Taurine protects against As2O3-induced autophagy in livers of rat offsprings through PPARγ pathway

Chronic exposures to arsenic had been associated with metabolism diseases. Peroxisome proliferator-activated receptor gamma (PPARγ) was found in the liver, regulated metabolism. Here, we found that the expression of PPARγ was decreased, the generation of reactive oxygen species (ROS) and autophagy were increased after treatment with As2O3 in offsprings’ livers. Taurine (Tau), a sulfur-containing β–amino acid could reverse As2O3-inhibited PPARγ. Tau also inhibit the generation of ROS and autophagy. We also found that As2O3 caused autophagic cell death and ROS accelerated in HepG2 cells. Before incubation with As2O3, the cells were pretreated with PPARγ activator Rosiglitazone (RGS), we found that autophagy and ROS was inhibited in HepG2 cells, suggesting that inhibition of PPARγ contributed to As2O3-induced autophagy and the generation of ROS. After pretreatment with Tau, the level of PPARγ was improved and the autophagy and ROS was inhibited in As2O3-treated cells, suggesting that Tau could protect hepatocytes against As2O3 through modulating PPARγ pathway.

Scientific RepoRts | 6:27733 | DOI: 10.1038/srep27733 The peroxisome proliferator-activated receptors (PPARs) were a family of nuclear fatty acid receptors that regulated tissue specific cellular metabolism and differentiation 14 . In humans, there were three PPAR isoforms, α , β and γ , which respond to a discrete set of ligands 15 . Peroxisome proliferator-activated receptor-γ (PPARγ ) was a ligand-activated transcription factor of the nuclear hormone receptor super family. PPARγ was highly expressed in adipocytes and the liver 10,14,16 . These nuclear receptors when activated directly bind to DNA and regulated gene expression through transcriptional activation (also called master regulators of transcriptional cascades). PPARγ was involved in a variety of physiological processes, including the regulation of the metabolism, inflammation, cellular growth and differentiation 17,18 . Previous study had found that the disruption of PPARγ might result the activation of autophagy 19 . In our study, we explored the relationship between PPARγ and the autophagy induced by arsenic in offsprings' livers.
PPARγ was activated by the thiazolidinediones (TZDs), a group of drugs widely used in patients in the management of type 2 diabetes (T2D), as they regulate glucose metabolism, adiposeness, differentiation, and the expression of several genes including antioxidant defenses 14,20 . However, TZDs were once withdrawn from the market or had restricted prescription because they provoked adverse effects such as weight gain, edema, liver injury, cancer, and heart failure 21 . Taurine (Tau), a sulfur-containing β -amino acid, was a major free intracellular amino acid present in many tissues of human and animals 22 . It was mainly distributed in the brain, heart, kidney, and reproductive organs, with many physiological activities 23 . Biosynthesis and dietary intake were the only sources of Tau in our bodies. Tau was synthesized from methionine and cysteine mainly in the liver and the biosynthetic capacity of Tau was very low in human 24,25 . Its recognized metabolic function in liver was conjugation with bile acids, which was important for bile secretion and lipid digestion 24,26 . Recent studies reported that Tau supplementation could prevent diabetes mellitus, insulin resistance and its complications 9,24 . Tau was a key determinant of oxidative phosphorylation and it played an essential role in mitochondria. It was reported that mitochondrial ROS generation was enhanced in taurine-deficient heart 27,28 . Nrf2 played a critical role in defending against oxidative stress and inflammation 29 . When oxidative stress was stimulated, Nrf2 would bind to antioxidant response elements (ARE) 30 . Eventually, it led to the restoration of cellular redox homeostasis 31 . The thioredoxin (Trx) system also played the antioxidant role. The cysteine disulfide bridges in oxidized proteins were reduced by Trx system 32 . It was found that PPARγ could up-regulate Nrf2 33,34 . Our previous study had found that Tau protected against As 2 O 3 -induced autophagy in pancreas of rat offsprings through Nrf2/Trx pathway 35 . Therefore, we investigated whether Tau executed the action of protecting hepatocytes against As 2 O 3 through modulating PPARγ /Nrf2 pathway in this study.

Results
As 2 O 3 changed offsprings' livers morphology. Pathological analysis showed that the size of hepatocyte was decreased after treatment with As 2 O 3 (Fig. 1). We found that hepacyte edema occurred in As 2 O 3 -treated offsprings' livers. The size of As 2 O 3 -treatedhepatocyte was increased and the cellular edema was disappeared after pretreatment with Tau.
The effects of As 2 O 3 on offsprings' livers weight and body weight. The offsprings' liver weight gains were significantly increased after treatment with As 2 O 3 , but As 2 O 3 did not affect the body weights (Table 1). After pretreatment with Tau, the liver weights were significantly decreased (Table 1). These changes might be related with the cellular edema in offsprings' livers. As 2 O 3 caused autophagosome accumulated in livers. To investigate whether autophagy was involved in As 2 O 3 -induced toxicity, we utilized transmission electron microscopy to observe the ultrastructure in As 2 O 3 -treatedlivers. Morphological hallmark of autophagy was the presence of autophagosomes ( Fig. 2A). Quantification of the autophagosomes numbers per cell demonstrated that As 2 O 3 increased autophagosomes number significantly and in a dose-dependent manner. The number of autophagosomes in As 2 O 3 -treated rats was obviously decreased by pretreatment with Tau (Fig. 2B).

As 2 O 3 activated autophagy in offsprings' livers.
In this study, we evaluated the expression of autophagy biomarkers, LC3-II and P62, by Western blot analysis (Fig. 3A,C). The level of LC3-II was increased dramatically and the level of P62 was decreased in As 2 O 3 -treated livers as shown in Western blot assay. After pretreatment with Tau, the expression of LC3-II was decreased and the expression of P62 was increased dramatically in As 2 O 3 -treated cells (Fig. 3B,D), this gave us a clue to investigate the correspondence of autophagy and cytotoxicity at different As 2 O 3 concentration and whether Tau could protect against the autophagy induced by As 2 O 3 .     13 . To investigate the level of oxidative stress, we used MDA test kit to measure the level of MDA. We found that the level of MDA was increased significantly after treatment with As 2 O 3 . After pretreatment with Tau, the level of MDA was decreased in the livers of As 2 O 3 -treated rats (Fig. 7). It suggested that treatment with As 2 O 3 could accelerate the generation of ROS, and pretreatment with Tau could withstand the oxidative stress induced by As 2 O 3 .  36 . The cell viability of As 2 O 3 -treated HepG2 cells was increased by knockdown of Atg5 with Atg5 siRNA (Fig. 8). This suggested that autophagy was the major cause of the cell death induced by As 2 O 3 .
The inhibition of PPARγ contributed to As 2 O 3 -induced cell death. The treatment of HepG2 cells with 1 μ M As 2 O 3 for 24 h resulted in cell death in this study. The cell viability of As 2 O 3 -treated HepG2 cells was elevated significantly after pretreatment with RGS, at a concentration of 100 μ M (Fig. 9). It suggested that inhibition of PPARγ contributed to As 2 O 3 -induced cell death.

Tau reduced cell death of As 2 O 3 -treated HepG2 cells.
We found that the autophagy in livers was decreased significantly after pretreatment of Tau. To investigate the action of Tau in vitro, the As 2 O 3 -treated HepG2 cells were pretreated with Tau at a concentration of 20 μ M. The cell viability was elevated significantly (Fig. 10). It suggested that Tau reduced the cell death in As 2 O 3 -treated HepG2 cells.

RGS and Tau increased the expression of PPARγ in As 2 O 3 -treated HepG2 cells. The expression
of PPARγ level was decreased significantly after treatment with 1 μ M As 2 O 3 for 24 h in this study (Fig. 11A). After pretreatment of RGS and Tau respectively, the level of PPARγ was increased significantly in As 2 O 3 -treated HepG2 cells (Fig. 11B). The activation of PPARγ and Tau reduced the generation of ROS in As 2 O 3 -treated HepG2 cells. The generation of ROS was increased significantly after treatment with 1 μ M As 2 O 3 for 24 h in this study ( Fig. 12A). After pretreatment with RGS, the generation of ROS was decreased significantly in As 2 O 3 -treated HepG2 cells (Fig. 12A). It suggested that the activation of PPARγ could reduce the generation of ROS induced by As 2 O 3 . After pretreatment with Tau, the generation of ROS was also decreased significantly in As 2 O 3 -treated HepG2 cells (Fig. 12B). It suggested that Tau had the same effect with RGS. The up-regulation of PPARγ could withstand the generation of ROS induced by As 2 O 3 .   The activation of PPARγ and Tau accelerated the expression of Nrf2 and Trx proteins in As 2 O 3 -treated HepG2 cells. The expression of Nrf2 and Trx proteins were decreased significantly after treatment with 1 μ M As 2 O 3 for 24 h in this study (Fig. 13A). After pretreatment of RGS, the expression of Nrf2 and Trx were increased significantly in As 2 O 3 -treated HepG2 cells (Fig. 13B,C). It suggested that the activation of PPARγ could accelerate the expression of Nrf2 and Trx. After pretreatment with Tau respectively, the expression of Nrf2 and Trx were also increased significantly (Fig. 13B,C). It suggested that Tau had the effect of up-regulating PPARγ and Tau could withstand the reduction of Nrf2 and Trx induced by As 2 O 3 .  RGS and Tau reduced autophagy in As 2 O 3 -treated HepG2 cells. In the study, the level of LC3-II was increased significantly and the expression of P62 was decreased in As 2 O 3 -treated HepG2 cells (Fig. 14A,C). After pretreatment with RGS, we found that autophagy was inhibited in As 2 O 3 -treated cells (Fig. 14C,D), suggesting that inhibition of PPARγ contributed to As 2 O 3 -induced autophagy. The level of autophagy was decreased after pretreatment with Tau in As 2 O 3 -treated cells (Fig. 14C,D), suggesting that Tau could protect hepatocytes against As 2 O 3 through modulating PPARγ -autophagy pathway.

Discussion
Drinking water contaminated with inorganic arsenic was a major threat to human health with more than 100 million people worldwide exposed to levels that exceed the World Health Organization's (WHO) recommended limit of 10 μ g As/L 37 . Arsenic affected a multitude of biological systems, however, the mechanism by which arsenic elicits its toxic effects remains largely unknown. Numerous studies had been conducted to elucidate the molecular events associated with arsenic exposure, and resulting data suggested multiple mechanisms. Previous studies showed that mice might be less susceptible than human to arsenic toxicity, partly due to a faster metabolism and clearance of arsenic 38 . Therefore, it was necessary to use higher exposure concentration of arsenic than the environmentally relevant concentrations in mouse experiment 39 . The As 2 O 3 concentration used in this study ranged from 2 mg/kg BW to 8 mg/kg BW. Autophagy was a gatekeeping mechanism for stabilizing cell homeostasis. Studies had shown that autophagy played important roles in physiology and pathophysiology 40 . Our previous study found that arsenic accelerated autophagosome formation and caused autophagic cell death in INS-1 cells 13 . Autophagy had recently been shown to be involved in metabolism, triggering great interests in elucidating the underlying mechanism and testing the  feasibility of targeting autophagy for the prevention and treatment of obesity and related metabolic disorders 41 . In addition to LC3-II, P62 was another autophagy specific substrate. P62 was degraded in autolysosomes 42 . In this study, Western blot analysis revealed that As 2 O 3 accelerated the expression of LC3-II in offsprings' livers and the level of P62 was decreased significantly in HepG2 cells after treatment with As 2 O 3 . It was a reliable indicator for the activation of autophagy in As 2 O 3 -treated offsprings' livers. As 2 O 3 -induced autophagy was prevented by Tau, suggesting that As 2 O 3 -induced injury of livers might have the connection with autophagy and Tau might prevent livers from As 2 O 3 -induced autophagy.
PPARγ , which belonged to a family of nuclear hormone receptors that regulated the function and expression of complex gene networks, especially involved in cell proliferation and differentiation, glucose metabolism and homeostasis, insulin sensitivity and lipid metabolism 43 . It was reported that after treatment with RSG observably attenuated GCI-induced elevation of the LC3-II and Beclin-1 hippocampus of brain, these observations suggested that RSG might exert its inhibitory effect by inactivating neuronal autophagy through decreasing Beclin-1 and LC3-II, and thus achieve a cerebral protective effect 44 . Prenatal arsenic exposure had been associated with altered gene expression in human cord blood leukocytes and various target tissues in rodents 3 . We found that As 2 O 3 reduced the expression of PPARγ protein and inhibited the expression of PPARγ gene in offsprings' livers. It suggested that the reduction of PPARγ expression was involved in As 2 O 3 -induced autophagy in offsprings' livers. After pretreatment with RGS, the expression of PPARγ was increased dramatically and the level of autophagy was decreased in HepG2 cells. It suggested that the inhibition of PPARγ contributed to As 2 O 3 -induced autophagy.
Arsenic was able to induce the generation of ROS 12 . In our previous studies, we found that arsenic could induce autophagic cell death through ROS pathway. Nrf2 was the key factor of the oxidative stress reaction. The Trx system was composed of NADPH, TrxR and Trx. It was also a crucial line of defense against ROS through its  activity of disulfide reductase 45 . In this study, we found that after treatment with As 2 O 3 , the expression of Nrf2 and Trx were decreased obviously. The level of MDA was increased significantly in As 2 O 3 -treated livers. We used the activator of PPARγ to investigate whether the up-regulation of PPARγ could accelerate the expression of Nrf2 and Trx. We found that after pretreatment with RGS, the levels of Nrf2 and Trx were both increased obviously. It suggested that the inhibitor of PPARγ contributed to ROS-dependent autophagic injury in As 2 O 3 -treated offsprings' livers.
It was reported that Tau was a non-essential free amino acid, which was one of the chemical components abundantly present in Lyciumbarbarum and crosses the blood-retinal barrier. A dietary source of Tau was essential for those animals (e.g. cat and humans), which cannot synthesize sufficient Tau and where greater consumption of Tau was required, such as in diabetes. Furthermore, several studies had reported that Tau potentiates the effect of insulin and possibly affected the insulin receptor. In addition, one study had indicated that high concentrations (20 mM) of Tau are capable of enhancing the phosphatidylinositide 3 (PI3)-kinase/Akt signaling pathway responsible for insulin-mediated stimulation of glucose transporter activity and glucose uptake 9,16,26 . Tau recognized metabolic function in liver was conjugation with bile acids, which was important for bile secretion and lipid digestion 46 . Tau supplementation might prevent alterations or restore endocrine pancreatic mass in malnutrition, obesity, T1D and T2D 9 . In this study, Tau prevented the stimulation of autophagy and the inhibition of PPARγ in As 2 O 3 -treated offsprings' livers. Tau also reduced the generation of ROS by accelerating the expression of Nrf2 and Trx. The expression of PPARγ was improved dramatically and the level of autophagy was decreased after pretreatment of Tau in As 2 O 3 -treated HepG2 cells. After pretreatment with Tau, the generation of ROS was decreased obviously. It suggested that Tau could accelerate the expression of PPARγ and withstand the hepatotoxicity induced by As 2 O 3 .

Conclusion
In this study, we found that arsenic induced autophagy in offsprings' livers through ROS pathway. The reduction of PPARγ level played a crucial role in this process. Tau could reverse arsenic-inhibited PPARγ . Tau could inhibit the generation of ROS and autophagy in arsenic-treated offsprings' livers. We found that As 2 O 3 caused autophagic cell death through ROS pathway in HepG2 cells. The inhibition of PPARγ contributed to As 2 O 3 -induced autophagy and generation of ROS. Tau could protect hepatocytes against As 2 O 3 hepatotoxicity through modulating PPARγ -ROS-autophagy pathway.     Transmission electron microscopy. Parts of liver were fixed with 2% glutaraldehyde for 2 h, and then post fixed in 1% osmium tetroxide for 1 h. Dehydration was done in increasing concentration of ethanol followed by propylene oxide. While incubated in 70% ethanol, the pellet was stained en bloc with 1% uranyl acetate. Finally the pellet was embedded in Epon resin. Ultrathin sections were post stained with uranyl acetate and Reynold's lead citrate routinely. Electron micrographs were taken with JEM 1400 transmission electron microscope at 80 kV.

Ethics statement. The Animal Ethics
Western blot. At the end of the designated treatments, the tissues were washed twice with ice-cold PBS and completely lysed in lysis buffer of a protein extraction kit (Keygen Biotech). The tissues lysate was centrifuged at 14000 rpm for 15 min at 4 °C, and the supernatant containing the total protein was collected. The concentration of total protein was quantified using BCA method. SDS-polyacrylamide gel electrophoresis was performed, and the proteins were then transferred to a nitrocellulose membrane. After blocking with 10% non-fat milk, the blots were incubated with primary antibodies against LC3-II (Sigma), PPARγ (Proteintech), Nrf2 (Proteintech), Trx (Abcam) or internal control β -actin (Santa). Blots were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma) followed by detection with a SuperSignal West Pico Kit (Thermo Scientific) according to the manufacturer's instructions. The expected protein bands were detected using Bio-Rad ChemiDoc TM MP imaging system. Relative abundance was measured with Gel-Pro Analyzer 4.0 software. The results were representative of three independent experiments.

RT-PCR.
Total RNA was isolated using RNAiso Plus (TaKaRa). RT-PCR was performed using PrimeScript ™ RT reagent Kit (TaKaRa) and Trans Start Top Green Qpcr Super Mix (TRANSGEN BIOTECH). Relative expression of target gene was calculated with Δ Δ CT method.
MTT assay. The cytotoxicity of As 2 O 3 was detected by MTT assay. HepG2 cells (1 × 10 5 /ml) were seeded in 96-well plates and treated with 1 μ M-8 μ M As 2 O 3 for 24 h. After treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was added, and the cells were incubated for 4 h at 37 °C. The supernatant was discarded, and 1% DMSO was added. The plate was gently agitated until the blue formazan crystals were fully dissolved. The absorbance at 570 nm was read using a Bio-Rad Microplate Reader, and the cell viability (%) was calculated using the following equation: (A570 of treated sample/A570 of control) × 100. Statistical analysis. The data were expressed as the means ± standard deviation (SD) from at least three independent experiments performed in triplicate and analyzed using the SPSS 13.0 statistical software. The comparisons between groups were analyzed using one-way ANOVA and Student-Newman-Keuls (SNK) test, and P < 0.05 was considered statistically significant.