Resveratrol and resveratrol-hydroxypropyl-β-cyclodextrin complex recovered the changes of creatine kinase and Na, K-ATPase activities found in the spleen from streptozotocin-induced diabetic rats

Type 1 diabetes (T1D) is the result of the selective destruction of the pancreatic β-cells by T cells of the immune system. Although spleen is a secondary lymphoid organ, it is also involved in the T1d pathogenesis. However, the alterations in a variety of cellular processes of this disease need to be further understood. We aimed to analyze the benefits of resveratrol, and its complexed form on diabetic complications in the spleen of rats. To this end, we investigated important enzymes of phosphoryl transfer network, and Na, K-ATPase activity. Wistar rats were divided into non-diabetic groups: Control, ethanol, Resveratrol, Hydroxypropyl-β-cyclodextrin, Resveratrol-hydroxypropyl-β-cyclodextrin, and diabetic groups with the same treatments. diabetes was induced by a single dose of 60 mg/kg of streptozocin intraperitoneally, and treatments by intragastric gavage once daily for 60 days. Hyperglycemia reduced creatine kinase activity, which was reversed by the administration of resveratrol. Na, K-ATPase activity was greatly affected, but it was reversed by resveratrol and resveratrol-hydroxypropyl-β-cyclodextrin. This suggest an energetic imbalance in the spleen of diabetic rats, and in case this also occurs in the diabetic patients, it is possible that resveratrol supplementation could be beneficial to the better functioning of the spleen in diabetic patients.


INTRODUCTION
diabetes mellitus (dm) is a chronic metabolic disease of high complexity, being responsible for health problems worldwide.The dm type 1 (dm1) is characterized by a decompensated energy metabolism, which causes total or partial autoimmune destruction of insulin-producing cells, resulting in high blood glucose levels (Bluestone et al. 2010, Nyaga et al. 2018).The spleen has a relevant participation in the metabolic changes of this disease since the splenocytes produce cytokines that contribute to the β-pancreatic cells destruction (Thorvaldson et al. 2005, García-Galicia et al. 2014, Tong et al. 2015).It is also reported that splenectomy may lead to diabetes (Kabelitz et al. 2008(Kabelitz et al. , ley et al. 2012)).
Streptozotocin (STZ) is usually used to induce type 1 diabetes in animal models (eriksson et al. 2000(eriksson et al. , lenzen 2008(eriksson et al. , Ghosh et al. 2018)).'Streptozotocin diabetes' is caused by specific necrosis in the pancreatic β-cells inducing glucotoxicity (Wu and Yan 2015).Some natural antioxidants have been reported to exhibit their therapeutic effects in 'streptozotocin diabetes' (morishita 2004, Rugde et al. 2007, Ghosh et al. 2018).Resveratrol is a natural phytoalexin present in a wide variety of plants, grapes, and red wine has been linked with beneficial activities towards health because it acts as an antioxidant, preventing and treating metabolic diseases such as diabetes (Chen et al. 2011, Chang et al. 2012).mice treated with resveratrol showed a significant reduction in the death of splenocytes.Resveratrol-induced autophagy of these cells, and the degradation of intracellular proteins and lipids provides energy (ATP) for the cell, preventing the activation of the apoptotic damage (Kim et al. 2007) and metabolic diseases such as dm (Gurusamy et al. 2010(Gurusamy et al. , lee et al. 2011)).
According to Baur and Sinclair (2006), resveratrol has a rapid metabolization due to its low bioavailability in the organism, being rapidly eliminated when administrated orally.So, an alternative for increasing the bioavailability is to complex it with cyclodextrin (Cd), which is capable of forming inclusion complexes with hydrophobic substances (Teixeira et al. 2013, Torres et al. 2018).Besides, the bioavailability may be enhanced by increasing the solubility and stability, and dissolution rate (Trollope et al. 2014, Kumar et al. 2017).
Adenylate kinase (AK), pyruvate kinase (PK), and creatine kinase (CK) are the key enzymes participating in a network of production and cellular energy homeostasis (dzeja and Terzic 2003, matté et al. 2007, Alekseev et al. 2012).Another relevant enzyme for the organism is the Na + , K + -ATPase, responsible for generating and maintaining the ionic gradients through the cell membrane (Pressley et al. 2005, Jaitovich andBertorello 2006).The nutrients and energy flow maintenance into the cell is essential for the homeostasis and cell work.The energy metabolism reduction can lead to a decrease in ATP synthesis.In this context, it has been shown that the energy metabolism deficiency may be implicated in the pathogenesis of several metabolic conditions (mattevi et al. 1996, Alves-Filho and Pålsson-mcdermott 2016, Xiao et al. 2018).
One of the pathways of cellular energy production is the glycolytic route.PK transfers phosphoryl group of the phospoenolpyruvate (PeP) to AdP resulting pyruvate and ATP.Aizawa et al. (2003) demonstrated apoptotic cells in the splenic red pulp of the PK-deficient patient.This indicates that PK may be considered a key enzyme for the cell metabolism.CK isoforms are part of a buffering system of cellular energy.It occurs between production and consumption of ATP in different compartments such as mitochondria and cytosol, contributing to homeostasis (Wallimann et al. 1992, 1998, Yan 2016).Among alternative phosphotransfer pathways, the AK-catalysed circuit, in normal conditions, maintains the concentrations of ATP, AdP, and AmP to equilibrium.Impaired AK activity leads to an alteration in cellular functions.
In addition, AK can take over the function of other kinases, such as CK, in pathological conditions (dzeja et al. 2011(dzeja et al. , Wujak et al. 2015)).The Na + , K + -ATPase plays an important role in the maintenance of the Na + across the cell membrane.It generates an electrochemical gradient through the plasmatic membrane (Erecińska and Silver 1994, Jaitovich andBertorello 2006) that is used for cellular volume regulation, osmotic balance, and to transport molecules (e.g.glucose) bonded to the co-transport of Na + (Kaplan 2002).This transport is essential for cellular functioning since more than one-third of hydrolyzed ATP is to pump ions across membranes.Shi et al. (2013) suggested that complications of type 1 diabetes may be partly due to the impaired Na + , K + -ATPase activity and that this dysfunction may be one of the mechanisms of type 1 diabetes-induced complications.The association between resveratrol with insulin may be an excellent treatment for type-1 diabetes.It was suggested, at least in part, by anti-hyperglycemic, and antioxidant potential of these molecules, preserving regulation of Na + , K + -ATPase (Bashir 2018).Thus, protecting the effective functioning of Na + , K + -ATPase from the complications caused by type 1 diabetes could contribute to slow the progression of these injuries in this disease.
It is known that splenocytes are involved in the diabetes development, however, few reports are found about the activity of the AK, PK, CK, and Na + , K + -ATPase enzymes in the spleen in the presence and absence of resveratrol, which has been considered a hypoglycemic substance.The current study aimed to investigate the role of resveratrol-hydroxypropyl-β-cyclodextrin, and its free form on the activities of important enzymes of the energy metabolism in the spleen of normal and hyperglycemic rats.

INClUSION COmPleX
Resveratrol (C 14 H 12 O 3 ; molecular weight = 228.25 g/mol; purity > 98%) and 2-hydroxypropyl-βcyclodextrin (HPβCD) were obtained from Sigma-Aldrich.0.1 mM HPβCD solution in water (8 ml) at 40 °C was prepared by vigorous stirring in UlTRA-TURRAX High-Speed Homogenizer T 25 ºC (IKA, UK) at 3,200 rpm and an equimolar amount of RSV (0.1 mm) was directly added to the suspension.The RSV was previously dissolved in ethanol (2 ml).After stirring for one minute, the suspension was filtered through 0.45 µm cellulose acetate membrane filter to removed undissolved particles.The solvent was removed by evaporation.The evaporation was performed using rotary evaporator at 40 °C for about 10 min under vacuum for 8 h.Since resveratrol is a photosensitive substance, the procedure was performed in a dark environment (Nishihira et al. 2017).

CHARACTeRIZATION OF THe INClUSION COmPleX
Fourier Transformed Infra-Red Spectroscopy (FT-IR) analyses were performed Perkin elmer (Spectrum One), R, HP and the complex spectra were collected using an FT-IR in a spectral region between 4,000 and 450 cm -1 .Samples were mixed in a pestle with potassium bromide (KBr) (1:100) and pressed in a hydraulic press (10 tons for 2 min) to small tablet and were placed in the infrared beam.Nuclear magnetic resonance (NmR) spectra were performed in BRUKeR dPX-400 (Bruker, Germany).Fifteen grams of samples were dissolved in dmSO-d6 using tetramethylsilane (TmS) as internal reference standard.The characterization was carried out in the laboratório de Química of the Universidade Federal de Santa maria (UFSm).Cienc (2019) 91(3)  e20181330 4 | 12 ANImAl mOdel

An Acad Bras
Seventy male Wistar rats from the Central Animal House of UFSm were submitted to a period of 15 days for adaptation.The animals with 60 days old and weighing an average and standard deviation of 216 ± 28 g were used in this experiment.The rats were kept in cages, four animals each, housed at a constant temperature and controlled humidity (23 ± 1 ºC and 70%, respectively) on a light/ dark cycle of 12 h.They had free access to commercial ration and water.All animal procedures were approved by the Animal ethics Committee from Franciscan University (protocol under number: 14/2012).
eXPeRImeNTAl INdUCTION OF dIABeTeS Hyperglycemia was provoked by a single intraperitoneal injection of 60 mg streptozotocin (STZ)/kg body mass of rat, dissolved in 0.1 m sodium citrate buffer (pH 4.5), after fasting period of 12 h.The animals to normoglycemic group received an equivalent volume of sodium citrate buffer.STZ-treated rats received 5% glucose solution instead of water for 24 h after diabetes induction to avoid death due to hypoglycemic shock (Avila-Acevedo et al. 2012).Blood samples were taken from the tail vein five days after STZ or vehicle administration to measure glucose levels.Glycemia was determined by using a commercial glucometer (Accu-Chek Performa, Roche®, USA).Animals with fasting glycemia higher than 200 mg/ dl (Barbalho et al. 2011) were used for the diabetic group (Fig. 1).
TReATmeNT WITH ReSVeRATROl After measuring glycemia, seventy rats were primarily divided into two groups (normal and hyperglycemic).The normoglycemic rats were divided into five groups, with seven animals each: Control (C); ethanol (e); Resveratrol (R); Resveratrol-hydroxypropyl-β-cyclodextrin (RHP) and Hydroxypropyl-β-cyclodextrin (HP).The hyperglycemic rats were divided into five groups, with seven animals each: diabetic Control (dC); diabetic ethanol (de); diabetic Resveratrol (DR); Diabetic Resveratrol-hydroxypropyl-βcyclodextrin (dRHP) and diabetic Hydroxypropylβ-cyclodextrin (DHP).Still on the fifth day was initiated the sixty-day chronic treatment with 1 daily administration, by intragastric gavage of water to the controls C and d (Figure 1).Resveratrol was dissolved in ethanol at 15% of aquose solution.A solution of 1 mg R/kg body mass was administered to R and dR groups and 15% ethanol solution to e and de groups. 1 mg of R in the complex/ kg body mass to RHP and dRHP groups and the equivalent concentration of HP to the one found in the complex to HP and dHP groups.RSV dosage choice of 1 mg/kg was based on previous studies, which authors observed hypoglycemic effect even in low dose (Chang et al. 2011(Chang et al. , 2012)).

TISSUe PRePARATION
The rats were killed by decapitation without anesthesia, and the spleen was rapidly dissected, weighted and kept chilled until homogenization, which was performed using a ground glass type Potter-elvejhem homogenizer (1:10 w/v) in buffer (pH 7.4) containing 0.32 M sucrose, 1 mM eGTA, 10 mm Tris-HCl.The homogenized was centrifuged at 800 × g for 10 min at 4 °C, and part of the supernatant was aliquotted to determine the AK and CK total activity.The other part of the supernatant was centrifuged at 10,000 × g for 15 min at 4 °C, and the supernatant was separated to determine the PK activity.Another spleen fraction was homogenized in buffer medium, pH 7.5 and centrifuged at 800 x g for 10 min at 4 °C.
The supernatant was used for the determination of the Na + , K + -ATPase enzymatic activity.The supernatants were stored for no more than 1 week at -80 °C when the assay was not carried out immediately.(1999).The reaction mixture contained 20 mm glucose, 100 mm KCl, 20 mm HePeS, 1 mm edTA, 4 mm mgCl 2 , 2 mm NAdP + , 4.5 U/ml of HK, 2 U/mL of G6PD, and 1 µg of protein homogenate.The reaction was initiated by adding 2 mm AdP.The reduction of NAdP + was followed at 340 nm for 3 min in a spectrophotometer.AdP, NAdP + , G6Pd, and HK were dissolved in water.
Reagents concentration and assay time (3 min) were chosen to assure the linearity of the reaction.The results were expressed in nanomol of ATP formed per min per mg of protein.
PYRUVATe KINASe ACTIVITY PK activity was assayed according to Feksa et al. (2003).The assay consisted of 0.1 m Tris/HCl buff er, pH 7.5, 0.16 mM NADH, 10 mM MgCl 2 , 75 mm KCl, 5.0 mm AdP, 0.1% (v/v) Triton X-100, 7 U of L-lactate dehydrogenase, and 10 µL of the mitochondria-free supernatant in a final volume of 0.5 ml.After 10 min of pre-incubation at 37 ºC, the reaction was started by the addition of 1 mm phosphoenol pyruvate.Results were expressed as µmol of pyruvate per min per mg of protein.
CReATINe KINASe ACTIVITY CK activity was assayed in the mixture containing the following final concentrations: 65 mM Tris-HCl buffer, pH 7.5, 9 mm mgSO 4 ,7 mM phosphocreatine, and 1 µg of protein in a final volume of 0.1 mL.After 10 min of preincubation at 37 ºC, the reaction was started by the addition of 0.3 µmol of ADP.The reaction was stopped after 10 min by the addition of 1 µmol of ρ-hydroxymercuribenzoic acid.The incubation time and the reagent concentrations were chosen to assure linearity of the enzymatic reaction.Appropriate controls were carried out to measure chemical hydrolysis of phosphocreatine.The creatine formed was measured according to the method of Hughes (1962).The color was formed by the addition of 0.1 mL 2% α-naphthol and 0.1 mL 0.05% diacetyl in a final volume of 1 ml and read after 20 min at 540 nm.Results were expressed as nanomol of creatine formed per min per mg of protein.
NA + , K + -ATPase ACTIVITY The reaction mixture for Na + , K + -ATPase activity assay contained 5.0 mm mgCl 2 , 80.0 mm NaCl, 20.0 mm KCl, and 40.0 mm Tris-HCl, pH 7.4, in a final volume of 200 mL.After 10 min of preincubation at 37 ºC, the reaction was started by the addition of ATP to a final concentration of 3.0 mM and was incubated for 20 min.Controls were carried out under the same conditions with the addition of 1.0 mm ouabain.Na + , K + -ATPase activity was calculated by the diff erence between the two assays (Wyse et al. 1999).Released inorganic phosphate (Pi) was determined by the method of Chan et al.
(1986), and enzyme specific activity was expressed as nmol of Pi released per min per mg of protein.

PROTeIN CONTeNT
Protein content of spleen homogenates was determined by the methods of lowry et al. (1951) to AK, PK and CK and Bradford (1976) to Na + , K + -ATPase, using serum bovine albumin as the standard.

BlOOd GlUCOSe leVelS
Before the experiment, there were no signifi cant diff erences between blood glucose of groups.Sixty days after to start the treatments, all rats were weighed, killed, and the blood was collected for serum levels glucose analysis.This was determined in an automated analyzer Vitros 250 (Ortho-Clinical diagnostics), using kits Johnson & Johnson ® by the method chemical dried.

STATISTICAl ANAlYSIS
Results were presented as the mean ± standard deviation (Sd).Assays were performed in duplicate or triplicate, and the mean or median was used for statistical analysis.data were analyzed using oneway analysis of variance (ANOVA) followed by the post-hoc Tukey's multiple comparison tests when F values were signifi cant.Diff erences between groups were rated signifi cantly at p < 0.05.All analyses were carried out using the GraphPad software.

eFFeCTS OF TReATmeNTS ON BlOOd GlUCOSe leVelS
Streptozotocin diabetes showed a significant increase in the blood glucose compared to normal levels [F 9,43 = 288.4],(p = 0.000).In relation to treatments, neither resveratrol free form, nor resveratrol-hydroxypropyl-β-cyclodextrin was able to revert hyperglycemia (Figure 2).

eFFeCTS OF TReATmeNTS ON eNZYme ACTIVITIeS
The AK activity was neither changed by streptozotocin diabetes, nor treatments in the spleen when compared to controls [F 9,60 = 1.5], (p = 0.153) (Figure 3a).In the PK activity, ANOVA shows that there was no signifi cant diff erence (p > 0.05) between the non-diabetic and the diabetic control.All treatments reduced PK activity, when compared to control (Figure 3b).The CK activity was reduced by hyperglycemia when compared to the control group (p < 0.05).The administration of the ethanol, and resveratrol prevented it.Already, the administration of resveratrol-hydroxypropylβ-cyclodextrin (RHP), as free cyclodextrin (HP) reduced enzyme activity in normoglycemic rats (p < 0.001) (Figure 3c).Na + , K + -ATPase activity increased with administration of free resveratrol in normoglycemic animals (p < 0.001).
Hyperglycemia caused an expressive reduction of the activity of the Na + , K + -ATPase activity (p < 0.001), which was prevented by free and resveratrolhydroxypropyl-β-cyclodextrin (DR and DRHP groups) administration.Ethanol, and HPβCD failed to prevent it, indicating that only treatments with the presence of resveratrol prevented this change (Figure 3d).

BOdY ANd SPleeN WeIGHT
All diabetic groups reduced body weight when compared to non-diabetic groups (p < 0.001).

DISCUSSION
The present study shows a reduction in the activity of some important spleen enzymes caused by hyperglycemia, which may alter the energetic homeostasis of this tissue in type 1 diabetes.CK, and Na + , K + -ATPase activities were recovered by resveratrol, and resveratrol-hydroxypropyl-βcyclodextrin, and its free form, respectively.The spleen is a secondary lymphoid organ involved in the type 1 diabetes pathogenesis (Ghosh et al. 2018).This disease is marked by alterations in a variety of cellular processes that need to be further understood (Badr et al. 2015).For the fi rst time, we measured activities of the phosphotransfer network, and Na + , K + -ATPase in the spleen of hyperglycemic rats, in the presence of the resveratrol complexed with HPβCD, or its free form.Here, streptozotocin increased blood glucose, and reduced body and spleen weight, as expected.Unfortunately, resveratrol treatment (complexed or not) not reverted hyperglycemia.Curiously, hyperglycemia was more severe with ethanol and HPβCD treatment.Persistent hyperglycemia can lead to ketoacidosis, a serious condition associated with very high blood glucose levels in type 1 diabetes.Besides, it can happen associated with alcohol abuse in diabetics (Bradford et al. 2017, Umpierrez andFreire 2002), suggesting that ethanol treatment could have developed this condition in our diabetic animals.moreover, a positive effect of resveratrol, against possible death of splenocytes, was observed.even though the treatments did not aff ect the weight of spleens in the normoglycemic rats, hyperglycemic spleen had a reduction in the weight with all treatments, except resveratrol.Similar protection was shown by Szkudelski and Szkudelska (2011), which reported a signifi cant reduction in the death of splenocytes from mice treated with resveratrol.Besides, weight loss of the spleen may be related to body weight reduced of the diabetic animals, once 'streptozotocin diabetes' significantly decreases body weight of the rats (Zafar and Naeem-ul-Hassan Naqvi 2010).
Intracellular processes regulate the mechanisms involved in energy homeostasis.For a correct functioning of the cell energy system is necessary that energy compounds, such as ATP, are synthesized and delivered to the consumption spots in an appropriate speed for its use rate (dzeja et al. 2002).Coupling of spatially separated intracellular ATP producing,  PK is a critical enzyme to energy metabolism in the mammalian tissues.It is a crucial enzyme of the glucose metabolism, the main pathway that provides energy for cell function (Hall and Cottam 1978).In this study, all treatments reduced PK activity in all groups.We observed that RHP did not show the expected effects, once the administration of this complex decrease, even more, the enzyme activity, when compared to resveratrol and HP separately.Deficiency of this enzyme causes a decline in ATP production, leading to the destruction of red blood cells present in the spleen (Rüfer and Wuillemin 2013).
We also tested the effects of hyperglycemia and treatments on the total CK activity in the spleen.We found that RHP, and HP administration decreased the CK activity, independent of the hyperglycemia.However, resveratrol, and ethanol reverted this effect in hyperglycemic animals.The reduction of CK activity by hyperglycemia was not compensated by other enzymes evaluated in this work, such as PK, AK and Na + , K + -ATPase, which leads us to believe that there is an injury to the spleen tissue.
Na + , K + -ATPase activity was significantly inhibited by hyperglycemia.Resveratrol complexed or not, reversed it, indicating that resveratrol can recover the changes of CK, and Na + , K + -ATPase activities in the spleen provoked by diabetes.Resveratrol is suggested as a preventive agent for cerebral ischemia.The administration of resveratrol before cerebral ischemia, brought Na + , K + -ATPase activity in the cortex, and hippocampus back to normal levels (Simão et al. 2011).Furthermore, it is suggested that the elimination of ROS contributes, in part, to the resveratrol-induced neuroprotection.However, more tests are necessary to elucidate the activity of Na + , K + -ATPase in the spleen, once these enzymatic dosages performed could have been distorted by interferents, like another cell line (erythrocytes), due to its function of the blood filtering.
Although the formulations were tested in a particular organ in this work, the measurements of enzymatic activities evaluated in this study are unique in the literature.The presence of PK, AK, and CK already been confirmed in different animal species including human (Böckelmann and Ritter 1968, Brock 1970, Fauquier et al. 2008), however, more studies are required for a better understanding about splenic bioenergetics.Furthermore, additional data are necessary to reinforce the efficacy of the performed analysis and to certify that the data obtained truly belongs to the spleen enzymes, once it is a path organ for other cells that could influence the results.

CONCLUSION
Based on this study, we concluded that resveratrol, and resveratrol-hydroxypropyl-β-cyclodextrin can recover the enzymatic changes in the spleen provoked by hyperglycemia in rats.Therefore, resveratrol, as a potential therapeutic agent, may hold promise auxiliary supplementation to the better functioning of the spleen in diabetic patients.