The effect of rivaroxaban and dabigatran on glycaemic parameters and lipid profile in high-fat diet and low-dose streptozotocin-induced diabetes mellitus in male Wistar rats

INTRODUCTION

Diabetes mellitus (DM) is a group of metabolic illnesses defined by high blood sugar levels caused by a complex interaction of hereditary and environmental variables. The causes contributing to elevated blood sugar levels include reduced insulin secretion, poor glucose utilisation and increased glucose generation. In addition, the metabolic dysregulation in DM leads to secondary pathophysiological changes across various organ systems, impacting individuals and healthcare systems globally.^[1] The global prevalence of diabetes has increased significantly over the past 30–40 years, and estimates indicate that it will continue to climb. From more than 100 million in 1995 to 250 million by 2025.^[2] The International Diabetes Federation forecasts that by 2040, 642 million people will be affected by diabetes.^[1] In India, the prevalence of diabetes is estimated at 7.3%, with pre-diabetes affecting 10.3% of the population, translating to 69.2 million individuals with diabetes and 77.2 million at risk.^[3]

Inflammation is an important part of the pathophysiology of diabetes. Decreased insulin secretion and increased insulin resistance result from inflammation, where cytokines affect beta cell function, leading to secretory dysfunction and increased apoptosis. The glucotoxicity and lipotoxicity as an outcome further increase the inflammatory process. Elevated white blood cell counts, particularly neutrophil counts, are indicators of inflammation and are associated with declining insulin sensitivity, diabetes and cardiovascular issues. Inflammatory markers such as C-reactive protein (CRP), interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-a) are elevated in patients with metabolic syndrome and type 2 diabetes (T2DM).^[4,5] The NLRP3 inflammasome generates interleukin 1 beta (IL-1b) significantly contributes to obesity and diabetes.^[6] Adipocytes, central to inflammation and insulin resistance in T2DM, secrete adipocytokines such as leptin, adiponectin and resistin, influencing insulin secretion and resistance. Adipose tissue also releases dipeptidyl peptidase 4, enhancing the degradation of glucagon-like peptide-1 and affecting beta cell function.^[4] Obesity, characterised by elevated nonesterified fatty acids, glycerol, hormones, cytokines and pro-inflammatory markers, impairs b islet cells’ function in the pancreas.^[7]

Thrombotic events significantly contribute to mortality in diabetic individuals, with cardiovascular issues responsible for 75% of all deaths, and cerebrovascular and peripheral vascular complications accounting for the remaining 25%.^[8] The vascular endothelium is damaged in diabetes, leading to augmented platelet and clotting factor activation, marking a hypercoagulable state. Laboratory analyses consistently show heightened coagulant potential, chronic platelet activation and diminished fibrinolytic potential in diabetic individuals, reinforcing the hypercoagulable nature of diabetes.^[8]

Rivaroxaban and dabigatran are anticoagulants with proven anti-inflammatory effects, reducing levels of inflammatory markers in diabetes. Studies have shown that dabigatran inhibits nuclear factor kB, TNF-a, IL-1b and IL-6 activities and increases catalase and superoxide dismutase activities.^[9] Rivaroxaban treatment reduces biomarkers such as D-dimer, thrombin antithrombin complex, high-sensitivity CRP and high-sensitivity IL-6 (hsIL-6).^[10] Given their anti-inflammatory properties and the lack of comprehensive studies on their direct effects on diabetes, this research aims to evaluate the impact of rivaroxaban and dabigatran on glycaemic parameters, lipid profile and inflammatory markers in a rodent model of diabetes induced by a high-fat diet (HFD) and low-dose streptozotocin (STZ).

MATERIALS AND METHODS

This was an experimental study involving healthy adult male Wistar rats. The total sample size was 38. The animals were separated into five groups of eight each, with the exception of the usual control group, which included six rats. The number of rats in the diabetic groups was higher to compensate for the expected mortality. The study was approved by the institutional animal ethics committee. The study was conducted as per the guidelines of the committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi. 3–4-month-old adult healthy male Wistar rats with a mean weight of 200 ± 20g were procured from the central animal house of J.N. Medical College, KAHER, Belagavi. The animals were kept in cages made of polypropylene with stainless steel top grills that provided food and water ad libitum. The rodents had unlimited access to food and water. The meal was delivered in the form of pellets. Paddy husk was utilised as bedding in the cages. The animals were acclimatised to a 12:12 h light-dark cycle for 10 days before the day of experimentation. The rats were divided into normal control (NC), diabetic control (DC) and treatment groups.

The rats of the required weight range were kept fasting overnight in the cages, a day before initiation of the study. Of the 38 rats, 6 were assigned to the NC group. The rest of the 32 rats were fed an HFD for 14 days. The NC rats were fed with the standard chow pellet.

On day 15, blood glucose levels were measured for all rats using the tail veins with a 30G insulin needle before their induction of Diabetes to check for the impact of HFD on the blood levels.

Type 2 DM was induced in rats as per the existing literature using low-dose STZ (35 mg/kg) and HFD.^[11,12]

40 mg STZ was weighed in a glass beaker, and the beaker was covered with aluminium foil. Fresh citrate buffer of 0.05M at a pH of 4.5 was prepared immediately before injection. The 0.05M citrate buffer was prepared by mixing 0.05M sodium citrate and 0.05M citric acid in a ratio of 2:3, and the pH was adjusted to 4.5. The sodium citrate buffer was used to dissolve STZ to a final concentration of 40 mg in 8 mL just before injection. The STZ solution was made right before injection and given out 5 min after it was dissolved. Using a 1 mL syringe and 23G needle, STZ was injected intraperitoneally (i.p.) into the rats belonging to various experimental groups at 35 mg/kg (6 mL/kg). An equal volume of citrate buffer was injected i.p. into the NC rats. After injection, STZ treated rats were given 5% glucose instead of water for 24 h to minimise hypoglycaemic shock-related mortality. Confirmation of the diabetes after 72 h of STZ administration, the fasting blood glucose (FBG) levels of all rats were measured using a glucometer from tail vein blood samples. Diabetic rats were defined as those with FBG levels of more than 200 mg/dL and were included in further study.^[13,14] Diabetes was induced successfully in all rats.

The confirmation day of diabetes was considered day one of diabetes. The NC and DC groups received saline as a vehicle. The standard group received metformin (MF), and the treatment groups received the study drugs Rivaroxaban and Dabigatran.

The study included several groups with specific treatments and doses. Group I, the NC group (n = 6), received only the vehicle at a dose of 1 mL. Group II, the DC group (n = 8), also received the vehicle at a dose of 1 mL. Group III consisted of diabetic rats treated with MF at a dose of 180 mg/kg (n = 8). Group IV included diabetic rats treated with rivaroxaban at a dose of 1.8 mg/kg (n = 8). Group V consisted of diabetic rats treated with dabigatran at a dose of 27 mg/kg (n = 8).

All the drugs were administered orally as a single daily dose from day 1 to day 42.

Parameters for the study were measured at various time points using specific methods. Body weight was recorded at baseline, after 14 days of HFD, 21 days after DM induction, and at the end of the study using a weighing balance. FBG levels were measured at baseline, after 14 days of HFD, 72 h after STZ administration, 21 days after DM induction and at the end of the study, using rat tail vein samples tested after overnight fasting with a glucometer. The lipid profile, including total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides, was measured at baseline and the end of the study by collecting blood samples from the rat tail vein. Inflammatory markers, specifically IL-1b, IL-6 and TNF-a, were measured at the end of the study by analysing blood samples collected from cardiac puncture using Enzyme-linked immunosorbent assay (ELISA) kits.

On day 43 of the study, thiopentone sodium at a dose of 120 mg/kg was given as an intraperitoneal injection.^[15] After this, cardiac puncture was performed for blood collection. The blood collected was then used to measure the serum lipid levels and was also centrifuged to create a homogenate to measure the levels of inflammatory markers using ELISA. Animals were then sacrificed as per the CPCSEA guidelines.

RESULTS

This study was carried out to evaluate the effect of dabigatran and rivaroxaban on glycaemic parameters, lipid profile and inflammatory markers in HFD and low-dose STZ-induced DM in male Wistar rats.

Data collected in the study were compiled into an Excel sheet and were analysed using GraphPad Prism Version 10.0. Data is expressed as Mean ± SEM. Parameters such as body weight, blood glucose, lipid levels and inflammatory markers were analysed using appropriate statistical tests. P < 0.05 was considered statistically significant.

Body weight

Body weight was measured at baseline, following 14 days of the HFD, 21 days after treatment, and at the end of the study.

The mean body weights of all the groups were comparable at baseline. After 14 days of the HFD, the mean body weights (in grams) of NC, DC, MF, rivaroxaban and dabigatran groups were 251 ± 5.865, 260 ± 2.853, 244 ± 10.71, 258 ± 4.7 and 248.3 ± 4.78, respectively. Two-way ANOVA showed no statistically significant difference between the groups [Graph 1].

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Graph 1:

The Effect of Metformin, Rivaroxaban and Dabigatran on Body Weight in High Fat Diet and Streptozotocin induced diabetes in Wistar rats. Values are expressed as Mean ± SEM, Analysis was done by ANOVA and post hoc Tukey’s test P < 0.0001, indicates the significant difference between diabetic control and treatment groups. #P < 0.001 indicates the significant difference between diabetic control and normal control group.

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On Day 21 of treatment, the mean body weights (in grams) of NC, DC, MF, rivaroxaban and dabigatran groups were 290 ± 3.990, 232.3 ± 2.684, 275.3 ± 9.531, 273 ± 4.54 and 268.8 ± 5.219, respectively. Two-way ANOVA revealed a statistically significant weight reduction between groups with P < 0.0001. Post hoc Tukey’s test showed that there was a statistically significant difference between the DC group in comparison with treatment groups (P < 0.0001) [Graph 1].

At the end of the study mean body weights (in grams) of the NC, DC, MF, rivaroxaban, and dabigatran groups were 337.5 ± 3.819, 180.8 ± 4.729, 304.6 ± 10.25, 286.7 ± 3.727, 292.4 ± 5.34, respectively. Two-way ANOVA revealed a statistically significant weight reduction between groups with P < 0.0001. Post hoc Tukey’s test showed that there was a statistically significant difference between the DC group in comparison with treatment groups (P < 0.0001) and also between the DC and NC (P < 0.001) [Graph 1].

FBG at various intervals

FBG was measured at baseline, following 14 days of the HFD, 72 h after STZ injection, 21 days after treatment and at the end of the study.

The mean FBG of all the groups was comparable at baseline. Two-way ANOVA revealed that there was no significant difference between various groups at baseline and after 14 days of HFD administration. Following 72 h of STZ administration, the mean FBG values (mg/dL) of DC, MF, rivaroxaban and dabigatran groups were 103.7 ± 1.909, 341.5 ± 12.51, 316.5 ± 10.99, 316.5 ± 13.93, 322.6 ± 13.27, respectively with no statistical difference detected between them using two-way ANOVA [Graph 2].

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Graph 2:

The Effect of Metformin, Rivaroxaban and Dabigatran on Fasting Blood Glucose in High Fat Diet and Streptozotocin induced diabetes in Wistar rats. Values are expressed as Mean ± SEM, Analysis was done by ANOVA and post hoc Tukey’s test *P < 0.0001, indicates the significant difference between diabetic control and treatment groups.

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After 21 days of treatment the mean FBG values (mg/dL) of DC, MF, rivaroxaban, and dabigatran groups were 368.5 ± 12.35, 288.8 ± 10.21, 310.4 ± 10.43 and 313.8 ± 13.02 respectively Two way ANOVA revealed a statistically significant reduction in blood glucose levels between groups with P < 0.0001. Post hoc Tukey’s test showed that there was a statistically significant difference between the DC group in comparison with the treatment groups (P < 0.0001) [Graph 2].

At the end of the study, the mean FBG values (mg/dL) of NC, DC, MF, rivaroxaban and dabigatran groups were 102.3 ± 1.838, 397.5 ± 10.06, 275.9 ± 9.33, 299.3 ± 13.54 and 296.4 ± 12.12, respectively. Two-way ANOVA revealed a statistically significant reduction in blood glucose levels between groups with P < 0.0001. Post hoc Tukey’s test showed that there was a statistically significant difference between the DC group in comparison with treatment groups (P < 0.0001) [Graph 2].

DISCUSSION

The purpose of this study was to assess the impact of anticoagulant medications, rivaroxaban and dabigatran, on glycaemic parameters in HFD and STZ-induced diabetes model in adult male Wistar rats. The anti-inflammatory effects of rivaroxaban and dabigatran are well established. However, the evidence on the effects of rivaroxaban and dabigatran on blood glucose levels is inconclusive, which has been the objective of the present study.

An HFD induces insulin resistance and/or glucose intolerance in rats, similarly to humans. STZ induces b-cell failure through mechanisms such as deoxyribonucleic acid alkylation, nitric oxide release and reactive oxygen species generation.^[16] The HFD STZ model was utilised to induce diabetes in the current investigation because it closely resembles the natural pathophysiology of actual diabetes. A state of permanent hyperglycaemia develops in the rats 48 h after STZ injection, and this is considered the start of the disease. Furthermore, 72 h post-injection is considered the best time point for measuring the blood glucose level to confirm the diagnosis of diabetes.

The current investigation found that rivaroxaban and dabigatran were effective at preventing diabetes-induced dramatic weight loss and were comparable to MF therapy. As diabetes progressed, there was significant weight reduction in the untreated diabetic rats compared to the non-diabetic animals. Such a drastic weight loss was also observed in studies done by Cheng et al.^[17] and Mestry et al.^[18] This can be explained on the basis that STZ-induced diabetes is accompanied by a substantial decrease in body weight as a result of hyperglycaemia, hypoinsulinaemia, muscle wasting and protein loss.^[17,19] Treatment of diabetic rats significantly improved body weight, indicating that muscle tissue damage caused by hyperglycaemia was prevented.

A state of hyperglycaemia was successfully generated following a single dose of STZ in this study. The FBG levels of untreated diabetic rats were consistently raised throughout the trial. The FBG of the rats treated with MF was substantially lower than that of the untreated rats halfway through the study. Rivaroxaban and dabigatran were also successful in bringing down the glucose levels, though the difference was not statistically significant. At the end of 6 weeks, rivaroxaban and dabigatran significantly reduced the FBG in comparison with the DC rats. Moreover, the results of rivaroxaban and dabigatran were comparable to MF therapy. These findings are in alignment with studies by Liu et al., and Cheung et al., who have demonstrated the reduction in blood glucose by rivaroxaban and dabigatran, respectively.^[20,21]

Diabetes induction worsened the lipid profile in our study. The untreated rats had significantly high total cholesterol, triglyceride values compared to the non-diabetic rats, along with low HDL values. LDL values were also significantly higher compared to the MF and dabigatran groups. In our study, MF monotherapy significantly reduced the total cholesterol, triglycerides and LDL levels compared to the untreated diabetic rats. Given that MF is known to improve lipid profiles, this result is in line with previous studies.^[22,23] The impact of rivaroxaban and dabigatran on the lipid profile has not been studied in detail. Our study demonstrated that MF, rivaroxaban and dabigatran reduced the total cholesterol significantly in comparison to the DC group by the end of the study. These findings are similar to studies done by Rocha et al. and Luo et al.^[24,25] MF and dabigatran reduced triglycerides significantly in comparison to DC; however, rivaroxaban did not affect it. These findings are similar to studies done by Rocha et al. and Gillani et al.^[24,26]

In our study, all three treatment groups were able to significantly lower serum IL-1, IL-6 and TNF levels in diabetic rats when compared to untreated diabetic rats. MF has previously been reported to lower these inflammatory markers in animal models of diabetes. In a rodent study by Kotb El-Sayed et al., it was observed that MF treatment significantly reduced the serum TNF and IL-6 compared to the DC rats.^[27] In a 2018 study by Kotb et al., MF significantly reduced serum IL-6, TNF-a when compared to untreated diabetic rats.^[28] Our study also confirmed the beneficial effect of MF on these inflammatory markers.

We found that rivaroxaban and dabigatran were effective in reducing these inflammatory parameters. The anti-inflammatory characteristics of rivaroxaban and dabigatran are well documented. The study by Song et al. demonstrated that dabigatran is a powerful inhibitor of IL-6, IL-1 and TNF-a. Dabigatran significantly inhibits the levels of proinflammatory cytokines IL-1b, IL-6 and TNF-a in acute myocardial infarction rabbits.^[9]

Rivaroxaban reduced the expression of inflammatory molecules in the aorta, including TNF-a, which is a key proinflammatory cytokine.^[9] Research conducted in vitro on mouse peritoneal macrophages or the murine macrophage cell line RAW264.7 revealed that factor Xa stimulated pro-inflammatory activation of macrophages, leading to an increase in IL-1b production. This effect was inhibited when rivaroxaban was present.^[9] In patients with atrial fibrillation undergoing planned cardioversion, rivaroxaban treatment resulted in a significant reduction in levels of hs IL-6 from baseline to the end of treatment.^[10]

The findings of the present study thus support that rivaroxaban and dabigatran exert beneficial effects on glycaemic parameters of diabetes that could be due to their effect on inflammatory markers. However, further studies are required to establish the effect of rivaroxaban and dabigatran in combination with standard anti diabetic drugs like MF to support their addition in the treatment of diabetes.

Limitations and future recommendations include doing studies of rivaroxaban and dabigatran in combination with MF, including histopathological examination and homeostasis model assessment-insulin resistance as investigations.

CONCLUSION

The present study showed that the treatment of diabetic rats with rivaroxaban and dabigatran improved the HFD STZ-induced biochemical alterations in glycaemic parameters and inflammatory markers. Furthermore, MF and rivaroxaban and dabigatran were found to be equally efficacious across all trial variables. Based on the findings of this study, it can be concluded that rivaroxaban and dabigatran may be a promising option for the management of Type 2 Diabetes. Future research of rivaroxaban and dabigatran in combination with standard antidiabetic drugs like MF in patients with diabetes, as well as clinical trials with larger sample sizes, needs to be considered.