Research Article
Alleviation of hyperlipidemia, insulin resistance, and myopathy by nano selenium/ nano CoQ10 platform with simvastatin in hyperlipidemic rats; comprehensive outlook
https://doi.org/10.21203/rs.3.rs-2385794/v1
This work is licensed under a CC BY 4.0 License
published 22 Sep, 2023
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Hyperlipidemia
simvastatin
solid lipid nanoparticles
selenium
co enzyme Q10
The world witnessed a rapid transition in dietary habits from healthy, homemade food to fast and junk food [1]. Indeed, all recent dietary traditions lead to the rapid rise of preceding factors to several metabolic diseases such as hypocholesteremia, insulin resistance (IR), and coronary heart diseases [2]. However, patients who suffer from hypocholesteremia are most likely to have other cardiovascular diseases (CVDs). CVDs are one of the major causes of death one-third of total deaths around the world with 17.8 million deaths in 2017 [3, 4]. Atherosclerosis and ischemic heart disease (IHD) are widely reported in hypercholesterolemic patients [5]. Statins such as simvastatin (SV) are the first treatment option for hypercholesterolemia through direct inhibition of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase enzymes [6].
SV therapy may oppose several challenges due to its lower bioavailability which is less than 5%. It is poorly absorbed from the gastrointestinal (GI) tract. Approximately 95% of an oral dose is not absorbed [7]. In addition, statin treatment is associated with musculoskeletal damage; severe changes in liver function in up to 1–3% of patients[8]. The long-term use of statins is associated with disruption of insulin signaling that may result in insulin resistance [9, 10]. There is no full explanation that can be used to answer the question related to the mechanism of statin-induced myopathy[11]. Earlier studies reported that statins-induced myopathy through inhibition of mitochondrial synthesis of CoQ10, which ultimately interferes with respiratory chains and the capacity for energy production by mitochondria [12]. No conclusive data confirm that mitochondrial myopathy is because of the reduction of intramuscular levels of CoQ10[13]. Side effects of different drugs in the conventional formulation may be alleviated by supplementation of nutraceuticals and herbs [14]. Therefore, Nutraceuticals attract special attention from researchers, stakeholders as well as consumers because of their potential effects on the prevention or treatment of different pathological conditions [15, 16]. However, reshaping and resizing nutraceuticals in the nanoscale may provide feasible solutions to improve the bioavailability and therapeutic efficacy of diverse drugs and nutrients [14, 17].
Coenzyme Q10 is the most commonly fat-soluble vitamin, especially in the heart, liver, kidney, and brain [18]. CoQ10 is the third of the top widely used dietary supplements and a potential candidate for treatment in most common causes of death [19]. Selenium is a fascinating microelement. It is a major constituent of several enzymes such as glutathione peroxidase (GPx), therefore it plays an essential role in all major functions [20]. Several reports encapsulated Coenzyme Q10 in nanoparticles to overcome its hydrophobicity limitation that negatively affects its bioavailability. Moschetti, et al. incorporated Coenzyme Q10 in phosphatidylcholine and apolipoprotein nanoparticles. The loaded nanoparticles allowed a significant increase in the drug uptake in mitochondria after cell incubation besides enhancing oxidative phosphorylation and improving the maximal oxygen consumption rate. These results opened the door to studying the effect of CoQ10 uptake on patients on statin therapy[21]. Another study reported by Lohan, et al encapsulated CoQ10 in ultra-small lipid nanoparticles to improve skin penetration and disposition. The developed nanoparticles limited the formation of reactive oxygen species resulting from UV irradiation, which usually leads to cell damage, skin aging, or skin cancer. The XTT cell viability study proved that the developed formulation was non-toxic with an antioxidant potential when tested on the HaCaT after cellular exposure to UVA and UVB irradiation[22]. Another extensive study to improve the skin penetration of CoQ10 for the treatment of androgenic alopecia was conducted by El-Zaafarany, et al. CoQ10 was encapsulated in liposomes, transfersomes, ethosomes, cerosomes and transethosomes. Transethosomes were selected due to their physical properties (146 nm, -55 mV, entrapment efficiency of 97.63, and skin deposition > 95% ). The clinical study on androgenic alopecia patients showed a good clinical response upon dermoscopic examination for patients who used CoQ10 transethosomes[23]. Keck, et al 2014, reported the incorporation of CoQ10 in ultra-small nanostructured lipid carriers. This study reported a comparison between classical nanostructured lipid carriers (230 nm), nanoemulsion, and ultra-small nanostructured lipid carriers (85 nm). The developed formulation showed the highest release, a better antioxidant effect, and the best skin penetration[24].
Selenium is one of the vital micronutrients for any living organisms, which motivate researchers to study its pharmacological role especially with its unique properties and biocompatibility. Selenium supplements are currently prescribed as preventive treatment for infection and immune and neural related diseases due its antioxidant activity[25]With the integration of nanotechnology in the biomedical field, selenium nanoparticles synthesis was extensively reported, and its biological properties were investigated[26]. The crystal structures of t-Se and m-Se are the main common forms of selenium nanoparticles. Although nanosized elements showed good pharmacological activity, several reports demonstrated that the safety of selenium nanoparticles increased with size increases[25, 26] Han et al reported the antibacterial activity of selenium nanoparticles, where the nanoparticles were prepared using chemical reduction technique. The nanoparticles showed a promising result against different multidrug resistance bacteria beside the synergistic effect with linezolid[27]. Also, antioxidant activity of selenium nanoparticles were investigated by Lee et al, where apoptosis and intracellular Reactive oxygen species levels of C2C12 cells after treatment with selenium nanoparticles[28].
The aim of the present work is to address the therapeutic value of nanoencapsulated platform of CoQ10 or selenium in hyperlipidemia and functionalize them in the alleviation of simvastatin-induced myopathy and adverse reactions in hyperlipidemic rats.
Wistar albino male rats (150–200) g were received from the laboratory animal farm of the National Research Center (Dokki, Giza, Egypt). Animals were kept in filter-top in polycarbonate cages (5 rats/ cage) after random assignment to experimental groups Rats were kept at well-controlled laboratory conditions including room temperature (25 ± 2 ºC), 12/12h light-dark cycle, rodent diet pellets, and free access to water ad libitum. The experimental work started after one week of acclimatization. All experimental procedures followed the guidelines and code of ethics released by the research ethics committee, faculty of Pharmacy, Cairo University, Cairo, Egypt, and followed the rules of the Laboratory Animals welfare [88]. The experimental design and procedure followed the three Rs’ rule (Replace, Reduce, Refine) requirements. Any unAnimals were treated gently; squeezing, pressure, pain, malnutrition, abnormal cold or heat, injury, illness, and tough maneuvers were avoided.
Simvastatin was obtained from Global Napi Pharmaceuticals (Giza, Egypt), and prepared by suspension in distilled water in concentration (20 mg/kg) [21], Whereas, CoQ10 was gifted from Arab Company for Pharmaceutical and Medicinal plants (Cairo, Egypt) Furthermore, Selenium powder was obtained from Egyptian European pharmaceutical Industries (Alexandria, Egypt). Compritol 888 ATO (glyceryl behenate, a mixture of ∼15% mono-, 50% di- and 35% triglycerides of behenic acid) and Gelucire 40/14 (PEG glyceride) were generous provided from Gatteffose, France. Poloxamer 407 has been obtained from BASF (Florham Park, NJ). Other chemicals were purchased in high analytical grade from Sigma-Aldrich for Chemicals (St. Louis, MO,USA)
Either coenzyme Q10 or selenium solid lipid nanoparticles prepared by hot melt ultrasonication method as previously reported[29]. Coenzyme Q10 or selenium (0.5% w/v) was mixed with molten lipids (1% compritol, and 3.04% gelicure) at 75°C till homogenous mixture obtained. 3% w/v Poloxamer 407 solution was prepared at 75°C PBS (pH 7.4). The poloxamer solution was added gradually on molten lipid phase with the aid of homogenization (GLH 850, Omni inc.,USA) at 5,000 to 15,000 rpm and at 75°C. The optioned suspension was sonicated (Model LC 60/H, Elma, Germany) for 3 min at 50% amplitude. Afterward, the obtained nanosuspension was cooled gradually until reach room temperature. The obtained nanosuspension was stored in 2–8°C till further investigation.
Malvern Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) were used to estimate particle size and Zeta-potential. All measurements were repeated at least three times to determine means and standard deviations.
The Entrapment Efficiency (EE %) was measured using indirect technique, where the unentrapped drug was measured in supernatant after centrifugation (Model 3K 30, Sigma, Germany) at 18000 rpm for 45 min at 4°C. The free selenium ions in the supernatant were measured using previously reported spectrophotometric method after a complexation of selenium ions with 4,5-diamino-6-hydroxy-2-mercapto pyrimidine (DAHMP) at 458 nm using a spectrophotometer (Shimadzu UV 1650, Japan) [30]. Free Coenzyme Q10 was measured using HPLC (Agilent, USA), mobile phase 1-propanol: methanol 60:40 v/v containing, flow rate 1.4 ml/min, and wavelength at 275 nm.
An electron microscope (Nova Nano SEM, FEI, USA) was used to study the morphological pattern of different particles. The sample was prepared with gold sputtering times for 20 s.
The dialysis bag diffusion technique was done for 24 hrs using a dialysis membrane (Mw cutoff 12 kDa; Severa) to evaluate the in-vitro drug release profiles of coenzyme q10 or selenium [10].
According to Onyeali et al. 2010 [31], High fat diet (20% w/w ) was formulated by adding 200 grams of unsaturated fats (margarine) to 800 g of the pellet. unsaturated fats (margarine) contain vitamin A, vitamin D3, and anti-oxidants according to attached information on the pack. The high-fat diet was dried by keeping it in a dry place (22–25°C) for 12 hours and stored in polyethylene or glass containers.
Animals were assigned for daily ingestion of 20 g of HFD for 112 days to induce hyperlipidemia [32].
Sixty male adult rats (8–10 rats/ group) were randomly assigned into:
Group I; Nano-vehicle control group: normal rats were treated daily with oral preparation of vehicle (20mg/Kg) for last 30 days of the in-vivo experiment.
Group II; HFD: rats were subjected to the high fat diet (HFD) (20% of the total amount of the daily required food) for 112 days[31].
Group III; (HFD + SV): rats with hyperlipidemia and treated daily with oral formulation of simvastatin (20 mg/kg) over a period of last 30 days [33].
Group IV; (HFD + SV and coQNPs): rats with hyperlipidemia and treated daily with combination of oral preparation of simvastatin (20 mg/Kg) and nano-coQ10 (10mg/Kg) for last 30 days of the in-vivo experiment [34].
Group V; (HFD + SV and SeNPs): hyperlipidemic rats treated with combination of simvastatin (20 mg/Kg) and nano-selenium (0.1 mg/Kg/day, P.O.) over a period of last 30 days [35].
Group VI: (HFD + SV + coQNPs and SeNPs): hyperlipidemic rats treated daily with combination of simvastatin (20 mg/Kg), nano-coQ10 (10 mg/Kg) and nano-selenium (0.1 mg/Kg) for last 30 days of the in-vivo experiment.
Every week, the changes in animal body weight were calculated. The in-vivo study was terminated by fasting animals overnight after delivering the last dose of the treatment regimen. Animals were treated with ketamine (12.5 mg/kg) and xylazine (1.5 mg/kg) for anesthesia and the blood samples were collected by using non-heparinized microhematocrit capillary tubes from the retro-orbital vein using [36]. Blood samples were collected for serum separation by cooling centrifugation at 3,000 rpm for 15 min and stored at -20°C until analysis. The sera were used for estimation serum level of total cholesterol (TC), triglycerides (TG), high-density lipoproteins cholesterol (HDL-c), glucose, creatine kinase, creatinine, urea, alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, and alkaline phosphatase (ALP) (Biomed Diagnostics, Egypt), insulin (DRG International, Inc., USA ), troponin and myoglobin (Cusabio biotech co., Wuhan, China ) according to the instructions of the analytical kits. Immediately after collection of the blood samples, animals were euthanized by overdose of anesthesia and the liver and quadriceps muscle were detached and distributed into two portions. The first portion of liver tissue were prepared for homogenization by tissue homogenizer (Heidolph, DIAX 900, Germany) to obtain (20% w/v). The supernatant was separated from homogenate by centrifugation for 15 minutes (4,000 rpm at 4°C). These supernatants were used for estimation of malondialdehyde (MDA) level, glutathione (GSH), and superoxide dismutase (SOD) activities (Biodiagnostics, Cairo, Egypt) according to the instruction of the kits. The other part of liver and quadriceps muscle were mounted in 10% formalin in saline for both histological and immunohistochemical analysis.
The results of the current study were articulated as means ± standard error of the mean (SE). GraphPad software was used for statistical analysis (software 2003, version 3.06 Inc., San Diego, USA). One-way Analysis of Variance (ANOVA) followed by Dunnett’s multiple comparisons test has been used to determine whether a statistical difference has existed among different groups or not [37]. One way ANOVA has been used for analyzing the monthly total body weight (TBW) of rats then Tukey’s multiple comparisons test was performed. All statements of significance were based on the probability of P˂ 0.05.
Table 1shows the successful preparation of blank solid lipid nanoparticles (SLNs) with particle size 250.2 nm and zetapotential − 17.3 mV. Encapsulation of Coenzyme Q10 slightly decreased particle size to reach 213.9 nm and zeta-potential of -13.49 mV, while encapsulation of selenium increased particle size to reach 296.72 nm and zeta potential of -6.12 mV.
F# |
Particle Size (nm) |
PDI |
Zeta-potential (mV) |
EE (%) |
---|---|---|---|---|
Blank SLNs |
250.2 ± 5.2 |
0.52 ± 0.10 |
-17.30 ± 1.95 |
- |
Coenzyme Q10-SLNs |
213.9 ± 6.3 |
0.35 ± 0.15 |
-13.49 ± 3.85 |
91.20 ± 2.14 |
Selenium SLNs |
296.72 ± 6.3 |
0.49 ± 0.12 |
-6.12 ± 2.85 |
94.89 ± 1.54 |
Table.2: Effect of simvastatin (20mg/kg) and its combination with nano-coQ10 (10mg/kg) and/or nano-selenium (0.1mg/kg) on body weight, lipid profiles and AIX in hyperlipidemic rats.
Data is described as means±S.E.M. (n = 8 rats); a: Significantly different from nano-vehicle value at p<0.05; b: Significantly different from HFD value at p<0.05; c: Significantly different from simvastatin value at p<0.05
The entrapment efficiency of coenzyme Q 10 was 91.2%, which measured indirectly. On the other hand, selenium encapsulation increased particle size to 296.72 nm, while zeta-potential was decreased to -6.12 mV due to adsorption of selenium ions on SLNs surface. The spectrophotometric method was used to measure the entrapment efficiency percentage, where 94.89% was entrapped inside SLNs (Table 1).
SLNs were visualized using SEM to investigate particle morphology (Fig. 1a). SEM micrograph demonstrated spherical shape particles. The particle size from SEM images showed a relevant range aligned with reported particle size using Zetasizer.
The in vitro release study of both drugs was conducted in PBS. Coenzyme Q10 showed a rapid release profile, where ~ 85% was released in 12 h. On the other hand, selenium released showed a slower profile, where ~ 50% was released in 12h and ~ 75% released in 24h. This could be attributed to interaction with the electrostatic charge obtained from selenium ions and negatively charged lipid nanoparticles (Fig. 1b).
Animals treated with HFD exhibited an obvious increment of ΔTBW by 29.9% in comparison to animals treated with a nano-vehicle group. Meanwhile, treatment with SV, co-administration of SV and coQNPs, concurrent administration of SV and SeNPs and combined therapy of SV and coQNPs and SeNPs resulted in significant decrease in ΔTBW by 48.6%, 51.0%, 61.8% and 60.9%, respectively, in comparison to HFD treated rats (Figure. 2). Fortunately, all combination groups reduced ΔTBW by 4.6%, 25.7% and 24.0%, respectively in comparison to SV treated animals (Table 2).
Effect of simvastatin, nano-coQ10 and/or nano-selenium on lipid profiles and atherogenic index (AIX) in hyperlipidemic rats
Alteration in lipid profile markers was depicted after daily consumption of HFD for 112 days, thus a remarkable elevation in serum level of total cholesterol was noticed in hyperlipidemic rats by 37.4% in comparison to nano-vehicle control group. While, co-administration of either SV alone or in combination with coQNPs or/and SeNPs resulted in enhancement in the anti-hypercholesteremic efficacy. Moreover, co-administration of SV and coQNPs, co-administration of SV and SeNPs and combination therapy of SV and coQNPs and SeNPs provoked more pronounced reduction in serum T.C level 8.9%, 9.1% and 9.9%, respectively, as compared to SV treated group. Animals treated with HFD showed a marked rise in serum T.G level by 62.0% as compared to nano-vehicle group. Combined therapy of SV and SeNPs succeeded to reach the normal value of T.G level and showed a significant reduction of T.G by 16.9% in comparison to SV treated group (Table 2). While, treatment with SV plus coQNPs and SeNPs showed marked increase in serum HDL-c level by 171.5%, respectively, relative to HFD control group. Serum LDL-c level was distinctly augmented by HFD by 85.8%, while the combined therapy of SV, coQNPs and SeNPs showed significant decrease in serum LDL-c level by 42.2%, 49.7%, 51.4% and 53.1%, respectively in comparison to HFD treated rats. Hyperlipidemic rats showed a prominent increase in AIX ratio by 392.0%, while such increment in AIX was markedly decreased by treatment of treatment with SV alone or in combination with coQNPs or / and SeNPs by 75.6%, 79.9%, 81.2% and 81.9%, respectively. Furthermore, all combination treatments succeeded to restrain completely the normal ratio of AIX (Table. 2).
Effect of simvastatin, nano-coQ10 and/or nano-selenium on hepatic function tests, albumin and ALP in hyperlipidemic rats
Hyperlipidemic rats showed a significant elevation in serum ALT activity by 155.5% in comparison to nano-vehicle treated group. Moreover, SV alone or in combination with coQNPs/ or SeNPs and combined therapy of SV and coQNPs and SeNPs leading to significant decline in serum ALT activity. HFD markedly blunted serum AST activity by 84.6% when compared to nano-vehicle control group. While combined treatment of SV and SeNPs showed significant decrease in AST by 44.9%, respectively, as compared to HFD treated group. Hyperlipidemic rats showed a marked reduction in serum albumin and co-administration of SV and coQNPs/ or SeNPs normalized the serum albumin level as compared to nano-vehicle treated group. HFD exhibited a significant elevation in serum ALP. Meanwhile, treatment with SV alone or in combination with coQNPs or/ and SeNPs revealed significant decrease in serum ALP activity by 17.0%, 23.6%, 24.9% and 15.8%, respectively, as compared to hyperlipidemic control group. Furthermore, it was observed that co-administration of SV and coQNPs and concurrent administration of SV and SeNPs had better effect on serum ALP activity by 8.0% and 9.6%, respectively, as compared to SV treated group. The results were graphically illustrated in Fig. 2 (a,b,c,d).
Effect of simvastatin, nano-coQ10 and/or nano-selenium on insulin, blood glucose and kidney function test in hyperlipidemic rats
Serum glucose level was augmented in hyperlipidemic rats by 104.3% in comparison to nano-vehicle treated rats. Treatment with SV alone or in combination with coQNPs or/ and SeNPs showed significant decrease in glucose by 44.8%, 49.4%, 42.3% and 44.4%, respectively in comparison to HFD treated group. However, Hyperlipidemic rats showed a significant elevation in serum insulin level by 56.3% relative to nano-vehicle control group. Such increase was markedly hampered by treatment with SV alone or in combination with coQNPs or/ and SeNPs leading to pronounced reduction insulin level. Elevation in serum creatinine level was revealed after induction of hyperlipidemia. While, treatment with either SV alone or with coQNPs or/ and SeNPs showed significant reduction in serum creatinine. SV and coQNPs or SeNPs succeeded to normalize serum creatinine level. Hyperlipidemic rats revealed a marked spike in serum urea level, meanwhile, treatment with SV, co-administration of SV and coQNPs or/ and SeNPs showed significant decrease in urea level in hyperlipidemic rats. It was observed that combined treatment of SV with coQNPs or SeNPs showed a restoration to the normal level of serum urea as compared to nano-vehicle group. HFD group significantly blunted BUN ratio, but this increase in BUN was markedly hampered by treatment with SV individually or in presence of coQNPs or / and SeNPs. It was observed that co-administration of SV and coQNPs or SeNPs showed significant reduction in BUN ratio by 50.6% and 52.6%, respectively (Table. 3).
Increased inflammatory response was depicted after daily consumption of HFD evidenced by a remarkable elevation in liver MDA content. While, treatment with SV in combination with coQNPs or / and SeNPs exhibited a marked reduction in MDA level of hepatic tissues. Treatment with SV and coQNPs or/ and SeNPs revealed a distinct decrease in liver MDA content by 60.4% and 14.5%, respectively, as compared to SV treated group. Furthermore, concurrent administration of SV and SeNPs showed a restoration of the normal liver MDA content along with a significant reduction by 63.5% as compared to SV treated group. Hyperlipidemic rats showed a marked depletion in liver GSH content, though it was replenished by treatment with SV alone or in combination with coQNPs or/ and SeNPs resulting in significant elevation in liver GSH. Treatment with SV and coQNPs or SeNPs succeeded to restore liver GSH content to normal value. A marked reduction in liver SOD activity was noticed in hyperlipidemic rats. Meanwhile treatment with SV in combination with coQNPs or / and SeNPs showed significant elevation in liver SOD activity (Table. 4).
Serum creatine kinase (CK) activity was markedly augmented by HFD. This was reversed by co-administration of SV and coQNPs or SeNPs causing considerable reduction in serum CK activity by 11.3% and 16.1%, respectively. Paradoxically, treatment with SV beside coQNPs and SeNPs showed a significant elevation in serum CK activity by 6.1% when compared to SV treated group. Hyperlipidemic rats provoked a pronounced spike in serum myoglobin (MYO) level by 523.5% when compared to nano-vehicle group. While, treatment with SV with coQNPs or/ and SeNPs showed significant decrease in serum MYO level. Unfortunately, combined treatment of SV with coQNPs and SeNPs showed a marked rise in serum MYO level by 29.6%. Serum troponin (Tn-T) level was significantly elevated in hyperlipidemic rats. On the other hand, Tn-T was notably lowered by the treatment with SV alone or in combination with coQNPs or/ and SeNPs. Furthermore, concurrent administration of SV and SeNPs completely restrained the normal serum Tn-T level with a significant reduction by 45.2% as compared to SV treated group (Table 5).
Histological studies revealed that nano-vehicle control rats exhibited normal architecture of the central, portal veins and blood sinusoide with a slight congestion (Figure. 3A). HFD showed deterioration of hepatic tissues characterized by deposition of fatty droplets in the parenchyma associated with few inflammatory cells infiltration in the portal area as well as congestion in the portal veins and sinusoids (Figure. 3-B). While, cross-section of hepatic tissue of SV treated rats revealed vacuolar degeneration that was detected in diffuse manner all over the hepatocytes in the parenchyma with few inflammatory cells infiltration in the portal area (Figure. 3-C). Liver tissue section of combination group of SV and coQNPs showed vacuolar degeneration was noticed in diffuse manner all over the hepatocytes in the parenchyma (Figure. 3-D). SV and SeNPs treatment exhibited fatty change in the hepatocytes in a focal manner associated with portal vein congestion (Figure. 3-E). SV treatment in combination with coQNPs and SeNPs resulted to vacuolar degeneration in the hepatocytes associated with few inflammatory cells infiltration in the portal area (Figure. 3-F).
Nano-vehicle control rats showed no histopathological alterations in their quadriceps muscle (Figure. 5-A). HFD showed deposition in fat droplets between the atrophied muscles (Figure. 4-B) that characterized by focal Zenkers necrosis in some few other bundles. Treatment of SV showed a marked deposited fat cells in the area between the muscle and atrophied cells (Figure. 4-C). Similarly, quadriceps muscle section of rats treated with combination group of SV and coQNPs showed normal muscle structure (Figure. 4-D). SV and SeNPs combination group did not show any histopathological alterations (Figure. 4-E). Furthermore, Combination therapy of SV and coQNPs and SeNPs showed muscular atrophy which was detected in focal manner associated with focal deposition of the fat droplets in between the bundles (Figure.4-F). Histological changes in sections of hepatic tissues and quadriceps muscles were quantitatively illustrated at Table 6.
Table.3: Effect of simvastatin (20mg/kg) and its combination with nano-coQ10 (10mg/kg) and/or nano-selenium (0.1mg/kg) on insulin, blood glucose levels and kidney function test in hyperlipidemic rats.
Data is described as means±S.E.M. (n = 8 rats); a: Significantly different from nano-vehicle value at p<0.05; b: Significantly different from HFD value at p<0.05; c: Significantly different from simvastatin value at p<0.05
Table 6: The effect of different treatments on histological patterns of hepatic tissues and quadriceps muscles
Table (7): Effect of different treatment on immunopathological reaction of caspase-3 in hepatic tissues and quadricepts muscles
Where:
+ve: immunoreaction with caspase 3.
-ve: No immunereactiuon.
Photomicrograph of a section of hepatic tissue of rats stained with caspase-3, animals treated with nano-vehicle showed no interaction between hepatic tissues after staining with caspase 3 (Figure.5-A). Photomicrograph of a hepatic section of rat treated with HFD stained immunohistochemically for caspase-3 showed an intense positive result (discoloration with dark brown) in many hepatocytes due to immunoreaction after staining with caspase 3 (Figure.5-B). SV treated rat showed an intense positive result that indicated by dark brown discoloration (Figure. 5-C). The combined treatment with SV and coQNPs showed a significant reduction of number of positive cells that stained with caspase-3 (Figure. 5-D). Photomicrograph of a section of hepatic tissue of combined therapy of SV and SeNPs showing reduction in intensity of positive cells when compared to SV treated rat (Figure. 5-E). Photomicrograph of a section of hepatic tissue of concurrent administration of SV and coQNPs and SeNPs then stained immunohistochemically for caspase-3 showing increase in number and intensity of positive results of hepatocytes as compared to SV treated rat (Figure.5-F).
Animals treated with nano-vehicle only showed no reaction with caspase 3 that indicated by negative results of quadriceps muscles (Figure.6-A). Cross section of quadriceps muscle of animals treated with HFD exhibited with a marked positive reaction with immunohistochemichal stain caspase 3 that indicated by dark brown discoloration in tremendous numbers of muscles filaments (Figure.6-B). Immunohistochenichal staining with caspase 3 showed distinct dark brown discoloration of quadriceps muscle of animals treated with SV (Figure.6-C). Cross section of immunohistochemichal stained of quadriceps muscles with caspase 3 that removed from animals treated with combination of SV and coQNPs showed marked decrease of positive cells (Figure.6-D). SV and SeNPs succeeded to reduce the number of positive cells that react with caspase 3 stain (Figure.6-E). Cross section of quadriceps muscle of animals treated with SV, coQNPs and SeNPs after direct immunohistochemichal staining showed a marked changes of number of positive cells that react with caspase 3 that indicated by dark brown discoloration (Figure.6-F). Immunohistological changes were scored at table 7.
Nanomedicine is a vital approach to improve the drug use and enhance the safety of most of existed drugs[38]. Although there are several hurdles in translation of nanomedicine from bench to bedside[39]. SLNs were prepared by hot homogenization/sonication method by emulsification of solid lipid with poloxamer solution. The combination of homogenization and sonication reduced the particles to reach nanosize. Blank nanoparticles were prepared based our previous report, where1% w/v Compritol, 3% w/v Gelucire® 44/14, and 3% w/v poloxamer 407 were used as optimum parameters [10].
In the present work, HFD was associated with severe alterations in lipid profile biomarkers manifested by marked elevation in the atherogenic index (AIX), total cholesterol (T.C), triglycerides (T.G), and low-density lipoprotein cholesterol (LDL-c) in addition to a deficiency of density lipoprotein cholesterol (HDL-c). The outcomes of the current study were in the same context as earlier experiments [40, 41]. The results are in line with liver histopathological findings, which noticed a diffusion of fatty droplets that accompanied all hepatocytes [42].
Biochemical examination of insulin and blood glucose levels revealed that animals treated with HFD exhibited marked elevation of both biomarkers. These findings may be attributed to the masking effect of HFD on insulin receptor and promotion of insulin resistance [43]. HFD potentiates the key cytokines such as IκB kinase (IKK), cJun-N-terminal-kinase (JNK), and protein kinase C (PKC)that promote insulin resistance and interference with insulin signaling[44]. The present investigation showed that HFD induced a marked elevation of liver function tests (ALT, AST and ALP) associated with marked reduction in albumin level. Thus indicating hepatic steatosis and liver injury and this finding was in line with many previous studies [45, 46]. The current study showed atrophied muscles were filled with fatty droplets and Zenker's necrosis in some other bundles. The histological changes were in the same line with earlier studies [42]. Animals treated with HFD showed muscular dysfunction that indicated by a significant elevation of serum level of CK, troponin and myoglobin. It has been well reported that consumption of a high-fat diet (HFD) was associated with impairment of alternative splicing process of pre-mRNA of troponin-T with stimulation of protein expression of myoglobin and activation of CK enzymes of skeletal muscles [47].
The data of the present work showed that HFD induced severe alterations of kidney function biomarkers via severe elevation in all indicators including creatinine, urea and BUN ratio. These results were affirmed by outcomes that showed HFD activates PI3K/Akt and mitogen-activated protein kinase (MAPK), leading to renal impairment and glomerular hypertrophy [48].
HFD consumption precipitate oxidative stress that indicated to series of inflammatory reaction associated with promotion of cytokines such as nuclear Factor κB (NFκB), activation of genetic expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) [49]. Immunohistochemical examination for apoptosis by determining caspase-3 reactivity revealed a marked positive interaction due to existence of caspase-3 all over hepatocytes and myocytes in the case of hyperlipidemic rat’s tissues. Mountain of evidence speculated on the elevation of MDA is associated with a higher cleaved of caspase-3 and apoptotic cells [50].
The present work showed that SV (20 mg/kg) treated rats exhibited a distinct drop of lipid profile including T.C, T.G and LDL-c. These results were in consistent with the previous work [51].
In the present work, blood glucose level and insulin were significantly decreased by simvastatin treatment in animals pretreated with HFD. These results were in agreement with previous work [52], which reported the inhibitory role of statins on dipeptidyl peptidase IV (DPP-IV). DPP-IV is a serine protease enzyme and catalyze the decomposition of both glucagon like peptide 1 (GLP-1) and glucose-dependent insulin-tropic polypeptide (GIP). GLP-1 and GIP are key factors in maintaining normal blood glucose levels through preserving 70% of insulin secretion after meals.
Hepatic injury was manifested by significant increment with serum level of ALT, AST, ALP and reduction of albumin due to simvastatin treatment. Previous studies exhibited SV treatment was associated with hepatitis, cholestatic jaundice, cirrhosis, hepatic failure, and hepatic necrosis that indicated to destruction of hepatic tissues [53]. It was in agreement with our finding in histopathological examination that showed progressive parenchymal liquefied degeneration in addition to limited infiltration of inflammatory cells around the portal vein.
Laboratory findings of the present study revealed that animals fed HFD and treated with SV hyperlipidemic animals treated with SV suffered from myopathy that was manifested by a significant increment of serum CK, myoglobin and troponin as compared to normal groups. There is no full explanation of the mechanism of statin-induced rhabdomyolysis, but previous studies speculated on the role of monocarboxylate transported in up taking and accumulation of stains in skeletal muscles [54], besides to deterioration of cell membrane fluidity and stability because of shortage of membrane cholesterol [55] that associated with dysfunction of chloride channel conductivity versus cytoplasmic overload due to malfunction of mitochondria [56]. The research outcome of an earlier study accomplished by westwood et al., 2005 revealed that patients who were treated with statins for a long time suffer from muscle aches due to deficiency of mevalonate and isoprenoids [57, 58]. Indeed, mevalonate deficiency will open the door in front of unwanted reactions such as lack of potent antioxidant, cell membrane destabilization due to coenzyme Q10/ubiquinone insufficiency.;[59], lack of enzymatic activity of isoprenylation of selenocysteine-tRNA because of shortage of selenoproteins resources [60], inactivation of the prenylation/geranylgeranylation of proteins through reducing in farnesyl and geranylgeraniol pyrophosphates[59, 61, 62] and agitation of N-linked glycosylation within proteins[63]. Consequently, fat cells deposition within quadriceps muscles and atrophied cells of histopathological studies provide further explanation for this phenomenon.
Immunohistochemical studies of liver and quadriceps muscles affirmed the apoptotic effects of simvastatin that were indicated by a positive reaction with caspase 3. These results were in agreement with earlier studies that speculated on the role of simvastatin by downregulation of PI3K/Akt pathway, Akt/protein kinase B (PKB), which controls cell survival and proliferation [64].
All kidney function biomarkers as creatinine, urea and BUN were founded to be increased as compared to corresponding normal control groups. It has been well reported that renal failure is most likely associated with rhabdomyolysis [65].
Simvastatin treatment showed a pronounced improvement of antioxidative stress biomarkers. These outcomes were in agreement with previous studies that revealed statins treatment associated with attenuation of lipid peroxidation [66]. Meanwhile, clinical research showed a positive impact of simvastatin treatment in hypercholesterolemic and diabetic patients, but without benefits on healthy volunteers [67].
Coenzyme Q10 (coQ10) is one of the most potent antioxidant and plays an essential role in oxidative phosphorylation in mitochondria [68]. Using coQ10 has been limited due to physical characters including poor solubility and high molecular weight that lead to lower bioavailability [19]. Therefore, great efforts have been made to develop novel formulations to enhance the pharmacokinetic properties of coQ10 [69]. Indeed, nanoformulation is one of the strategies that succeeded in improving drug bioavailability in different nutrients and minerals such as coQ10 and selenium [70]. Nano-coQ10 was developed by hot melt technique. Animals treated with nano-coQ10 showed a pronounced improvement in drug bioavailability in comparison to suspended formulation of the parent drug [71].
The current results showed that the combination of SV and coQNPs exhibited a significant decrease in serum LDL-c, T.C, and T.G with a significant increment in HDL-c and a normalized AIX. These findings was in line with previous studies [72, 73]. Moreover, clinical research reported that the endothelial function was improved in dyslipidemic patients who used coQ10 regularly due to anti-athergonic activities especially in conduit arteries[74].
Earlier study reported that coQ10 able to enhance the mitochondrial fatty acid oxidation and vascular protection by excessive degradation of T.G-rich lipoprotein. This anti-oxidant activity of coQNPs was compatible with our results, thus, it was founded that combination regimen of SV and coQNPs reduced MDA significantly with a normalized levels of GSH and SOD [75]. Other vascular protecting mechanism was showed by limiting the inactivation of endothelial NO in response to superoxide radicals [76]. Therefore, inactivation of endothelium-derived relaxing factor and/or fibrosis of arteriolar smooth muscle will be reduced [77].
There are consistent results highlighted to the relation between statin therapy and reduction of serum level of coQ10 [78]. There are mounting evidences that kink between deficiency of coQ10 in one side and skeletal muscles dysfunction and insulin resistance in another side due to the essential role of skeletal muscles in glucose uptake and insulin regulation [79]. This data would explain our results that serum glucose and insulin level was normalized by administration of the combined therapy of SV and coQNPs. Furthermore, SV and coQNPs reduced alkaline phosphatase activity with normalized alanine amino transferase (ALT), aspartate amino transferase (AST) and albumin with a distinct decrease of CK activity, MYO and troponin relative to SV. CoQ10 reduce statin side effects and have anti-obesity effect[80]. A study of Abdelbaset et al., (2014) repurpose coQ10 in lowering cholesterol level without elevation of CK in comparison to statin-induced myopathy[81]. These results were confirmed by the histological and immunohistochemichal studies in the present work.
Selenium (Se) is an essential micronutrient and plays an important role in sevral biological reactions. Selenium is major component of different proteins; selenocysteine-containing proteins (selenoproteins) such as glutathione peroxidases (GPx), iodothyronine deiodinases, and thioredoxin reductases. Selenoproteins exhibited homeostatic activity in redox system, metabolic function of thyroid hormone, and cellular surviving in front of oxidative stress and inflammatory reactions [82]. Therapeutic applications of selenium was limited due to the potential toxicity associated with any slight increase in the daily dose, therefore Selenium nano-particles (SeNPs) provides an inventible solution to overcome the toxic limitation of selenium [83].
In the present work, the combination therapy of SV and SeNPs exhibited a pronounced anti-hyperlipidemic activity relative to SV alone associated with a marked reduction in liver function biomarkers which is in line with Dkhil et al., (2016) who verified that SeNPs ameliorated effects of hepatic disturbances, histopathology and oxidative stress, which evidenced by our histopathological and immnuohistochemical findings[84].
In the current study, the combined therapy of SV and SeNPs succeeded to restore the normal values of glucose and insulin. Earlier studies advocate selenium use in diabetes due to activation of Akt and other kinases that control insulin signaling and carbohydrate metabolism. There are several explanation of hypoglycemic action of selenium including prevention of glucose transport from GIT tract and enhancement of urinary excretion [85]. Furthermore, Febiyanto et al., (2018) donated that selenium improve insulin sensitivity and glucose transport in experimental animal model of diabetes [86].
All the previous mentioned studies donated that SeNPs has lower side effects which manifested by a normalized muscle function biomarkers and kidney function. The muscular histopathological findings showed a normal structure with lesser caspase-3 immunoreactivity as compared to SV. It was noted that SeNPs possesses equal efficacy of selenium with much lower toxicity[86].
The most obvious effect of combination group of SV and SeNPs is the restoration of the oxidative stress biomarkers. Hassan et al., (2021) stated that selenium able to scavenge free radical and terminate the oxidative stress through selenoenzymes such as GPx [87].
The research outcomes of the current study showed combined treated of SV with coQNPs and SeNPs exhibited a powerful anti-hyperlipidemic and anti-hyperglycemic activity through a significant reduction in T.C, T.G, LDL-c and AIX in addition to an increment in HDL-c, while, serum glucose and insulin was normalized. A significant increase in ALT, AST was detected relative to SV group with no significant alterations in ALP and albumin was founded between SV and this combined therapy. This hepatic effect was confirmed by histopathological examination. An intense caspase-3 expression was founded in rat’s quadriceps muscle as compared to SV treated group.
A very few studies determined the possible therapeutic efficacy and side effects of this triple combination. Bogsrud et al., (2013) reported that no significant chnges in combined treatment with atorvastatin, coQ10 and selenium on SIM compared with the placebo. In contrast, our results revealed a marked increase in CK, MYO and Tn-T as compared to treatment with SV alone [88]. This un-preferable effect was evidenced by our histological and immunohistochemical findings. Histopathological findings in rat’s quadriceps showed muscular atrophy in focal manner with focal deposition of the fat droplets in between the bundles. An increase in number and intensity of apoptotic cells was founded in quadriceps skeletal muscles as compared to SV treated group. While, No significant difference was determined between combination therapy of SV and coQNPs and SeNPs and treatment with SV in kidney function biomarkers as creatinine, urea and BUN. On the other hand, concurrent administration of SV and coQNPs and SeNPs reduced MDA content with increased GSH content in addition to a normalized SOD activity. We suggest that this triple combination suffers from certain interaction led to pro-oxidation which worsen the side effects of simvastatin in addition to interaction circumstances.
In conclusion, Selenium, and coenzyme Q10 are currently prescribed as preventive treatment due to their antibacterial and antioxidant activities. the current study provides a comprehensive outlook on SLNs platform as a drug delivery system that improves therapeutic values of selenium and coenzyme Q10. SV in combination with coQNPs or SeNPs succeeded to normalize all biochemical parameters and histological patterns in comparison to SV treatment only. These findings provide new insights regarding potential adverse effects in a combination of SV with coQNPs and SeNPs for future translational research or clinical applications.
SeNPs |
nanoencapsulated platform of selenium |
coQNPs |
nanoencapsulated platform of Coenzyme Q10 |
(SV) |
simvastatin |
HFD |
High fat diet |
ΔTBW |
Total B.W difference |
T.C |
Total Cholesterol |
ALP |
Alkaline Phosphatase |
Tn-T |
Serum troponin |
ETHICAL STATEMENT:
o Ethics approval and consent to participate:
The current study was performed according to approval released by the ethics committee of the faculty of pharmacy, Cairo University (Permit Number: PT 1353).
o Consent for publication:
All authors have read and approved the submitted manuscript.
o Availability of data and materials:
Data will be available on request to authors.
o Competing interests:
All authors declare that they have no competing of interest.
o Funding:
The authors confirm that the current study had not received any funding support from any funding agency, and it is self-funded work.
o Authors' contributions:
o Acknowledgment
Authors express their thanks to Dr. Adel Bekier, for his generous support in histopathological examination in this manuscript.
o Authors' information:
published 22 Sep, 2023
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