Effect of Pioglitazone on Expression of Interleukin-17A, Toll-like receptor 2, Inducible nitric oxide synthase 2 and Arginase 1 in Alzheimeric-model of rats Brain.

Introduction: Neuroinammation is actively involved in neurological disorders such as Alzheimer's disease and it is one of the primary and permanent features of Alzheimer's disease. PPARγ receptors are widely distributed in the brain and it is very important for learning. Agonists of this receptor such as pioglitazone can have benecial effects in the treatment of Alzheimer's disease. In this study, the effect of pioglitazone (PPARγ pathway agonist) on gene expression of Toll-like receptor 2, interleukin A, inducible nitric oxide synthase 2, and arginase-1 in the hippocampus Alzheimer's model of male rats was investigated. Methods: In this study, 35 male Wistar rats were randomly divided into ve groups of seven. The groups include control, Alzheimer's Sham group scopolamine (3 mg/ kg) +DMSO, and three groups receiving scopolamine+ pioglitazone with doses 5, 10, and 20 mg/kg for 12 days. All injections are intraperitoneal. In the end, hippocampal tissue was extracted to evaluate the expression of the desired genes by Real-time PCR and nally, SPSS software was used for statistical analysis of data. Results: It was observed that the gene expression of Toll-like receptor 2, interleukin A and inducible nitric oxide synthase 2 in the Sco+DMSO group increased compared to the control group (except in the arginase-1 gene) and also the gene expression of these factors in the groups receiving pioglitazone decreased compared to the Sco+DMSO (except in the arginase-1 gene). Conclusion: Pioglitazone can decrease the gene expression of Toll-like receptor 2, interleukin A and inducible nitric oxide synthase 2 and increase the gene expression of arginase-1 in the rat brain. So pioglitazone is useful for the reduction of inammation in the brain.


Background
The quality of life in Alzheimer's patients is low and often current therapy regimens are not su cient to treat the disease. More than 70% of dementia cases are Alzheimer's disease (AD), and in the 1990s, approximately 20.2 million cases of AD were reported. At the end of 2020, about 50 million people were reported to have AD and by the end of 2050, that number is expected to may reach 125 million [1].
Pathologically, AD is marked by the presence of amyloid β (Aβ) deposition and neuro brillary tangles, the cholinergic system dysfunction, increase of oxidative stress, activation of microglia and, steady neuroin ammation [1]. In ammatory markers detection in AD shows a potential role for neuroin ammation in the pathogenesis of this disease [2]. Neuroin ammation, the major checkpoint in AD's pathogenesis, is marked by an increase in proin ammatory cytokines, immune cells in ltration, and activated glial cells [3]. One of the main causes of neuroin ammation in AD is microglial activation [1] which is associated with the secretion of in ammatory cytokines and chemokines [4]. Microglia produces interleukin-17 (IL-17) protein [5] and this cell expresses toll-like receptors [6], inducible nitric oxide synthase (iNOS) [7]. Additionally, arginase 1 is another microglial activation marker that competes with iNOS for L-arginine and inhibits nitric oxide production [8].
Microglia is the rst line of defense versus pathogens and other brain tissue injuries [9]. Microglia express types of toll-like receptors such as toll-like receptor 2 (TLR2) in the brain [6] which is a member of patternrecognition receptors in the innate immune system [10]. Toll-like receptors play a critical role in AD [6] and the gene of TLR2 is highly expressed in AD in the brain areas containing plaque formation [9]. This receptor has a dual role in AD [9] including the clearance of Aβ accumulation [11,12], and induction of expression of pro-in ammatory molecules in response to Aβ [11]. Indeed, stimulation of TLR2 by activating microglia induces Aβ phagocytosis [9], and also, upregulating this receptor may be a treatment option in AD by regulating the phagocytic activity of microglia [13].
IL-17, a pro-in ammatory cytokine [14], acts as a central regulator of the proin ammatory reaction in the brain [5,15]. IL-17 mRNA expresses in astrocytes and oligodendrocytes and IL-17 protein produces in microglia [5]. In AD, IL-17 is produced from microglia and its expression is increased in the hippocampus, cerebrospinal uid, and serum after Aβ-42 peptide injection. But the exact mechanism of upregulation of IL-17 in the brains of AD patients is still unknown [16].
iNOS express in central nervous system cells such as neurons and microglia [7,17] and is involved in microglial activation [17]. In vivo and in vitro studies demonstrated that iNOS can induce neuronal apoptosis and also induce apoptosis in macrophages, astrocytes, and differentiated PC12 cells. Thus, neuronal damage in AD can reduce by inhibition of neuronal iNOS expression [18].
Hydrolysis of L-arginine to L-ornithine and urea is performed by arginase, which is a manganese metalloenzyme [19]. This enzyme is associated with central nervous system disorders including AD, multiple sclerosis, diabetic retinopathy, and retinopathy of prematurity. Two different genes encode two isoforms of arginase including arginase 1 and arginase 2 [20]. Brain-in ltrating macrophages on microglia express arginase 1 after central nervous system damage and these macrophages rely on arginase 1 expression to indicate a repair role to ameliorate injuries [21]. In an AD mouse model, IL-4 injection was induced arginase 1+ microglia expression, which remarkably reduced Aβ plaque accumulation under an IL-1β dependent neuroin ammation [22].
To treat hyperglycemia in type 2 diabetes is used pioglitazone which is an agonist of the peroxisome proliferator-activated receptor (PPAR) [23,24]. PPAR is a ligand-activated transcription factor [25] which have three types including α, δ/β, and γ [26]. These receptors have anti-in ammatory and antioxidant effects in brain damage or in neurodegenerative disorders including AD, ischemic stroke, and Parkinson's diseases [25]. PPARγ express at the neuronal level in the rat hippocampus and cortex [27] and could be a new therapeutic approach for the treatment of AD [28]. Additionally, these receptors with expression in microglia and astrocytes exert anti-in ammatory effects in the central nervous system [29]. The activation of these receptors in some cells, including macrophages, monocytes, and microglial cells reduces proin ammatory cytokine and iNOS expression [18]. It is reported that pioglitazone activates the PPAR-γ receptor and has a protective effect versus neuronal degeneration and cognitive de cits induced by binge alcohol with inhibition of pro-in ammatory cytokines in rats [30].
Pioglitazone, a thiazolidinedione derivative, inhibits Pam3CSK4-induced TLR2 expression in human monocytes and db/db mice which indicates anti-in ammatory effects of this drug [31]. Also, the level of in ammatory bone destruction decreased with pioglitazone and this drug decrease the circulating and local expression of IL-17 in rats with adjuvant-induced arthritis [32].
Scopolamine, a non-selective muscarinic cholinergic antagonist, impairs memory and cognitive functions in human and animals [33], and this drug increase pro-in ammatory cytokines, and in ammation [33,34].
Scopolamine may induce neuroin ammation by increasing the level of oxidative stress and proin ammatory cytokines in the hippocampus [35].
Although there are many studies on PPARγ and pioglitazone, there are few studies on the effect of pioglitazone on anti-in ammatory and pro-in ammatory factors in the AD model of rats. In the current study, we used a scopolamine-induced model of AD to show the treatment effect of pioglitazone on neuroin ammation and to provide a feasible theoretical basis and therapeutic target for AD treatment.

Animals
Adult male Wistar rats at 10 weeks of age were obtained from Pishro mehravaran azma pars (Babul, Iran), kept individually, and had access to free food and water. Rats acclimatized for 1 week in an animal room at 22±3 °C temperature with a 12/12-h light-dark cycle. All experimental methods were performed in accordance with the National Institutes of Health Guide (NIH Publications No. 8023, revised 1978) for the Care and Use of Laboratory Animals. This study was approved by the Ethics Committee of Golestan University of Medical Sciences, Gorgan, Iran (Ethics number: ir.goums.rec.1399.346).

Experimental design
A total of 35 Wistar rats were randomly divided into 5 groups; each group included 7 rats, as follows: Group 1: A control group that did not receive any drug.
Group 2 (Sco+DMSO group): A group that received scopolamine at a dose of 3 mg/kg/day [36, 37] for one day and DMSO injection for 12 consecutive days. Group 3 (Sco+Pio 5 mg/kg group): A group that received scopolamine at a dose of 3 mg/kg/day for one day and was treated with pioglitazone 5 mg/kg/day for 12 consecutive days.
Group 4 (Sco+Pio 10 mg/kg group): A group that received scopolamine at a dose of 3 mg/kg/day for one day and was treated with pioglitazone 10 mg/kg/day for 12 consecutive days.
Group 5 (Sco+Pio 20 mg/kg group): A group that received scopolamine at a dose of 3 mg/kg/day for one day and was treated with pioglitazone 20 mg/kg/day for 12 consecutive days.
All injections were intraperitoneal and pioglitazone was dissolved in DMSO.
48 hours after the last drug injection and after sacri ced rats, the hippocampus was rapidly isolated from rat brain tissue and used to study gene expression by real-time polymerase chain reaction. (RT-PCR) method.

RNA extraction and RT-PCR
The mRNA expression of IL-17A, TLR2, iNOS2, and arginase 1 were speci ed with the RT-PCR method. Total RNA was extracted from hippocampus tissue with the use of the Pars Tous RNA isolation Kit (Mashhad, Iran) according to the manufacturer's protocols. Their concentration was measured using

Statistical analysis
Data were reported as the mean ± SD. Data were statistically analyzed by ANOVA using the SPSS software version 16.0 (Armonk, USA). Differences among means of multiple groups were compared with Tukey's test, and P < 0.05 was statistically signi cant.

Effect of pioglitazone on the mRNA expression of IL-17A
Administration of scopolamine to rats increased signi cantly the mRNA expression of IL-17A in the Sco+DMSO group compared with the control group ( Fig. 1, P < 0.05). Injection of pioglitazone (5, 10, and 20 mg/kg) to rats decreased signi cantly the mRNA expression of IL-17A compared with the Sco+DMSO group ( Fig. 1, P < 0.05). Effective dose was 20 mg/kg pioglitazone.
Effect of pioglitazone on the mRNA expression of TLR2 mRNA expression of TLR2 increased signi cantly in the Sco+DMSO group compared with a control group (Fig. 2, P < 0.05). Treatment of rats with different doses of pioglitazone (5, 10, and 20 mg/kg) decreased mRNA expression of TLR2 (Fig. 2). But this reduction was not signi cant compared with the Sco+DMSO group (Fig. 2) and also this reduction in mRNA expression of TLR2 was signi cant compared with the control group (Fig. 2, P < 0.05).

Effect of pioglitazone on the mRNA expression of iNOS2
Scopolamine administration could increase the mRNA expression of iNOS2 in the Sco+DMSO group compared with the control group (Fig. 3, P < 0.05). Pioglitazone could decrease the mRNA expression of iNOS2 compared with the Sco+DMSO group (Fig. 3, P < 0.05). Also, this reduction in the mRNA expression of iNOS2 was signi cant compared with a control group (Fig. 3, P < 0.05).

Effect of pioglitazone on the mRNA expression of arginase 1
The mRNA expression of arginase 1 reduced signi cantly with scopolamine injection in Sco+DMSO group compared with control group (Fig. 4, P < 0.05). Pioglitazone Injection (5, 10 and 20 mg/kg) to rats increased signi cantly the mRNA expression of arginase 1 compared with Sco+DMSO group (Fig. 4, P < 0.05). Effective dose was 20 mg/kg pioglitazone.

Discussion
Our results demonstrated that the treatment of pioglitazone, which is one of the PPARγ agonists, led to a decrease in the mRNA expression of IL-17A, TLR2, and iNOS2 and an increase in the mRNA expression of arginase 1 in the rat hippocampus.
Important factors for induction/stimulation of AD are pro-in ammatory cytokines [39] such as IL-17A [40]. IL-17A, a signature cytokine of T helper 17 cells, is involved in in ammation [41] and may have an important role in AD pathogenesis [41,42]. Behairi et al. (2015) and Hamdan et al. (2014) showed baseline levels of IL-17A and the serum level IL-17A were signi cantly higher in AD patients compared with controls [40,42,43]. Also, Liu et al, (2021) [44] reported an increase in IL-17 in the hippocampus of APP/PS1 transgenic mice, and IL-17 induced an increase of Aβ levels in mice. We also found that mRNA expression IL-17A increased in the hippocampus of scopolamine-treated rats.
Intrahippocampal injection of Aβ42 into rats increased the expression of IL-17A in the hippocampus and also increased the cerebrospinal uid and the serum of IL-17A in AD occurrence and development [45]. One of the plasma biomarkers for diagnosis of AD and neocortical Aβ load is the plasma IL-17A levels [46,47]. These ndings suggest that blockage of IL-17 may reduce Aβ-induced neurotoxicity and cognitive impairment in AD [44].
Nishimori et al. found that in response to bacterial entry into the intestinal mucosa, they produced IL-17A by direct TLR2 activation [48]. It is reported that energy deprivation up-regulated TLR2 in neurons and this may play a role in the AD pathogenesis [49]. TLR signaling have both bene cial and deleterious in AD and TLR2 expression can increase in the temporal cortex in AD [50,51]. Also, TLR2 up-regulated markedly compared to the control in a transgenic mouse model of AD [52]. It is reported that TLR2 de ciency decreased Aβ deposition and neuroin ammation in a mouse model of AD [53]. Also, TLR2 de ciency decreased Aβ-induced in ammation, increased Aβ clearance [54] in cultured microglia, and ameliorated tauopathies in mice, which shows useful effects of TLR2 in AD [55,56]. Additionally, we found that mRNA expression TLR2 increased in the hippocampus of AD model rats.
iNOS expression can upregulate with IL17A, which is a proin ammatory cytokine [57]. iNOS appears to be a major initiator of Aβ deposition and AD progression, thus a treatment option for AD may be inhibition of iNOS [58]. It is reported that intracerebroventricular Aβ1-40 injection increase iNOS expression. The iNOS inhibition or the iNOS genetic deletion signi cantly ameliorates Aβ1-40-induced impairment of learning and memory. The expression of iNOS has been strongly linked with AD pathology [59]. As we showed in this study, injection of scopolamine into rats to model AD increased the mRNA expression iNOS in the rat hippocampus.
In humans, arginase 1 is a cytosolic enzyme expressed in various areas of the brain especially in hippocampal neurons [19]. Arginase 1 is a marker of in ammation and stable arginase 1 expression reduces tau pathology and in ammatory response [60]. This enzyme has an important role in AD pathogenesis [19] so Kan et al ., found that arginase 1 is extremely expressed in Aβ deposition areas [61]. It is reported that arginase 1 insu ciency in lysozyme M increased Aβ deposition, activated microglia, and behavioral impairments [21]. In this research we showed that scopolamine could decrease the mRNA expression arginase 1 in the rat hippocampus.
In ammatory responses can modulate with PPARγ agonists in the brain [18]. The PPARγ agonist such as pioglitazone is FDA approved drug for diabetes treatment and has been used for treatment aims in animal models of central nervous system damage [62]. It is reported that the administration of pioglitazone improved in ammatory markers by reducing tumor necrotic factor α levels [63]. Also, it found that pioglitazone has an anti-in ammatory effect in the animal models of sub-acute and chronic in ammations [64]. As well as, regulating the PPARγ/NF-κB pathway by pioglitazone nanoliposomes reduce the in ammatory response in sepsis-induced acute lung injury [65]. Also, pioglitazone administration reduces in ammation following traumatic brain injury [62]. Also, we found that in this study, pioglitazone can decrease in ammation in rat hippocampus after scopolamine injection.
One of the ways in which pioglitazone (1 μM) can induce an anti-in ammatory effect is by inhibiting the Pam3CSK4-induced TLR2 expression in human monocytes and db/db mice [31]. Also, we found that pioglitazone (5, 10, and 20 mg/kg) can decrease the mRNA expression of TLR2 in scopolamine-treated rats.
Pioglitazone (20 mg/kg, p.o. daily for 14 days), can inhibit the iNOS expression in mice model of AD [66]. In another study, pioglitazone could inhibit iNOS production in lipopolysaccharide-stimulated microglial cells [67]. As we found in this study, treatment with pioglitazone at different doses (5, 10, and 20 mg/kg, for 12 days) decreased the mRNA expression iNOS in rat hippocampus. It is reported that pioglitazone could decrease iNOS expression and increase the arginase 1 expression [68,69]. An in vivo study showed that expression of arginase 1 can increase in aortic macrophages isolated from pioglitazone-nanoparticle-treated mice [70]. As well, we showed that treatment with pioglitazone increases the mRNA expression of arginase 1 in scopolamine-treated rats.
These studies further suggest a potential bene t of pioglitazone in managing neuroin ammation, with reduction of pre-in ammation genes expression and increase of anti-in ammation genes expression.

Conclusion
It can be concluded that Pioglitazone with decreases the gene expression of Toll-like receptor 2, interleukin A and inducible nitric oxide synthase 2 and with increases the gene expression of arginase-1 in the rat brain, can reduce the in ammation in the rat's brain. So, Pioglitazone is useful for the protection of the brain against in ammation.

Consent for publication
Not applicable.

Availability of data and materials
You can request data information via the email below: mejahanshahi@yahoo.com Effect of different doses of pioglitazone (5, 10 and 20 mg/kg) on the mRNA expression of IL-17A in rat hippocampus. * means P < 0.05. Figure 2