The levels of visfaTin and Toll-like recepTors in arTerial hyperTension and Type 2 diabeTes melliTus

hypertension and type 2 diabetes mellitus (DM) remain widespread diseases that are becoming more prevalent. the role of visfatin and toll-like receptor (tLr) molecules in the pathogenesis of these diseases requires further research. our aim was to study changes in visfatin and tLr levels in patients with hypertension and type 2 diabetes. Fifty-one patients were examined and divided into two groups: group 1 included 27 patients with hypertension and group 2 included 24 people with hypertension and type 2 DM. the control group included 18 practically healthy people. all individuals underwent general blood test, coagulogram, biochemical blood test, enzyme immunoassay to determine the level of visfatin and tLr in the blood serum and echocardiography. hypertrophy of the walls of the left ventricle (LV) was observed in patients of two observed groups. the most common type of LV geometry was concentric hypertrophy (41.2%). the level of visfatin was significantly higher in patients of group 1, while in patients of group 2 it was decreased (P ˂ 0.05

hypertension and type 2 diabetes mellitus (DM) remain widespread diseases that are becoming more prevalent.the role of visfatin and toll-like receptor (tLr) molecules in the pathogenesis of these diseases requires further research.our aim was to study changes in visfatin and tLr levels in patients with hypertension and type 2 diabetes.Fifty-one patients were examined and divided into two groups: group 1 included 27 patients with hypertension and group 2 included 24 people with hypertension and type 2 DM. the control group included 18 practically healthy people.all individuals underwent general blood test, coagulogram, biochemical blood test, enzyme immunoassay to determine the level of visfatin and tLr in the blood serum and echocardiography.hypertrophy of the walls of the left ventricle (LV) was observed in patients of two observed groups.the most common type of LV geometry was concentric hypertrophy (41.2%).the level of visfatin was significantly higher in patients of group 1, while in patients of group 2 it was decreased (P ˂ 0.05) and the level of TLR was increased (P ˂ 0.05).The elevated level of TLR in the serum of patients with hypertension can be considered a factor of low-grade inflammation, especially in combination with type 2 DM.The increase in the concentration of visfatin in hypertension serves as a more sensitive marker compared to tLr regarding the risk of developing comorbid cardiovascular pathology.the therapeutic treatments of patients with type 2 DM cause a reduction in the concentration of visfatin induced by hypertension.k e y w o r d s: hypertension, type 2 diabetes mellitus, visfatin, toll-like receptors.
H ypertension remains the main cause of cardiovascular pathology with an increas ing trend worldwide.In particular, hyper tension affects 30 to 45% of adults and may cause disability and premature death due to severe compli cations (stroke, myocardial infarction, heart failure, chronic kidney failure).Therefore, at present, the re lationship between hypertension and cardiovascular diseases requires close attention from the point of view of public health and requires timely prevention and effective treatment [1][2][3][4][5].
The prevalence of type 2 diabetes mellitus (DM) is progressively increasing throughout the world.Despite numerous studies, treatment options are still limited.Type 2 DM often occurs in patients with metabolic disorders and especially in obesity due to the development of insulin resistance and an imbalance of pro-and anti-inflammatory factors [6][7][8][9].
The danger of elevated blood glucose levels in type 2 DM in patients with hypertension is associa ted with the occurrence of neuropathy, damage to the glomeruli of the kidneys with the development of nephropathy and retinopathy.The presence of type 2 DM is associated with atherosclerosis and cardiovas cular diseases, as well as cognitive impairment, Alz heimer's disease and vascular dementia [9,10].
Adipose tissue is an organ that is responsible for the synthesis of adipocytokines, which participate in metabolic homeostasis and are the link between obesity and comorbid pathologies.An important pro-inflammatory adipokine that attracts attention is visfatin, which performs a regulatory function in many physiological and pathological processes, and doi: https://doi.org/10.15407/ubj96.02.051 exhibits an insulin-mimetic effect through binding to the insulin receptor-1 and affects the development of insulin resistance [6,11,12].
In the context of metabolic diseases, more evi dence is accumulating about the influence of lowgrade inflammation, excessive expression of proinflammatory cytokines, cellular infiltration and oxidative stress on the course of hypertension and type 2 DM [13].These conditions are accompanied by an increase in the level of visfatin [14].Currently, the association between circulating visfatin and car diovascular pathology is being intensively investi gated [15].
Visfatin is preB cell colonyenhancing factor (PBEF), nicotinamide phosphoribosyltransferase (NAMPT), NAmPRTase, EC = 2.4.2.12, PBEF1.Nowadays the terms visfatin, PBEF and NAMPT are used interchangeably.Visfatin has been proposed as an insulinmimicking adipocytokine predominantly secreted from visceral adipose tissue and correlated with obesity.Visfatin is actively produced by vis ceral adipose tissue and its level correlates with body mass index (BMI) and is associated with the pro gression of obesity.Visfatin is a proinflammatory marker of adipose tissue associated with systemic in sulin resistance and hyperlipidemia [11,12].In addi tion, synthesis of visfatin occurs in cardiomyocytes, skeletal muscles, liver and brain cells.Visfatin acts as a PBEF, which also participates in the regulation of the synthesis of interleukins (IL-1β, IL-6 and tu mor necrosis factor -α). Visfatin exhibits pleiotropic effects (being a cytokine, hormone and enzyme), has both intracellular and extracellular functions and is involved in the synthesis of nicotinamide [12].
A progressive decrease in the level of visfatin leads to severe multiorgan insulin resistance [6].In the context of metabolic diseases, elevated levels of visfatin are considered a marker of endothelial dys function, inflammation and destabilization of athero sclerotic plaques.At the same time, visfatin exhibits an insulin-mimetic effect, which leads to a decrease in the level of glucose and stimulates its utilization in adipocytes and myocytes [6].An increased level of visfatin reflects a higher activity of the inflam matory process, which is associated with the forma tion of carotid atherosclerotic plaques in patients with type 2 DM.Among the biochemical effects of visfatin, which leads to endothelial dysfunction, the effect on the activity of nitric oxide synthase and the activation of oxidative stress due to the increase in the formation of superoxide anion is shown [16].The increased serum level of visfatin can be considered a potential risk marker for the development of comor bid cardiovascular pathology [17,18].
Tolllike receptors (TLR) belong to integral membrane glycoproteins that are involved in re sponse to pathogens and damage, as well as stimu lating the production of antimicrobial peptides, cy tokines and chemokines that neutralize pathogens and initiate inflammation [9].The expression and activation of epithelial TLR depend on their loca tion.In vascular endothelial cells, TLR activation is a link in the inflammatory response of the micro circulation [9].It is believed that TLR may be in volved in the pathogenesis and development of type 2 DM, as well as its complications.TLR regulates the creation of vasoactive lipids and reactive oxygen species, which leads to the release of proinflamma tory cytokines, such as tumor necrosis factor-α and interleukin-1β [9].Recent studies have shown that visfatin induces TLR4-mediated-NF-κB signaling activation [19,21].
The insulin-like effect of visfatin leads to a de crease in glucose levels.However, research results are contradictory with both positive and negative effects found.Relationships and changes in visfatin and TLR levels in patients with hypertension and type 2 DM require further research.The aim of this research is to study changes in the levels of visfatin and TLR in patients with hypertension and type 2 DM.

material and methods
Patient selection.Fiftyone inpatients at the Lviv St. Panteleimon Clinical Hospital were exam ined.They were divided into two groups, group 1 in cluded 27 patients with hypertension and group 2 in cluded 24 people with hypertension and type 2 DM.The control group included 18 practically healthy people.All patients were urgently hospitalized and provided with appropriate treatment to stabilize their condition.Patients were treated according to the ex isting pathologies, namely, with antihypertensive drugs (ACE inhibitors, sartans) and hypoglycemic drugs (insulin, metformin) for the immediate stabi lization of each patient's condition.
ethical approval.Before the examination, all patients signed a voluntary consent to participate in the study, which was approved by the commit tee on the ethics of scientific research, experimental develop ments and scientific works of the Danylo Ha lytsky Lviv National Medical University (protocol No 8 dated 09.26.2022).
Inclusion and exclusion criteria.The inclu sion criteria in the study were age of patients 40-75 years with a diagnosis of hypertension, type 2 DM.Patients with decompensated concomitant diseases, existing mental disorder, alcohol and drug addiction were excluded from the study.
Patients' examination.All study participants underwent a general physical examination with measurement of blood pressure (BP), anthropomet ric measurements with calculation of BMI, complete blood count (erythrocytes, hemoglobin, leukocytes, platelets using Convergys® X5 Main Unit reagents on automatic hematological analyzer Convergys X5 Convergent Technologies GmbHCo.KG (Germany)), coagulogram (prothrombin time, prothrombin index, fibrinogen, international normalized ratio, using Hu man reagents on automatic coagulometer HumaClot Pro HumanGmbH (Germany)), biochemical blood test (glucose, alanine transaminase (ALT), aspartate transaminase (AST), creatinine, urea, glomerular filtration rate (GFR), total bilirubin, total protein), as well as enzyme immunoassay to determine the level of visfatin and TLR in blood serum using Human Visfatin Intracellular (CLOUDCLONE CORP., Houston, USA) and Human TLR4 (ab277392, Ab cam, Cambridge, UK) ELISA Kits.GFR was calcu lated according to the formula CKDEPI Creatinine Equation (2021).In addition, an echocardiographic examination was performed to determine the thick ness of the interventricular septum (IVS), the pos terior wall of the left ventricle (PWT), the size of the chambers of the left atrium (LA), right ventricle (RV), left ventricle (LV), calculation of the myocar dial mass index of the left ventricle (LVMMI) and relative LV wall thickness (RWT), as well as estab lishing the left ventricular ejection fraction (LVEF) and the type of LV geometry, taking into account indicators of RWT and LVMMI.
Diagnosis establishment.The diagnosis of hypertension was established in patients with BP greater than 140/90 mm Hg, as well as in persons with a previously established diagnosis who received antihypertensive drugs.Type 2 DM confirmation is based on the determination of the glucose level of capillary blood, the results of the glucose tolerance test as well as in patients with a previously estab lished diagnosis following current protocols.
Statistical analysis.Statistical analysis of the obtained results was carried out using Microsoft Excel (2010) and GraphPad Prism 8.01.1 licensed software.All data are presented as mean values with standard deviation and as median and percen tiles according to the normality of the distribution, which was determined by the threesigma rule.Stu dent's ttest, chisquare and ANOVA test were used to determine the reliability of intergroup differences.MannWhitneyWilcoxon test was used to assess the difference between two groups with non-Gaussian distribution of parameters.Correlations were studied with the calculation of the Pearson correlation coef ficient.Significance was considered at P < 0.05.

results and discussion
During the patients' examination, no difference was found in gender, age and anthropometric parameters, which indicates the homogeneity of the examined groups.It was established that sys tolic BP was significantly higher in individuals of group 1 (P ˂ 0.05), while diastolic BP did not show a differen ce (P ˃ 0.05) (Table 1).Heart rate (HR) as well as systolic and diastolic BP were significant ly higher in patients of the two main groups com pared with practically healthy individuals of the control group (P ˂ 0.05).There was no statistically significant difference in the duration of anamnesis of hypertension in patients of the observed groups (group 1 -14.1 ± 4.2 years, group 2 -14.8 ± 5.0 years, P > 0.05, Student's ttest).
The prothrombin index and hemoglobin were significantly lower (P ˂ 0.05), the blood glucose level (P ˂ 0.01) and the fibrinogen level was higher (P ˂ 0.01) in patients of group 2 (Table 2).
Glucose level was significantly higher in the group with hypertension and type 2 DM (P ˂ 0.01) as well as increased urea level (Table 3).The GFR was significantly lower in groups 1 and 2 (P ˂ 0.05), as well as a decrease in its level in patients of the two groups, which is probably associated with the development of hypertensive and diabetic nephropa thy [10].
Structural and functional changes of the myo cardium are characteristic of the long course of hy pertension, characterized by myocardial hypertro phy [21].There was an increase in the thickness of the IVS (P < 0.01), the size of the LA (P < 0.01), an increase in the LVMMI in patients of two experi mental groups and an increase in the size of the LV in patients of groups 1 and 2 (P < 0.01) (Table 4).The most common type of LV geometry was concentric hypertrophy (41.2%).These changes were caused by longterm pressure overload of the LV, which is typi cal for hypertension.

t a b l e 1. comparison of gender, age and physical parameters of patients of the examined groups with hypertension and type 2 diabetes mellitus
Notes: BP -blood pressure; BMI -body mass index; DM -diabetes mellitus; P C1 -Pvalue when comparing the control group with group 1; P C2 -Pvalue when comparing the control group with group 2; P 12  The level of visfatin was significantly highest in patients with hypertension, while it was decreased in patients with hypertension and type 2 DM com pared to group 1 (P ˂ 0.05), which could be due to the effect of drug treatment with insulin and met formin aimed at correcting carbohydrate metabolism (Fig. 1).The existing results are contradictory be cause according to the researchers, the level of vis fatin was increased in individuals with type 2 DM [22].Taking into account the data of other studies, it is known that antihypertensive, hypolipidemic and hypoglycemic drugs can both decrease and increase the content of the studied protein [8,23].
The insulin-mimetic effect of visfatin has at tracted attention since it became possible to identify an adipokine that allows the lowering of the blood glucose level; however, numerous studies indicate ambiguous relationships between the level of visfatin and blood glucose [6].In an in vivo study by Haider and colleagues, it was established that intravenous infusion of glucose increased the level of visfatin in healthy individuals, while infusion of insulin and somatostatin, on the contrary, decreased the level of this enzyme [6].Inhibition of visfatin synthesis in plasma by oral administration was greater in over weight and female patients, whereas intravenous glu cose infusion, induction of osmotic stress (by manni tol administration) and use of sex steroids (estradiol and testosterone) did not promote visfatin synthesis in this group of persons [12].
Intracellular enzymatic effects of visfatin affect the synthesis of NAD + , which makes it an important

t a b l e 2. comparison of the results of general blood test and coagulogram of patients of the examined groups
Notes: DM -diabetes mellitus; INR -international normalized ratio; P C1 -Pvalue when comparing the control group with group 1; P C2 -Pvalue when comparing the control group with group 2; P 12  regulator particularly of sirtuins, polyADPribose polymerases and proteins [24].Sirtuins are a class of NADdependent proteins that have the properties of histone deacetylases and monoribosyltransfera ses.Visfatin, in turn, possessing ribosyltransferase activi ty, catalyzes the formation of NAD + from nico tinamide, i.e., serves as the main enzyme of its reuti lization reactions (salvage) [19].Because the activity of sirtuins depends on the content of NAD + , NADH and nicotinamide or a combination of these parame ters, it is visfatin that can serve as a decisive factor in their production.Analyzing the results, it can be as sumed that the increased level of visfatin in patients with hypertension has both a direct effect on meta bolic processes and possibly mediates its metabolic effects through the formation of sirtuins.Visfatin, regulating the level of NAD + , also affects the func tioning of polyADPribose polymerases (enzymes that participate in the post-translational modification of proteins using NAD + ), regulate cell division, DNA repair and RNA transcription [25,26].
The extracellular effects of visfatin are associated with the modulation of the immune response.Visfatin has been shown to regulate about 50 N.Pokrovska, S. Mahiiovych, I. Fomenko et al.

t a b l e 3. comparison of the results of general blood test and coagulogram of patients of the examined groups
Notes: ALT -alanine aminotransferase; AST -aspartate aminotransferase; DM -diabetes mellitus; GFR -glomerular filtration rate; P C1 -Pvalue when comparing the control group with group 1; P C2 -Pvalue when comparing the control group with group 2; P 12 [19].In addition, it was estab lished that visfatin induces the production of mono cyte chemoattractant protein 1 (MCP-1) [27] and the expression of matrix metalloproteinases [28].Vifatin is associated with the activation of many inflamma tory pathways, in particular NF-κB -the activated intracellular pathway [29].Inflammasome activation is an important factor in the pathogenesis of adipose tissue inflammation, insulin resistance and meta bolic diseases associated with obesity and impaired visfatin synthesis [30].Visfatin has been shown to induce endothelial dysfunction in the early stages of obesity via the NLRP3 inflammasome [31].Visfa tininduced vascular dysfunction in mice was also found to involve the NLRP3 inflammasome and IL-1ß through a tolllike receptor 4 (TLR4)dependent NAMPT signaling pathway [14].However, in our study no correlation was found between the level of visfatin and the concentration of TLR in the blood serum of patients with hypertension and type 2 DM.A significant increase in the level of TLR in patients of group 2 (P ˂ 0.05) can be explained by an increase in the activity of the inflammatory process,

t a b l e 4. comparison of the results of the morpho-functional characteristics of the myocardium in patients with hypertension and type 2 DM
Notes: DM -diabetes mellitus; IVS -interventricular septum, LA -left atrium; LV -left ventricle; LVEF -left ven tricular ejection fraction; LVMM -myocardial mass index of the left ventricle; LVMMI -myocardial mass index of the left ventricle; PWT -posterior wall thickness; RWT -relative wall thickness; P C1 -Pvalue when comparing the control group with group 1; P C2 -Pvalue when comparing the control group with group 2; P 12  which is confirmed by established positive correla tions between the level of TLR and leukocytes in the blood (r = 0.480; P ˂ 0.05) and BMI (r = 0.428; P ˂ 0.05) (Fig. 2).
An increase in the level of TLR was also as sociated with an increase in the size of the LA (r = 0.795; P ˂ 0.01).It is known that TLR activation is enhanced under conditions of hyperglycemia, and can be a trigger for the release of pro-inflammatory cytokines [32].
There is some data about the relation between the increasing level of visfatin and obesity, low grade inflammation and insulin resistance [33].However, it is known that insulin can affect the level  of visfatin causing its decrease.The patients received therapy with insulin to lower their blood glucose lev els.Insulin may promote the anti-inflammatory ef fect and suppress the generation of O 2 radicals and oxidative stress [33].Nevertheless, no difference was found in visfatin concentration in patients with type 2 DM and healthy individuals [19].At the same time, one more study shows the association between the GFR and level of visfatin.It was shown that in patients with GFR more than 75 ml/min/1.73m 2the level of visfatin was lower, while in individuals with decreased GFR visfatin concentration tended to increase.In our study, the group of patients with hypertension showed a significantly lower level of GFR and a higher level of visfatin [34].However, the level of TLR was increased and its concentra tion was not affected despite the treatment.All blood samples were taken 3-5 days after hospitalization and receiving therapy.We consider that visfatin is a more sensitive marker that immediately tends to decrease after using insulin for diabetes treatment compared to TLR concentration that remained in creased.Further research is needed to establish more precise mechanisms.
To summarize, hypertension and type 2 DM affect the concentration of visfatin and TLR.The highest level of visfatin in blood serum was found in patients with hypertension (P ˂ 0.05), while a lower level of this marker was observed in people with a comorbid course of hypertension and type 2 DM, which is probably related to the peculiarities of drug treatment of the main pathologies using metformin and insulin.The TLR level is significantly higher in patients with a combined course of hypertension and type 2 DM (P ˂ 0.05) as well as established correla tions with the level of blood leukocytes (r = 0.480; P ˂ 0.05) and BMI (r = 0.428; P ˂ 0.05) due to pos sible consequence of the synthesis of pro-inflamma tory cytokines and activation of the inflammatory process in conditions of hyperglycemia.
The longterm course of hypertension in pa tients of two groups led to the occurrence of mor phofunctional changes in the myocardium, namely, hypertrophy of the walls of the LV myocardium and an increase in the size of the LV.Under conditions of chronic LV pressure overload, concentric hypertro phy was the most frequently determined type of LV geometry.It was observed in 41.2% of all patients, due to the anamnesis of hypertension.
conclusions.The elevated level of TLR in the serum of patients with hypertension can be conside red a factor of low-grade inflammation, especially in combination with type 2 DM.The increase of vis fatin concentration in hypertension serves as a more sensitive marker compared to TLR regarding the risk of developing comorbid cardiovascular pathology.The therapeutic approaches for treating patients with type 2 DM cause the reduction of visfatin concentra tion induced by hypertension.

Fig. 1 .
Fig. 1. comparison of visfatin levels in patients of the examined groups (Mann-Whitney-Wilcoxon test)

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Pvalue when comparing group 1 with group 2, using ANOVA test; *chisquare test