Long‐term abuse of caffeine sodium benzoate induces endothelial cells injury and leads to coagulation dysfunction

Our hospital admitted a patient who had difficulty in coagulation even after blood replacement, and the patient had abused caffeine sodium benzoate (CSB) for more than 20 years. Hence, we aimed to explore whether CSB may cause dysfunction in vascular endothelial cells and its possible mechanism. Low, medium, and high concentrations of serum of long‐term CSB intake patients were used to treat HUVECs, with LPS as the positive control. MTT and CCK8 were performed to verify CSB's damaging effect on HUVECs. The expression of ET‐1, ICAM‐1, VCAM‐1, and E‐selectin were measured by ELISA. TUNEL assay and Matrigel tube formation assay were carried out to detect apoptosis and angiogenesis of HUVECs. Flow cytometry was applied to analyze cell cycles and expression of CD11b, PDGF, and ICAM‐1. Expression of PDGF‐BB and PCNA were examined by western blot. The activation of MAPK signaling pathway was detected by qRT‐PCR and western blot. Intracellular Ca2+ density was detected by fluorescent probes. CCK8 assay showed high concentration of CSB inhibited cell viability. Cell proliferation and angiogenesis were inhibited by CSB. ET‐1, ICAM‐1, VCAM‐1, and E‐selectin upregulated in CSB groups. CSB enhanced apoptosis of HUVECs. CD11b, ICAM‐1 increased and PDGF reduced in CSB groups. The expression level and phosphorylation level of MEK, ERK, JUN, and p38 in MAPK pathway elevated in CSB groups. The expression of PCNA and PDGF‐BB was suppressed by CSB. Intracellular Ca2+ intensity was increased by CSB. Abuse of CSB injured HUVECs and caused coagulation disorders.

MAPK signaling pathway was detected by qRT-PCR and western blot.Intracellular Ca 2+ density was detected by fluorescent probes.CCK8 assay showed high concentration of CSB inhibited cell viability.Cell proliferation and angiogenesis were inhibited by CSB.ET-1, ICAM-1, VCAM-1, and E-selectin upregulated in CSB groups.CSB enhanced apoptosis of HUVECs.CD11b, ICAM-1 increased and PDGF reduced in CSB groups.The expression level and phosphorylation level of MEK, ERK, JUN, and p38 in MAPK pathway elevated in CSB groups.The expression of PCNA and PDGF-BB was suppressed by CSB.Intracellular Ca 2+ intensity was increased by CSB.Abuse of CSB injured HUVECs and caused coagulation disorders.

K E Y W O R D S
caffeine sodium benzoate, calcium, coagulation, HUVEC, MAPK

| INTRODUCTION
Caffeine sodium benzoate (CSB), also called sodium caffeine benzoate, Annaca, or caffeine and sodium benzoate, is a mixture of caffeine and sodium benzoate according to the National Library of Medicine in the National Center for Biotechnology Information.It is employed to address acute respiratory depression triggered by an overdose of CNS depressant drugs and to prevent vascular headaches, among other indications. 1However, longterm intake may induce various side effects such as hyperventilation, visual disturbances, cardiac arrhythmias, hearing loss, and even addiction. 2In Inner Mongolia, a large number of farmers, miners, and other heavy manual laborers abuse veterinary CSB to relieve fatigue and get excited.The number of patients with acquired hemophilia in Inner Mongolia has increased sharply in recent years.All of the patients have the habit of taking CSB.A rare case of acquired factor XIII deficiency was admitted to the department of transfusion medicine in our hospital.Although the coagulation indices recovered to normal levels after blood replacement, the patient still showed continuous wound bleeding and had difficulty in coagulation.It was found that the patient had abused CSB for more than 20 years.Based on these facts, we speculated that taking CSB may cause dysfunction in vascular endothelial cells.
Coagulation is a dynamic process 3 involving activation, adhesion, and aggregation of platelets, and deposition and maturation of fibrin. 4The endothelium is key to vascular homeostasis between hemorrhage and thrombosis by acting as an active signal transducer in circulation. 5t is able to produce a wide range of factors such as ET-1 (endothelin-1), ICAM-1 (intercellular adhesion molecule 1), VCAM-1 (vascular cell adhesion molecule 1), and E-selectin that regulate vascular tone, cellular adhesion, smooth muscle cell proliferation, and thromboresistance. 6Among the factors, procoagulants and anticoagulants must keep in balance. 7Endothelial cells may be injured by bacterial or viral infection, hyperlipidemia, hypoxia, environmental irritants such as tobacco, and so on. 8Healthy endothelial cells express antiplatelet and anticoagulant agents and when vascular injury happens, cellular and protein materials congregate at the injury site to stop bleeding. 9owever, in patients who have long-term abused CSB, the function of endothelial cells cannot be changed from anticoagulant reaction to procoagulant reaction in time, resulting in coagulation dysfunction.Research about CSB's effect on coagulation is still in the exploratory stage.In this study, we aim to explore whether CSB causes dysfunction to endothelial cells and discover the related signal pathways, which will aid to treat diseases induced by CSB and prevent CSB abuse.

| Sample preparation
The protocols of this research were examined and approved by the Ethical Committee of Inner Mongolia Autonomous Region Cancer Hospital, Affiliated People's Hospital of Inner Mongolia Medical University.All participants signed the informed consent prior to the experiments.All the information was the same as mentioned in previous study. 10e prepared CSB serums in gradient concentrations by mixing serums of patients under long-term CSB inhalation (n = 35) and those of healthy volunteers (n = 35) at different ratios.The blood samples were collected and prepared as mentioned in previous study. 10CSB high concentration serum was the stock solution of long-term inhalation of CSB patients' serum (the annual exposure to CSB was 215.69 g [range: 112-351 g]).Healthy volunteers' serum was mixed with long-term inhalation of CSB patients' serum at 1:1 and 2:1 (v/v) to make CSB medium and low concentrations serum, respectively.
Human umbilical vein endothelial cells (HUVECs) are isolated from the vein of the umbilical cord and are commonly used in physiological and pharmacological research, such as coagulation, angiogenesis, fibrinolysis, and macromolecule transport.In this study, HUVECs were bought from Hunan Fenghui Biotechnology Co., Ltd.(Hunan, China).HUVECs were cultured with media with lipopolysaccharides (LPS), low, medium, and high concentrations of CSB serums.we named these groups as control group, LSP group (positive control group), low (CSB) group, medium (CSB) group, and high (CSB) group.

| Cell viability assays
MTT 11 (chemically 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was used to determine the dose-and time-effects of LPS on HUVECs.Each group was subjected to a different treatment: one group was treated with concentration, and the other group was treated with time.Additionally, all samples were replicated three times.In the dose-effect experiment, HUVECs were treated with LPS at five concentrations (0.0, 1.0, 5.0, 10.0, 50, and 100 mg/ml) for 48 hr.In the time-effect experiment, HUVECs were treated with 10.0 mg/ml LPS for 0, 24, 48, and 72 hr.Then they were inoculated to 96-well plates at a concentration of about 3,000 cells per well.Ten microliter MTT of 10 mg/ml was added to the cells and the plates were cultured for 4 hr.One hundred microliters of DMSO solution was the media added to the media and the plates were shaken for 10 min to fully dissolve the formazan crystals.In the end, the absorbances were measured at 490 nm by a microplate reader (Bio-tek, USA).
After the dose and treatment time of LPS were determined, we applied CCK-8 (Cell Counting Kit-8) to obtain the viability of HUVECs after treated with CSB of low, medium, and high concentrations.One hundred microliters of cell suspension (1,000 cells/ well) was added into a 96-well plate.Then the plates were incubated for 24 hr at 5% CO 2 and 37 C.Ten microliter CCK8 solution was added into each well and the incubation was continued for one more hour.Finally, we observed the absorbance value at 450 nm by a microplate reader (Bio-tek, USA).

| Flow cytometry
Flow cytometry was applied to test cell cycles of HUVECs after being treated with different concentrations of CSB serums.Cell cycle staining Kits (CCS012) were bought from MultiSciences (Lianke) Biotech Co., Ltd. in Hangzhou, Zhejiang Province, China.The protocol was performed following the instructions.We analyzed the cell suspensions by BD FACSCalibur™ Flow Cytometer (E97501093, BD Biosciences, USA).
Flow cytometric analyses were also carried out to test the expression of CDllb, PDGF, and ICAM-1 in HUVECs treated with different concentrations of CSB.All groups of cells were collected by centrifugation and the supernatant was discarded.Then the cells were resuspended to 100 μl by flow cytometry staining buffer (S1001, MultiSciences (Lianke) Biotech Co., Ltd. in Hangzhou, Zhejiang Province, China) and 5 μl of CD11b Monoclonal Antibody (12-0549-42, Invitrogen, USA) or CD54 (ICAM-1) Monoclonal Antibody (12-0549-42, Invitrogen, USA) or Anti-PDGF B antibody (ab51869, Abcam, UK).After the cells had been incubated in dark at 4 C for 30 min, we added 1 ml of flow cytometry staining buffer to each tube, centrifuged at 300g for 5 min, discarded the supernatant, and resuspended in 500 μl flow cytometry staining buffer.Finally, the cell suspensions were analyzed by BD FACSCalibur™ Flow Cytometer.

| TUNEL assay
To detect apoptosis of HUVECs treated with CSB, we performed TUNEL (terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling) assay to measure DNA double-stranded cleavage.One Step TUNEL Apoptosis Assay Kits (C1089) were bought from Beyotime Technology Co., Ltd. in Shanghai, China.The cell slides were placed in 24-well plate and cells were seeded 50%-80% full overnight.After treating the cells by low, medium, and high concentrations of CSB and LPS, we aspirated the culture medium, washed three times with PBS, and added 1 ml of 4% paraformaldehyde (P0099, Beyotime Technology Co., Ltd., Shanghai, China) to fix for 30 min.After fixation, the cells were washed one time with PBS and incubated for 5 min in 0.3% Triton X-100 (T8200, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) solution in PBS, then washed twice with PBS.The labeling reaction was performed using 100 μl of TUNEL detection solution to each sample and incubated for 1 hr at 37 C.After rinsing three times with PBS, a drop of Anti-Fade Mounting Medium (With DAPI) (S2110, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was put on each slide to stain nuclei and then covered with cell slides.Pictures were taken under a fluorescent microscope (BH2-RFCA, Olympus Corporation, Japan) after drying in dark.

| Matrigel tube formation assay
We carried out a Matrigel tube formation assay to confirm CSB's inhibition effect on HUVEC angiogenesis.Matrigel (356,234, Corning Inc., USA) was put into a 4 C refrigerator 24 hr in advance and thawed for later use.The passage number of cells was 2-6.The next day, a pre-chilled 24-well plate was put on the ice box, and the Matrigel was diluted with ECM basal medium (35,195, ScienCell Research Laboratories, inc., CA, USA) 1:1 and centrifuged.Then we added 100 μl Matrigel into each well of the 24-well plate and placed it into an incubator (HF151 [UV], Shanghai Lishen Science Instrument Limited Company, Shanghai, China) for 60 min to form gels. Trypsinized to dislodge the cells after HUVECs reached 80% confluent and collected them for counting.Added 200 μl of HUVEC cell suspension to a 24-well plate, with a concentration of about 100,000 cells.Then the plate was incubated for 4 hr at 37 C and the formation was observed.

| Real-time quantitative PCR
In order to confirm the activation of the MAPK signaling pathway by CSB, we performed qRT-PCR to determine the mRNA expressions of MEK, ERK, JNK, and p38.The total RNA was extracted by Redzol (SBS Genetech Co. Ltd., Beijing, China), then reverse transcription was done using cDNA Reverse Transcription Kit (QP057, iGene Biotechnology Co., Ltd., Guangzhou, China).The primers' sequences are shown in Table 1, and we used GAPDH as the internal control gene. 2 Â SYBR Green qPCR Master Mix (None ROX) (Wuhan Servicebio Technology Co., Ltd., Wuhan, China) was contained in the mixture.Then, we conducted qRT-PCR in iQ5 Real-Time PCR Detection System (Applied Biosystems, MA, USA)

Gene
Primers' sequences with the program of 95 C for 30 s; 95 C for 15 s and 40 cycles of 60 C for 30 s.All data were analyzed using the 2 ÀΔΔCT method.

| Fluorescence staining of Ca 2+
We carried out fluorescence staining to visualize the activation of Ca 2+ .HUVECs treated with different concentrations of CSB were incubated for 30 min at 37 C in the dark with Fluo-3 AM (121714-22-5, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).After removing Fluo-3 AM, the cells were washed three times with HBSS (HEPES buffer saline H1045 D-Hank's, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).Then the cells were covered with HBSS solution containing 1% FBS and incubated at 37 C for 30 min.Fluorescence images were captured by a fluorescence microscope (BH2-RFCA, OLYMPUS, Japan).

| High concentration of CSB suppressed the cell viability of HUVECs
To determine the dose-effect and time-effect of LPS on HUVECs, we measured cell viability by MTT assay.As the LPS concentration increased, the cell viability dropped.When the concentration was as high as 50 mg/ml, the declining trend became smooth with IC50 of 10.74 mg/ml (Figure 1a).Meanwhile, the viability decreased to half after 50.98 hr (Figure 1b).Hence, LPS was proved to have negative effect on HUVECs' viability and proliferation and could be used as a positive control.Next, a pattern of decreased cell viability was observed with increasing concentrations of CSB serums in the treated cells.(Figure 1c), suggesting that CSB could injure HUVECs.

| Cell proliferation of HUVECs was inhibited after the treatment of CSB
Flow cytometry analysis of cell cycle (shown in Figure 2) revealed that treatment with CSB resulted in a significant increase in the number of cells in the G0/G1 stage and a corresponding decrease in the S stage compared to the control group (Figure 2a).The differences between the CSB-treated groups and the control group, as well as between different concentrations of CSB-treated groups in the G0/G1 stage, were found to be statistically significant (Figure 2b).These findings suggested that CSB treatment resulted in cell cycle arrest before DNA synthesis, leading to a reduction in the proliferation of cells.Furthermore, the concentration-dependent increase in the percentage of cells in the G0/G1 stage and the decrease in the S stage suggested that CSB inhibited cell proliferation in a concentration-dependent manner.

| CSB exerted pro-apoptotic responses to HUVECs and inhibited angiogenesis
TUNEL assay was performed to detect whether CSB induced apoptosis in HUVECs.The percentage number of apoptotic cells in the CSB groups was significantly higher than control groups, suggesting CSB's anti-proliferative and pro-apoptotic effects on HUVECs.Apoptosis rate increased significantly after HUVECs being treated with CSB, but the concentrations did not make any differences (Figure 3).
What was more, in vitro angiogenesis was assessed by Matrigel tube formation assay.The branching of HUVECs treated with CSB was less compared with cells in control group (Figure 4a).The tube formation of HUVECs was significantly declined after treated with CSB, and high concentration influenced significantly more than low concentration (Figure 4b).Hence, CSB was able to exert pro-apoptotic responses to HUVECs and anti-angiogenesis effect of CSB was revealed.

| CSB regulated crucial genes related to coagulation
We performed ELISA to explore how CSB affected the expression of coagulation-related genes, ET-1 (Figure 5a), ICAM-1(Figure 5b), VCAM-1 (Figure 5c), and E-selectin (Figure 5d).The expression pattern of all genes showed that the CSB-treated groups exhibited significantly higher expression levels compared to the control groups, and their expressions increased with higher CSB concentrations of the treatment.
PCNA and PDGF-BB were closely related to coagulation, thus we detected their expression in HUVECs after CSB treatment by western blot.The result was coherent with the patient's symptom of difficulty in hemostasis.CSB was found to significantly inhibit the expression of both PCNA and PDGF-BB, and this inhibition was concentration-dependent, with higher concentrations resulting in greater inhibition compared to lower concentrations (Figures 6a,b and S1).What was more, flow cytometric analyses were applied to measure the expression of CDllb, PDGF, and ICAM-1.Stained cells treated with CSB gave shift (increase) in fluorescence intensity to the right, showing higher expression of CD11b and ICAM-1.Meanwhile, the fluorescence intensity of stained cells treated with CSB shifted to the left compared to control group (Figure 6c), suggesting CSB's inhibiting effect on the PDGF.High concentration of CSB affected significantly more on the expression of these proteins than low concentration of CSB (Figure 6).Therefore, CSB regulated the expression of genes related to coagulation.

| CSB treatment activated MAPK and calcium signaling pathways in HUVECs
MAPK signaling pathway played an important role in complex cellular programs such as proliferation, differentiation, transformation, and apoptosis. 12Especially, MAPKs function essentially in regulating platelet.Each MAPK pathway regulates different platelet function. 13To gain insight into the mechanism of how CSB affected coagulation, we explored the expression of p-MEK, MEK, p-ERK, ERK, p-JNK, JNK, p-p38, and p38 in MAPK signaling pathway.According to qRT-PCR results, low concentration of CSB made no significant difference in the expression of MEK, ERK, and JNK, but medium and high concentrations of CSB increased the expression of MEK, ERK, and JNK.The expression of p38 was significantly higher after CSB treatment than control group (Figure 7a).Furthermore, the results of western blot   showed that p-MEK, p-ERK, p-JNK, and p-p38 expressed significantly higher after treatment by CSB, suggesting phosphorylation of MEK, ERK, JNK, and p38 was induced by CSB in HUVECs.The expression increased with increasing concentration of CSB (Figures 7b and  S2).These results indicated that MAPK signal pathway was activated after treating with CSB in HUVECs.
Also, we detected calcium signaling pathway in the CSB functioning process.As Figure 8a showed, the green fluorescence in HUVECs became stronger after CSB treatment, suggesting calcium signaling pathway was activated by CSB.However, the sensitivity of Ca 2+ activation did not appear to be significantly affected by CSB concentration changes, as there was no statistically significant difference observed between the various concentrations of CSB tested (Figure 8b).
In this study, CSB has been proven to cause damage to HUVECs by enhancing apoptosis and inhibiting cell viability, proliferation, and angiogenesis.After CSB treatment, coagulation-related genes were significantly aberrantly expressed, besides, MAPK signaling pathway and calcium signaling pathway were significantly activated.
PDGFs, namely platelet-derived growth factors, participate in wound healing and regulation of blood vessel tonus, and plays a vital role in cell proliferation, cell migration, and angiogenesis.Also, PDGFs help to heal wounds and repair damage to blood vessel walls.PCNA, namely proliferating cell nuclear antigen, is a protein working as a DNA sliding clamp and functioning well in regulating cell proliferation. 14In the result of western blot in our study, the expression of PDGF and PCNA were both downregulated by CSB, reflecting the damage and inhibition of cell proliferation of HUVECs.E-selectin, which served as a marker for endothelial apoptosis in certain diseases 15 was shown to be upregulated by CSB via ELISA.TUNEL assay proved that the apoptosis was induced in HUVECs after being treated with CSB.
Mitogen-activated protein kinase (MAPK) signaling pathways are essential to regulate varieties of cellular processes, such as proliferation, differentiation, apoptosis, and stress responses. 16In mammals, MAPK pathways are typically grouped into three main subfamilies: ERKs, JNKs, and p38/SAPKs, 17 mainly function in cell growth and differentiation, 18 apoptosis and metabolism, 19 and apoptosis and cell cycle regulation, 20 respectively.In our study, HUVEC proliferation was inhibited and apoptosis was stimulated by CSB, so we decided to check the expression of genes in MAPK pathway, including these subgroups.According to qRT-PCR, MEK, ERK, JNK, and p38 were upregulated by CSB, and the result of western blot revealed that their phosphorylation levels were also upregulated by CSB, suggesting activation of these pathways.MEK1 and MEK2 mediate ERK1 and ERK2 activation during adhesion and growth factor signaling. 21affeine shows an active effect on p38 pathway and dual effect on ERK pathway according to its concentration. 22owever, sodium benzoate reduces MAPK signals in the kidneys of AdCKD mice. 23The activation of MAPK pathways makes sense combined with our results of inhibited cell viability and cell cycles, and stimulated apoptosis.MAPK pathway not only works in the damage of vascular endothelial cells 24 but also functions in the protection of vascular endothelium. 25The involving mechanism is quite complicated and requires more research.
We found calcium signaling pathway was activated as well after CSB-treated HUVECs.Ca 2+ plays a major role in the regulation of coagulation cascade which is fundamental in hemostasis. 26Besides platelet activation, Ca 2+ is responsible for the complete activation of several coagulation factors, such as coagulation Factor XIII (FXIII). 27ccording to Sneha Singh et al., FXIII-A calcium binding sites could be putative pharmacologically targetable regions. 28The rare case which gave rise to this study was acquired factor XIII deficiency, causing difficulty in hemostasis even when coagulation indices recovered to normal after treatment.Our study proved Ca 2+ was activated by CSB, although Ca 2+ was prone to promote coagulation.However, it is not hard to understand the fact.CSB consists of caffeine and sodium benzoate.We are familiar with caffeine, it is a kind of blood thinner.Caffeine has the capacity to inhibit the degradation or metabolism of anticoagulants such as warfarin and enhance anticoagulant effects. 29Sodium benzoate has an anticoagulant effect and an inhibitory effect on erythrocyte sedimentation. 30It might be the reason why Ca 2+ was activated but coagulation was inhibited by CSB.In the future, we plan to dig deeper in the direction of the related mechanisms using animals, constructing relations between drugs and detailed physiological effects, and distinguishing functions of different proportions of caffeine and sodium benzoate.

| CONCLUSIONS
In this study, we identified CSB serum causes damages to vascular endothelial cells and induces coagulation disorders through activated MAPK signaling pathway and calcium signaling pathway.Our findings suggested that CSB was able to inhibit cell viability, proliferation, and angiogenesis of HUVECs while apoptosis can be enhanced by CSB.Coagulation disorders may be induced by these damages.MAPK and calcium signaling pathway functioned in CSB's impacts on HUVECs.The findings in our study will aid to treat blood diseases caused by CSB and prevent CSB abuse.

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I G U R E 1 Cell viability analysis.(a) Analysis on HUVECs treated by different concentrations of LPS via MTT assay.(b) Analysis on HUVECs treated by LPS at different duration of time via MTT assay.(c) Analysis on HUVECs treated by different concentrations of CSB via CCK-8 assay.

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I G U R E 2 Cell cycle analysis of HUVECs by flow cytometry.(a) Cell cycle analysis of control, LPS, low, medium, and high concentrations of CSB groups respectively.(b) Summary of cell cycle analysis of all groups.(*p < .05;**p < .01;***p < .001;****p < .0001,ns indicates non-significance, # indicates the p-values for comparisons between groups and LPS group).