Introduction

Conventional total knee arthroplasty causes a huge trauma to the body, and its blood loss is about 1~1.5 L. Postoperative patients may need blood transfusion for anemia. However, blood transfusion may induce direct risks including immune response and intravascular hemolysis, thereby seriously endangers the health of patients and increases medical costs [1]. In addition, severe trauma such as total knee arthroplasty can also lead to systemic vascular endothelial cell damage, ultimately leading to Acute Traumatic Coagulopathy (ATC), cellular inflammatory response and endothelial cell barrier dysfunction, which is called "Endotheliopathy Of Trauma (EoT)"[2]. It has been reported that as many as 1/4 to 1/3 of trauma patients have developed coagulopathy before surgery, and if EoT cannot be diagnosed or treated timely in the early phase, it would develop into multiple organ failure, and the mortality rate is extremely high. Therefore, early intervention of EoT is a key factor of improving the prognosis of patients with severe trauma.

As a synthetic anti fibrinolytic drug, TXA is widely used in clinical trauma treatment. Current clinical randomized studies showed that early administration of TXA can play a better hemostatic effect and reduce complications [3]. TXA treatment after 6 hours of trauma is often not ideal. Previous studies have shown that TXA has anti fibrinolytic and anti-inflammatory effects, but the exact mechanism of TXA remains unclear [3]. In recent years, it has been reported that TXA can protect epithelial cells through protecting the epithelial polysaccharide-protein complex layer in the hemorrhagic shock intracavitary induced intestinal epithelial cell injury model [4]. However, the role of TXA in EoT and the timing of drug administration are still unclear. In this study, EoT model was established by applying H₂O₂ on primary human umbilical vein endothelial cells in vitro culture and explored the effect and mechanism of TXA on EoT at different time points.

Methods

Ethics Statement

The study has gotten approval from Medical Ethics Committee of Shandong Medical College Affiliated Hospital, and all participants provided written informed consent.

Cell Culture

Human Umbilical Vein Endothelial Cells (HUVECs) were provided by Inner Mongolia Medical University, and then cultured in ECM medium (Gibco, USA) containing 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, USA) and 1% penicillin–streptomycin mixture (Hyclone Laboratories, Logan, USA). Cultures were maintained in an incubator with 5% CO₂ at 37^°C.

Grouping Criteria

The cell concentration was adjusted to 1×10⁶/L, and 200 μl of the cell suspension was inoculated into 96-well plates and randomly divided into normal control group, H₂O₂ group, H₂O₂ +TXA (0 min) group, H₂O₂+TXA (30 min) group, H₂O₂+TXA (60 min) group and H₂O₂+TXA (90 min) group. All the latter 5 groups were added with 100 μmol/L H₂O₂ for 2 h and added TXA (150 μmol/L) at four time points (0 h, 30 min, 60 min and 90 min) after the H₂O₂ application.

Fluorescence Observation

Briefly, cells were washed with PBS three times and then fixed in 70% ethanol. After that, the fixed cells were washed twice with PBS and stained in Hoechst 33258 (Beijing Biosea Biotechnology, Beijing, P.R. China), incubated for 10 min at room temperature in the dark, and washed with PBS three times. Added 10 μl anti-fluorescence attenuating seal to the sterilized slide and then immediately subjected to the OLYMPUS inverted fluorescence microscope (Olympus, Japan).

Cell Viability Assay

Cell viability was assessed with the cell counting kit-8 (CCK-8, Boster, China) assay according to the manufacturer’s guidelines. HUVECs from each group were starved in 2% FBS for 12 h. Cells were then treated with 10 μL CCK-8 solution for 3 h, and the absorbance was read at a 450-nm wavelength.

Western Blotting Assay of ICAM and MMP-9 protein expression in cells

Western blotting assay was established using Bio-Rad Bis-Tris Gel system and performed as previously described [5]. ICAM and MMP-9 protein proteins in the cells were electrophoresed on polyacrylamide gels, and the gels were transferred onto Polyvinylidene Difluoride (PDVF) membranes (Mil lipore) that were incubated with relevant antibodies. Anti-ICEM anti- MMP-9 and β-actin monoclonal antibody were purchased from Epitomics Biotechnology (Epitomics, USA). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, USA) for 1 hour at room temperature. The protein signals were detected by enhanced chemiluminescence Western blotting substrate (Pierce, Thermo Fisher Scientific, USA).

ELISA Analysis

Supernatants of cell culture were harvested and the presence of tPA, PAI-1, polyligand, hyaluronic acid and TNF-α were measured by commercial ELISA kit (Kai Ji Biotechnology, China) according to the manufacturer’s instructions, and then the absorbance value were obtained at 450 nm of the microplate reader (ELx808, BioTek Instruments, USA). Standard curves were prepared and then tPA, PAI-1, Polyligand, hyaluronic acid and TNF-α content values were calculated, respectively.

Statistical Analysis

Statistical analysis was performed using SPSS 16.0 software. All measurement data obtained in this study were described as mean ± standard deviation. The ANOVA variance SNK-q method was used to analyze the difference between the mean of each two groups. P < 0.05 was considered significantly different.

Result

Cell Viability

As shown in Figure 1, the nucleus of HUVECs in the normal control group showed uniform low-intensity fluorescence. After H₂O₂ treatment, a large number of cells showed new-moon form at the edge of chromosome, which indicated typical apoptotic cell changes, and the cell viability rate was significantly lower than that of the control group (P < 0.001, Figure 2). Compared with H₂O₂ group, cell viability in H₂O₂+TXA (0 min) group and H₂O₂+TXA (30 min) group were significantly higher (P < 0.01, Figure 2). The cell viability of H₂O₂+TXA (60 min) group and H₂O₂+TXA (90 min) group was not significantly different from that of H₂O₂ group (P > 0.05, Figure 2).

The effect of TXA on the expression of ICAM and MMP-9 protein in HUVECs

As shown in Figure 3, Table 1 and Table 2, the expression of ICAM and MMP-9 protein were significantly up-regulated after H₂O₂ treatment (P < 0.001). Compared with H₂O₂ group, the expression of ICAM and MMP-9 protein in H₂O₂+ TXA (0 min) and the H₂O₂+TXA (30 min) group was significantly decreased (P < 0.001). There was no significant difference in the H₂O₂+TXA (60 min) group and H₂O₂+TXA (90 min) group (P > 0.05).

Effect of TXA on the expression of tPA, PAI-1, polyligand, hyaluronic acid and TNF-α in HUVECs

As shown in Figure 4ACDE, the expression of tPA, polyligand, hyaluronic acid and TNF-α was significantly up-regulated in H₂O₂ group, compared with the control group (P < 0.001), while the expression in the H₂O₂+TXA (0 min) group and the H₂O₂+TXA (30 min) group were significantly decreased compared with the H₂O₂ group (P < 0.001). Polyligand, tPA, hyaluronic acid and TNF-α in the H₂O₂+TXA (60min) group and the H₂O₂+TXA (90min) group were not expressed differently from that in the H₂O₂ group (P > 0.05).

As shown in Figure 4B, H₂O₂ significantly inhibited the expression of PAI-1 in HUVECs compared with the control group. The difference between the two groups was significantly different (5972.4 ± 1029.7 vs. 4613.5 ± 872.1, P < 0.05). The expression of PAI-1 in the H₂O₂+TXA (0min) group and the H₂O₂+TXA (30 min) group were (5869.7 ± 1425.2) pg/ml and (6127.5 ± 1147.6) pg/ml, respectively, significantly up-regulated compared with the H₂O₂ group (P < 0.05). The expression of PAI-1 in H₂O₂+TXA (60min) group and H₂O₂+TXA (90min) group was not significantly different from H₂O₂ group (P > 0.05).

Discussion

As a strong oxidant, H₂O₂ plays an important role in regulating cellular oxidative signaling pathways. However, it has been reported that a certain concentration of H₂O₂ can cause EoT in endothelial cells [6]. H₂O₂ can significantly induce apoptosis in endothelial cells and play a key role in the microcirculation and vascular dysfunction [7]. In this study endothelial cells after treated by 100 μmol/L H₂O₂ were observed the dense staining of many nuclei, which indicated that H₂O₂ could induce damage and death to a large number of endothelial cells.

As a widely used clinical coagulation drug, it has been found that the application of TXA within 3 hours after hemorrhagic trauma can effectively decrease the mortality caused by bleeding. In addition, a prospective clinical trial reported that within 1 hour of acute trauma, the use of TXA has a significant effect on decreasing mortality [3,4]. A recent study of rodent shock models has shown that the mechanism of TXA action is related to the inhibition of the expression of disintegrin, metalloproteinase and tumor necrosis factor by TXA [4,8]. In present study, TXA was applied to the in vitro EoT model at different time points and early TXA administration especially within 1h was found to effectively inhibit cell damage, but not in the late administration,

In this study we found that TXA can inhibit the expression of tPA, multi-ligand glycans, hyaluronic acid, TNF-α, ICAM and MMP-9 but promote PAI-1 expression to reduce endothelial cell injury. As a superfamily of adhesion molecules in endothelial cells, ICAM is up-regulated by inflammatory factors and closely related with mediating cellular inflammatory responses [9,10]. tPA and PAI-1 act as regulators of synthesis and secretion in endothelial cells and play an important role in regulating the balance between the body's fibrinolytic system and the coagulation system. tPA can specifically bind fibrin in thrombus while simultaneously activates lysozyme and fibrinolytic enzyme in the blood and inhibits platelet aggregation. As a rapid inhibitor of tPA, PAI-1 can form a complex binding to tPA, thereby exerting an inhibitory effect on tPA [11,12]. As one of the representative inflammatory cytokines, TNF-α plays an important role in mediating various cell damage and inflammatory reactions in endothelial cells. Down-regulation of TNF-α expression can inhibit adhesion molecules and other cellular inflammation [13]. The polysaccharide-protein complex in endothelial cells acts as a skeleton of the endothelial cell membrane, plays an important barrier function in maintaining vascular permeability, and transmitting extracellular signals into cells [14,15]. Since the polysaccharide-protein complex is difficult to be observed in vivo, the detection of the multi-ligand glycan and hyaluronic acid can indirectly reflect the degradation of the polysaccharide-protein complex. It can also be used as an early marker of endothelial cell damage caused by traumatic shock, as they are often down-regulated in patients with severe trauma [16]. In addition, polysaccharide-protein complexes in vascular endothelial cells can be degraded by extracellular proteases including MMPs after the stimulation of inflammatory factors. Therefore, improving or reversing the degradation process of polysaccharide-protein complex in vascular endothelial cell is expected to reduce mortality and improve prognosis in patients with severe trauma.

Conclusion

The clinical application of TXA requires early administration in patients with acute hemorrhagic trauma and TXA administration pk within one hour can effectively protect the endothelium from inflammatory factors and maintain the normal function of endothelial cells.

Figure 1: Cell Viability under Fluorescence Observation. HUVECs were stained by Hoechst and observed after a magnification of 400 times by fluorescence microscope. Nucleus was concentrated and dense, and the color was white.

Figure 2: Cell viability test. Cell viability was detected with CCK-8. Values are the mean ± SD; ^***P < 0.05.

Figure 3: Western blot analysis of the effect of TXA administration on ICAM and MMP-9 protein expression in HUVECs at different time points, with β-actin as an internal reference.

Figure 4: ELISA assay for the expression of tPA, PAI-1, polyligand, hyaluronic acid and TNF-α in HUVECs in vitro, ^*P < 0.05, ^***P < 0.001.

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Citation: Liang HH, Ding F, Wang CJ, Sharen GW (2019) Protective effect of TXA on human umbilical vein endothelial cell injury. J Orthop Res Ther 11: 1142. DOI: 10.29011/2575-8241.001142