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Research Article
Inhibition of selective factor Xa suppresses type 1 diabetes development in mice via the CXCL16 chemokine
expand article infoMohamed S. Abdel-Bakky‡, Hamdoon A. Mohammed‡, Elham Amin‡
‡ Qassim University, Buraydah, Saudi Arabia

Corresponding author: Mohamed S. Abdel-Bakky ( m.abdelbakky@qu.edu.sa )

Academic editor: Rumiana Simeonova
© 2024 Mohamed S. Abdel-Bakky, Hamdoon A. Mohammed, Elham Amin.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Abdel-Bakky MS, Mohammed HA, Amin E (2024) Inhibition of selective factor Xa suppresses type 1 diabetes development in mice via the CXCL16 chemokine. Pharmacia 71: 1-10. https://doi.org/10.3897/pharmacia.71.e137797
Open Access

Abstract

Type 1 diabetes mellitus (T1DM) is associated with enhanced thrombosis potential. This research aims to investigate the role of coagulation system activation and CXCL16 in T1DM progression and the possible preventive effect of the selective Factor Xa inhibitor, Fondaparinux (Fond). Forty male Wistar albino mice were classified into 4 groups. Control, Fond, Streptozotocin (STZ), and Fond + STZ groups. Biochemical, hematological, and histopathological parameters, expressions of CXCL16, insulin, fibrinogen, and PAR-2, were investigated. A significant increase in blood glucose level and expressions of CXCL16, fibrinogen, and PAR-2, together with histological and nuclear changes in the pancreatic islets, was observed in STZ-treated mice, accompanied by a reduction in the serum insulin level. Fond improved the deleterious effects resulting from STZ treatment. In conclusion, coagulation system activation and CXCL16 play a critical role in the initiation of T1DM and could be used as therapeutic targets for T1DM prevention.

Keywords

CXCL16, Fibrinogen, Fondaparinux, insulin, Type 1 diabetes

Introduction

The main role of the normal coagulation system is to prevent excessive bleeding and to repair the damaged sites. However, the normal coagulation system can be activated in systemic circulation under certain circumstances, while formed red blood cells contain high levels of enzymes that catalyze the deposition of fibrin (Lipinski 2012). Inflammatory and immune stimuli often trap the blood-containing tissue factor, which is released in concentrated microenvironments by perivascular cells or secreted into the bloodstream (Duque et al. 2021). In addition to acute tissue damage caused by tissue injury, inflammation, and infection, microvascular dysfunction and systemic endothelial damage are also important initial events in the progression of microvascular and macrovascular complications caused by T1DM and T2DM (Duque et al. 2020). As long as vascular injury is not present in systemic circulation, activation of the coagulation system modulates systemic inflammation and host defense and, via signaling pathways, plays a role in insulin control. The coagulation system activated in various body districts of patients with T1DM and T2DM may be involved in endothelial barrier dysfunction and insulin resistance; therefore, a suggested close link between coagulation system activation and cardiovascular diseases has been developed.

However, the activation of factor VII and TF-VII coagulation initiated under the pathogenetic conditions of type 1 diabetes has become one of the latest recognized and undoubtedly important factors. It is considered to be contribute to the up-regulation of apoptosis and immune system activity, exacerbating the situation and under conditions of the disease, not only intensifying the damaging of islets, contraction of insulin gene expression, beta-cell “tiredness” or “exhaustion,” but also leading to the irreversible destruction of insulin-generating cells and hyperglycemia (Roep 1973; von Scholten et al. 2021). The current study is the first that elucidates the effect of selective inhibition of Factor Xa in T1DM development rather than as a complication.

In multiple low-dose streptozotocin-induced T1DM models, specific inhibition of the extrinsic coagulation factor, tissue factor (TF), and/or blocking of factor VII and the antibody-blocking of FXIIa decrease the risk of the formation of diabetes (Giwa et al. 2020; Zhou et al. 2020). Coagulation system activation in T1DM is thought to be a response to endothelial damage (Jasser-Nitsche et al. 2020). Some findings indicate thrombin involvement in the development of insulin resistance and β-cell apoptosis (Wautier and Wautier 2021). Recently, it has been observed that children previously diagnosed with T1DM had elevated coagulation activity and increased synthesis of FVII and FVIII, the main coagulation factors predominantly synthesized in the liver (Ali & Becker, 2024). Additionally, FVII and FVIII levels in a subgroup of at-risk autoimmune diabetes patients had a correlation with an increase in glucose level during the oral glucose tolerance test (Grant et al. 1988). Finally, coagulation system activation in T1DM has been described, but whether its activation may initiate T1DM is still unclear. The current work is designed to clarify the possible effect of coagulation system activation in the initiation of T1DM. Furthermore, the possible protective effect of the antithrombin III (ATIII)-mediated selective inhibition of Factor Xa agent, fondaparinux, against streptozotocin (STZ)-induced T1DM in mice was investigated, together with its possible mechanism.

Materials and methods

Chemicals and antibodies

Streptozotocin and DAPI were obtained from Sigma-Aldrich (Missouri, USA). Fluoromount G was provided by Biozol Diagnostica Vertrieb GmbH (Eching, Germany). Bovine serum albumin (BSA) was obtained from BIO MARK laboratories (Maharashtra, India). Mouse monoclonal CXCL16, fibrinogen, PAR-2, and insulin antibodies were acquired from Santa Cruz Biotechnology (TX, USA). Cyanin red (Cy3)-conjugated goat anti-mouse, Alexafluor 488-conjugated goat anti-mouse, and Alexa Fluor 488-conjugated goat anti-rabbit secondary antibodies were obtained from Jackson Immunoresearch (PA, USA). The remaining chemicals used were of the highest analytical grades available.

Animals and work design

Male Wistar albino mice (23 ± 2 weeks old, weighing 27–30 g) were obtained from the Animal House of Qassim University, Qassim, Saudi Arabia. Mice were kept at 25 °C, about 45% humidity, and were kept in a standard food and water ad libitum. The investigational process was accomplished in accordance with Saudi Arabia ethical guidelines and the NIH Guidelines for the Care and Use of Lab Animals. This study was reviewed and approved by [the ethical committee at Qassim University, Qassim, Saudi Arabia] with an ethical approval number: [24-89-23], dated [May 6, 2024]. Forty mice were divided into 4 weight-matched groups, each of 10 animals. Group 1: Served as a control group and administered saline only for 12 days, starting on day 4 of the experiment. Group 2: Fondaparinux control group; mice received fondaparinux daily (5 mg/kg, i.p.) for 12 days from day 4 (Abdel-Bakky et al. 2023). Group 3: (Diabetic mice); animals were injected with STZ 55 mg/kg, i.p., in citrate buffer (50 mmol/L, pH 4.5) daily for 5 consecutive days starting from the 1st day until the 5th day (Abdel-Bakky et al. 2022). Group 4: (Diabetic + Fondaparinux); mice were injected with Fondaparinux (5 mg/kg, i.p.) for 12 days from the 4th day to the end of the experimentation and STZ 55 mg/kg, i.p. daily from day 1 for 5 consecutive days.

Blood sampling and preparation

At the end of the experimentation, animals were anesthetized, and the blood was collected from the retro-orbital plexus, then separated into 2 aliquots. One portion was taken on EDTA tubes and was used for the assessment of blood parameters. The second portion was preserved for centrifugation for 20 min at 4000 rpm to isolate serum for insulin assessment.

Tissue sampling preparation

Animals were anesthetized and euthanized by cervical decapitation at the end of the experiment. Pancreatic tissues were collected, dissected, preserved in Davidson’s buffer, and blocked in paraffin. Kidney tissues were used for histopathological analysis and immunofluorescence detection of PAR-2, CXCL16, fibrinogen, and insulin.

Disposal of carcasses

Dead mice during the experimental procedure, sacrificed mice, and waste of isolated tissues at the end of the experiment were collected in a plastic bag and sealed tightly. The tightly sealed bags were removed to the animal house and stored in a freezer (-20 °C). Finally, all the remains were collected by the Veterinary Hospital (Buraidah) from the college and disposed of by incineration.

Determination of biochemical parameters

Serum creatinine, BUN, urea, ALT, and AST levels were performed using colorimetric assay kits (Bio-Diagnostic Co., Egypt) consistent with the manufacturer’s instructions.

Determination of complete blood count (CBC)

Hematology parameters including lymphocyte % (Lymph%), granulocyte % (Gr%), mononuclear cell % (Mon%), total WBC, RBC number, hemoglobin (Hgb), hematocrit (Hct), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), red cell distribution width (RDW), mean platelet volume (MPV), platelet count (PLT), and platelet distribution width (PDW) were tested using the ABX Micros 60 hematology analyzer (HORIBA Medical, France), which counts and sizes blood cells.

Estimation of blood glucose and serum insulin levels

For diabetes initiation confirmation, blood samples from all groups were collected and tested for serum insulin and blood glucose at the end of the work. Assessment of the level of blood glucose was achieved on a drop of blood using a glucometer (Uright, Taiwan). Serum insulin level was measured using a mouse insulin ELISA kit from Biovision Inc. (CA, USA).

Histopathological study

Pancreatic tissue samples were fixed in Davidson`s solution, rinsed with tap water, and dehydrated using a graded concentration of alcohol. The clearing of tissue was done using xylol and incubated in melted paraffin for 24 hours. The tissues were blocked in paraffin and were cut at 4 microns using microtomes on glass slides. Tissues were deparaffinized, rehydrated, and hematoxylin and eosin stained for examination of the architectures. The examination and figure capture were performed blindly through the light microscope as described by (Çakir Güney et al. 2022).

Immunofluorescence analysis

The immunofluorescence method was performed to analyze the expression of insulin, CXCL16, fibrinogen, and PAR-2 in the pancreatic tissue samples, as described earlier (Hammad et al. 2013). In brief, after the steps of deparaffinization using an oven at 20 °C for 20 min and xylene incubation for 30 min, sections were rehydrated in descending graded ethanol concentration. Sections were buffered and washed (0.05% tween 20 dissolved in phosphate buffer, pH 7.4), followed by heating in sodium citrate buffer (pH 6) in the microwave at 500 Watts for 15 min and letting the solution cool down. After washing, sections were fixed with methanol for 10 min and incubated with blocking buffer (10% horse serum in 1% bovine serum albumin dissolved in PBS) for 1 hour at RT. Following washing, the slides were incubated with the primary antibodies (Abs) for 3 hours at 37 °C and overnight at 4 °C. After washing, pancreatic slides were exposed to Cy3-conjugated goat anti-mouse, Alexa Fluor 488-conjugated goat anti-rabbit, or Alexa Fluor 488-conjugated goat anti-mouse secondary antibodies (Jackson Immuno-research (PA, USA)) at RT for 30 min. Following washing, slides were nuclear counterstained by incubation with DAPI for 3 min. At the end, slides were mounted, and figures were captured using fluorescence microscopy (Leica DM5000 B). Assessment of fluorescence intensity and histogram by calculating the average intensity of at least 6 fields for each section using Image-J/NIH software.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Qassim University, Qassim, Saudi Arabia, with an ethical approval number: 24-89-23, dated May 6, 2024.

Statistical analysis

The resulted data were denoted as mean ± SEM, and statistics were performed using version 8 from GraphPad Prism® (GraphPad Software Inc., California, USA). A one-way ANOVA test was used to find out significant differences for all sets, followed by Tukey’s post-ANOVA for multiple comparisons, where the differences were considered significant at probability < 0.05.

Results

Effect of different treatments on hematological parameters in STZ-induced T1DM in mice

Results presented in Table 1 showed no significant changes in hematological parameters were seen in the Fond-treated group except with WBCs (8.05 ± 0.24 109 cell/L) (p < 0.05) compared to control mice (9.141 ± 0.25 109 cell/L). Alternatively, STZ-treatment exhibited significant decrease in WBCs count (5.18 ± 0.26 109 cell/L), RBCs count (7.41 ± 0.30 1012 cell/L), percentage of HCT (36.45 ± 1.47%), HGB level (10.5 ± 0.59 mg/dl), MCV size (43.34 ± 1.201 μm3), MCH (16.46 ± 0.27 Pg), and MCHC level (27.48 ± 0.59 g/dL) compared to control mice (9.141 ± 0.25 109 cell/L, 10.20 ± 0.45 1012 cell/L, 46.86 ± 1.84%, 14.01 ± 0.23 mg/dl, 54.4 ± 1.83 μm3, 20.12 ± 0.96 pg, and 34.89 ± 1.15 g/dl, respectively). On the other hand, STZ-treatment demonstrated a significant increase in PLT count (1061 ± 40.06 109/L) and percentage of Lymph (85.53 ± 1.08%) compared to control animals (670.6 ± 11.27 109/L and 71.53 ± 0.92%, respectively). Fond treatment in the presence of STZ significantly amended WBCs count (7.25 ± 0.33 109 cell/L), RBCs count (10.62 ± 0.30 1012 cell/L), percentage of HCT (52.22 ± 2.108%), HGB level (15.2 ± 0.32 mg/dl), Lymph% (78.15 ± 1.88%) and MCV size (49.58 ± 0.54 μm3) compared to STZ-treated mice group (5.18 ± 0.26 109 cell/L, 7.41 ± 0.30 1012 cell/L, 36.45 ± 1.47%, 10.5 ± 0.59 mg/dl, 85.53 ± 1.08%, and 43.34 ± 1.201 μm3, respectively).

Table 1.
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Effect of different treatments on hematological parameters in STZ-induced T1DM in mice.

Control Fond STZ Fond + STZ
WBCs (109 cell/L) 9.141 ± 0.25 8.05 ± 0.24* 5.18 ± 0.26*** 7.25 ± 0.33***,##
RBCs (1012 cell/L) 10.20 ± 0.45 10.03 ± 0.46 7.41 ± 0.30*** 10.62 ± 0.30###
PLT (109/L) 670.6 ± 11.27 775.1 ± 29.77 1061 ± 40.06*** 1104 ± 52.43***
HCT (%) 46.86 ± 1.84 45.86 ± 0.93 36.45 ± 1.47*** 52.22 ± 2.108###
HGB (mg/dl) 14.01 ± 0.23 13.96 ± 0.47 10.5 ± 0.59*** 15.2 ± 0.32###
Lymph % 71.53 ± 0.92 71.57 ± 0.88 85.53 ± 1.08*** 78.15 ± 1.88**,##
MCV (μm3) 54.4 ± 1.83 51.05 ± 0.83 43.34 ± 1.201*** 49.58 ± 0.54*,##
MCH (Pg) 20.12 ± 0.96 18.8 ± 0.57 16.46 ± 0.27** 17.73 ± 0.75
MCHC (g/dL) 34.89 ± 1.15 33.8 ± 1.06 27.48 ± 0.59*** 30.48 ± 1.31*

Data in the table are represented as means ± SEM (n = 10). WBCs: white blood cells; RBCs: red blood cells; PLT: platelets; HCT: hematocrit; HGB: hemoglobin; Lymph: lymphocytes; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration. Where *P < 0.05, **P < 0.01, and ***P < 0.001 are considered statistically significant compared to the control group, while ##P < 0.01 and ###P < 0.001 are considered statistically significant compared to the STZ-treated group using one-way ANOVA followed by a Tukey-Kramer post-test for multiple comparison (P < 0.05).

Effect of fond and STZ on liver and kidney toxicity parameters in STZ-induced T1DM in mice

Table 2 demonstrated normal levels of creatinine (0.08 ± 0.005 mg/dl), BUN (20.82 ± 0.99 mg/dl), urea (41.77 ± 1.38 mg/dl), ALT (38.14 ± 3.17 U/L), and AST (69.7 ± 3.02 U/L). On the other hand, STZ-treated group showed significant elevation in creatinine (0.192 ± 0.005 mg/dl), BUN (38.57 ± 1.49 mg/dl), urea (76.53 ± 3.13 mg/dl), ALT (275 ± 14.6 U/L), and AST (291.9 ± 22.58 U/L) when equated to control group (0.08 ± 0.005 mg/dl, 20.82 ± 0.99 mg/d), 41.77 ± 1.38 mg/dl, 38.14 ± 3.17 U/L, and 69.7 ± 3.02 U/L, respectively). Treating mice with Fond in the presence of STZ significantly corrected levels of BUN (27.65 ± 1.31 mg/dl), urea (59.85 ± 2.73 mg/dl), ALT (213.9 ± 10.9 U/L), and AST (231 ± 11.5 U/L) compared to STZ-treated mice. Notably, Fond treatment in the presence of STZ showed no significant change in the level of creatinine (0.198 ± 0.007 mg/dl) compared to STZ treatment alone (0.192 ± 0.005 mg/dl).

Table 2.
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Effect of Fond and STZ on liver and kidney toxicity parameters in STZ-induced T1DM in mice.

Control Fond STZ Fond + STZ
Creatinine mg/dl 0.08 ± 0.005 0.09 ± 0.006 0.192 ± 0.005*** 0.198 ± 0.007***
BUN (mg/dl) 20.82 ± 0.99 22.27 ± 0.98 38.57 ± 1.49*** 27.65 ± 1.31**,###
Urea mg/dl 41.77 ± 1.38 43.39 ± 2.49 76.53 ± 3.13*** 59.85 ± 2.73***,###
ALT (U/L) 38.14 ± 3.17 47.29 ± 2.35 275 ± 14.6*** 213.9 ± 10.9***, ###
AST (U/L) 69.7 ± 3.02 57.29 ± 2.35 291.9 ± 22.58*** 231 ± 11.5***,#

Data in the table are represented as means ± SEM (n = 10). Where **P < 0.01 and ***P < 0.001 are considered statistically significant compared to the control group, while ###P < 0.001 is considered statistically significant compared to the STZ-treated group using one-way ANOVA followed by a Tukey-Kramer post-test for multiple comparison (P < 0.05).

Effect of STZ with or without fond on levels of serum insulin and blood glucose

Results in Fig. 1 demonstrated normal serum levels and blood glucose in the saline and Fond-treated mice groups. Whereas the STZ group exhibited a significant elevation in glucose and reduction in insulin levels compared to control mice. The fond + STZ group displayed a significant reduction in glucose level accompanied by a significant increase in insulin level in comparison to the STZ-treated group.

Figure 1. 

Effect of STZ with or without Fond on blood glucose level (A) and serum insulin level (B). Data represent mean ± SEM. Statistical calculations were carried out using one-way ANOVA and Tukey-Kramer as post ANOVA for multiple comparisons, where ***P < 0.001 is considered different significantly in comparison with normal mice, #P < 0.01 and ##P < 0.001 significantly different are considered different significantly in comparison with STZ-treated mice.

Effect of fond on general architectures of pancreatic tissues from STZ-induced T1DM

As noted in Fig. 2A, B, sections of the control and Fond groups showed normal-sized islets of Langerhans with a normal number of beta cells, normal exocrine areas, and normal interstitial blood vessels. STZ-treated group showed few small-sized hypocellular pale-staining islets of Langerhans with a small number of β cells, normal exocrine areas, normal pancreatic duct, and normal interstitial blood vessels. Fond-treatment in the presence of STZ denoted improvement of the pathological features as compared to pancreatic tissues of the STZ-treated group.

Figure 2. 

Effect of Fond on general architectures of pancreatic tissues from STZ-induced T1DM. Tissues from control and Fond groups show average-sized islets of Langerhans with an average number of beta cells (black arrow), average exocrine areas (blue arrow), and average interstitial blood vessels (red arrow). STZ-treated mice showing few small-sized hypocellular pale-staining islets of Langerhans with a small number of b cells (black arrow), average exocrine areas (blue arrow), average duct (yellow arrow), and average interstitial blood vessels (red arrow). Fond-treatment in the presence of STZ represented improvement of the pathological features as compared to pancreatic tissues of the STZ-treated group. (H&E x 400).

Pancreatic β cells insulin, PAR-2, and fibrinogen protein expression in Fond-treatment with or without STZ

Control and Fond-treated mice showed constitutive expression of insulin with a normal, average-sized islet of Langerhans. The STZ-treated mice group displayed a low number of pancreatic β cells and therefore low expression of insulin protein. Treating mice with Fond in the presence of STZ represented higher insulin protein expression in pancreatic β cells as compared to pancreatic tissues of the STZ-treated group (Fig. 3).

Figure 3. 

Insulin protein expression in the pancreatic beta cells in Fond-treated in the presence or absence of STZ using immunofluorescence technique (A), fluorescence intensities for all groups were measured and blotted in graph (B) and represented as fluorescence histogram (C). Control and Fond-treated mice showing constitutive expression of insulin in a normal, average-sized islet of Langerhans. STZ-treated group showing a low number of pancreatic b islets and therefore low expression of insulin protein. Treating mice with Fond in the presence of STZ represented higher insulin protein expression in pancreatic b islets as compared to pancreatic tissues of the STZ-treated group.

Investigation of protease-activated receptor 2 (PAR-2, Fig. 4) and fibrinogen (Fig. 5) protein expression (green fluorescence) in normal and Fond-treatment displayed normal expression in a normal average-sized islet of Langerhans.

Figure 4. 

Expression of protease-activated receptor 2 (PAR-2) protein (green fluorescence) in the pancreatic islets in Fond-treated with or without STZ treatment using immunofluorescence technique (A) Fluorescence intensities for all groups were measured and blotted in a bar chart (B) and represented as a fluorescence histogram (C). Control and Fond-treated mice showing basal expression of PAR-2 in a normal, average-sized islet of Langerhans. STZ-treated group showing strong expression of PAR-2 protein as measured in the islets. Treating mice with Fond in the presence of STZ represented lowered PAR-2 protein expression in pancreatic b islets as compared to pancreatic tissues of the STZ-treated group. PI = pancreatic islets.

Figure 5. 

Fibrinogen protein expression (green fluorescence) in the pancreatic islets of Fond-treated mice with or without STZ treatment using immunofluorescence technique (A) Fluorescence intensities for all groups were measured and blotted in a bar chart (B) and represented as a fluorescence histogram (C). Control and Fond-treated mice showing basal expression of fibrinogen in a normal, average-sized islet of Langerhans. The STZ-treated group showed strong expression of fibrinogen protein as measured in the islets in each field. Treating mice with Fond in the presence of STZ represented lower fibrinogen protein expression in pancreatic b islets as compared to pancreatic tissues from the STZ-treated group. PI = pancreatic islets.

The STZ-treated group showed strong expression of both proteins as measured in the pancreatic islets. Treating mice with Fond in the presence of STZ significantly lowered PAR-2, and fibrinogen protein expression in β cells in comparison to the pancreatic tissues of STZ mice. Figs 3, 4, 5 presented expression of insulin, PAR-2 and fibrinogen, respectively, in all groups using the immunofluorescence technique (A). Fluorescence intensities for all groups were measured and blotted in a graph (B) and represented as a fluorescence histogram (C).

Effect of STZ with/without fond on insulin (red fluorescence) and CXCL16 (green fluorescence) pancreatic islet expression using double immunofluorescence analysis

Results in Fig. 6 displayed a constitutive expression of insulin (red fluorescence) and CXCL16 (green fluorescence) in control and Fond-treated mice in the islets of Langerhans. The STZ-treated group showed a low number of β cells and therefore low expression of insulin protein as compared to the pancreatic tissues of control mice. In contrast, CXCL16 was strongly expressed in STZ treatment in comparison to normal tissues. Treatment of mice with Fond in the presence of STZ denoted significant increase in β cell insulin along with significant reduction in CXCL16 compared to STZ-treated mice. Obviously, both insulin and CXCL16 were co-localized in the pancreatic β cells. Fig. 6 represents the expression of insulin and CXCL16 in all groups using the immunofluorescence technique (A). Fluorescence intensities for insulin (B) and CXCL16 (C) in all groups were measured and blotted in a graph.

Figure 6. 

Effect of STZ with/without Fond on insulin (red fluorescence) and CXCL16 (green fluorescence) protein expression in mice pancreatic tissues using double immunofluorescence staining, 400X (A) Fluorescence intensity graphical presentation for insulin (B) and CXCL16 (C) in mice tissues were analyzed and blotted. Data in the graphs represent Mean ± SEM, where ***P < 0.001 significantly when compared with control mice, ##P < 0.01 considered significantly different from STZ-treated animals, using one-way ANOVA followed by Tukey Kramer multiple comparisons.

Nuclear morphology of pancreatic islets from STZ-treatment with or without fond using DAPI staining

DAPI staining of nuclei from normal and Fond groups showed normal nuclear morphology, size, and architecture. STZ-treated mouse sections displayed DNA condensation, apoptotic nucleus, and DNA fragmentation compared to the normal nucleus from the saline-treated group. Mice treated with Fond in the presence of STZ represented fewer nuclear changes and consistent morphology (Fig. 7).

Figure 7. 

Nuclear morphology of pancreatic islets (indicated by insulin localization with green fluorescence) from STZ-treated mice in the presence or absence of fond using DAPI staining. DAPI staining of nucleus from STZ-treated mice sections showing DNA condensation (yellow arrow), apoptotic nucleus (red arrow), and DNA fragmentation (green arrow) in the yellow box zoom area. Sections from Control, Fond, and Fond + STZ showing normal nuclear DAPI staining. The yellow box denotes the magnified zone, while the red circle states the pancreatic islets. Scale bar: 25 μm.

Discussion

Millions of people worldwide have been affected by type 1 diabetes mellitus (T1DM). It disrupts hemostasis by altering levels of coagulation system proteins and activating platelets (Sobczak and Stewart 2019). After coagulation activation, factor VII (FVII) binds to the extrinsic coagulation factor, tissue factor (TF), and produces a complex. This complex leads to the activation of FIX and FX and consequently stimulates FV. Both FX and FV activation lead to prothrombin cleavage and thrombin generation with activated platelets. Correspondingly, thrombin leads to activation of FVIII, forming a complex with FIXa. Generation of enough thrombin converts fibrinogen from its soluble form into insoluble fibers of fibrin (Ariëns et al. 2002). Earlier efforts have focused on the hypercoagulability complications in T1DM; however, no previous research has studied whether the coagulation system activation is a leading cause of T1DM or not. In the current study, we developed T1DM using i.p. injection with STZ low doses for 5 consecutive days. STZ strongly developed a significant low level of serum insulin, hyperglycemia, increased platelet count (thrombocytosis), and low WBC count (leukopenia), similar to an earlier study (Rehman et al. 2023). STZ is a known chemical that causes T1DM in the models of experimental animals through its selective cytotoxicity on β-cell islets of the pancreas, resulting in low insulin secretion and increased levels of blood glucose (Grieb 2016).

The current study’s results demonstrated a significant reduction in hematocrit (HCT), MCH, MCHC, and MCV, which is consistent with earlier studies (Soma-Pillay et al. 2016; Rehman et al. 2023). Furthermore, it was previously stated that platelets play an important role in blood clotting by producing insoluble fibers of fibrin at the injured blood vessels and therefore halting bleeding (Oyedemi et al. 2010). In this regard, the current findings of an elevated count of platelets and lymphocyte percent in T1DM nicely match the results of (Rehman et al. 2023).

Because diabetes is a metabolic disease, it causes a certain degree of hepatic and renal cell injury. The measurement of hepatic toxicity parameters like serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) or renal toxicity markers including creatinine, blood urea nitrogen (BUN), and blood urea are considered valuable evidence for this toxicity. Herein, STZ induced T1DM and significantly increased (p < 0.001) liver toxicity markers (ALT and AST) and renal toxicity markers (creatinine, BUN, and urea) in comparison to the normal group. These data are in great accordance with earlier studies done by (Adisa et al. 2010; Oyedemi et al. 2010).

Additionally, STZ treatment showed histopathological changes in the form of decreased size of pancreatic islets and number of islet cells, as confirmed with H&E staining. Indeed, the few pancreatic islet cell numbers were correlated with the decreased expression of the specific marker, insulin, for β cells. The observed low number of β cells in STZ-treated mice matches the biochemical observation, including high blood glucose level and low serum insulin level. The morphometric and immunofluorescence results recorded herein confirm the findings presented earlier by (Ahmadi et al. 2010; Abunasef et al. 2014) who mentioned the disruption and reduced number of β-cell islets in an experimental animal model of STZ treatment. On the other hand, the results of the current work included an increase in the expression of both PAR-2 and fibrinogen in β cells of the pancreas, which provides a clear indication that the coagulation system is greatly activated during the development of T1DM. This may indicate the involvement of the coagulation system in β cell death, which might be attributable to cutting off the blood supply to pancreatic islets, leading to ischemia and ultimately β cell death. This could be further evidenced by the significant increase level of insulin in the serum and the significant reduce level of blood glucose after the use of the anticoagulant drug, Fondparinux (Fond). Fond in the current study has improved the deleterious STZ consequences in the form of elevated blood glucose level, reduced serum insulin level, platelet count, as well as elevated insulin expression in the pancreatic β cells resulting from STZ treatment. In addition, it reduced PAR-2 and fibrinogen and prevented DNA condensation, fragmentation, and apoptosis induced by STZ-treatment. These results confirm the former statement that Fond can promote atherosclerotic lesions stability in mice deficient with apolipoprotein E. This may be due to decreased MMP-9 and MMP-13 synthesis (Zuo et al. 2015). Furthermore, Ono reported that Fond repressed glomerular hypertrophy, urinary protein, and extracellular matrix deposition protein deposition in diabetic mice (Ono 2012).

CXCL16 chemokine is an important mediator in many diseases related to inflammation, such as rheumatoid arthritis, prostate cancer, or glomerulonephritis (Darash-Yahana et al. 2009). It works by binding to its specific receptor, CXCR6, expressed in many cells, especially leukocytes (Matloubian et al. 2000; Sheikine and Sirsjö 2008). CXCL16 is found in two distinct forms: the transmembrane form, which is expressed in platelets, inflamed endothelial cells (Abel et al. 2004), macrophages (Shimaoka et al. 2000), T cells (Yamauchi et al. 2004), and smooth muscle cells (Wågsäter et al. 2004) and functions as a receptor for ox-LDL (Abdel-Bakky et al. 2022). The soluble form is released after its cleavage by metalloproteinase ADAM10 (Abel et al. 2004), which binds to CXCR6-expressing immune cells in many cells, including platelets (Borst et al. 2012). In the current study, CXCL16 was significantly increased in the pancreatic β cells in tissues from STZ-treated mice compared to normal non-treated mice. These results might suggest that STZ-treatment enhances platelet activation and adhesion via increased CXCL16 expression in pancreatic β cells. On the procoagulant surface of activated platelets, coagulation factors form a mesh of cross-linked fibrin. These findings were supported by the study of Bost et al. (2012), which demonstrated that activation and adhesion of platelets were observed by CXCL16 through the CXCR6-dependent PI3-kinase/Akt signaling pathway, suggesting a critical role of CXCL16 in thrombo-occlusive diseases. Furthermore, it was previously noted that the platelet CXCL16 may act as a stimulus for thrombotic tendency (Guan et al. 2022).

Conclusion

The current study highlighted, for the first time, that disturbances in the coagulation system may exhibit an important role, not only in diabetes complications but also in its development. This finding is confirmed by using the anticoagulant drug, Fond. Moreover, Fond successfully alleviates CXCL16 and markedly decreases T1DM-specific markers. Targeting CXCL16 and the coagulation system could be a good therapeutic option for diabetes-high-risk individuals.

Additional information

Conflict of interest

All authors declare that there are no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical statements

The authors declared that no clinical trials were used in the present study.

The authors declared that no experiments on humans or human tissues were performed for the present study.

The authors declared that no informed consent was obtained from the humans, donors or donors’ representatives participating in the study.

Experiments on animals: The ethical Committee at Qassim University, Qassim, Saudi Arabia] with an ethical approval number: [24-89-23], dated [May, 6 2024].

The authors declared that no commercially available immortalised human and animal cell lines were used in the present study.

Funding

The authors gratefully acknowledge Qassim University, represented by the Deanship of Scientific Research, for the financial support for this research under the number (2023-SDG-1-HMSRC-35681) during the academic year 1445 AH / 2023 AD.

Author contributions

M.S.A collected data, performed in vivo studies, performed the mouse model, performed statistical analysis, biochemical assay, and drafted the manuscript. E.A.S. participated in the design of the study and the practical study, overall manuscript revision and conducted the immunofluorescence imaging and analysis. H.A.M participated in the manuscript editing and overall manuscript revision. All authors approved the final version of the manuscript to be published.

Consent to participate

Not applicable.

Consent to publish

Not applicable.

Author ORCIDs

Mohamed S. Abdel-Bakky https://orcid.org/0000-0002-0426-5458

Data availability

The authors declare that the data supporting the findings of this study are available within the paper.

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