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mTOR pathway
Journal of Functional Foods 66 (2020) 103828
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Polysaccharide from the seeds of Plantago asiatica L. alleviates nonylphenol induced reproductive system injury of male rats via PI3K/Akt/mTOR pathway ⁎
Fenfen Lia, Danfei Huanga, , Weiyu Yanga, Xiaozhen Liub, Shaoping Niea, Mingyong Xiea,
T
⁎
a
State Key Laboratory of Food Science and Technology, China-Canada Joint Lab of Food Science and Technology (Nanchang), Nanchang University, 235 Nanjing East Road, Nanchang 330047, China b Engineering Research Center of Heath Food Design & Nutrition Regulation, School of Chemical Engineering and Energy Technology, Dongguan University of Technology, Dongguan 523808, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Reproductive system injury Nonylphenol Polysaccharide Plantago asiatica L. Apoptosiss and autophagy Oxidative stress
The preventive role of polysaccharide from the seeds of Plantago asiatica L. (PLCP) against reproductive system injury induced by nonylphenol (NP) was investigated. Male Sprague-Dawley rats were exposed orally to NP 1 h after PLCP intragastric administration continuously for 45 days. The results showed that NP induced significant decrease in relative weight of testes and epididymis, changed testicular histomorphology, and disrupted hormone secretions. Moreover, oxidative stress occurred after NP exposure by overproduction of Malondialdehyde, lipid peroxidation, inducible nitric oxide synthase, and low activities of superoxide dismutase and catalase. Furthermore, the induction of apoptosis and disturbance of PI3K/Akt/mTOR pathway by NP were also proved. Interestingly, PLCP alleviated NP toxicity by improving antioxidant enzymes, recovering hormone secretions, inhibiting apoptosis and autophagy, which involves PI3K/Akt/mTOR pathway. These findings provide a good direction that PLCP may be a good candidate to help body defend against toxic substance, particularly endocrine disrupting chemicals induced reproductive toxicity.
1. Introduction Recently, the increasing incidence of reproductive disorders in the human male has attracted researchers’ attention. A large amount of evidence has proved that the trend of decreasing male fertility might be as a result of exposure to environmental toxicants (Mathur & D'Cruz, 2011). Epidemiological researches have indicated that environmental contaminants are contributed to the poor sperm quality (Swan et al., 2003) and high risk of cryptorchidism (Weidner, Møller, Jensen, & Skakkebæk, 1998). The spermatogenesis damages are even permanent (Eaton et al., 1986). Nonylphenol (NP), a degraded product of nonylphenol ethoxylate, is persistent and toxic. It has been widely used in the manufacture of nonionic surfactants, emulsifiers, stabilizer polymers wetting, pesticides and lubricants (Soares, Guieysse, Jefferson, Cartmell, & Lester, 2008). Because of its lipophilic characteristic, NP could be accumulated in adipose tissue and then enter the food chain. Once it enters into the body, it will induce many sublethal effects especially on the reproductive system due to its high accumulation (Abd-Elkareem, Khalil,
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& Sayed, 2018; Noorimotlagh, Haghighi, Ahmadimoghadam, & Rahim, 2017). It was reported that significant damage in the structure and function of testis, apoptosis and oxidative stress in epididymis occurred after exposure to NP orally for 90 days (Chen et al., 2014). The level of serum testosterone, sperm counts in the tail of the epididymis and the quantity and motility of daily gonepoiesis in the testes of F1 male rats dramatically decreased after NP exposure were also proved (Jie, Fan, & Liu, 2008). In addition, NP could also result in lower activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione reductase, and glutathione peroxidase (GPx) while higher lipid peroxidation in the epididymal sperm comparing to normal rats (Aly, Domènech, & Banjar, 2012; Chitra, Latchoumycandane, & Mathur, 2002). Moreover, some researchers found that excess NP exposure triggered apoptosis, autophagy and reproductive injury involving the PI3K/Akt/mTOR pathway both in vivo and in vitro (Huang et al., 2016). Recently, investigations on the alleviation of NP toxicity by phytochemicals were gradually increased, but most of them were merely limited to small molecules (Chitra & Mathur, 2004; Sayed & Soliman, 2017; Sayed, Mohamed, Ismail, Abdel-Mageed, & Shoreit,
Corresponding authors. E-mail addresses: [email protected] (D. Huang), [email protected] (S. Nie), [email protected] (M. Xie).
https://doi.org/10.1016/j.jff.2020.103828 Received 14 May 2019; Received in revised form 13 October 2019; Accepted 28 January 2020 1756-4646/ © 2020 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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2.3. Animal experimental design
2016). Polysaccharides are macromolecules that widely distributed in nature. A variety of researches has found that polysaccharides possess diverse bioactivities including detoxification (Li, Lu, Zhang, Lu, & Liu, 2008; Zhu, Pan, Zheng, Cui, & Cao, 2012). Tang et al. (2017) have found that Lycium barbarum polysaccharide could alleviate the decrease in sperm count and repair testicular damage of zebrafish caused by NP. Plantago asiatica L. is a traditional medicinal and edible plant in China for its variously beneficial functions, such as diuresis, anti-inflammation and antiasthma (Marlett & Fischer, 2003). Recently, our group has obtained a polysaccharide from the seeds of Plantago asiatica L. (PLCP) through extraction, separation and purification, and determined its structural characterization. Our results showed that the polysaccharide was consisted of β-1,4-linked Xylp backbone with highly branched chains. β-T-linked Xylp, α-1,3-linked Araf and α-T-linked Araf were the main components among branched chains. α-T-linked GlcAp was also detected in the terminal residue (Yin, Lin, Li, et al., 2012; Yin, Li, Nie, & Xie, 2013; Yin, Lin, Nie, Cui, & Xie, 2012). In addition, we found that PLCP possesses high viscosity (Yin et al., 2015) and good laxative characteristics (Wu, Tian, Xie, & Li, 2007). In the current study, we aimed to investigate whether PLCP alleviate reproductive system injury of rats induced by NP and discuss its potential mechanisms.
After one week of adaptation, the rats were randomly divided into six groups (12 for each group) as follows: control, corn oil, NP, Low + NP, Middle + NP and High + NP. The last three groups were given by gavage once a day with different doses of PLCP (75, 150, 300 mg/kg body weight) while the first three groups were administrated orally with distilled water. One hour later after the gavage of PLCP, the last four groups were given oral administration of NP (50 mg/ kg body weight), meanwhile control group and corn oil group were given distilled water and corn oil respectively. This process lasted for 45 consecutive days. Then all rats were weighted and sacrificed under isoflurane anesthesia followed by blood collection from retro-orbital plexus. The testis and epididymis were dissected and weighted in order to calculate the organ/body weight ratios for each rat. After weighing, one of the two testes and epididymis of each rat was immediately placed in 10% neutral formalin for histological analysis, while the other one stored at −80 °C for subsequent determination. 2.4. Histopathologic analysis The testis and epididymis fixed in formalin were dehydrated in graded ethanol, and cleared in xylene followed by embedding in paraffin blocks. Then they were cut into 4 µm thickness and stained with hematoxylin and eosin (H/E) according to standard procedures. Sections were visualized by using an Aperio LV1 Digital Pathology Slide Scanner (Leica, IL, US) under a magnification of 400.
2. Materials and methods 2.1. Chemicals and reagents
2.5. Determination of sex hormone
Nonylphenol (CAS No. 84852-15-3) was purchased from Aladdin (Shanghai, China) and dissolved in corn oil (Jinlongyu, China). The seeds of Plantago asiatica L. were obtained from Ji’an (Jiang Xi, China) and PLCP was prepared as described in precious literature (Yin, Nie, Zhou, Wan, & Xie, 2010). The main component of PLCP was mainly composed of arabinose (32.2%) and xylose (61.1%) with a molecular weight of 1894 kDa (Yin, Lin, Li, et al., 2012; Yin et al., 2010). Rat testosterone, estrogen, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) ELISA kits were purchased from Shanghai Westang Biotech CO. Ltd (Shanghai, China). Assay kits of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione (GSH), Malondialdehyde (MDA), lipid peroxidation (LPO), inducible nitric oxide synthase (iNOS), and total nitric oxide synthase (tNOS) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Whole protein extraction kit was obtained from KeyGEN BioTECH (Jiangsu, China). BCA protein assay kit was purchased from Beyotime Biotechnology (Shanghai, China). The primary antibodies of Cleaved-Caspase 3, PI3K p85, LC3A/B, Cytochrome C, Bax, p-Akt (Ser473), Akt, p-TSC2, TSC2, p-mTOR, and mTOR were purchased from Cell Signaling Technology (Beverly, MA, USA). Fas antibody was obtained from Abcam (Cambridge, MA) and Bcl-2 from Boster (Wuhan, China). The primary antibody of β-actin and secondary antibodies of horseradish-labeled goat anti-mouse IgG and horseradishlabeled goat anti-rabbit IgG were purchased from ZSGB-BIO (Beijing, China).
Serum was obtained by centrifuging at 3000 rpm at 4 °C for 15 min after blood coagulation. Then the levels of testosterone, estrogen, FSH and LH in serum were determined directly by using commercially available ELISA kits. Whereas for the testis, the supernatant was obtained by homogenizing testis in pre-cooling normal saline and centrifuging, which was used to determine the level of testosterone in testis. 2.6. Assay of antioxidant biomarkers and LPO expressions 100 mg testicular and epididymis tissue was weighed and put into 2 mL eppendorf tube containing pre-cooling normal saline (1:9, mg/ µL). Subsequently they were homogenized by using high speed tissue grinder at 60 Hz for 60 s and centrifuged at 2500 rpm for 10 min. The supernatant was used to determine the activities of CAT, SOD, GPx, GSH, iNOS, tNOS and the contents of MDA, LPO, according to the manufacturer’ directions respectively. The whole processes were conducted at 4 °C. 2.7. Western blotting analysis The protein of testis was obtained by using whole protein extraction kit and quantified by BCA protein assay kit. Then the lysate protein was mixed with 5 × SDS-PAGE protein loading butter (4:1, v/v) and denaturalized at 100 °C for 5 min. The protein was separated by 10% SDSPAGE for Cleaved-Caspased 3, PI3K, p-Akt, Akt, Fas and 7% SDS-PAGE for p-TSC2, TSC2, p-mTOR, mTOR, and then transferred to PVDF membrane. The membrane was incubated with primary antibodies against Cleaved-Caspase-3, PI3K, LC3A/B, Cytochrome C, Bax, Bcl-2, pAkt, Akt, Fas, p-TSC2, TSC2, p-mTOR and mTOR (1:1000 dilution) overnight at 4 °C after blocking for 1 h with 5% BSA at room temperature. Then washing off unbound primary antibody and incubated with horseradish peroxidase-conjugated secondary antibody (1:10000 dilution) for 1 h at room temperature. The intensity images were obtained by reacting with BeyoECL Plus and exposure in BIO-RAD Chemi XRS+ gel image system after washing off unbound secondary antibody.
2.2. Animals Male Sprague-Dawley (SD) rats (150-170 g) were purchased from Hunan SJA Lab Animal Ltd. (certificate number SCXK (xiang) 2011003, Hunan, China). All rats were housed in a 12 h light-dark cycle with a relative humidity of 55 ± 10% and a temperature of 23 ± 2 °C for one week of adaptation. The rats were fed with freedom. All procedures were followed NIH rules for the care and use of laboratory animals and permitted by the Nanchang University Animal Ethic Review Committee (license No: SYXK(gan)2015-0001).
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Table 1 Relative weight of testes and epididymis after exposure to NP and treatment with PLCP. Organ
Relative weight (mg/g) Control
testes epididymis
Corn Oil b
8.38 ± 0.38 1.54 ± 0.14b
NP b
8.41 ± 0.35 1.53 ± 0.15b
Low + NP a
7.89 ± 0.39 1.37 ± 0.10a
Middle + NP b
8.52 ± 0.61 1.51 ± 0.19b
b
8.67 ± 0.51 1.53 ± 0.19b
High + NP 8.56 ± 0.60b 1.53 ± 0.17b
Data are represented as mean ± SD. Values with different superscript letter in the same row for each parameter are significantly different (p < 0.05).
2.8. Statistical analysis Data were presented as mean ± SD. IBM SPSS statistics 19.0 software was used for analyzing the data. All values were analyzed by means of univariate analysis of variance (ANOVA) using a one-way factorial design. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Effect of PLCP on relative organ weights of reproductive system The relative organ weights of testes and epididymis in each group are presented in Table 1. There was no significant difference in relative organ weights between control and corn oil group. However, the relative weights of both testes and epididymis in NP treatment group were significantly lower than that of control group (p < 0.05). The relative organ weights in PLCP administration group showed a significant difference compared to the NP group but no to control group. 3.2. Effect of PLCP on histological morphology of testes and epididymis Histological morphology changes of testes and epididymis were investigated by using H & E staining. A cross section of testis from control group and corn oil group showed normal seminiferous tubules with all stages of spermatogenic cells closely arranged and lots of mature sperm concentrated in the middle lumen of seminiferous tubules (Fig. 1Aa & Ab). However, histopathological changes were found in the testes of NP exposed rats. Spermatogenic cells in the seminiferous tubules were disorderly arranged with reduced cell layer and enlarged lumen in the NP group compared to control group (Fig. 1Ac). When the rats were pretreated with PLCP, the testicular damage caused by NP was significantly alleviated. While no obvious histological pathological changes were observed in the epididymis among the groups (Fig. 1B). The epididymides of all groups were filled with abundant sperms.
Fig. 1. Histological morphology of testes (A) and epididymis (B) stained with H & E after exposure to NP and treatment with PLCP (400×). a: Control; b: Corn oil; c: NP; d: Low + NP; e: Middle + NP; f: High + NP Arrow indicates spermatogenic cells at all levels.
administration could dramatically increase the activities of these antioxidant enzymes (Table 2).
3.3. Effect of PLCP on hormone expressions
3.5. Effect of PLCP on MDA, LPO contents and iNOS and tNOS activities
The results of hormone expressions in serum and testis were shown in Fig. 4. Testosterone expressions in serum and testis were both remarkably diminished in the NP group compared to the control group and corn oil group (p < 0.05). However, PLCP administration could restore the level of testosterone (Fig. 2A & E). Higher expressions of estrogen, FSH and LH occurred in the NP group compared to the control group and corn oil group, while PLCP treatment significantly decreased the over-expressions caused by NP (Fig. 2B, C & D).
Treatment with 50 mg/kg of NP induced significant increase in testis MDA, LPO contents and iNOS and tNOS activities, as well as in epididymis MDA, LPO contents and iNOS activities compared to control group and corn oil group (Table 3) (p < 0.05). Interestingly, PLCP administration showed dramatically lower activities both in testis and epididymis than those of the NP group.
3.4. Effect of PLCP on antioxidant enzymes and GSH activity
Apoptosis and autophagy associated proteins were determined by using western blotting. Protein expression of Cleaved-Caspase-3 was dramatically higher in the NP group compared to the control group and corn oil group (Fig. 3A & F). In addition, significant increase of Fas (Fig. 3B & G) and Cytochrome C (Fig. 3C & H) expression and decrease of Bcl-2/Bax ratio (Fig. 3D & I) were observed in the NP group. Furthermore, the ratio of LC3-Ⅱ/Ⅰ was dramatically high in the NP group (Fig. 3E & J). However, PLCP administration could greatly inhibit the higher expression of apoptosis and autophagy associated proteins
3.6. Effect of PLCP on NP induced apoptosis and autophagy in testis
As shown in Table 2, CAT and SOD activities in testis were both significantly lower in the NP group when compared to control group and corn oil group (p < 0.05). However, compared with NP group, pre-treatment with PLCP resulted in increased CAT and SOD activities. Nevertheless, there was no significant difference in testicular GPx and GSH activities among the groups. With regard to epididymis, NP exposure also attenuated CAT, SOD and GPx activities, while PLCP 3
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Fig. 2. Sex hormone levels in serum and testis after exposure to NP and treatment with PLCP. A: testosterone expression in serum. B: estrogen expression in serum. C: FSH expression in serum. D: LH expression in serum. E: testosterone expression in testis. Data are represented as mean ± SD. Values with different superscript letter are significantly different (p < 0.05).
were down-regulated in the NP group, whilst that of Akt, p-TSC2, TSC2 and mTOR was up-regulated (Fig. 4). Moreover, PLCP treatment showed great increase in PI3K, p-Akt and p-mTOR expressions and decrease in Akt, p-TSC2, TSC2 and mTOR compared to the NP group.
caused by NP.
3.7. Effect of PLCP on PI3K/Akt/mTOR signaling pathway The protein expressions of PI3K, p-Akt and p-mTOR in the testis 4
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Table 2 Antioxidant enzymes activities in testis and epididymis after exposure to NP and treatment with PLCP. Groups
Testis
Epididymis
CAT U/mg prot Control Corn Oil NP Low + NP Middle + NP High + NP
25.04 23.63 13.12 24.16 18.81 21.81
± ± ± ± ± ±
3.29c 2.96c 3.38a 4.98c 4.60b 1.93bc
SOD U/g prot 117.30 122.54 106.69 117.12 117.77 106.46
± ± ± ± ± ±
GPx U/mg prot 7.02b 13.62b 10.56a 11.32b 7.23b 5.25a
25.61 25.82 25.62 24.78 24.44 23.15
± ± ± ± ± ±
GSH nmol/mg prot
2.58a 3.12a 4.02a 4.45a 2.52a 1.60a
18.14 19.17 16.84 20.39 16.88 18.27
± ± ± ± ± ±
6.15a 3.46a 3.56a 5.08a 7.14a 4.76a
CAT U/mg prot
SOD U/g prot
31.48 ± 3.26a 33.61 ± 3.56a 5.07 ± 2.24c 6.50 ± 2.00c 27.91 ± 4.18b 27.20 ± 3.22b
109.33 109.15 101.65 108.05 110.96 103.28
GPx U/mg prot
± ± ± ± ± ±
5.72a 6.15a 5.45c 8.75ab 5.81a 2.67bc
25.56 31.72 13.89 27.75 31.38 36.37
± ± ± ± ± ±
3.99c 4.35ab 6.09d 4.52bc 3.99ab 6.54ab
Data are represented as mean ± SD. Values with different superscript letter in the same column for each parameter are significantly different (p < 0.05).
4. Discussion
no obvious histological pathological changes were observed. There were several possible reasons for this phenomenon. One is that the testicular spermatogenic epithelium is more sensitive to exogenous toxicants than the epididymal epithelium. Sertoli cells and some spermatogenic cells in developmental stages are very sensitive to poisons, thus morphological changes are inclined to occur after injury. Another reason is that not like testicular spermatogenic epithelium, the epididymis epithelium does not possess active cell characteristics of proliferation and differentiation. Once spermatogenic epithelium of testis is damaged or interfered, its spermatogenesis is affected and easy to observe. NP has estrogenic effect as a kind of endocrine disrupting chemicals. It potentially interferes with hormonal regulations and homeostasis, therefore alters the health of aquatic organisms and human (Bonefeldjørgensen, Long, Hofmeister, & Vinggaard, 2007). Sexual hormones such as testosterone, FSH and LH are crucial endocrine factors in controlling testicular functions (Ramaswamy & Weinbauer, 2014). In the current study, the level of testosterone in the serum and testis showed a sharp decline, while estrogen, FSH and LH expression in serum exhibited a marked increase in NP treated group. Previous studies found that P450c17 is a pivotal enzyme in testosterone synthesis, which is inhibited by NP (Laurenzana et al., 2002). Thus the decrease of testosterone expression level in NP group might due to the inhibition of P450c17. FSH and LH was regulated by testosterone through a negative feedback mechanism. So the decrease of testosterone resulted in the increase of FSH and LH secretion in NP group. Our results were consistent with that of Han et al. (2004), who reported low testosterone while high FSH and LH expressions in the serum after NP exposure. When NP exposed rats were treated with PLCP, all these hormones trended to normal levels, indicating PLCP could alleviate the inhibition of P450c17 caused by NP. Oxidative stress results from the imbalance between reactive oxygen species (ROS) and antioxidants in the body. Many researches had demonstrated that NP triggered oxidative stress in testis and epididymis (Aly et al., 2012; Duan et al., 2017). In this study, the expressions of MDA, LPO, iNOS in the testis and epididymis were increased respectively in NP group, while reduced by pretreatment of PLCP. High levels
NP is known as an environmental endocrine disruptor. Human and wildlife are inevitably exposed to NP as it is distributed diffusely in surface water, groundwater, soils, atmosphere, and human foodstuffs (Vidal-Liñán, Bellas, Salgueiro-González, Muniategui, & Beiras, 2015). A large amount of studies confirmed that NP exposure disrupts the functions of reproductive system (Lerner, Björnsson, & Mccormick, 2007). In the present study, we demonstrated the protective effects of PLCP pretreatment on NP exposure induced reproductive system injury of male rats. Previous research found the viscosity of chitosan may be contributed to the promotion of dioxins excretion when comparing the effects of oral various dietary fibers on the fecal excretion rate of dioxins (Kohda, Inoue, Noda, & Saito, 2012). Viscous fibres exert metabolic functions that appear to have their effect by reducing absorption rate of small molecular substances from the small intestine (Hu, Nie, & Xie, 2018; Jenkins, Marchie, Augustin, Ros, & Kendall, 2004; Yin, Wang, Lin, Xie, & Nie, 2016). Therefore, the protection effects of PLCP may be attributed to its high viscosity, which may slow NP absorption. The results showed that the relative weight of the testes and epididymis was dramatically reduced after 50 mg/kg body weight NP exposure orally, indicating the inhibition of spermatogenesis occurred (Aly et al., 2012). Interestingly, all different dose of PLCP remitted the decrease of relative weight caused by NP. We proposed that PLCP could restore spermatogenesis, then increase daily sperm production and number of sperms. To confirm our speculation, we further observed the morphological changes in testis and epididymis. The histopathologic evaluation of testis showed there was a normal structure in corn oil group, indicating that corn oil gavage treatment does not affect the spermatogenic function of the testis. However, severe disorganization, enlarged lumen and loss of maturation of germ cells were observed in NP treated group, while PLCP pretreatment group presented a normal morphology. This means that the testicular tissue showed obvious spermatogenic dysfunction after NP exposure and PLCP pretreatment alleviate the injury, which confirmed the speculation about the changes of relative weight. In terms of epididymis, all groups were filled with abundant sperms that
Table 3 MDA, LPO, iNOS and tNOS contents in testis and epididymis after exposure to NP and treatment with PLCP. Groups
Testis
Epididymis
MDA nmol/mg prot Control Corn Oil NP Low + NP Middle + NP High + NP
0.80 0.82 1.02 0.85 0.79 0.72
± ± ± ± ± ±
0.08ab 0.06ab 0.14c 0.09b 0.13ab 0.09a
LPO nmol/mg prot 0.14 0.16 0.25 0.13 0.17 0.18
± ± ± ± ± ±
0.07ab 0.03ab 0.06c 0.05a 0.02ab 0.04b
iNOS U/mg prot 0.42 0.43 0.64 0.54 0.42 0.53
± ± ± ± ± ±
tNOS U/mg prot
0.08a 0.09a 0.09c 0.10b 0.08a 0.06b
0.86 0.85 0.96 0.88 0.82 0.85
± ± ± ± ± ±
0.06a 0.07a 0.11b 0.08a 0.14a 0.07a
MDA nmol/mg prot
LPO nmol/mg prot
9.51 ± 3.24c 10.76 ± 3.51c 14.64 ± 5.21a 11.05 ± 4.08bc 10.54 ± 2.86c 14.34 ± 3.71ab
5.52 5.74 9.22 6.48 5.83 7.51
± ± ± ± ± ±
0.95b 2.18b 2.42a 2.66b 1.67b 1.55ab
iNOS U/mg prot 5.71 5.72 6.86 6.90 5.75 5.80
± ± ± ± ± ±
0.81b 0.80b 1.19a 1.47a 0.69b 0.62b
Data are represented as mean ± SD. Values with different superscript letter in the same column for each parameter are significantly different (p < 0.05). 5
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of polyunsaturated fatty acids which are necessary for fluidity in the sperm cell membranes, make the cell more susceptible to ROS attack and lipid peroxides generation (De & Gagnon, 2015). Over expressions of MDA and LPO lead to decreased fluidity of membrane and motility of sperm, which eventually reduce the fertilizing capacity. Moreover, iNOS, enhanced under oxidative stress (Kuo, Abe, & Schroeder, 1997), could produce high levels of NO, and then inhibit testosterone production in Leyding cells. Fortunately, there were various antioxidant enzymes and free radical scavengers to resist oxidative stress. The present work found that the activities of antioxidant enzymes including SOD and CAT decreased significantly in NP group. The reduction of these enzymes and increase of MDA, LPO, iNOS reflects that NP destructed the balanced antioxidant system. The previous works have demonstrated that PLCP possesses antioxidant properties (Ye, Hu, & Dai, 2011). In this study, we found PLCP could improve the activities of SOD and CAT in the testis and epididymis while decrease the expressions of MDA, LPO, iNOS, indicating that PLCP could alter the balance between ROS and antioxidant enzymes. Nevertheless, there was no clear doseeffect relationship from the results, which may be due to the narrow dose range of PLCP we chose and/or individual differences of rats. Thus, further studies need to be done to explore the dose-response relationship of PLCP. A large amount of evidence has proved that there is a direct connection between testicular oxidative stress and degenerated seminiferous tubules (Wang, 2010). Apoptosis is considered to play a pivotal role in the induction of testicular toxicity. Caspases, a family of cysteine proteases, are the central regulators of apoptosis. Once apoptosis signaling occurred, the caspases cleave and activate downstream effector caspases, which in turn execute apoptosis. Notably, the expression of Cleaved-Caspase-3 was activated by NP administration, indicating that NP induced the oxidative damage evoked apoptosis and disrupted the spermatogenic and androgenic process. Bcl-2 family proteins (Bcl-2, Bax) and Cytochrome C are also of importance in cell apoptosis (Sun et al., 2012). Mitochondrial membrane could be damaged and depolarized under high levels of ROS, which leads to Bcl-2 destabilization. Bcl-2, an anti-apoptotic protein, is performed by prevent bax gene expression and free radical generation. While Bax is a pro-apoptotic protein that promote the release of Cytochrome C from mitochondria and eventually activated Caspase 3. Therefore, the low ratio of Bcl-2 to Bax and high level of Cytochrome C illustrated NP induced the apoptosis mediated by mitochondria. Moreover, Fas is the surface molecule that mediates the mitochondria mediated apoptotic pathway (Farley et al., 2006). PLCP decreased the high Fas, Cytochrome C and CleavedCaspase-3 expressions, as well as increased the ratio of Bcl-2/Bax caused by NP exposure, indicating PLCP may inhibit NP induced apoptosis via mitochondria mediated pathway. Apoptosis is complexly connected with autophagy (Roos, Thomas, & Kaina, 2015). In addition, ROS and cellular redox may have a direct effect on autophagy (Levine & Kroemer, 2008). LC3 is currently recognized as an autophagy marker with two types (LC3-Ⅰ, LC3-Ⅱ) (Cheng et al., 2018). LC3-Ⅰ would transform into LC3-Ⅱ once autophagy occurred. Thus the ratio of LC3-Ⅱ to LC3-Ⅰ could estimate the level of autophagy. Our result showed that NP exacerbated the ratio of LC3-Ⅱ/Ⅰ, which indicated that NP induced autophagy. PI3K/Akt/mTOR mediated autophagy is very important for spermatogenesis, and considered to stimulate translation of messages. However, ROS inhibited some phosphatases of the Akt pathway and affected regulatory proteins related to autophagy (Yoshimori & Noda, 2008). It was found mTOR expression was a central signaling node to maintain spermatogenesis and germ cell development (Boyer et al., 2016). Akt and TSC1/2 are the upstream inputs of mTOR. Increasing recent evidence indicated that NP suppressed the p-Akt, TSC2 and mTOR activity in testis (Dutton, 1980; Huang et al., 2016). The results in the present research may resulted from severe oxidative stress, which were in accordance with previous studies. Interestingly, administration of PLCP restored these changes (Fig. 5). All these results suggested that PLCP could restore NP induced
Fig. 3. Apoptosis in testis after exposure to NP and treatment with PLCP. A, B, C, D, E: Cleaved-Caspase-3, Fas, Cytochrome C, Bcl-2 and Bax, LC3-Ⅰ/Ⅱ expressions in testicular tissue. F, G, H, I, J: Quantitative analysis of CleavedCaspase-3, Fas Cytochrome C expressions, Bcl-2/Bax, ratio, LC3-Ⅱ/Ⅰ ratio in testicular tissue. Data are represented as mean ± SD. Values with different superscript letter are significantly different (p < 0.05).
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Fig. 3. (continued)
These findings provide a good direction that PLCP may be a good candidate to help the body defend against toxic substance, particularly endocrine disrupting chemicals induced reproductive toxicity.
apoptosis and autophagy, which involves PI3K/Akt/mTOR pathway. 5. Conclusion
6. Ethics Statements
In summary, our study investigated the effects of PLCP on NP induced reproductive system injury in SD rats. NP disrupted the prooxidant/antioxidant balance by altering the activities of antioxidant enzymes and increasing the levels of MDA, LPO, iNOS, broke the balance of hormones secretion, induced apoptosis of testis, disordered PI3K/Akt/mTOR pathway. PLCP is shown to alleviate the testicular toxicity caused by NP through its antioxidant activity and inhibition of apoptosis and autophagy, which involves PI3K/Akt/mTOR pathway.
All animals used in this experiment were approved by the institutional animal care and use committee, Nanchang University (license No: SYXK(gan)2015-0001). Additionally, these mice were cared for in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication 85-23, 1996). 7
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Fig. 5. Potential signaling pathway of PLCP on NP induced toxicity. Pretreatment of PLCP restored the high expression of Fas and oxidative stress caused by NP exposure, which resulted in Caspase-3 activation. In addition, PLCP increased PI3K expression, promoted phosphorylation of Akt and mTOR, thus inhibited the transformation of LC3-Ⅰ into LC3-Ⅱ. Furthermore, Akt activation inhibited Caspase-3 activation by inhibiting Bax transfer onto mitochondria and activating Bcl-2 that inhibited the release of Cytochrome C from mitochondria. All these results suggested that PLCP could restore NP induced apoptosis and autophagy, which involves PI3K/Akt/mTOR pathway.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This study was supported by the National Natural Science Foundation of China (Grant number 31860471), the National Key R&D Program of China (Grant number 2017YFF0207800) and the Postgraduate Innovation and Special Funds for Nanchang University (Grant number cx2016007). References Abd-Elkareem, M., Khalil, N. S. A., & Sayed, A. H. (2018). Hepatotoxic responses of 4nonylphenol on African catfish (Clarias gariepinus): Antixoidant and histochemical biomarkers. Fish Physiology & Biochemistry, 44(3), 969–981. Aly, H. A. A., Domènech, Ò., & Banjar, Z. M. (2012). Effect of nonylphenol on male reproduction: Analysis of rat epididymal biochemical markers and antioxidant defense enzymes. Toxicology and Applied Pharmacology, 261(2), 134–141. Bonefeldjørgensen, E. C., Long, M., Hofmeister, M. V., & Vinggaard, A. M. (2007). Endocrine-disrupting potential of bisphenol A, bisphenol A dimethacrylate, 4-nnonylphenol, and 4-n-octylphenol in vitro: New data and a brief review. Environmental Health Perspectives, 115(Suppl 1), 69–76. Boyer, A., Girard, M., Thimmanahalli, D. S., Levasseur, A., Céleste, C., Paquet, M., ... Boerboom, D. (2016). mTOR Regulates Gap Junction Alpha-1 Protein Trafficking in Sertoli Cells and Is Required for the Maintenance of Spermatogenesis in Mice1. Biology of Reproduction, 95(1), 1–11. Chen, L. W., Qing, W. A., Ling, C. X., Guo, Y., Yao, L., Ou, C. Y., ... Lin, T. H. (2014). 90d exposure to nonylphenol has adverse effects on the spermatogenesis and sperm maturation of adult male rats. Biomedical and Environmental Sciences, 27(11), 109–907. Cheng, B., Lu, J., Li, T., Meng, Z., Liu, M., Sun, M., & Guan, S. (2018). 1,3-Dichloro-2Propanol inhibits autophagy via P53/AMPK/mTOR pathway in HepG2 cells. Food and Chemical Toxicology, 122, 143–150. Chitra, K., Latchoumycandane, C., & Mathur, P. (2002). Effect of nonylphenol on the antioxidant system in epididymal sperm of rats. Archives of Toxicology, 76(9), 545–551. Chitra, K. C., & Mathur, P. P. (2004). Vitamin E prevents nonylphenol-induced oxidative stress in testis of rats. Indian Journal of Experimental Biology, 42(2), 220–223. De, L. E., & Gagnon, C. (2015). Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes. Journal of Andrology, 13(5), 368–378. Duan, P., Hu, C., Butler, H. J., Quan, C., Chen, W., Huang, W., ... Shi, Y. (2017). 4Nonylphenol induces disruption of spermatogenesis associated with oxidative stress-
Fig. 4. PI3K/Akt/mTOR signaling pathway in testis after exposure to NP and treatment with PLCP. A: Protein expressions in testicular tissue. B: Quantitative analysis of PI3K, p-Akt, and Akt expressions in testicular tissue. C: Quantitative analysis of p-TSC2, TSC2, p-mTOR and mTOR expressions in testicular tissue. Data are represented as mean ± SD. Values with different superscript letter are significantly different (p < 0.05).
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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Polysaccharide from the seeds of Plantago asiatica L. alleviates nonylphenol induced reproductive system injury of male rats via PI3K/Akt/mTOR pathway ⁎
Fenfen Lia, Danfei Huanga, , Weiyu Yanga, Xiaozhen Liub, Shaoping Niea, Mingyong Xiea,
T
⁎
a
State Key Laboratory of Food Science and Technology, China-Canada Joint Lab of Food Science and Technology (Nanchang), Nanchang University, 235 Nanjing East Road, Nanchang 330047, China b Engineering Research Center of Heath Food Design & Nutrition Regulation, School of Chemical Engineering and Energy Technology, Dongguan University of Technology, Dongguan 523808, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Reproductive system injury Nonylphenol Polysaccharide Plantago asiatica L. Apoptosiss and autophagy Oxidative stress
The preventive role of polysaccharide from the seeds of Plantago asiatica L. (PLCP) against reproductive system injury induced by nonylphenol (NP) was investigated. Male Sprague-Dawley rats were exposed orally to NP 1 h after PLCP intragastric administration continuously for 45 days. The results showed that NP induced significant decrease in relative weight of testes and epididymis, changed testicular histomorphology, and disrupted hormone secretions. Moreover, oxidative stress occurred after NP exposure by overproduction of Malondialdehyde, lipid peroxidation, inducible nitric oxide synthase, and low activities of superoxide dismutase and catalase. Furthermore, the induction of apoptosis and disturbance of PI3K/Akt/mTOR pathway by NP were also proved. Interestingly, PLCP alleviated NP toxicity by improving antioxidant enzymes, recovering hormone secretions, inhibiting apoptosis and autophagy, which involves PI3K/Akt/mTOR pathway. These findings provide a good direction that PLCP may be a good candidate to help body defend against toxic substance, particularly endocrine disrupting chemicals induced reproductive toxicity.
1. Introduction Recently, the increasing incidence of reproductive disorders in the human male has attracted researchers’ attention. A large amount of evidence has proved that the trend of decreasing male fertility might be as a result of exposure to environmental toxicants (Mathur & D'Cruz, 2011). Epidemiological researches have indicated that environmental contaminants are contributed to the poor sperm quality (Swan et al., 2003) and high risk of cryptorchidism (Weidner, Møller, Jensen, & Skakkebæk, 1998). The spermatogenesis damages are even permanent (Eaton et al., 1986). Nonylphenol (NP), a degraded product of nonylphenol ethoxylate, is persistent and toxic. It has been widely used in the manufacture of nonionic surfactants, emulsifiers, stabilizer polymers wetting, pesticides and lubricants (Soares, Guieysse, Jefferson, Cartmell, & Lester, 2008). Because of its lipophilic characteristic, NP could be accumulated in adipose tissue and then enter the food chain. Once it enters into the body, it will induce many sublethal effects especially on the reproductive system due to its high accumulation (Abd-Elkareem, Khalil,
⁎
& Sayed, 2018; Noorimotlagh, Haghighi, Ahmadimoghadam, & Rahim, 2017). It was reported that significant damage in the structure and function of testis, apoptosis and oxidative stress in epididymis occurred after exposure to NP orally for 90 days (Chen et al., 2014). The level of serum testosterone, sperm counts in the tail of the epididymis and the quantity and motility of daily gonepoiesis in the testes of F1 male rats dramatically decreased after NP exposure were also proved (Jie, Fan, & Liu, 2008). In addition, NP could also result in lower activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione reductase, and glutathione peroxidase (GPx) while higher lipid peroxidation in the epididymal sperm comparing to normal rats (Aly, Domènech, & Banjar, 2012; Chitra, Latchoumycandane, & Mathur, 2002). Moreover, some researchers found that excess NP exposure triggered apoptosis, autophagy and reproductive injury involving the PI3K/Akt/mTOR pathway both in vivo and in vitro (Huang et al., 2016). Recently, investigations on the alleviation of NP toxicity by phytochemicals were gradually increased, but most of them were merely limited to small molecules (Chitra & Mathur, 2004; Sayed & Soliman, 2017; Sayed, Mohamed, Ismail, Abdel-Mageed, & Shoreit,
Corresponding authors. E-mail addresses: [email protected] (D. Huang), [email protected] (S. Nie), [email protected] (M. Xie).
https://doi.org/10.1016/j.jff.2020.103828 Received 14 May 2019; Received in revised form 13 October 2019; Accepted 28 January 2020 1756-4646/ © 2020 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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2.3. Animal experimental design
2016). Polysaccharides are macromolecules that widely distributed in nature. A variety of researches has found that polysaccharides possess diverse bioactivities including detoxification (Li, Lu, Zhang, Lu, & Liu, 2008; Zhu, Pan, Zheng, Cui, & Cao, 2012). Tang et al. (2017) have found that Lycium barbarum polysaccharide could alleviate the decrease in sperm count and repair testicular damage of zebrafish caused by NP. Plantago asiatica L. is a traditional medicinal and edible plant in China for its variously beneficial functions, such as diuresis, anti-inflammation and antiasthma (Marlett & Fischer, 2003). Recently, our group has obtained a polysaccharide from the seeds of Plantago asiatica L. (PLCP) through extraction, separation and purification, and determined its structural characterization. Our results showed that the polysaccharide was consisted of β-1,4-linked Xylp backbone with highly branched chains. β-T-linked Xylp, α-1,3-linked Araf and α-T-linked Araf were the main components among branched chains. α-T-linked GlcAp was also detected in the terminal residue (Yin, Lin, Li, et al., 2012; Yin, Li, Nie, & Xie, 2013; Yin, Lin, Nie, Cui, & Xie, 2012). In addition, we found that PLCP possesses high viscosity (Yin et al., 2015) and good laxative characteristics (Wu, Tian, Xie, & Li, 2007). In the current study, we aimed to investigate whether PLCP alleviate reproductive system injury of rats induced by NP and discuss its potential mechanisms.
After one week of adaptation, the rats were randomly divided into six groups (12 for each group) as follows: control, corn oil, NP, Low + NP, Middle + NP and High + NP. The last three groups were given by gavage once a day with different doses of PLCP (75, 150, 300 mg/kg body weight) while the first three groups were administrated orally with distilled water. One hour later after the gavage of PLCP, the last four groups were given oral administration of NP (50 mg/ kg body weight), meanwhile control group and corn oil group were given distilled water and corn oil respectively. This process lasted for 45 consecutive days. Then all rats were weighted and sacrificed under isoflurane anesthesia followed by blood collection from retro-orbital plexus. The testis and epididymis were dissected and weighted in order to calculate the organ/body weight ratios for each rat. After weighing, one of the two testes and epididymis of each rat was immediately placed in 10% neutral formalin for histological analysis, while the other one stored at −80 °C for subsequent determination. 2.4. Histopathologic analysis The testis and epididymis fixed in formalin were dehydrated in graded ethanol, and cleared in xylene followed by embedding in paraffin blocks. Then they were cut into 4 µm thickness and stained with hematoxylin and eosin (H/E) according to standard procedures. Sections were visualized by using an Aperio LV1 Digital Pathology Slide Scanner (Leica, IL, US) under a magnification of 400.
2. Materials and methods 2.1. Chemicals and reagents
2.5. Determination of sex hormone
Nonylphenol (CAS No. 84852-15-3) was purchased from Aladdin (Shanghai, China) and dissolved in corn oil (Jinlongyu, China). The seeds of Plantago asiatica L. were obtained from Ji’an (Jiang Xi, China) and PLCP was prepared as described in precious literature (Yin, Nie, Zhou, Wan, & Xie, 2010). The main component of PLCP was mainly composed of arabinose (32.2%) and xylose (61.1%) with a molecular weight of 1894 kDa (Yin, Lin, Li, et al., 2012; Yin et al., 2010). Rat testosterone, estrogen, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) ELISA kits were purchased from Shanghai Westang Biotech CO. Ltd (Shanghai, China). Assay kits of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione (GSH), Malondialdehyde (MDA), lipid peroxidation (LPO), inducible nitric oxide synthase (iNOS), and total nitric oxide synthase (tNOS) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Whole protein extraction kit was obtained from KeyGEN BioTECH (Jiangsu, China). BCA protein assay kit was purchased from Beyotime Biotechnology (Shanghai, China). The primary antibodies of Cleaved-Caspase 3, PI3K p85, LC3A/B, Cytochrome C, Bax, p-Akt (Ser473), Akt, p-TSC2, TSC2, p-mTOR, and mTOR were purchased from Cell Signaling Technology (Beverly, MA, USA). Fas antibody was obtained from Abcam (Cambridge, MA) and Bcl-2 from Boster (Wuhan, China). The primary antibody of β-actin and secondary antibodies of horseradish-labeled goat anti-mouse IgG and horseradishlabeled goat anti-rabbit IgG were purchased from ZSGB-BIO (Beijing, China).
Serum was obtained by centrifuging at 3000 rpm at 4 °C for 15 min after blood coagulation. Then the levels of testosterone, estrogen, FSH and LH in serum were determined directly by using commercially available ELISA kits. Whereas for the testis, the supernatant was obtained by homogenizing testis in pre-cooling normal saline and centrifuging, which was used to determine the level of testosterone in testis. 2.6. Assay of antioxidant biomarkers and LPO expressions 100 mg testicular and epididymis tissue was weighed and put into 2 mL eppendorf tube containing pre-cooling normal saline (1:9, mg/ µL). Subsequently they were homogenized by using high speed tissue grinder at 60 Hz for 60 s and centrifuged at 2500 rpm for 10 min. The supernatant was used to determine the activities of CAT, SOD, GPx, GSH, iNOS, tNOS and the contents of MDA, LPO, according to the manufacturer’ directions respectively. The whole processes were conducted at 4 °C. 2.7. Western blotting analysis The protein of testis was obtained by using whole protein extraction kit and quantified by BCA protein assay kit. Then the lysate protein was mixed with 5 × SDS-PAGE protein loading butter (4:1, v/v) and denaturalized at 100 °C for 5 min. The protein was separated by 10% SDSPAGE for Cleaved-Caspased 3, PI3K, p-Akt, Akt, Fas and 7% SDS-PAGE for p-TSC2, TSC2, p-mTOR, mTOR, and then transferred to PVDF membrane. The membrane was incubated with primary antibodies against Cleaved-Caspase-3, PI3K, LC3A/B, Cytochrome C, Bax, Bcl-2, pAkt, Akt, Fas, p-TSC2, TSC2, p-mTOR and mTOR (1:1000 dilution) overnight at 4 °C after blocking for 1 h with 5% BSA at room temperature. Then washing off unbound primary antibody and incubated with horseradish peroxidase-conjugated secondary antibody (1:10000 dilution) for 1 h at room temperature. The intensity images were obtained by reacting with BeyoECL Plus and exposure in BIO-RAD Chemi XRS+ gel image system after washing off unbound secondary antibody.
2.2. Animals Male Sprague-Dawley (SD) rats (150-170 g) were purchased from Hunan SJA Lab Animal Ltd. (certificate number SCXK (xiang) 2011003, Hunan, China). All rats were housed in a 12 h light-dark cycle with a relative humidity of 55 ± 10% and a temperature of 23 ± 2 °C for one week of adaptation. The rats were fed with freedom. All procedures were followed NIH rules for the care and use of laboratory animals and permitted by the Nanchang University Animal Ethic Review Committee (license No: SYXK(gan)2015-0001).
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Table 1 Relative weight of testes and epididymis after exposure to NP and treatment with PLCP. Organ
Relative weight (mg/g) Control
testes epididymis
Corn Oil b
8.38 ± 0.38 1.54 ± 0.14b
NP b
8.41 ± 0.35 1.53 ± 0.15b
Low + NP a
7.89 ± 0.39 1.37 ± 0.10a
Middle + NP b
8.52 ± 0.61 1.51 ± 0.19b
b
8.67 ± 0.51 1.53 ± 0.19b
High + NP 8.56 ± 0.60b 1.53 ± 0.17b
Data are represented as mean ± SD. Values with different superscript letter in the same row for each parameter are significantly different (p < 0.05).
2.8. Statistical analysis Data were presented as mean ± SD. IBM SPSS statistics 19.0 software was used for analyzing the data. All values were analyzed by means of univariate analysis of variance (ANOVA) using a one-way factorial design. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Effect of PLCP on relative organ weights of reproductive system The relative organ weights of testes and epididymis in each group are presented in Table 1. There was no significant difference in relative organ weights between control and corn oil group. However, the relative weights of both testes and epididymis in NP treatment group were significantly lower than that of control group (p < 0.05). The relative organ weights in PLCP administration group showed a significant difference compared to the NP group but no to control group. 3.2. Effect of PLCP on histological morphology of testes and epididymis Histological morphology changes of testes and epididymis were investigated by using H & E staining. A cross section of testis from control group and corn oil group showed normal seminiferous tubules with all stages of spermatogenic cells closely arranged and lots of mature sperm concentrated in the middle lumen of seminiferous tubules (Fig. 1Aa & Ab). However, histopathological changes were found in the testes of NP exposed rats. Spermatogenic cells in the seminiferous tubules were disorderly arranged with reduced cell layer and enlarged lumen in the NP group compared to control group (Fig. 1Ac). When the rats were pretreated with PLCP, the testicular damage caused by NP was significantly alleviated. While no obvious histological pathological changes were observed in the epididymis among the groups (Fig. 1B). The epididymides of all groups were filled with abundant sperms.
Fig. 1. Histological morphology of testes (A) and epididymis (B) stained with H & E after exposure to NP and treatment with PLCP (400×). a: Control; b: Corn oil; c: NP; d: Low + NP; e: Middle + NP; f: High + NP Arrow indicates spermatogenic cells at all levels.
administration could dramatically increase the activities of these antioxidant enzymes (Table 2).
3.3. Effect of PLCP on hormone expressions
3.5. Effect of PLCP on MDA, LPO contents and iNOS and tNOS activities
The results of hormone expressions in serum and testis were shown in Fig. 4. Testosterone expressions in serum and testis were both remarkably diminished in the NP group compared to the control group and corn oil group (p < 0.05). However, PLCP administration could restore the level of testosterone (Fig. 2A & E). Higher expressions of estrogen, FSH and LH occurred in the NP group compared to the control group and corn oil group, while PLCP treatment significantly decreased the over-expressions caused by NP (Fig. 2B, C & D).
Treatment with 50 mg/kg of NP induced significant increase in testis MDA, LPO contents and iNOS and tNOS activities, as well as in epididymis MDA, LPO contents and iNOS activities compared to control group and corn oil group (Table 3) (p < 0.05). Interestingly, PLCP administration showed dramatically lower activities both in testis and epididymis than those of the NP group.
3.4. Effect of PLCP on antioxidant enzymes and GSH activity
Apoptosis and autophagy associated proteins were determined by using western blotting. Protein expression of Cleaved-Caspase-3 was dramatically higher in the NP group compared to the control group and corn oil group (Fig. 3A & F). In addition, significant increase of Fas (Fig. 3B & G) and Cytochrome C (Fig. 3C & H) expression and decrease of Bcl-2/Bax ratio (Fig. 3D & I) were observed in the NP group. Furthermore, the ratio of LC3-Ⅱ/Ⅰ was dramatically high in the NP group (Fig. 3E & J). However, PLCP administration could greatly inhibit the higher expression of apoptosis and autophagy associated proteins
3.6. Effect of PLCP on NP induced apoptosis and autophagy in testis
As shown in Table 2, CAT and SOD activities in testis were both significantly lower in the NP group when compared to control group and corn oil group (p < 0.05). However, compared with NP group, pre-treatment with PLCP resulted in increased CAT and SOD activities. Nevertheless, there was no significant difference in testicular GPx and GSH activities among the groups. With regard to epididymis, NP exposure also attenuated CAT, SOD and GPx activities, while PLCP 3
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Fig. 2. Sex hormone levels in serum and testis after exposure to NP and treatment with PLCP. A: testosterone expression in serum. B: estrogen expression in serum. C: FSH expression in serum. D: LH expression in serum. E: testosterone expression in testis. Data are represented as mean ± SD. Values with different superscript letter are significantly different (p < 0.05).
were down-regulated in the NP group, whilst that of Akt, p-TSC2, TSC2 and mTOR was up-regulated (Fig. 4). Moreover, PLCP treatment showed great increase in PI3K, p-Akt and p-mTOR expressions and decrease in Akt, p-TSC2, TSC2 and mTOR compared to the NP group.
caused by NP.
3.7. Effect of PLCP on PI3K/Akt/mTOR signaling pathway The protein expressions of PI3K, p-Akt and p-mTOR in the testis 4
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Table 2 Antioxidant enzymes activities in testis and epididymis after exposure to NP and treatment with PLCP. Groups
Testis
Epididymis
CAT U/mg prot Control Corn Oil NP Low + NP Middle + NP High + NP
25.04 23.63 13.12 24.16 18.81 21.81
± ± ± ± ± ±
3.29c 2.96c 3.38a 4.98c 4.60b 1.93bc
SOD U/g prot 117.30 122.54 106.69 117.12 117.77 106.46
± ± ± ± ± ±
GPx U/mg prot 7.02b 13.62b 10.56a 11.32b 7.23b 5.25a
25.61 25.82 25.62 24.78 24.44 23.15
± ± ± ± ± ±
GSH nmol/mg prot
2.58a 3.12a 4.02a 4.45a 2.52a 1.60a
18.14 19.17 16.84 20.39 16.88 18.27
± ± ± ± ± ±
6.15a 3.46a 3.56a 5.08a 7.14a 4.76a
CAT U/mg prot
SOD U/g prot
31.48 ± 3.26a 33.61 ± 3.56a 5.07 ± 2.24c 6.50 ± 2.00c 27.91 ± 4.18b 27.20 ± 3.22b
109.33 109.15 101.65 108.05 110.96 103.28
GPx U/mg prot
± ± ± ± ± ±
5.72a 6.15a 5.45c 8.75ab 5.81a 2.67bc
25.56 31.72 13.89 27.75 31.38 36.37
± ± ± ± ± ±
3.99c 4.35ab 6.09d 4.52bc 3.99ab 6.54ab
Data are represented as mean ± SD. Values with different superscript letter in the same column for each parameter are significantly different (p < 0.05).
4. Discussion
no obvious histological pathological changes were observed. There were several possible reasons for this phenomenon. One is that the testicular spermatogenic epithelium is more sensitive to exogenous toxicants than the epididymal epithelium. Sertoli cells and some spermatogenic cells in developmental stages are very sensitive to poisons, thus morphological changes are inclined to occur after injury. Another reason is that not like testicular spermatogenic epithelium, the epididymis epithelium does not possess active cell characteristics of proliferation and differentiation. Once spermatogenic epithelium of testis is damaged or interfered, its spermatogenesis is affected and easy to observe. NP has estrogenic effect as a kind of endocrine disrupting chemicals. It potentially interferes with hormonal regulations and homeostasis, therefore alters the health of aquatic organisms and human (Bonefeldjørgensen, Long, Hofmeister, & Vinggaard, 2007). Sexual hormones such as testosterone, FSH and LH are crucial endocrine factors in controlling testicular functions (Ramaswamy & Weinbauer, 2014). In the current study, the level of testosterone in the serum and testis showed a sharp decline, while estrogen, FSH and LH expression in serum exhibited a marked increase in NP treated group. Previous studies found that P450c17 is a pivotal enzyme in testosterone synthesis, which is inhibited by NP (Laurenzana et al., 2002). Thus the decrease of testosterone expression level in NP group might due to the inhibition of P450c17. FSH and LH was regulated by testosterone through a negative feedback mechanism. So the decrease of testosterone resulted in the increase of FSH and LH secretion in NP group. Our results were consistent with that of Han et al. (2004), who reported low testosterone while high FSH and LH expressions in the serum after NP exposure. When NP exposed rats were treated with PLCP, all these hormones trended to normal levels, indicating PLCP could alleviate the inhibition of P450c17 caused by NP. Oxidative stress results from the imbalance between reactive oxygen species (ROS) and antioxidants in the body. Many researches had demonstrated that NP triggered oxidative stress in testis and epididymis (Aly et al., 2012; Duan et al., 2017). In this study, the expressions of MDA, LPO, iNOS in the testis and epididymis were increased respectively in NP group, while reduced by pretreatment of PLCP. High levels
NP is known as an environmental endocrine disruptor. Human and wildlife are inevitably exposed to NP as it is distributed diffusely in surface water, groundwater, soils, atmosphere, and human foodstuffs (Vidal-Liñán, Bellas, Salgueiro-González, Muniategui, & Beiras, 2015). A large amount of studies confirmed that NP exposure disrupts the functions of reproductive system (Lerner, Björnsson, & Mccormick, 2007). In the present study, we demonstrated the protective effects of PLCP pretreatment on NP exposure induced reproductive system injury of male rats. Previous research found the viscosity of chitosan may be contributed to the promotion of dioxins excretion when comparing the effects of oral various dietary fibers on the fecal excretion rate of dioxins (Kohda, Inoue, Noda, & Saito, 2012). Viscous fibres exert metabolic functions that appear to have their effect by reducing absorption rate of small molecular substances from the small intestine (Hu, Nie, & Xie, 2018; Jenkins, Marchie, Augustin, Ros, & Kendall, 2004; Yin, Wang, Lin, Xie, & Nie, 2016). Therefore, the protection effects of PLCP may be attributed to its high viscosity, which may slow NP absorption. The results showed that the relative weight of the testes and epididymis was dramatically reduced after 50 mg/kg body weight NP exposure orally, indicating the inhibition of spermatogenesis occurred (Aly et al., 2012). Interestingly, all different dose of PLCP remitted the decrease of relative weight caused by NP. We proposed that PLCP could restore spermatogenesis, then increase daily sperm production and number of sperms. To confirm our speculation, we further observed the morphological changes in testis and epididymis. The histopathologic evaluation of testis showed there was a normal structure in corn oil group, indicating that corn oil gavage treatment does not affect the spermatogenic function of the testis. However, severe disorganization, enlarged lumen and loss of maturation of germ cells were observed in NP treated group, while PLCP pretreatment group presented a normal morphology. This means that the testicular tissue showed obvious spermatogenic dysfunction after NP exposure and PLCP pretreatment alleviate the injury, which confirmed the speculation about the changes of relative weight. In terms of epididymis, all groups were filled with abundant sperms that
Table 3 MDA, LPO, iNOS and tNOS contents in testis and epididymis after exposure to NP and treatment with PLCP. Groups
Testis
Epididymis
MDA nmol/mg prot Control Corn Oil NP Low + NP Middle + NP High + NP
0.80 0.82 1.02 0.85 0.79 0.72
± ± ± ± ± ±
0.08ab 0.06ab 0.14c 0.09b 0.13ab 0.09a
LPO nmol/mg prot 0.14 0.16 0.25 0.13 0.17 0.18
± ± ± ± ± ±
0.07ab 0.03ab 0.06c 0.05a 0.02ab 0.04b
iNOS U/mg prot 0.42 0.43 0.64 0.54 0.42 0.53
± ± ± ± ± ±
tNOS U/mg prot
0.08a 0.09a 0.09c 0.10b 0.08a 0.06b
0.86 0.85 0.96 0.88 0.82 0.85
± ± ± ± ± ±
0.06a 0.07a 0.11b 0.08a 0.14a 0.07a
MDA nmol/mg prot
LPO nmol/mg prot
9.51 ± 3.24c 10.76 ± 3.51c 14.64 ± 5.21a 11.05 ± 4.08bc 10.54 ± 2.86c 14.34 ± 3.71ab
5.52 5.74 9.22 6.48 5.83 7.51
± ± ± ± ± ±
0.95b 2.18b 2.42a 2.66b 1.67b 1.55ab
iNOS U/mg prot 5.71 5.72 6.86 6.90 5.75 5.80
± ± ± ± ± ±
0.81b 0.80b 1.19a 1.47a 0.69b 0.62b
Data are represented as mean ± SD. Values with different superscript letter in the same column for each parameter are significantly different (p < 0.05). 5
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of polyunsaturated fatty acids which are necessary for fluidity in the sperm cell membranes, make the cell more susceptible to ROS attack and lipid peroxides generation (De & Gagnon, 2015). Over expressions of MDA and LPO lead to decreased fluidity of membrane and motility of sperm, which eventually reduce the fertilizing capacity. Moreover, iNOS, enhanced under oxidative stress (Kuo, Abe, & Schroeder, 1997), could produce high levels of NO, and then inhibit testosterone production in Leyding cells. Fortunately, there were various antioxidant enzymes and free radical scavengers to resist oxidative stress. The present work found that the activities of antioxidant enzymes including SOD and CAT decreased significantly in NP group. The reduction of these enzymes and increase of MDA, LPO, iNOS reflects that NP destructed the balanced antioxidant system. The previous works have demonstrated that PLCP possesses antioxidant properties (Ye, Hu, & Dai, 2011). In this study, we found PLCP could improve the activities of SOD and CAT in the testis and epididymis while decrease the expressions of MDA, LPO, iNOS, indicating that PLCP could alter the balance between ROS and antioxidant enzymes. Nevertheless, there was no clear doseeffect relationship from the results, which may be due to the narrow dose range of PLCP we chose and/or individual differences of rats. Thus, further studies need to be done to explore the dose-response relationship of PLCP. A large amount of evidence has proved that there is a direct connection between testicular oxidative stress and degenerated seminiferous tubules (Wang, 2010). Apoptosis is considered to play a pivotal role in the induction of testicular toxicity. Caspases, a family of cysteine proteases, are the central regulators of apoptosis. Once apoptosis signaling occurred, the caspases cleave and activate downstream effector caspases, which in turn execute apoptosis. Notably, the expression of Cleaved-Caspase-3 was activated by NP administration, indicating that NP induced the oxidative damage evoked apoptosis and disrupted the spermatogenic and androgenic process. Bcl-2 family proteins (Bcl-2, Bax) and Cytochrome C are also of importance in cell apoptosis (Sun et al., 2012). Mitochondrial membrane could be damaged and depolarized under high levels of ROS, which leads to Bcl-2 destabilization. Bcl-2, an anti-apoptotic protein, is performed by prevent bax gene expression and free radical generation. While Bax is a pro-apoptotic protein that promote the release of Cytochrome C from mitochondria and eventually activated Caspase 3. Therefore, the low ratio of Bcl-2 to Bax and high level of Cytochrome C illustrated NP induced the apoptosis mediated by mitochondria. Moreover, Fas is the surface molecule that mediates the mitochondria mediated apoptotic pathway (Farley et al., 2006). PLCP decreased the high Fas, Cytochrome C and CleavedCaspase-3 expressions, as well as increased the ratio of Bcl-2/Bax caused by NP exposure, indicating PLCP may inhibit NP induced apoptosis via mitochondria mediated pathway. Apoptosis is complexly connected with autophagy (Roos, Thomas, & Kaina, 2015). In addition, ROS and cellular redox may have a direct effect on autophagy (Levine & Kroemer, 2008). LC3 is currently recognized as an autophagy marker with two types (LC3-Ⅰ, LC3-Ⅱ) (Cheng et al., 2018). LC3-Ⅰ would transform into LC3-Ⅱ once autophagy occurred. Thus the ratio of LC3-Ⅱ to LC3-Ⅰ could estimate the level of autophagy. Our result showed that NP exacerbated the ratio of LC3-Ⅱ/Ⅰ, which indicated that NP induced autophagy. PI3K/Akt/mTOR mediated autophagy is very important for spermatogenesis, and considered to stimulate translation of messages. However, ROS inhibited some phosphatases of the Akt pathway and affected regulatory proteins related to autophagy (Yoshimori & Noda, 2008). It was found mTOR expression was a central signaling node to maintain spermatogenesis and germ cell development (Boyer et al., 2016). Akt and TSC1/2 are the upstream inputs of mTOR. Increasing recent evidence indicated that NP suppressed the p-Akt, TSC2 and mTOR activity in testis (Dutton, 1980; Huang et al., 2016). The results in the present research may resulted from severe oxidative stress, which were in accordance with previous studies. Interestingly, administration of PLCP restored these changes (Fig. 5). All these results suggested that PLCP could restore NP induced
Fig. 3. Apoptosis in testis after exposure to NP and treatment with PLCP. A, B, C, D, E: Cleaved-Caspase-3, Fas, Cytochrome C, Bcl-2 and Bax, LC3-Ⅰ/Ⅱ expressions in testicular tissue. F, G, H, I, J: Quantitative analysis of CleavedCaspase-3, Fas Cytochrome C expressions, Bcl-2/Bax, ratio, LC3-Ⅱ/Ⅰ ratio in testicular tissue. Data are represented as mean ± SD. Values with different superscript letter are significantly different (p < 0.05).
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Fig. 3. (continued)
These findings provide a good direction that PLCP may be a good candidate to help the body defend against toxic substance, particularly endocrine disrupting chemicals induced reproductive toxicity.
apoptosis and autophagy, which involves PI3K/Akt/mTOR pathway. 5. Conclusion
6. Ethics Statements
In summary, our study investigated the effects of PLCP on NP induced reproductive system injury in SD rats. NP disrupted the prooxidant/antioxidant balance by altering the activities of antioxidant enzymes and increasing the levels of MDA, LPO, iNOS, broke the balance of hormones secretion, induced apoptosis of testis, disordered PI3K/Akt/mTOR pathway. PLCP is shown to alleviate the testicular toxicity caused by NP through its antioxidant activity and inhibition of apoptosis and autophagy, which involves PI3K/Akt/mTOR pathway.
All animals used in this experiment were approved by the institutional animal care and use committee, Nanchang University (license No: SYXK(gan)2015-0001). Additionally, these mice were cared for in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication 85-23, 1996). 7
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Fig. 5. Potential signaling pathway of PLCP on NP induced toxicity. Pretreatment of PLCP restored the high expression of Fas and oxidative stress caused by NP exposure, which resulted in Caspase-3 activation. In addition, PLCP increased PI3K expression, promoted phosphorylation of Akt and mTOR, thus inhibited the transformation of LC3-Ⅰ into LC3-Ⅱ. Furthermore, Akt activation inhibited Caspase-3 activation by inhibiting Bax transfer onto mitochondria and activating Bcl-2 that inhibited the release of Cytochrome C from mitochondria. All these results suggested that PLCP could restore NP induced apoptosis and autophagy, which involves PI3K/Akt/mTOR pathway.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This study was supported by the National Natural Science Foundation of China (Grant number 31860471), the National Key R&D Program of China (Grant number 2017YFF0207800) and the Postgraduate Innovation and Special Funds for Nanchang University (Grant number cx2016007). References Abd-Elkareem, M., Khalil, N. S. A., & Sayed, A. H. (2018). Hepatotoxic responses of 4nonylphenol on African catfish (Clarias gariepinus): Antixoidant and histochemical biomarkers. Fish Physiology & Biochemistry, 44(3), 969–981. Aly, H. A. A., Domènech, Ò., & Banjar, Z. M. (2012). Effect of nonylphenol on male reproduction: Analysis of rat epididymal biochemical markers and antioxidant defense enzymes. Toxicology and Applied Pharmacology, 261(2), 134–141. Bonefeldjørgensen, E. C., Long, M., Hofmeister, M. V., & Vinggaard, A. M. (2007). Endocrine-disrupting potential of bisphenol A, bisphenol A dimethacrylate, 4-nnonylphenol, and 4-n-octylphenol in vitro: New data and a brief review. Environmental Health Perspectives, 115(Suppl 1), 69–76. Boyer, A., Girard, M., Thimmanahalli, D. S., Levasseur, A., Céleste, C., Paquet, M., ... Boerboom, D. (2016). mTOR Regulates Gap Junction Alpha-1 Protein Trafficking in Sertoli Cells and Is Required for the Maintenance of Spermatogenesis in Mice1. Biology of Reproduction, 95(1), 1–11. Chen, L. W., Qing, W. A., Ling, C. X., Guo, Y., Yao, L., Ou, C. Y., ... Lin, T. H. (2014). 90d exposure to nonylphenol has adverse effects on the spermatogenesis and sperm maturation of adult male rats. Biomedical and Environmental Sciences, 27(11), 109–907. Cheng, B., Lu, J., Li, T., Meng, Z., Liu, M., Sun, M., & Guan, S. (2018). 1,3-Dichloro-2Propanol inhibits autophagy via P53/AMPK/mTOR pathway in HepG2 cells. Food and Chemical Toxicology, 122, 143–150. Chitra, K., Latchoumycandane, C., & Mathur, P. (2002). Effect of nonylphenol on the antioxidant system in epididymal sperm of rats. Archives of Toxicology, 76(9), 545–551. Chitra, K. C., & Mathur, P. P. (2004). Vitamin E prevents nonylphenol-induced oxidative stress in testis of rats. Indian Journal of Experimental Biology, 42(2), 220–223. De, L. E., & Gagnon, C. (2015). Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes. Journal of Andrology, 13(5), 368–378. Duan, P., Hu, C., Butler, H. J., Quan, C., Chen, W., Huang, W., ... Shi, Y. (2017). 4Nonylphenol induces disruption of spermatogenesis associated with oxidative stress-
Fig. 4. PI3K/Akt/mTOR signaling pathway in testis after exposure to NP and treatment with PLCP. A: Protein expressions in testicular tissue. B: Quantitative analysis of PI3K, p-Akt, and Akt expressions in testicular tissue. C: Quantitative analysis of p-TSC2, TSC2, p-mTOR and mTOR expressions in testicular tissue. Data are represented as mean ± SD. Values with different superscript letter are significantly different (p < 0.05).
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