstrated that serum SDC1 and HS were signi cantly higher in trauma group patients than those in the control group, indicating that multiple trauma patients also had signi cant elevated EG degradation.
AKI has high morbidity and mortality in hospitalized patients, and various factors such as traumatic bleeding, uid imbalance and in ammatory mediators are considered to be the potential pathogenesis of AKI 24 . In our study, SDC1 and HS were signi cantly correlated with renal function and metabolic indicators in trauma group patients, and the EG high degradation group had a higher incidence of AKI, indicating that abnormal EG degradation after trauma is a risk factor for early occurrence of AKI.
Post-traumatic bleeding is the primary cause of death for trauma patients, and about one-third of trauma patients combined TIC, which signi cantly increased the risk of death 25 . EG has the effects of inhibiting platelet adhesion on the endothelial surface, anti-microthrombosis and maintaining coagulationanticoagulant balance. Post-traumatic bleeding, in ammation and other factors lead to abnormal degradation of EG, which may participate in the TIC mechanism 26 . This study revealed SDC1 and HS were both correlated with blood loss and coagulation indexes including Hb, PLT, PT, APTT, AT-III and Ca 2+ . The high degradation group had signi cantly worse blood loss and coagulation indexes, and a higher incidence of TIC. SDC1 was a risk factor and predictive index for early TIC. Abnormal EG degradation may be involved in the pathophysiological process of TIC.
Due to the electrochemical properties of highly sulfated GAG side chain complex of EG, the permeability of EG to solute molecules is dependent on molecular size and its negative charge, which plays a role in isolating water and maintain the gel-like structure of EG, maintaining the low permeability of albumin and preventing the extravasation of intravascular liquid 12 . The increase of EVLW is the basic pathophysiological change in the early stage of lung injury. studies of isolated animal lungs and isolated human donor lungs indicated that LUSS 27 can accurately re ect the degree of extravascular lung water 28,29 , and more clinical evidence supported the application of lung ultrasound in critical diseases 30 .
This study found that SDC1 and HS were both positively correlated with EVLW and negatively correlated with oxygenation index. Compared with the low degradation group, high degradation group had higher EVLW and lower oxygenation index, suggesting that EG degradation was related to early lung injury after trauma. The integrity of EG barrier structure and function is of great signi cance for the study of lung injury mechanism and lung protection after trauma 31 .
This study showed that SDC1 and HS were signi cantly positively correlated with NT-pro BNP and Mb, but cardiac function after trauma was not evaluated, so the signi cance was not analyzed.
Studies have shown that abnormal expressions of IL-6, IL-8 and TNF-α may be involved in EG degradation 19,23 . We observed serum IL-6 and TNF-α levels were signi cantly elevated in trauma group, and the levels were both positively correlated with SDC1 and HS levels, suggesting that post-traumatic in ammatory responses may be involved in the abnormal EG degradation mechanism, although various pathways could be associated with EG degradation, theoretically.
It should be noted that there were also some limitations in our study. First, the selected cases were patients with multiple traumas admitted to EICU, which may have selection bias and information bias. Second, sample size was small, and the statistical results may not be robust enough. In consideration of the complexity of multiple traumas, meanwhile, transfusion and emergency surgery further increase the heterogeneity of the research objects, enhancing sample size and observing longer periods would be more meaningful to demonstrate the mechanism and in uence of EG degradation in multiple trauma patients.

Conclusions
In summary, we demonstrated signi cant elevated EG degradation in multiple trauma patients, and the association between EG degradation indicators and multiple organ function impairment, including coagulation, kidney, metabolism and respiratory system. Our study indicated SDC1 is a valuable predicter of early TIC, AKI and 28-day mortality in multiple trauma patients. Availability of data and material All data generated or analyzed during this study are including in this article.

Competing interests
The authors declare no completing interests.

Funding
This study was supported by Nantong Health and Wellness Commission (MB2019012), and Nantong Science and Technology Bureau (HS2018002), Jiangsu, China.
Authors' contributions F.Li and F. Qi contributed to the conception and design of the study; F. Qi, H. Zhou, and P. Gu performed the experiment, F. Qi and F. Li performed the data analyses and drafted the manuscript. BF. Zhu, JR. Chen, and JS. Zhang contributed to manuscript revision. All authors commented on and approved the nal manuscript. SD denotes standard deviation.
IQR denotes interquartile range.   AUC denotes area under the curve.