Research Article

 

Synthetic Salicylates and/ or Pioglitazone Ameliorative Effect in Type 2 Induced Diabetic Rats

 

Yousef M. Shehata1, Amany I. Ahmed1, Nada Y. Nasr1, Ahmed Hamed Arisha2*

1Department of Biochemistry, Faculty of Veterinary Medicine, Zagazig University, 44519 Zagazig, Egypt; 2Department of Physiology, Faculty of Veterinary Medicine, Zagazig University, 44519 Zagazig, Egypt.

 

Received | September 19, 2019; Accepted | October 26, 2019; Published | December 12, 2019

*Correspondence | Ahmed Hamed Arisha, P.h. D. Department of Physiology, Faculty of Veterinary Medicine, Zagazig University, 44519 Zagazig, Egypt; Email: [email protected]

Citation | Shehata YM, Ahmed AI, Nasr NY, Arisha AH (2019). Synthetic salicylates and/ or pioglitazone ameliorative effect in type 2 induced diabetic rats. Adv. Anim. Vet. Sci. 7(s2): 137-144.

DOI | http://dx.doi.org/10.17582/journal.aavs/2019/7.s2.137.144

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright © 2019 Shehata et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

INTRODUCTION

 

Type 2 diabetes mellitus (T2DM) is the most common metabolic disorder, characterized by insulin resistance followed by pancreatic islet beta-cell failure leading to hyperglycemia (Asmat et al., 2016). Insulin resistance is defined as a decreased response of peripheral tissues to normal levels of circulating insulin. Insulin resistance is usually compensated by hyperinsulinemia. In these regards, animal models are cornerstone to our ever-advancing knowledge on the pathophysiology of T2D and related complications (King, 2012). Elevated daily consumption of fructose has been recently linked to the type 2 diabetes. Furthermore, high fat high fructose (HFHF) was used to induce T2D (Pyo and Lee, 2014). This HFHF T2D model gives significant inputs about the pathophysiological alterations associated with the early stage of T2D; hyperglycemia, insulin resistance, and abnormal lipid profiles (Samuel, 2011). Additionally, inflammation plays a crucial role in both insulin resistance and pancreatic beta-cell failure (Pechlivani and Ajjan, 2018). T2D is considered as inflammatory disorder characterized by increasing circulating levels of cytokines, chemokines and C-reactive protein (CRP) (Donath and Shoelson, 2011). Increase of insulin signaling impairment in adipocytes; the release of cytokines and chemokines from adipose tissues into the circulation stimulate inflammation in pancreatic islet and other tissues (Donath and Shoelson, 2011). The production of inflammatory molecules associated with insulin resistance such as tumor necrosis factor-alpha (TNF-α), Interleukin-6 (IL-6), IL-1β, and IL-8 stimulate NF-kβ pathway stimulating vicious loop of insulin resistance (Hameed et al., 2015). Adiponectin which is a protective adipokine, acts as anti-inflammatory cytokine and enhances insulin sensitivity (Rehman and Akash, 2016). It inhibits the expression of TNF-α, IL-6 and hepatic gluconeogenesis and stimulates glucose uptake in skeletal muscles and fatty acid oxidation in liver and skeletal muscles. Adiponectin acts as a biomarker for insulin resistance (Park et al., 2015).

 

Pioglitazone, one of thiazolidinediones (TZDs) or glitazones, is considered insulin sensitizers and therefore used in the treatment of T2D. Pioglitazone has anti-diabetic effect through an increased transactivation of peroxisome proliferator activated receptors-gamma (PPAR-γ agonists), a reduced gluconeogenesis and increase glucose and lipid metabolism (Nanjan et al., 2018). A decreased insulin resistance (Rehman and Akash, 2016), results in an increased glucose uptake through GLUT-4 present in skeletal muscles and adipose tissue (Mirza et al., 2019). On the other hand, Salicylates is one of non-steroidal anti-inflammatory drugs that is widely used for antiplatelet therapy in cardiovascular diseases (Majithia and Bhatt, 2018). Salicylates has antidiabetic effect, improve insulin sensitivity and glucose metabolism through mechanisms that are yet to be further explored (Bellucci et al., 2017; Rehman and Akash, 2016). This work aimed to investigate the biochemical and molecular mechanisms related to the antidiabetic effect of both low and high doses of salicylates as well as pioglitazone, either alone or in combination, in diabetic male albino rats.

 

MATERIAL AND METHODS

 

Chemicals and reagents

A synthetic salicylate (acetylsalicylic acid; aspirin) was purchased from Sigma-Aldrich chemical Co., St. Louis. Mo. (USA). Pioglitazone (PIO) was purchased from Hikma Pharmaceuticals, Egypt and stored in the dark at -4ºC. A stock solution of PIO was prepared by dissolving it in dimethyl sulfoxide (DMSO; Sigma-Aldrich). Working solutions of PIO (0, 5, 10, 20 and 40 μmol/L) were prepared by diluting the stock solution in a DMSO medium. The DMSO concentration in the working solutions was < 0.1%. The same concentration of DMSO (< 0.1%) was used in the control group.

 

Experimental design

Seventy male adult albino rats (150-200g) were acquired from the central animal house of the Faculty of Veterinary Medicine, Zagazig University, Egypt. Rats were acclimatized for 2 weeks under standard laboratory conditions prior to any treatments. Rats were kept at a suitable temperature of 20-25 °C, 12-hour light-dark cycle and free access to food and water. The experimental protocol was approved by the ethics committee of Institutional Animal Care and Use (IACUC) number (ZU-IACUC/2/F/34/2018), Zagazig University, Egypt. After 2 weeks of adaptation, rats were subdivided into 2 groups. The first one served as a normal control group (G1, 10 rats); rats were supplied with a typical rat chow for 16 weeks (full experimental period) in addition to pure water. The other 60 rats were fed high fat and high fructose (HFHF) diet formulated as per previously described composition (El-Naggar and El-Dawy, 2019; Pyo and Lee, 2014).

 

Rats were monitored weekly for both body weight and fasting blood glucose. At 13 weeks, rats (HFHF dietary group) with blood glucose level less than 200mg/dl were considered non-diabetic and were excluded. Only rats which displayed blood glucose levels more than 200 mg/dl and hyper-insulinemia for 7 days were considered resistant and were further confirmed for insulin resistance (IR) using a homeostasis model assessment (HOMA-Insulin Resistance index; HOMA-IR) (Wilson and Islam, 2012). Diabetic rats in the HFHF induced diabetes were then randomly subdivided into six groups and were given different treatments for the following 4 weeks: group 2 (G2): diabetic received no treatment, group 3 (G3): diabetic and daily treated orally with low-dose salicylates at 10 mg/kg (Patumraj et al., 2000; Pyo and Lee, 2014), group 4 (G4): diabetic and daily treated orally with high-dose salicylates at 120 mg/kg (Yuan et al., 2001), group 5 (G5): diabetic and daily treated orally with pioglitazone at 10 mg/kg (Lee et al., 2007; Schaalan, 2012), group 6 (G6): diabetic and daily treated orally with both low-dose salicylates (10 mg/kg) and pioglitazone (10 mg/kg), group 7 (G7): diabetic and daily treated orally with both high-dose salicylates (120 mg/kg) and pioglitazone (10 mg/kg). The dose was modified weekly in relation to changes in weight gain to sustain analogous dosage per kg body weight of rat throughout the study period.

 

Sampling

Rats were sacrificed via cervical decapitation, blood was collected, serum and plasma were separated by centrifugation at 3000 rpm for 20 mins and sent for the different biochemical and hormonal assays. A 50 mg of liver tissue was collected and used for gene expression analysis.

 

Biochemical analysis

Serum glucose was determined by oxidase method using Spectrum Diagnostics glucose kit (Spectrum Diagnostics, Egypt) (El-Gayar et al., 2012). Estimation of serum insulin level by ELISA rat insulin kits using Ray Bio kit (RayBiotech, Inc., USA). Lipid profile was measure using Spectrum diagnostic kit (Spectrum Diagnostics, Egypt) following manufacturer instructions. The direct enzymatic colorimetric liquid method was used for determination of high density lipoprotein -c (HDL-c) and low-density lipoprotein -c (LDL-c) calculated according to friedewald formula (Friedewald et al., 1972). Evaluation of triacylglycerol (TAG) using GPO-PAP enzymatic colorimetric method (Bucolo and David, 1973), cholesterol liquizyme CHOD-PAP (Penttilä et al., 1981) and free fatty acid (FFA) were determined spectrophotometrically by enzymatic colorimetric method using Biovision FZscreen TM (BioVision, Inc., USA). Serum concentrations of TNF-α, IL-6 and adiponectin were determined by the commercially available rat ELISA kits from MyBioSource (MyBioSource, USA) following manufacturer instructions.

 

Relative quantitative RT-PCR analysis

A detailed description of the protocols used has been previously reported (Arisha et al., 2019; Arisha and Moustafa, 2019; Khamis et al., 2020). The real-time RT-PCR was performed in aMx3005P Real-Time PCR System (Agilent Stratagene, USA) using 5x HOT FIREPol EvaGreen qPCR Mix Plus (Solis Bio Dyne, Tartu, Estonia) and specific primers for the target genes (Table 1). The relative expression of each of the gene normalized to the housekeeping GAPDH was reported as fold change by 2−ΔΔCT (Livak and Schmittgen, 2001).

 

Statistical analysis

Statistical analysis was carried out using one-way ANOVA (SPSS 16, Chicago, Ill). Data were expressed as Mean±SEM where P< 0.05 was statistically significant.

 

RESULTS

 

Effect of administered salicylates and/or pioglitazone on the glycemic index and HOMA-IR

Dietary supplementation HFHF diet induces the development of type 2 diabetes characterized by elevated glucose, abnormal insulin concentrations and insulin resistance compared to the control group (Table 2). However, the plasma glucose and insulin concentrations of the diabetic group treated with salicylates (low and high dose) and/or pioglitazone were significantly (P < 0.01) lower than the diabetic group. The degree of insulin resistance was estimated at the baseline by an insulin score (HOMA-IR). Low HOMA-IR values indicate high insulin sensitivity, whereas high HOMA-IR values indicated low insulin sensitivity. As shown in Table 2, HOMA-IR was significantly (P < 0.01) higher in the HFD group than others but HOMA-IR of the diabetic group supplemented with salicylates (low and high dose) and/or pioglitazone were significantly lower than the diabetic group.

 

Effect of administered salicylates and/or pioglitazone on the lipid profile and FFA levels

The diabetic group showed significant (P < 0.01) increase in serum levels of FFAs and development of diabetic dyslipidemia that characterized by increase of TC, TAG, LDL-c, VLDL-c and decrease of HDL-c when compared to the normal control group. The HFHF group treated with salicylates (low and high dose) and/or pioglitazone showed significant decrease in serum levels of FFA, TC, TAG, LDL-c and VLDL-c with increase serum levels of HDL-c when compared to the HFHF group.

 

Effect of administered salicylates and/or pioglitazone on pro inflammatory cytokines and adiponectin levels

The diabetic group showed significant (P < 0.05) increase in serum levels of TNF-α, IL-6 and decrease serum adiponectin level when compared to the normal control group. Diabetic group treated with salicylates (low and high dose) and/or pioglitazone showed significant (P < 0.05) decrease in serum concentration of TNF-α, IL-6 with increase serum levels of adiponectin when compared with HFHF group.

 

Effect of administered salicylates and/or pioglitazone on hepatic mRNA expression of PPAR- γ and NFKB-p65

Diabetic group showed significant (P < 0.01) decrease in PPAR-γ gene expression with significant (P < 0.001) increase in NF-KB gene expression when compared with normal control group (Figure 1 A&B). Administration of salicylates and/or pioglitazone to diabetic rats showed significant (P < 0.01) increase in PPAR-γ gene expression with significant (P < 0.001) decrease in NF-KB gene expression when compared with diabetic untreated group (Figure 1 A&B).

 

DISCUSSION

 

Salicylates has antidiabetic effect by unclear mechanism and its clinical use has been limited due to its adverse effects that are associated with high dosage (Sami et al., 2012). It may act as antidiabetic through inhibition of Ikkβ/NF-kβ and, especially at high dosage, subsequently reduced proinflammatory cytokines (Rehman and Akash, 2016) and plasma free fatty acids (Rumore and Kim, 2010). Also, An et al., 2009. showed that salicylate decrease proinflammatory cytokines and increase PPAR-γ and adiponectin and therefore result in an inhibited interaction between adipocytes and proinflammatory mediators (An et al., 2009). A study suggests that anti-inflammatory effect of NSAIDs may result an activation of PPAR-γ and inhibition of pro-inflammatory gene expression (Landreth and Heneka, 2001). This study provides a great deal of evidence that low dose salicylates alone is an effective drug for diabetes (Abdin et al., 2010). However, the study outcomes contradicts another observation that showed greater impact

 

Table 1: Sequence of the primers used in the RT-PCR experiments.

 

Gene	Forward primer (5′–3′)	Reverse primer (5′–3′)	Accession no	Product size	Reference
NF-kβ p65	CAGGACCAGGA ACAGTTCGAA	CCAGGTTCTG GAAGCTATGGAT	NM_199267.2	150	(Yang and Yuan, 2018)
PPAR-ϒ	CCTGAAGCT CCAAGAATACC	GATGCTTTA TCCCCACAGAC	NM_013124.3	153	(Chistyakov et al., 2014)
Gapdh	GGCACAGTCAA GGCTGAGAATG	ATGGTGGTGA AGACGCCAGTA	NM_017008.4	143	(Yang et al., 2015)

 

Table 2: Means ±SEM of serum levels of fasting blood glucose, insulin, HOMA-IR in rats fed high fat high fructose diet and treated with salicylates and/ or pioglitazone

 

	Control	Diabetes	Low salicylates	High salicylates	Pioglitazone	Low salicylates+ pioglitazone	High salicylates+pioglitazone
Glucose (mg/dl)	92 .80 ± 1.52g	221.50 ± 2.72a	190.15 ± 1.20b	165.40 ± 3.48c	150.95 ± 0.66d	144.30 ± 0.57e	130.40 ± 0.88f
Insulin (μU/ml)	10.41 ± 0.15d	12.13 ± 0.12a	13.51 ± 0.28b	13.40 ± 0.25b	13.33 ± 0.38b	12.36 ± 0.35bc	12.00 ± 0.25c
HOMA-IR	2.38 ± 0.14e	6.63 ± 0.30a	6.34 ± 0.12b	5.46 ± 0.16b	4.96 ± 0.42c	4.40± 0.16d	3.86 ± 0.26d

 

abcdefg Means ± SEM at the same row and bearing different superscripts are significantly different at p ≤ 0.05.

 

Table 3: Means ±SEM of lipid profile (TC-TG-LDL-HDL-VLDL) in rats fed high fat high fructose diet and treated with salicylates and / or pioglitazone.

 

	Control	Diabetes	Low salicylates	High salicylates	Pioglitazone	Low salicylates+ pioglitazone	High salicylates+ pioglitazone
TC (mg/dl)	84.33 ± 2.33d	112.13 ± 1.16a	98.66 ± 0.88b	95.33 ± 0.33b	90.66 ± 0.66c	86.68 ± 0.65d	84.00 ± 0.57d
TAG (mg/dl)	140.33 ± 0.88g	180.00 ± 0.57a	171.67 ± 0.89b	167.00 ± 1.15c	154.33 ± 0.67d	150.00 ± 0.57e	144.67 ± 0.33f
LDL-c (mg/dl)	41.266± 2.15cd	66.33 ± 0.98a	50.46 ± 1.047b	43.43 ± 0.23c	43.80 ± 1.68c	35.80 ± 1.24d	29.70 ± 3.75e
VLDL-c (mg/dl)	28.06 ± 0.40g	36.80 ± 0.57a	34.33 ± 0.40b	33.10 ± 0.63c	30.70 ± 0.33d	29.70 ± 0.86e	28.90 ± 0.69f
HDL-c (mg/dl)	15.05 ±0 .57c	10.02 ±0.53e	13.66 ±0.33cd	16.02 ± 2.06bc	18.50 ± 0.28bc	20.83 ± 0.45ab	25.46 ± 3.30a
FFA (mmol/l)	0.510 ± 0.26d	0.990 ± 0.30a	0.780 ± 0.24b	0.773 ± 0.040b	0.710 ± 0.24b	0.610 ± 0.84c	0.576 ± 0.20d

 

AbcdefgMeans ± SEM at the same row and bearing different superscripts are significantly different at p ≤ 0.05.

 

Table 4: Means ±SEM of FFA, adiponectin, TNF-α and IL-6 in rats fed high fat high fructose diet and treated with salicylates and/ or pioglitazone.

 

	Control	Diabetes	Low salicylates	High salicylates	Pioglitazone	Low salicylates+ pioglitazone	High salicylates + pioglitazone
Adiponectin (ng/ml)	2.70 ± 0.54d	1.64 ± 0.44g	2.05 ± 0.64f	2.42 ± 0.18e	3.33 ± 0.43c	3.77 ± 0.16b	3.93 ± 0.13a
TNF- α (pg/ml)	30.60 ± 0.26g	50.84 ± 0.30a	39.56 ± 0.12c	36.83 ± 0.44d	41.83 ± 0.40b	33.26 ± 0.24e	31.96 ± 0.20f
IL-6 (pg/ml)	41.160 ± 0.53d	48.66 ± 0.69a	44.733 ± 0.35b	43.26 ± 0.17c	45.33 ± 0.14b	42.30 ± 0.11cd	41.90 ± 0.25d

 

Abcdefg Means ± SEM at the same row and bearing different superscripts are significantly different at p ≤ 0.05.

 

on fasting blood glucose at a high dose (Sami et al., 2012). Our results showed that low and high doses of salicylates ameliorate the insulin resistance, hyperglycemia, hyperlipidemia and reduce the inflammatory markers providing good glycemic control. These observations are consistent with other several studies including high doses (Hundal et al., 2002; Kim et al., 2001; Sami et al., 2012; Sethi et al., 2011; Sun et al., 2011; van Diepen et al., 2011; Yuan et al., 2001) and low doses (Amiri et al., 2015; Ibrahim, 2018; Lin et al., 2014). In contrast, early studies showed that salicylates result in an increased insulin resistance in diabetic patients although decrease of fasting blood glucose may be due to an increased plasma response to insulin (Bratusch-Marrain et al., 1985) and normal patient (Giugliano et al., 1982). This difference in the effect of salicylates may be due to the difference in the experimental model and species studied (Sami et al., 2012). In another study, where low salicylates were used, no difference in blood glucose level was recorded (Lin et al., 2014). Although Sun et al., 2011 reported that high doses of salicylates did not induce notable reduction in blood glucose level (Sun et al., 2011).

 

In our study we found that low and high dose salicylates caused a significant decrease in insulin level. Also, other studies showed that low salicylates ameliorate hyperinsulinemia (Amiri et al., 2015; Lin et al., 2014). In addition, another study illustrated that low salicylates caused an increase in serum insulin level that indicate development of insulin resistance although increase of PPAR-γ gene expression in male mice, whereas, in female mice, salicylates reversed glucose intolerance with no difference in insulin level (Zhou et al., 2019). Also, Sun et al., 2011, showed that high dose salicylates ameliorate hyperinsulinemia (Sun et al., 2011). In addition, it was reported that high salicylates caused significant increase of insulin secretion (Sethi et al., 2011). Also, an increase in PPAR-γ, that regulates β-oxidation in male mice, suggesting the development of insulin resistance (Zhou et al., 2019). Another study demonstrated that low dose improve hyperlipidemia via inhibition of NF-kβ (Lin et al., 2014). In contrast, further study showed that moderate and high dose caused an increase of TAG which may be due to short duration of salicylates administration to diabetic rats or to the experimental model or species difference (Sami et al., 2012). Our results showed that low and high salicylates caused inhibition of NF-kβ in a pattern demonstrated previously (Lin et al., 2014).

 

 

Our results showed that low and high salicylates decreased levels of FFA in diabetic rats as described previously using low (Abdin et al., 2010) and high dosages (Sun et al., 2011; Yuan et al., 2001). Furthermore, our results showed that low salicylates decrease levels of TNF-α and IL-6 similar to the one reported previously (Abdin et al., 2010). This is contrary to observation made in another study where low salicylates caused no alteration in TNF-α and IL-6 expression in male mice (Zhou et al., 2019). Our study showed that high salicylates decrease levels of TNF-α, IL-6 (Abdin et al., 2010) and TNF-α in diabetic rats (Sun et al., 2011).

 

The findings of this study showed that salicylates caused increase in gene expression of PPAR-γ as previously shown (Zhou et al., 2019). Salicylates might be a peroxisome proliferator and may activate PPAR-γ (Lehmann et al., 1997). Also, Fidaleo reported that salicylates might affect the related target gene expression of PPAR-γ in the liver and kidney of rats (Fidaleo et al., 2008). Activation of PPAR-γ may lead to inhibition of NF-kβ (Romeiro et al., 2008; Xue et al., 2010).

 

TZDs act as anti-inflammatory at higher drug concentration as repression occurs at higher drug concentration than that required for activation (Landreth and Heneka, 2001). Our results showed that pioglitazone improve hyperglycemia and hyperlipidemia and these results are in accordance with several studies (Ding et al., 2005; Refaat et al., 2016; Schaalan, 2012; Vijay et al., 2009). Also, hyperinsulinemia with significant decrease in HOMA-IR and FFA was observed in pioglitazone treated diabetic rats in accordance to previous studies (Refaat et al., 2016; Schaalan, 2012). Also, our study showed that pioglitazone showed inhibition of NF-kβ and increase adiponectin as shown in previous studies (Refaat et al., 2016; Sundararajan et al., 2005; Sung et al., 2006).

 

Combination of salicylates (low or high) and pioglitazone caused significant decrease in insulin resistance, fasting blood glucose, insulin, FFAs, TNF-α, IL-6, TC, TAG, LDL-c, and VLDL-c and also, caused significant increase of HDL-c and adiponectin providing good glycemic control. This augmented effect can be explained by an observation that both salicylates and pioglitazone can bind and activate PPAR-γ and therefore cumulatively can inhibit NF-kβ. Hence, salicylates has the ability to potentiate effect of pioglitazone or any PPAR-γ agonists and therefore can act alone as antidiabetic drug. Also, some other studies indicate that salicylates and pioglitazone is a good combination simply because pioglitazone potentiates the effects of salicylates by reduction of inflammation and inhibition of platelet function (Evangelista et al., 2005; Mongan et al., 2012). Pioglitazone can suppress the acute gastric mucosal injury in rats that is considered a serious adverse effect of salicylates therapy (Naito et al., 2001). Combination of salicylates and pioglitazone can enhance catalase activity in liver and lead to reduction of oxidative stress, inhibition of non-enzymatic glycation, and cataract formation in type 2 diabetes (Panahi et al., 2019).

 

Conclusions

 

Combination of salicylates and pioglitazone provide more beneficial effects, good glycemic control and decrease risk of cardiovascular complications when compared with pioglitazone alone. However, the role of salicylates in insulin resistance requires more basal and clinical studies due to significant difference between experimental studies.

Acknowledgments

 

The present study was a result of collaboration between the Departments of Biochemistry and Physiology, Faculty of Veterinary Medicine and the Medical Research Center at the Zagazig University, Egypt.

 

Authors Contribution

 

A.I.A, N.Y.N and A.H.A conceived the project, researched data, analyzed data and drafted the manuscript. Y.M.S and A.H.A researched the data and reviewed and edited the manuscript. All authors read and approved the final draft of the manuscript.

 

Conflict of interest

 

The authors declare no conflicts of interest, financial or otherwise.

 

References

 

• Abdin AA, Baalash AA, Hamooda HE (2010). Effects of rosiglitazone and aspirin on experimental model of induced type 2 diabetes in rats: focus on insulin resistance and inflammatory markers. J. Diab. complications 24: 168-178. https://doi.org/10.1016/j.jdiacomp.2009.01.005

• Amiri L, John A, Shafarin J, Adeghate E, Jayaprakash P, Yasin J, Howarth FC, Raza H (2015). Enhanced glucose tolerance and pancreatic beta cell function by low dose aspirin in hyperglycemic insulin-resistant type 2 diabetic Goto-Kakizaki (GK) rats. Cell. Physiol. Biochem. 36: 1939-1950. https://doi.org/10.1159/000430162

• An Y, Liu K, Zhou Y, Liu B (2009). Salicylate inhibits macrophage-secreted factors induced adipocyte inflammation and changes of adipokines in 3T3-L1 adipocytes. Inflamm. 32: 296-303. https://doi.org/10.1007/s10753-009-9135-1

• Arisha AH, Ahmed MM, Kamel MA, Attia YA, Hussein MMA (2019). Morin ameliorates the testicular apoptosis, oxidative stress, and impact on blood–testis barrier induced by photo-extracellularly synthesized silver nanoparticles. Environ. Sci. Pollut. Res. 26: 28749–28762. https://doi.org/10.1007/s11356-019-06066-1

• Arisha AH, Moustafa A (2019). Potential inhibitory effect of swimming exercise on the Kisspeptin–GnRH signaling pathway in male rats. Theriogenol. 133: 87-96. https://doi.org/10.1016/j.theriogenology.2019.04.035

• Asmat U, Abad K, Ismail K (2016). Diabetes mellitus and oxidative stress- a concise review. Saudi Pharm. J. 24: 547-553. https://doi.org/10.1016/j.jsps.2015.03.013

• Bellucci PN, González Bagnes MF, Di Girolamo G, González CD (2017). Potential effects of nonsteroidal anti-inflammatory drugs in the prevention and treatment of type 2 diabetes mellitus. J. pharm. Pract. 30: 549-556. https://doi.org/10.1177/0897190016649551

• Bratusch-Marrain P, Vierhapper H, Komjati M, Waldhäusl W (1985). Acetyl-salicylic acid impairs insulin-mediated glucose utilization and reduces insulin clearance in healthy and non-insulin-dependent diabetic man. Diabetologia. 28: 671-676. https://doi.org/10.1007/BF00291974

• Bucolo G, David H (1973). Quantitative determination of serum triglycerides by the use of enzymes. Clin. Chem. 19: 476-482.

• Chistyakov DV, Aleshin S, Sergeeva MG, Reiser G (2014). Regulation of peroxisome proliferator‐activated receptor β/δ expression and activity levels by toll‐like receptor agonists and MAP kinase inhibitors in rat astrocytes. J. Neurochem. 130: 563-574. https://doi.org/10.1111/jnc.12757

• Ding SY, Shen ZF, Chen YT, Sun SJ, Liu Q, Xie MZ (2005). Pioglitazone can ameliorate insulin resistance in low‐dose streptozotocin and high sucrose‐fat diet induced obese rats. Acta Pharmacologica Sinica 26, 575-580. https://doi.org/10.1111/j.1745-7254.2005.00090.x

• Donath MY, Shoelson SE (2011). Type 2 diabetes as an inflammatory disease. Nat. Rev. Immunol. 11: 98. https://doi.org/10.1038/nri2925

• El-Gayar KE, Ibrahim MA, Mohamed SH, Zakaria Z, Alhameed ASA (2012). Application of extracted peroxidase enzyme from turnip roots (BRASSICA NAPUS) in clinical diagnostic kit. Int. J. Cur. Res. Rev. 4: 1-2.

• El-naggar A, El-Dawy K (2019). Role of micro RNAs and its associated genes in prediction of obesity and diabetes mellitus (Type II). Zagazig Vet. J. 47: 57-67. https://doi.org/10.21608/zvjz.2019.6135.1010

• Evangelista V, Totani L, Rotondo S, Lorenzet R, Tognoni G, De Berardis G, Nicolucci A (2005). Prevention of cardiovascular disease in type-2 diabetes: how to improve the clinical efficacy of aspirin. Thromb. Haemost. 93: 8-16. https://doi.org/10.1160/TH04-07-0453

• Fidaleo M, Berardi E, Sartori C (2008). Differential modulation of PPARα and γ target gene expression in the liver and kidney of rats treated with aspirin. Exp. Toxicol. Pathol. 59: 391-397. https://doi.org/10.1016/j.etp.2007.11.011

• Friedewald WT, Levy RI, Fredrickson DS (1972). Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin. Chem. 18: 499-502.

• Giugliano D, Sacca L, Scognamiglio G, Ungaro B, Torella R (1982). Influence of acetylsalicylic acid on glucose turnover in normal man. Diabete metabolisme. 8: 279-282.

• Hameed I, Masoodi SR, Mir SA, Nabi M, Ghazanfar K, Ganai BA (2015). Type 2 diabetes mellitus: from a metabolic disorder to an inflammatory condition. World J. Diabetes. 6: 598. https://doi.org/10.4239/wjd.v6.i4.598

• Hundal RS, Petersen KF, Mayerson AB, Randhawa PS, Inzucchi S, Shoelson SE, Shulman GI (2002). Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes. J. Clin. Invest. 109: 1321-1326. https://doi.org/10.1172/JCI0214955

• Ibrahim DAA (2018). Dyslipidemic and protective effects of aspirin and clopidogrel in obese rats. Int. J. Pharm. Sci. Res. 9: 3647-3655.

• Khamis T, Abdelalim AF, Abdallah SH, Saeed AA, Edress NM, Arisha AH (2020). Early intervention with breast milk mesenchymal stem cells attenuates the development of diabetic-induced testicular dysfunction via hypothalamic Kisspeptin/Kiss1r-GnRH/GnIH system in male rats. Biochim. Biophys. Acta (BBA)- Mol. Basis Dis. 1866: 165577. https://doi.org/10.1016/j.bbadis.2019.165577

• Kim JK, Kim Y-J, Fillmore JJ, Chen Y, Moore I, Lee J, Yuan M, Li ZW, Karin M, Perret P (2001). Prevention of fat-induced insulin resistance by salicylate. J. Clin. invest. 108: 437-446. https://doi.org/10.1172/JCI11559

• King AJ (2012). The use of animal models in diabetes research. Br. J. Pharmacol. 166: 877-894. https://doi.org/10.1111/j.1476-5381.2012.01911.x

• Landreth GE, Heneka MT (2001). Anti-inflammatory actions of peroxisome proliferator-activated receptor gamma agonists in Alzheimer’s disease. Neurobiol. Aging. 22: 937-944. https://doi.org/10.1016/S0197-4580(01)00296-2

• Lee MY, Lee EY, Lee BJ, Won CS, Koh JH, Shin JY, Shin YG, Cho BP, Chung CH (2007). Beneficial effects of thiazolidinediones on diabetic nephropathy in OLETF rats. Yonsei Med. J. 48: 301-307. https://doi.org/10.3349/ymj.2007.48.2.301

• Lehmann JM, Lenhard JM, Oliver BB, Ringold GM, Kliewer SA (1997). Peroxisome proliferator-activated receptors α and γ are activated by indomethacin and other non-steroidal anti-inflammatory drugs. J. Biol. Chem. 272: 3406-3410. https://doi.org/10.1074/jbc.272.6.3406

• Lin HL, Yen HW, Hsieh SL, An LM, Shen KP (2014). Low‐dose aspirin ameliorated hyperlipidemia, adhesion molecule, and chemokine production induced by high‐fat diet in Sprague‐Dawley rats. Drug Dev. Res. 75: 97-106. https://doi.org/10.1002/ddr.21159

• Livak KJ, Schmittgen TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods. 25: 402-408. https://doi.org/10.1006/meth.2001.1262

• Majithia A, Bhatt DL (2018). Antiplatelet Therapy in Diabetes. Endocrinol. Metab. Clin. North Am. 47: 223-235. https://doi.org/10.1016/j.ecl.2017.10.009

• Mirza AZ, Althagafi II, Shamshad H (2019). Role of PPAR receptor in different diseases and their ligands: Physiological importance and clinical implications. Eur. J. Med. Chem. 166: 502-513. https://doi.org/10.1016/j.ejmech.2019.01.067

• Mongan J, Mieszczanska HZ, Smith BH, Messing SP, Phipps RP, Francis CW (2012). Pioglitazone inhibits platelet function and potentiates the effects of aspirin: a prospective observation study. Thromb. Res. 129: 760-764. https://doi.org/10.1016/j.thromres.2011.12.019

• Naito Y, Takagi T, Matsuyama K, Yoshida N, Yoshikawa T (2001). Pioglitazone, a specific PPAR‐γ ligand, inhibits aspirin‐induced gastric mucosal injury in rats. Aliment. Pharmacol. Ther. 15: 865-873. https://doi.org/10.1046/j.1365-2036.2001.00983.x

• Nanjan M, Mohammed M, Kumar BP, Chandrasekar M (2018). Thiazolidinediones as antidiabetic agents: A critical review. Bioorg. Chem. 77: 548-567. https://doi.org/10.1016/j.bioorg.2018.02.009

• Panahi Y, Yekta R, Dehghan G, Rashtbari S, Jafari NJ, Moosavi-Movahedi AA (2019). Activation of catalase via co-administration of aspirin and pioglitazone: Experimental and MLSD simulation approaches. Biochimie. 156: 100-108. https://doi.org/10.1016/j.biochi.2018.10.007

• Park SE, Park C-Y, Sweeney G (2015). Biomarkers of insulin sensitivity and insulin resistance: Past, present and future. Crit. Rev. Clin. Lab. Sci. 52: 180-190. https://doi.org/10.3109/10408363.2015.1023429

• Patumraj S, Tewit S, Amatyakul S, Jariyapongskul A, Maneesri S, Kasantikul V, Shepro D (2000). Comparative effects of garlic and aspirin on diabetic cardiovascular complications. Drug Deliv. 7: 91-96. https://doi.org/10.1080/107175400266650

• Pechlivani N, Ajjan RA (2018). Thrombosis and vascular inflammation in diabetes: mechanisms and potential therapeutic targets. Front. Cardiovasc. Med. 5: 1. https://doi.org/10.3389/fcvm.2018.00001

• Penttilä I, Voutilainen E, Laitinen P, Juutilainen P (1981). Comparison of different analytical and precipitation methods for direct estimation of serum high-density lipoprotein cholesterol. Scand. J. Clin. Lab. Invest. 41: 353-360. https://doi.org/10.3109/00365518109092057

• Pyo Y-H, Lee KW (2014). Preventive effect of Monascus-fermented products enriched with ubiquinones on type 2 diabetic rats induced by a high-fructose plus high-fat diet. J. Med. Food. 17: 826-829. https://doi.org/10.1089/jmf.2013.3001

• Refaat R, Sakr A, Salama M, El Sarha A (2016). Combination of vildagliptin and pioglitazone in experimental type 2 diabetes in male rats. Drug Dev. Res. 77: 300-309. https://doi.org/10.1002/ddr.21324

• Rehman K, Akash MSH (2016). Mechanisms of inflammatory responses and development of insulin resistance: how are they interlinked? J. Biomed. Sci. 23: 87. https://doi.org/10.1186/s12929-016-0303-y

• Romeiro NC, Sant’Anna CM, Lima LM, Fraga CA, Barreiro EJ (2008). NSAIDs revisited: Putative molecular basis of their interactions with peroxisome proliferator-activated gamma receptor (PPARγ). Eur. J. Med. Chem. 43: 1918-1925. https://doi.org/10.1016/j.ejmech.2007.11.031

• Rumore MM, Kim KS (2010). Potential role of salicylates in type 2 diabetes. Ann. Pharmacother. 44: 1207-1221. https://doi.org/10.1345/aph.1M483

• Sami HH, Saeed S, Ahmad IY, Saad D (2012). Aspirin and Blood Glucose and Insulin Resistance. Open J. Endocr. Metab. Dis. 2012: 2: 16-26.

• Samuel VT (2011). Fructose induced lipogenesis: from sugar to fat to insulin resistance. Trends Endocrinol. Metab. TEM. 22: 60-65. https://doi.org/10.1016/j.tem.2010.10.003

• Schaalan MF (2012). Effects of pioglitazone and/or simvastatin on circulating TNFα and adiponectin levels in insulin resistance. J. Immunotoxicol. 9: 201-209. https://doi.org/10.3109/1547691X.2012.660998

• Sethi A, Parmar HS, Kumar A (2011). The effect of aspirin on atherogenic diet‐induced diabetes mellitus. Basic Clin. Pharmacol. Toxicol. 108: 371-377. https://doi.org/10.1111/j.1742-7843.2010.00663.x

• Sun X., Han F, Yi J, Han L, Wang B (2011). Effect of aspirin on the expression of hepatocyte NF-κB and serum TNF-α in streptozotocin-induced type 2 diabetic rats. J. Korean Med. Sci. 26: 765-770. https://doi.org/10.3346/jkms.2011.26.6.765

• Sundararajan S, Gamboa J, Victor N, Wanderi E, Lust W, Landreth G (2005). Peroxisome proliferator-activated receptor-γ ligands reduce inflammation and infarction size in transient focal ischemia. Neurosci. 130: 685-696. https://doi.org/10.1016/j.neuroscience.2004.10.021

• Sung B, Park S, Yu BP, Chung HY (2006). Amelioration of age-related inflammation and oxidative stress by PPARγ activator: suppression of NF-κB by 2, 4-thiazolidinedione. Exp. Gerontol. 41: 590-599. https://doi.org/10.1016/j.exger.2006.04.005

• van Diepen JA, Vroegrijk IO, Berbée JF, Shoelson SE, Romijn JA, Havekes LM, Rensen PC, Voshol PJ (2011). Aspirin reduces hypertriglyceridemia by lowering VLDL-triglyceride production in mice fed a high-fat diet. Am. J. Physiol. Endocrinol. Metab. 301: E1099-E1107. https://doi.org/10.1152/ajpendo.00185.2011

• Vijay SK, Mishra M, Kumar H, Tripathi K (2009). Effect of pioglitazone and rosiglitazone on mediators of endothelial dysfunction, markers of angiogenesis and inflammatory cytokines in type-2 diabetes. Acta Diabetologica. 46: 27-33. https://doi.org/10.1007/s00592-008-0054-7

• Wilson RD, Islam MS (2012). Fructose-fed streptozotocin-injected rat: an alternative model for type 2 diabetes. Pharmacol. Rep. PR 64: 129-139. https://doi.org/10.1016/S1734-1140(12)70739-9

• Xue J, Hua Y, Xie M, Gu Z (2010). Aspirin inhibits MMP-9 mRNA expression and release via the PPARα/γ and COX-2/mPGES-1-mediated pathways in macrophages derived from THP-1 cells. Biomed. Pharmacother. 64: 118-123. https://doi.org/10.1016/j.biopha.2009.04.033

• Yang G, Zhou Z, Cen Y, Gui X, Zeng Q, Ao Y, Li Q, Wang S, Li J, Zhang A (2015). Death receptor and mitochondria-mediated hepatocyte apoptosis underlies liver dysfunction in rats exposed to organic pollutants from drinking water. Drug Design, Dev. Ther. 9: 4719. https://doi.org/10.2147/DDDT.S86843

• Yang Z-Y, Yuan C-X. (2018). IL-17A promotes the neuroinflammation and cognitive function in sevoflurane anesthetized aged rats via activation of NF-κB signaling pathway. BMC Anesthesiol. 18: 147-147. https://doi.org/10.1186/s12871-018-0607-4

• Yuan M, Konstantopoulos N, Lee J, Hansen L, Li Z-W, Karin M, Shoelson SE (2001). Reversal of obesity-and diet-induced insulin resistance with salicylates or targeted disruption of Ikkβ. Sci. 293: 1673-1677. https://doi.org/10.1126/science.1061620

• Zhou Y, Peng H, Liu Z, Zhang KK, Jendrusch C, Drake M, Hao Y, Xie L (2019). Sex-associated preventive effects of low-dose aspirin on obesity and non-alcoholic fatty liver disease in mouse offspring with over-nutrition in utero. Lab. Invest. 99: 244. https://doi.org/10.1038/s41374-018-0144-2