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Antioxidant and anti-caspase 3 effect of chitosan-Pinus merkusii extract nanoparticle against lead acetate-induced testicular toxicity in rat

Objective: To investigate the antioxidant and anti-caspase 3 effect of chitosan-Pinus merkusii extract nanoparticle on lead acetate-induced toxicity in rat testis. Methods: Chitosan-Pinus merkusii nanoparticles were identified by dynamic light scattering and scanning electron microscope. The male rats were divided into control group (rats were given with distilled water); lead acetate group [rats were injected with lead acetate 20 mg/kg body weight (BW) i..p.], and the treatment group (rats were given the chitosan-Pinus merkusii nanoparticle 150 mg; 300 mg; 600 mg/kg BW orally and were injected with lead acetate 20 mg/kg BW). The testis tissues were collected to evaluate the malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), histological evaluations of testis damage, and the caspase 3 mRNA expression was measured by reverse transcription-polymerase chain reaction. Results: The dynamic light scattering showed that the size of chitosan-Pinus merkusii nanoparticle was (530.2±38.2) nm. The scanning electron microscope images of the chitosan-Pinus merkusii nanoparticles showed an irregular shape, and the morphology surface showed the rough surface. The treatment with lead acetate resulted in significantly increasing MDA level and caspase 3 mRNA expression, and significantly decreasing level of SOD and GPx when compared with control group. The treatment with the chitosan-Pinus merkusii nanoparticle 600 mg/kg BW but not 150 and 300 mg/kg BW significantly decreased the MDA levels, caspase 3 mRNA expression, and also increased level of SOD and GPx when compared with lead acetate group. The lead acetate induced loss of the normal structure of testicular cells and necrosis, whereas treatment with chitosan-Pinus merkusii nanoparticle inhibited testicular cell necrosis. Conclusions: It can be concluded that chitosan-Pinus merkusii nanoparticle protects rat testis from oxidative damage and apoptosis caused by lead acetate, through increasing antioxidant and inhibiting caspase 3 expression.


Introduction
Lead is well known as one of most toxic heavy metals which can cause extensive environmental pollution and problems in human and animal health. The sources of lead toxicity mainly include industrial processes such as coal burning, lead-containing paint, lead smelting, lead-based pipes, grids, battery recycling, Objective: To investigate the antioxidant and anti-caspase 3 effect of chitosan-Pinus merkusii extract nanoparticle on lead acetate-induced toxicity in rat testis. Methods: Chitosan-Pinus merkusii nanoparticles were identified by dynamic light scattering and scanning electron microscope. The male rats were divided into control group (rats were given with distilled water); lead acetate group [rats were injected with lead acetate 20 mg/kg body weight (BW) i.p.], and the treatment group (rats were given the chitosan-Pinus merkusii nanoparticle 150 mg; 300 mg; 600 mg/kg BW orally and were injected with lead acetate 20 mg/kg BW). The testis tissues were collected to evaluate the malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), histological evaluations of testis damage, and the caspase 3 mRNA expression was measured by reverse transcription-polymerase chain reaction. Results: The dynamic light scattering showed that the size of chitosan-Pinus merkusii nanoparticle was (530.2±38.2) nm. The scanning electron microscope images of the chitosan-Pinus merkusii nanoparticles showed an irregular shape, and the morphology surface showed the rough surface. The treatment with lead acetate resulted in significantly increasing MDA level and caspase 3 mRNA expression, and significantly decreasing level of SOD and GPx when compared with control group. The treatment with the chitosan-Pinus merkusii nanoparticle 600 mg/kg BW but not 150 and 300 mg/kg BW significantly decreased the MDA levels, caspase 3 mRNA expression, and also increased level of SOD and GPx when compared with lead acetate group. The lead acetate induced loss of the normal structure of testicular cells and necrosis, whereas treatment with chitosan-Pinus merkusii nanoparticle inhibited testicular cell necrosis. Conclusions: It can be concluded that chitosan-Pinus merkusii nanoparticle protects rat testis from oxidative damage and apoptosis caused by lead acetate, through increasing antioxidant and inhibiting caspase 3 expression. and leaded gasoline [1]. The lead has non-biodegradable nature, so it is prolongedly present in the environment. Lead acetate, one of toxic heavy metals, accumulates in almost all the body tissues and causes multiple systemic toxicities to several organs such as liver [2], reproductive organs [3,4], heart [5] and kidneys [6].
The mechanism of lead caused-testicular toxicity can induce oxidative stress through increasing reactive oxygen species (ROS) [3,7]. The focus of research attention is oxidative stress which causes damage effects on the body and also on the death of cells. Oxidative stress will occur if ROS increases and antioxidants decrease in the cells. It has been reported that lead-induced overproduction of ROS such as superoxide ion, nitric oxide and hydroxyl radical could consequently lead to increasing lipid peroxidation, impairment of antioxidant enzymes such as decreasing superoxide dismutase (SOD), and glutathione peroxidase (GPx) [7,8]. In addition, free radicals are very reactive to lipids of membrane, protein, DNA and to be the main factors to cause testicular cell damage [9]. Many researchers have demonstrated that lead induced apoptosis in rat tissues including brain, testis, fibroblasts, liver and lung [8,10,11]. Xu et al [9] have demonstrated that lead can increase Bax/Bcl-2 ratio and caspase 3 activity which caused DNA damage and apoptosis in a cell.
Malondialdehyde (MDA) is a secondary product of lipid peroxidation, which can be used as a biomarker of lead acetateinduced testicular toxicity. The increase in MDA level showed decreased antioxidant activity to inhibit excessive free radicals formation [3,12].
Several antioxidants of natural product have protective effects against lead-induced oxidative damage and free radical generated formation in the body. Natural products are easily found as sources of antioxidants which contain a mixture of different chemical compounds to improve health and to treat diseases. The advantage of using natural products for the treatment of various diseases was low cost and little side effects compared with conventional drug [13].
This study has focused on natural products of chitosan-Pinus merkusii which have antioxidant activities to inhibit free radicalcaused testicular cell injury. It has been demonstrated that these antioxidant activities of Pinus plants were due to the phytochemicals possessed, including alkaloids, polyphenols, flavonoids, lignans, triterpenes, sterols, glycosides, triterpenoids, and saponins [16,17].
In recent years, synthesis of natural product nanoparticles is an interesting issue of the nanoscience and nanobiotechnology. The natural product nanoparticle has the opportunities to prevent and treat diseases in both human and animal. Nanoparticles vary in size but are generally ranging from 100 to 800 nm. Nanoparticle-based natural product has a potential future for enhancing the activity, drug stability and treatment efficacy, and overcoming problems associated with a pure natural product [24,25]. Chitosan, one of natural products, is widely used in pharmaceutical and biomedical applications [26,27].
Chitosan nanoparticles have been studied extensively by researchers for their controlled drug release properties and are used for both invitro and in vivo applications [28,29]. Chitosan nanoparticles are also non-toxic and have many biological activities such as antibacterial, antioxidant, antihyperlipidemic, anti-diabetic, anti-HIV, antiinflammatory, drug delivery, and immunoenhancing activities, which makes chitosan nanoparticle as an ideal delivery agent for application in medicine [30,31]. The aim of the research is to investigate the antioxidant and anti-caspase 3 activity of chitosan-Pinus merkusii extract nanoparticle against lead acetate-caused toxicity in rat testis.

Making process of chitosan-Pinus merkusii extract nanoparticles
Chitosan-Pinus merkusii extract nanoparticle was prepared using ionotropic gelation method [32]. The 0.2% (w/v) solution of chitosan was made in 0.1% (v/v) glacial acetic acid and then filtered.

Characterization of nanoparticles by scanning electron microscopy and dynamic light scattering
The surface morphological features such as particle size, shape and topography of the chitosan-Pinus merkusii extract nanoparticle were observed using scanning electron microscope.
Dynamic light scattering was done using Malvern instruments version 2.2. Average particle size of the chitosan-Pinus merkusii extract nanoparticle was determined.

Experimental animal
The male Wistar rats were used in this research with weighing about 200-250 g which were purchased from Gadjah Mada University, Yogyakarta, Indonesia. The rats were housed in plastic cages with a temperature at (24 ± 2) 曟 in an air-conditioned room and given feed and water ad libitum. This study was reviewed by

Experimental design
The 50 male rats were divided into: control group (rats were given with distilled water); lead acetate group [rats were injected i.p. with lead acetate 20 mg/kg body weight (BW)], and the treatment group (rats were given orally the chitosan-Pinus merkusii nanoparticle 150 mg/kg, 300 mg/kg, 600 mg/kg BW once a day for 11 days, and on 4th day rats were injected i.p. with lead acetate solution at a dose of 20 mg/kg BW one hour after the chitosan-Pinus merkusii extract nanoparticle). On day 11, the rats were sacrificed, and testis tissues were homogenized with 50 mM sodium phosphate buffer (pH 7.

Measurement of MDA
The supernatant of homogenate testis tissue was measured to determine MDA level by the thiobarbituric acid method, on the absorbance at 532 nm, which was expressed in nanomoles MDA/g tissue [5,33].

Measurement of antioxidant enzymes
The SOD activity was measured with a kit of SOD detection according to the manufacturer's instructions. The level of SOD was measured at 505 nm, which was expressed in U/mg protein [34].
The GPx activity was measured with a kit of GPx detection according to the manufacturer's instructions. The GPx was evaluated spectrophotometrically at 340 nm, which was expressed in U/mg protein [34].

RNA extraction and reverse transcription-polymerase chain reaction analysis
Total RNA was isolated from rat testis by using a solution of Trizol, re-suspended in 50 µL diethyl pyrocarbonate-treated water, and stored at 80 曟 . Synthesis of cDNAs for caspase 3 detection was made by using reverse transcription of Promega [11]. The used specific primers were 5-AGAGAACAATGGCGGATA-3 for forward and 5-CCAGTTGAGGGATGAAAG-3 for reverse, as control was carried out amplification of rat 毬 -actin mRNA in each sample using the primer: 5-GAGGCTCAGAGCAAGAGAGG-3 for forward, and

Histopathological examination of testis damage
The tissue of testis was fixed in a 10% solution of neutral buffered formalin, embedded in paraffin and stained with hematoxylin and eosin[3].

Statistical analysis
Data were shown as mean±standard deviation (mean±SD) and analyzed with one-way ANOVA. The statistical comparisons among the groups were carried out with an Fisher's least significant difference test (SPSS V. 17.0).

Characterization of chitosan-Pinus merkusii extract nanoparticles by scanning electron microscope
Scanning electron microscopy images of the chitosan-Pinus merkusii nanoparticles prepared using ionic gelation revealed that the nanoparticle surface showed the rough surface morphology and an irregular shape (Figure 1).

Characterization of chitosan-Pinus merkusii nanoparticles by dynamic light scattering
Dynamic light scattering showed that the average particle size of the chitosan-Pinus merkusii extract nanoparticles was (530.2±30.2) nm

Effects of chitosan-Pinus merkusii extract nanoparticle on expression of caspase 3 mRNA of lead acetate-induced testicular toxicity
The increasing of the expression of caspase 3 mRNA indicated cell apoptosis. Figure 3 showed

Effects of chitosan-Pinus merkusii extract nanoparticle on lead acetate-caused testis cell damage
Histological observations on the control group showed testicular cells were observable and they appeared with normal architecture of the testicular cells. The positive lead acetate group showed testicular cell damage (necrosis). In the rats, treated with chitosan-Pinus merkusii extract nanoparticle, the number and morphological integrity of testicular cells were preserved. Observations indicated that the testicular toxic effect of lead acetate was reduced by chitosan-Pinus merkusii extract nanoparticle (Figure 4).

Discussion
In recent years, the new advance of nanotechnology, synthesis of natural product nanoparticles is an interesting issue of the nanoscience and nanobiotechnology [24,28]. The natural product nanoparticle has the opportunities to prevent and treat diseases in both human and animal.
The chitosan has gained great attention in both the pharmaceutical and medical fields, which is used for herbal extract-loaded nanoparticles. Chitosan is a natural polysaccharide which is biodegradable and biocompatible [28,29]. In this study, we made Pinus merkusii extract encapsulated into chitosan nanoparticle using a cross-linking agent (sodium tripolyphosphate) by ionotropic gelation method, which has more advantages over Pinus merkusii extract.
The dynamic light scattering showed that the size of chitosan-Pinus  [3,14]. In the current study, lead acetate can decrease SOD and GPx activities in rat testis, whereas the administration of chitosan-

Pinus merkusii extract nanoparticle increases the levels of SOD
and GPx. The result shows that chitosan-Pinus merkusii extract nanoparticle acts as a scavenger for the oxygen-derived free radicals which can inhibit the formation of free radicals, thus protect lead acetate induced rat testis damage.
The decrease in lipid peroxidation due to chitosan-Pinus merkusii extract nanoparticle can increase the antioxidant defense system such as SOD and GPx which protect against free radical exposure.
The primary mechanism of action of chitosan-Pinus merkusii extract nanoparticle may involve the scavenging of free radicals which can inhibit free radical formation.
The lead toxicity can cause increase of oxidants product and decrease of the defense systems of antioxidant which may induce overproduction of ROS and damage of proteins, lipoproteins, lipids, mitochondria, and DNA, leading to testicular cell death through apoptosis or necrosis [9,10]. This study showed that in the lead acetate treatment group, the caspase 3 mRNA expression of testicular tissue was significantly increased compared with the control group. The caspase is an executioner caspase and as a hallmark of apoptosis [8].
Considering all these findings, it would be logical to conclude that lead acetate results in caspase-3 overexpression by triggering the apoptotic pathway. Dose dependent-manner of chitosan-Pinus merkusii extract nanoparticle decreased caspase 3 mRNA expression of testicular tissue in lead acetate treatment. The mitochondrial injury and changes in levels of apoptogenic proteins including Bcl-2, Bax, and caspase-3 may cause tissue cell apoptosis [9]. In lead toxicity, the expression levels of caspase-3 and Bax were significantly increased, while the level of Bcl-2 was significantly decreased. The caspase-3 mRNA expression plays an important role in the lead acetateinduced testis apoptotic cell.
In the present study, histopathological image of the lead acetate group showed destroyed seminiferous tubules, loss of spermatid and necrosis in rat testis compared with the control group. The testis damage (necrosis) was mild in the groups treated with chitosan-Pinus merkusii extract nanoparticle.
In conclusion, the present study indicates that the administration of chitosan-Pinus merkusii extract nanoparticle inhibits the effects of lead acetate-induced testicular toxicity, through increasing antioxidant and inhibiting caspase 3 mRNA expression. Furthermore, protective effects of chitosan-Pinus merkusii extract nanoparticle can be developed to treat patients with lead acetate-induced testicular toxicity.

Conflict of interest statement
The authors have no conflict of interest.