Modulation of cardio-metabolic disorders by Tocilizumab in rats with fructose-induced metabolic syndrome: Role of cardiac NLRP3 Inflammasome and TIMP1

: Introduction: Insufficient regulation of NLRP3 inflammasome and TIMP1 has a role in the pathogenesis of CVD. Moreover, tocilizumab has a cardio-metabolic protective effect mainly through improved metabolic indices, IL6, alongside cardiac TNFα, NLRP3, and TIMP1 activity. Aim of the study: The study aims to examine the effect of IL6 receptor blocker; tocilizumab on cardiovascular disorders (CVD) in rats with fructose-induced metabolic syndrome (MetS); elucidating how it works. Materials and Methods: Four groups of male albino rats were allocated into Control, Tocilizumab (8mg/kg/week intraperitoneal), Fructose (10-25%in drinking water), and Fructose +Tocilizumab. After seven weeks of the experiment, measurements of systolic blood pressure (SBP), heart rate (HR), serum fasting glucose, insulin, lipids, IL6 levels, and HOMA test for insulin resistance (IR) were done. Cardiac tissue concentrations of nucleotide-like receptor protein 3(NLRP3) inflammasome and tissue inhibitor metalloproteinase1 (TIMP1) were estimated. Finally, a histopathological heart examination was performed. Results: Treatment with tocilizumab significantly alleviated fructose-induced metabolic disorders such as increased HR, glycemic parameters, IR, lipid profile, and IL6. Concurrently, tocilizumab ameliorated cardiac NLRP3 and TIMP1 concentrations; normalizing the histopathological findings of inflammatory infiltration, interstitial fibrosis, and TNFα immunobiological reactivity. Conclusions: These results show promising cardiovascular protective effects of tocilizumab to be used clinically. However, further cardiovascular investigations such as ventricular contractility, ECG, and vascular reactivity may be required to interpret more benefits of tocilizumab for this purpose.


Introduction
Metabolic syndrome (MetS) is a multiple risk factor of biochemical, metabolic, physiological, and clinical disorders such as hyperglycemia, reduced insulin sensitivity, abnormal lipid profile, abdominal obesity, hypertension, and inflammation.These disorders can increase the risk of cardiovascular diseases (CVD) by twofold and the risk of diabetes by fivefold [1].
Fructose-induced MetS is strongly correlated with chronic inflammation, characterized by increased levels of blood mononuclear cells and enhanced systemic release of inflammatory cytokines [2].IL6 is crucial in several metabolic pathways.Increased levels of IL6 in MetS can impact several important conditions such as elevated hepatic glucose secretion, diminished insulin-mediated skeletal muscle glucose uptake, IR, and an increased likelihood of developing hypertension [3].
Tocilizumab, a monoclonal antibody that blocks IL6 receptors (IL6R), is employed to treat inflammatory and autoimmune disorders.
Tocilizumab can inhibit both the membranebound and soluble versions of IL6R, hence blocking the activity of inflammatory cells and reducing the levels of pro-inflammatory cytokines generated [4].
Inflammasomes are crucial intracellular multiprotein complexes of the innate immune system, involving a group of receptor proteins known as nucleotide-like receptor proteins (NLRP), involving the NLRP3 inflammasome.
The NLRP3 inflammasome plays a significant role in developing and advancing IR, type 2 diabetes mellitus (T2DM), and other metabolic diseases [5].
Matrix metalloproteinases (MMPs) are a class of enzymes that play a role in both normal tissue development and remodeling, as well as in the progression of abnormal conditions.The activity of MMP is regulated by the proteolytic impact of endogenous tissue inhibitors of metalloproteinases (TIMPs) [6].The expression of TIMP1 is substantially regulated by growth factors and cytokines, such as IL6 and TNFα.
Under specific pathological circumstances, there may be an occurrence of improper restructuring of cardiovascular tissue.This can happen either because of excessive production of metalloproteinase or inadequate control of TIMPs.Consequently, a range of disorders such as arthritis and CVD can develop [7].
Because chronic inflammation is observed as a key factor of cardio-metabolic disorders, especially T2DM, MetS, and CVD, treatment of inflammation could represent an important therapeutic target for MetS [8].This research intends to examine the impact of tocilizumab on cardio-metabolic disorders in male white rats with fructose-induced Mets.

Experimental Animals and Design
Twenty-four adult male Wister albino rats weighing 150±20 g was purchased from the National Research Institute, Cairo University, and observed for one week before the study, feeding normal standard diet & drinking tap water ad libitum.The animals were separated into four groups (n=6) as follows: Control and Tocilizumab (8mg/kg intraperitoneal once weekly [9]; rats received normal food and tap water.
Fructose group; rats were given 10% fructose in drinking water for the first 39 days, then fructose concentration was modified to 25% in the last 10 days [10,11], and Fructose +Tocilizumab; rats were given fructose and tocilizumab for seven weeks.

Blood Pressure and Heart Rate Measurements
At the end of the seventh week, the SBP & HR of all rats were assessed utilizing the LE 5001 non-invasive blood pressure meter (Panlab; Harvard Apparatus, Barcelona, Spain).The rats were inserted into the restrainer.The tail cuff/transducer was placed into the base of the tail area, the selector switch was switched on and the readings were displayed on the apparatus' screen [12].

Blood Sampling and Histological Study
Under light general anesthesia with diethyl ether, blood samples were collected from retro-orbital veins of fasting rats, centrifuged and serum was separated for determination of biochemical measurements.The rats were promptly euthanized by cervical dislocation.The heart of each rat was removed and rinsed with an ice-cold saline solution.
Subsequently, the heart was divided into two sections.
One section was frozen at -60 ºC for the quantification of NLRP3 and TIMP1 concentrations, while the other section was fixed in 10% formalin for histopathological analysis.We acquired serial slices that were 4 microns thick and stained them with concerning the following equation [14]: IR= Glucose (mg/dL) X Insulin (μU/mL)/405.

Statistical analysis
Statistical analysis was done using SPSS software Version 22. Data analysis was made utilizing one-way analysis of variance (ANOVA).The comparison among groups was done utilizing the post-hoc Tukey test.All data were expressed as mean± standard deviation (SD) and variances were regarded as significant at P-value <0.05.Pearson correlation was done to test the correlation among normally distributed variables.

Discussion
In this study, researchers induced a model of MetS by giving subjects fructose in their drinking water for seven weeks.This resulted in significant increases in serum metabolic indices, IL6, SBP, and HR, which is consistent with previous studies [11,12] Moreover, the lipogenic impact of fructose, which causes the accumulation of triglycerides and cholesterol in the liver, might result in glucose intolerance and reduced insulin sensitivity [16].
Research has demonstrated that specific health disorders, including atherosclerosis, cardiovascular events, hypertension, and MetS, are marked by a state of low-level inflammation triggered by IL6 and Tumor TNFα [3,17].The results suggest that tocilizumab, a medication utilized for inflammatory treatment, effectively ameliorates several cardio-metabolic alterations in fructose-fed rats, except higher SBP, in comparison to rats not administered the drug.
The findings indicate that tocilizumab may reduce the levels of IL6, which are elevated by fructose, by inhibiting the signal transduction of soluble IL6R.Consequently, this restraint may result in a decrease in cardiac TNFα and an enhancement in insulin sensitivity in this specific animal model [15].
The observed reduction in cardiac NLRP3 inflammasome levels in rats treated with fructose and tocilizumab is likely attributed to the ability of tocilizumab to inhibit IL6 inflammatory signaling.This aligns with a prior investigation, which showed that inhibiting IL6 resulted in a reduction in the activation of NLRP3 inflammasome in mice afflicted with collagen-induced arthritis [18].Additional studies have documented that tocilizumab simultaneously inhibits the activation of NLRP3 inflammasome and blocks IL6 receptors [19].
The function of MMPs, which are enzymes that break down proteins inside the extracellular matrix and basement membrane, is regulated by the equilibrium between active MMPs and TIMPs [11].The results of our study suggest that the intake of fructose is associated Hematoxylin and Eosin (H & E) to evaluate histological alterations in the myocardium.Additionally, we utilized Masson's trichrome stain to visualize fibrosis.Then immunohistochemical staining using TNFα antibodies was made.Moreover, the quantitative morphometric study was done by utilizing the "Toup view" image analyzer computer system (China).Serum glucose, TC, TG, and HDL are measured according to the manufacturer's instructions (Atlas Medical Co., Cambridge, UK).LDL levels were measured by the Friedewald equation [13]: LDL=TC-(TG / 5) -HDL.By using ELISA kits, the procedures and methods for measurement of serum insulin (Cat.No. MBS045315), IL 6 (Cat.No. MBS175908), and tissue NLRP3 (Cat.No. MBS 7612469) were according to the manufacturer's instructions of MyBiosource, Southern California, San Diego, USA, while TIMP1(Cat.No.: E -EL-R0540) was according to Elabscience Biotechnology, Wuhan, China.Calculation of IR was by homeostatic model assessment for insulin resistance (HOMA-IR) The present results of the Fructose group revealed a significant increase in TG, TC & LDL levels and a significant decrease in HDL as compared with Control (p <0.05).Results of Tocilizumab and Fructose +Tocilizumab showed significantly decreased TG, and LDL with increased HDL levels as compared with Fructose results, while there were insignificant changes in comparison to Control (p >0.05) (Table1).Compared withControl, there were significantly increased levels of IL6 and NLRP3inflammasome in the Fructose group while TIMP1 concentration significantly decreased (p <0.05), meanwhile, there was an insignificant difference between Tocilizumab and Control results.Fructose +Tocilizumab exhibited a significant decrease in IL6 and NLRP3 alongside increased TIMP1 levels as compared to the Fructose group (p <0.05) (Figure 1, 1C).We further explored the association among IR and serum IL6 as well as cardiac NLRP3 inflammasome of all groups; the results revealed a significant positive correlation of the parameters (p <0.05 and r>0.696), however, there was a significant inverse correlation among IR and cardiac TIMP1 level (p <0.05 and r < -0.696) (Figure 1D-1F).Our results showed a significant direct association between IL6 and cardiac NLRP3 concentrations (p <0.05 and r > 0.696), while there was a significant negative association among IL6 and TIMP1 levels (p <0.05 & r < -

Figure 1 :
Figure 1: The effect of Tocilizumab on tested variables.(A, B, &C) the effect of tocilizumab on IR, serum IL6 level and cardiac tissue concentration of NLRP3 and TIMP1 respectively; (D, E, F, G, &H) The correlation between IR and serum IL6 level, NLRP3 level and TIMP1 level, and between serum IL6 level and cardiac NLRP3 and TIMP1 level respectively.

Figure 2 :
Figure 2: A photomicrograph of H&E stained cardiac muscle sections from all experimental groups Control (A) and Tocilizumab treated group; (B) showing regular arrangement of the myocardial fibers, with centrally located cigar-shaped vesicular nuclei (red arrows).Fructose-treated group; (C) showed a distorted and irregular wavy appearance of myocardial fibers (arrowheads).Wide spaces (right-angled arrows) and inflammatory infiltration (curved arrows) are observed between the myocardial muscle fibers.Congested blood vessels (dashed arrow) could be also detected.in Fructose + Tocilizumab treated group; (D) most of the muscle fibers appeared normal with vesicular nuclei (red arrows).(H&E stain, scale bar = 50μm).
. The MetS model exhibited decreased cardiac levels of TIMP1, elevated NLRP3 inflammasome activity, increased TNFα immunoreactivity, and enhanced collagen deposition.Fructose consumption resulted in the emergence of IR and hyperglycemia, potentially caused by the decrease in insulin receptors, leading to a reduction in insulin-dependent glucose utilization and insulin sensitivity [15].
with a reduction in myocardial TIMP1 levels and an increase in collagen deposition.This might be attributed to the connection between elevated levels of MMP and heightened concentrations of inflammatory markers, which are associated with various metabolic abnormalities in the MetS model [6].Our study findings indicate a considerable rise in cardiac TIMP1 levels with the administration of tocilizumab, as compared to the fructose group.Nevertheless, no substantial alterations were detected in comparison to the standard control group.This impact may have been caused by the enhancement of inflammatory markers, such as IL6, TNFα, and NLRP3 inflammasome, which have a role in controlling TIMP1 expression.Furthermore, our investigation uncovered a notable and favorable correlation between IR and IL6 or NLRP3.This finding aligns with a prior study that demonstrated a direct correlation between the intensity of type 2 diabetes (T2DM) and heightened expression of the NLRP3 inflammasome in adipose tissue.The presence of amyloid polypeptide in pancreatic islets in T2DM may cause the disruption of lysosomes and an increase in oxygen radicals in pancreatic macrophages.This, in turn, activates the NLRP3 inflammasome [20].According to Yang et al. (2019), the NLRP3 inflammasome is involved in the advancement of IR and T2DM [5].The reciprocal relationship between IL6 and NLRP3 inflammasome in immune response and inflammation, along with the notably improved cardiac TNFα immunoreactivity, may elucidate the influence of tocilizumab's anti-inflammatory activities in our MetS model [21].Following a period of seven weeks of providing a diet high in fructose, the analysis of lipids revealed a notable rise in the levels of TC, TG, and LDL, accompanied by a drop in HDL levels.This agrees with previous findings [15, 22, 23].Dyslipidemia observed in this study may be caused by hyperglycemia, which alters the intermediary metabolism of lipids and proteins.This typical triad of dyslipidemia, which includes increased TG and LDL with reduced HDL, is commonly associated with IR in patients with MetS [24].Impaired lipid metabolism primarily raises levels of LDL in the bloodstream, while inflammatory processes aid in the development of atherosclerotic plaques, as NLRP3, which is known to be highly expressed in the aorta of individuals with atherosclerosis [25].In addition, in MetS, increased cytokine production such as IL6 and TNFα may alter TG metabolism, resulting in increased TG and VLDL levels through either excessive synthesis of VLDL or reduced clearance [26].The IL6 receptor-blocking effect of tocilizumab may stimulate lipid clearance by inducing the VLDL receptor, stimulating lipolysis in hepatic and adipose tissue, and decreasing hepatic lipid synthesis [26].This, together with modifying NLRP3 inflammasome and TNFα reactivity, could explain the improvement in lipid profile by tocilizumab.Furthermore, the histopathological results in cardiac tissues confirmed the ameliorated state with tocilizumab in fructose-fed rats.These clear improvements were the result of the improvement of all features of MetS.Conclusion The results of the present research provide evidence that the biological agent, tocilizumab, can reverse certain changes caused by fructose consumption.These findings confirm the role of IL6 and TNFα in the development of some aspects of the MetS model.Tocilizumab may modulate NLRP3 inflammasome and TIMP1, which are considered strong cardiac inflammatory sensors and potential cardio-protective markers.These promising results indicate that tocilizumab may have cardiovascular protective effects that could be beneficial for clinical use.
N= 6 rats/ group, Data are represented as mean ±SD, p-value at < 0.05 is considered significant.† Significant of all groups contrasted with the control group, ‡ Significant values in comparison with the fructose group, ¶ Significant values in comparison with the tocilizumab group.