Ameliorative Effects of Nano-Selenium Against Fluoride Stress Induced Hepatocytes Autophagy and Apoptosis in Mice

Background: Fluorine is widespread in the environment, and the injurious impacts of uoride underscore its signicance for public health. The long-term presence of uorine in environment could be a risk in hepatotoxicity for both human beings and animals. Important role of selenium in mitigation of heavy metal toxicity via regulating autophagy and apoptosis is well-known. Further, nano-Se is a common articial nano material, with higher biological activity and lower toxicity. The aim of the current study was to examine whether nano-Se supplementation can reduce the effects of uoride-induced hepatocytes autophagy and apoptosis. Results: Here, we report that uoride exposure induces apoptosis and autophagy with nucleus broken, dissolved and disappeared of hepatocyte, contributing to its hepatotoxicity. More importantly, Cyt-C and Beclin-1/Bcl-2 pathways are involved in the regulation of autophagy and apoptosis via targeting Caspase-9, Caspase-3, P53, Bax, LC3, ATG-5, P62 and mTOR expressions. Conclusion: Nano-Se is capable to alleviate uoride-induced hepatocyte damage, that selenium can be prefer to prevent chronic uorosis-induced autophagy and apoptosis by regulating Cyt-C and Beclin-1/Bcl-2 signaling pathway. In precisely, NaF-induced the liver injury by activating autophagy and apoptosis, which indicate that uorine exposure, pose an ecological risk to human beings and animals. Nano-Se has protective effects against uoride-induced hepatocytes.


Introduction
Fluorine is an important trace element for humans and in animals, which is widely distributed in natural rock, soil, and in industrial waste. However, the excessive long-term uorine exposure in the air (industrial exhaust), food, water, and even excessive use of toothpaste could be a risk for human beings [13].
Drinking water is main source of uorine exposure [16], that why cut value for uoride is 1.5 mg/L [16] according to World Health Organization (WHO). WHO reports indicated that about 200 million residents worldwide are using unsafe drinking water which has concentration well above the standard [16; 26].
Fluorosis is a worldwide endemic chronic systemic disease closely related to the long-term intake of excessive uorine in the geographical environment [29], mainly manifested as dental uorosis and skeletal uorosis [36]. The endemic uorosis is widely spread in many countries, like Thailand, India, Egypt, China, etc. [16; 32]. Human populations in many regions (e.g. Northwest, Southwest, Loess Plateau) of China suffer from the uorosis due to the long-term intake of uorine [21; 36; 40]. Previous study indicated that almost 19 million people suffered from uorosis, of which the skeletal uorosis has a icted about 1.2 million people in China caused mainly by uoride from coal-burning [35], which also disturb crops and livestock sector. Long-term consumption of food or water with high uoride content will also cause chronic poisoning of uorine (in ammation and degeneration of kidney, liver, intestine, brain, and lung) [9]. Among them, uoride has toxic effect on the liver, so excessive intake of uoride can lead to extensive degenerative changes in the liver ultimately reduce its detoxi cation function.
Oxidative Stress (OS) lead to the unbalanced oxidant-antioxidant status, and OS is initiating factor for the uorosis [31]. Abnormal imbalance in the redox state of normal cells will produce peroxides and free radicals which result in damaging the proteins, lipids and nucleic acids of cells. Fluorine can directly interfere with oxygen metabolism, leading to the increase in ROS. ROS can activate the release of Cyt-C in mitochondria, damage DNA molecules, and regulate the expression of apoptosis related genes [2; 3]. Previous report indicated that sodium uoride can promote Bax gene expression and inhibit the expression of BCI-2 gene to accelerate cell apoptosis, and the rate of cell apoptosis is positively correlated with uorine concentration [5]. Zhao et al., found that NaF induced mitochondrial damage and ROS contribute to hepatotoxicity and cell damage [47].
Autophagy, a highly conserved biological process that degrades damaged organelles and doublemembrane vesicles (autophagosomes), and then transported to lysosomes for future degradation, and considered important process in an array of cellular events (cell development, death, oxidative stress, toxic injury) [10; 19]. In the process of studying how uoride induces autophagy in rat HAT-7 cell, Zhang et al. found that the expressions of autophagy genes LC3, Beclin-1 and Alg5 were up-regulated and the phosphorylation level of mTOR protein was reduced in mouse Leydig cells exposed to uoride, which proved that uoride could affect cytotoxicity via autophagy [45]. In addition, NaF-induced autophagy via regulating the autophagy related genes expression, like p62, Beclin1 and LC3 [48].
Selenium is involved in the synthesis of various selenoenzyme and selenoproteins, including glutathione peroxidase, which catalyzes the transformation of peroxides into water or various alcohols in the body, and protecting bio lms from oxidative damage [9; 37]. Selenium has an alleviation effect on many heavy metals toxic elements such as cadmium, arsenic, mercury, lead and silver, etc. At the same time, and selenium can antagonize cell damage caused by uorosis, promote urinary uoride excretion, correct free radicals and lipid metabolism disorder and signi cantly inhibit uorine-induced lipid peroxidation. Previous study showed that the protective effect of selenium on uoride-induced cell injury is closely related to its antioxidant capacity [34; 37; 39]. Feng et al., reported that selenium eliciting antagonistic effects in uorosis by promote the removal of uoride, repair lipid peroxidation, and enhances autoxidation [6]. In particular, selenium produced antioxidant effects on uoride, relieves the toxic effect of uoride in liver, and inhibits apoptosis [23]. Nanomaterials have been widely used in human and animal clinical practice due to diverse properties including increase absorption rate, decrease toxicity, high bioavailability and strong oxidation resistance, as compared with other therapeutic materials. As the point of interest in nanotechnology treatment, selenium nanoparticles (SeNPs) have been used as protective agents in different murine experimental models due to unique biological activities and low toxicity. However, the research regarding mechanism of action of nano-Se in relieving the toxic effect of uoride has less reported, and its relevant mechanisms and biological functions connected with autophagy and apoptosis need to be further investigated.
Previous study reveals that uoride has hepatotoxic effects, and selenium has vital role in mitigation of heavy metal toxicity via regulating autophagy and apoptosis is well-known. Still, the underlying molecular mechanisms of nano-Se in reversing uoride-induced toxicity has rarely reported. The present study aimed to explore the autophagy and apoptosis in hepatocytes as well as the effects of nano-Se against uoride-induced hepatocytes damage to demonstrate the mechanism by which selenium prevents chronic uorosis-induced autophagy and apoptosis.

Experimental Groups and Mice Treatment
Total of 40 healthy Kunming mice were divided into four equal groups (n = 10) randomly. All animals were offered ad libitum regular diet and housed in a controlled environment (12-h light/dark cycle; temperature 23 ± 2 °C) as suggested by the Laboratory Animal Center of South China Agricultural University. First group was control group (n = 10) administered with a standard normal diet. Second group (n = 10) was treated with 24 mg/kg NaF dose via intragastric route, third group (n = 10) was treated with 1 mg/kg nano-Se dose via same route, and lastly group (n = 10) was treated with NaF + nano-Se with same doses. After 28 days, animals were sacri ced by cervical dislocation. Liver samples were quickly xed in paraformaldehyde (4%) for histopathology and immuno uorescence, and remaining samples were frozen in liquid nitrogen, until subsequent use and further analysis.

H&E Staining and Immunohistochemistry
The liver samples (n = 10) from each treatment group were processed for histological analysis via hematoxylin and eosin (H&E) according to our previous study [30; 44]. Brie y, the liver tissues were xed in paraformaldehyde (4%) for 48 h, and then dehydrated in gradients of ethanol, subsequently embedded in para n. The tissue sections with 4-5 µm-thick sections were placed on polylysine-coated slides for H&E staining and IHC. For H&E, the above slides were dried for 12 h at 37 °C, then stained with hematoxylin/eosin, and observed under a light microscopy. For immunohistochemical analysis, the slides were blocked by 3% hydrogen peroxide for 10-20 min after washed in PBS. After washing with PBS, horse serum (10%) was used on sections for 1 h at 37 °C, then the slides were incubated with primary antibody against Cyt-C and LC3 for 1.5-2.5 h at 25 °C and washed with PBS. Thereafter, the sections were incubated for 1.5 h with secondary antibodies at 37 °C to t the primary antibodies. The photomicrographs were obtained by using microscope (Olympus, Japan). For expression analysis of proteins, six elds of slides were selected and observed randomly, and the expression values were analyzed quantitatively using the Image-Pro ® Plus 6.0.

TUNEL analysis for apoptosis of hepatocytes
For the hepatocytes apoptosis assay, the TUNEL staining was used according reported protocol [38; 46].
Brie y, the para n sections were de-waxed with xylene and ethanol, and then slides were digested with proteinase K (20 µg/ml) at 37 °C for 15-30 min. The slides were lled with TdT and dUTP (1:9) for 2 h at 37 °C and washed with PBS three times. Finally, sections were stained with 4, 6-diamido-2-phenylindole dihydrochloride, and observed under a uorescence microscope. The value of positive nuclei was measured using the Image-Pro ® Plus 6.0.

RT-qPCR analysis
Speci c primers for evaluating the apoptosis (Caspase-3, Bax, Bcl2, P53, Cyt-c, Caspase-9, Bak1 and APAF1) and autophagy (Parkin, Pink1, Beclin-1, Atg-5, LC3, mTOR and P62) genes were chosen and designed according to published mice sequences (Table S1) using the Primer Premier 6.0 software (Premier, USA), and the housekeeping gene (GAPDH) was served as the control. Total RNA from the liver tissues were extracted using the TRIzol (Invitrogen). The 50 µl cDNA was obtained by using the commercial reverse transcription cDNA kit (AT301-02, TransGen Biotech, China). Then, the real-time qPCR (RT-qPCR) was performed in quadruplex with Step One-Plus™ Real-Time PCR System. Relative mRNA expression level was calculated using delta Ct (2 −ΔΔCt ) method as described by our previous study [43].

Western Blot analysis
Liver samples were collected and washed with PBS three times on ice, and homogenized in RIPA lysis buffer with 1% PMSF, and then centrifuged at 12,000 rpm for 10-15 min for collecting the supernatants for further experiments. The concentration of the total protein in liver samples was determined by BCA assay kit. The samples were boiled for 10 min with SDS-PAGE loading buffer. The protein was separated by SDS-PAGE on 10% polyacrylamide gel and then transferred to PVDF membrane according to the laboratory protocol. The membrane was incubated in 5% skimmed milk for 1.5 h at 25 °C and then incubated with diluted primary antibodies against ATG-5, Beclin-1, Caspase-3, Cyt-C, LC3, P62 and GAPDH (1:1000) at 4 °C for 12 h. Then, the membranes were washed for 3-5 times with TBST for 5 min each, followed by incubating with corresponding secondary antibody for 30 min at 25 °C. The images of proteins band were photographed with an imaging system (TransGen Biotech Co., China). Proteins levels were analyzed by detecting the optical density value via the Image J software (Bethesda, MD, USA).

Statistical Analysis
The statistical analyses were performed using Student's t-test by using SPSS 19.0 software. The results were shown the means ± standard deviation. *P < 0.05 was set as level of signi cance.
Effects of nano-selenium against uoride-induced hepatocytes histological changes in liver Autopsy affected the liver development (volume and weight decreased) of mice in the sodium uoride (NaF) group more prominently than the control group. However, non-signi cantly change in the nano-Se group was observed as compared with the control group, and the abnormal liver development in NaF induced mice by was alleviated by nano-Se treatment. As shown in Fig. 1A, the hepatocytes were arranged precisely without any visible structural damage in live condition in the normal mice. In the NaFtreated mice, hepatocytes were disturbed, and observed changes were disordered arrangement, swelling, granulosa degeneration, vacuolar degeneration and necrosis of hepatocytes, and even nucleus broken, dissolved and disappeared. However, nano-Se treatment enhances the degree of hepatocyte resist oxidative damage or apoptosis, that protect hepatocyte damage induced by NaF. These results imply that uoride induced hepatocytes apoptosis, and nano-Se is capable to alleviate uoride-induced hepatocyte damage that selenium can be chosen to prevent chronic uorosis-induced apoptosis.
TUNEL staining of liver tissues were analyzed to evaluate the hepatocyte apoptosis (Fig. 1B&D). No evident signs of hepatocytes damage were found, and TUNEL-positive cells were rarely detected in the control and nano-Se groups. In contrast, NaF treatment enhances the degree of hepatocyte apoptosis, and the percentages of TUNEL-positive increased by more than 7 fold as compared with normal mice in control. Fortunately, nano-Se could alleviate uorosis-induced hepatocytes injury in mice (Fig. 1C).
Cytochrome C (Cyt-C) important factor in mitochondria pathway in uoride-induced apoptosis Immunostaining showed that the Cyt-C protein level in NaF group was highly expressed (P < 0.05) vs control and nano-Se groups, while the expression level of Cyt-C was decreased after nano-Se plus NaF treatment group (Fig. 2A&B). Cyt-C expression by RT-qPCR and western blot were depicted in Fig. 2C&D. As well, the expression of Cyt-C in NaF group was signi cantly higher as compared to control and nano-Se groups, and nano-Se possibly could reduce Cyt-C expression in uorosis-induced hepatocyte apoptosis.
Beclin-1/Bcl-2 regulates autophagy and apoptosis in uoride-induced hepatocyte damage To assess whether the ameliorative effects of nano-Se against NaF-induced hepatocyte damage was associated with the activation of the Beclin-1/Bcl-2 pathways, the expression of the key marker protein LC3 of autophagy was studied. In comparison with control, the LC3 expression level was elevated in NaF group, and down-regulated in nano-Se group (Fig. 3). Together, the ratio of LC3II/LC3I protein expression was increased in NaF groups, while mRNA level of LC3 was increased in both NaF and nano-Se groups.
Forehead results indicate that the hepatocytes undergo autophagy after uoride treatment; in context the role of Beclin-1/Bcl-2 pathway remains unclear. For this purpose, the Beclin-1 and Bcl-2 mRNA and protein expression levels was further investigated. In comparison with control, the protein expressions of Bcl-2 and beclin-1 was increased in NaF treated hepatocytes. NaF vs nano-Se groups, Bcl-2 level was elevated and Beclin-1 level was reduced (P < 0.05), respectively. In conclusion outcomes spotlight the role of Beclin-1/Bcl-2: in regulation of autophagy in uoride-induced liver damage, and in nano-Se against defective autophagy in the anesis of liver damage. These results could contribute to understanding the mechanism of uoride-induced hepatotoxicity and the effects of nano-Se in alleviating uorosis.
Ameliorative effects of nano-selenium against uorideinduced hepatocytes apoptosis Fluorosis is a major public health problem, and excessive uoride exposure can induce apoptosis, and selenium has been used to alleviate this problem. To evaluate the ameliorative effects of nano-Se on hepatocyte apoptosis in uorosis-affected mice, the Caspase-3, Caspase-9, Bax, P53, Pink1, and Parkin genes expression were analyzed. In comparison with control, the expression levels of Caspase-3, Caspase-9, and Bax mRNA were signi cantly elevated in the NaF and nano-Se treated groups; while Bax mRNA expression was up-regulated in nano-Se group (Fig. 4). In addition, the apoptosis proteins (Caspase-3, P53, Pink1, and Parkin) highly expressions were observed in treatment groups. Conversely, compared to the NaF group, the above abnormally expressed genes were decreased with the supplementation of nano-Se. These results suggested that NaF exposure impaired the liver damage and apoptosis, as con rmed by Caspase-3, Caspase-9, Bax, P53, Pink1, and Parkin genes expression. Fortunately, nano-Se could prevent chronic uorosis-induced liver injury, in which anesis in apoptosis may also be considered as a key process in alleviating uorosis.

Nano-selenium alleviate autophagy in liver induced by uoride
Reported studies suggested that excessive uoride exposure induced liver damage and autophagy. In current study, the autophagy was observed in hepatocytes by con rming protein LC3 (autophagy key marker) after uoride treatment. However, the underlying mechanism of uoride-induced autophagy in hepatotoxicity remains unknown. Compared to control and nano-Se groups, non-signi cantly differences were noticed in ATG-5, mTOR, and P62 mRNA expressions in NaF treatment group; while genes expression was decreased in nano-Se treatment. Surprisingly, the protein expression of ATG-5 was decreased after NaF exposure, and was increased after nano-Se treatment. In conclusion, NaF triggered the autophagy in hepatocytes and produced hepatotoxicity, and nano-Se play protective role in reversing toxicity.

Discussion
Fluoride common pollutant in the environment, imitated from the uorine industry and used in the daily life, accompanied by the widespread presence in various geographical locations [13; 17]. Human population living in uoride contaminated environment (water, soil, food, inusterial waste, drugs, cosmetics) suffer from chronic skeletal uorosis and dental uorosis [49]. Rather than dense tissues, recently more attention has been paid to uoride toxicity connected with kidney, liver, and reproductive system. Liver is a major detoxifying organ for the metabolism in the body, so in vulnerable way damaged by poisons and pollutants. Reported that excessive exposure of uoride has hepatic toxic effect, including reduction in detoxi cation function, and studies have found that excessive uorine exposure can cause signi cant liver damage, like hepatocyte swelling, degeneration and even necrosis, central venous distention and congestion, cell organelle disturbance, [1; 33; 41; 50] along with accompanied by apoptosis [33; 41; 49].
High uoride exposure can inhibit the activity of cathepsin, trypsin and streptomycin, accompanied by promotion in the activity of transaminases (GOT and GPT); in addition, uoride can signi cantly increase the malondialdehyde (MDA) level, NO content and free OH radical production [28]. Meanwhile, the previous studies have con rmed that signi cantly higher ROS and MDA levels were found in the ovaries of the uoride treated mice, suggesting that uoride has led to oxidative damage in the ovaries of zebra sh [20]. According to the previous results, we know that of SOD, CAT, and GSH-Px were decreased, and their mRNA expressions down-regulated, which may lead to reduce in antioxidant activities in uoride exposure [20], and these effects can ultimately lead to changes in cell structure and function. The antioxidant enzymes (SOD, CAT, GPx and GSH), along with malondialdehyde (MDA), were studied as potential biomarkers in oxidative damage [51]. Meanwhile, previous report found that the oxidative stress genes (Gstp1, Ncf1, and Cygb) were signi cantly elevated in liver tissue in sheep exposed to chronic uoride, while GPx, SOD 1, and SOD 2 genes were signi cantly decreased, which suggested that uoride can induce hepatocyte injury [5]. In present study, we found that hepatocytes showed a disordered arrangement, swelling, granulosa degeneration, vacuolar degeneration and necrosis of hepatocytes, and even nucleus broken, dissolved and disappeared in the excess uoride exposed hepatocytes. Meanwhile, the NaF enhances the degree of hepatocyte apoptosis, and the percentages of TUNEL-positive increased by more than 7-fold. Serum antioxidant levels suggested that uoride could signi cantly reduce the level of SOD and GSH-PX, while MDA content was increased in the livers of uoride exposed mice.
Current reports indicate that the mechanism of uorosis accumulation induced-pathological damage is related to the increase of free radical level, manifested as disorder of free radical metabolism, decreased antioxidant capacity and increased oxidative stress level [14; 42]. Excessive intake of uorine directly attacks oxygen, which leads to the production of oxygen free radicals (OFR). Meanwhile, studies have shown that uorine attack antioxidant enzymes to inhibit their activity, which increased the oxygen free radicals and further leads to lipid peroxidation damage and cell death [42; 51]. Presently, free radical damage is considered as the main cause of cell death induced by uorosis. Selenium is an effective antioxidant and an important component of GSH-PX, which is considered as an essential element for both animals and humans. Selenium was reported to improve the antioxidant capacity in both animals and humans with uorosis; but the cell damage caused by uoride exposure is antagonized by selenium via scavenging OFR [7; 6]. Current study found that the liver injure caused by uorosis was characterized by the abnormal changes in antioxidant capacity with hepatocyte apoptosis, suggesting that uorosis induced liver damage may be caused by increasing the free radical levels to induce lipid peroxidation, reduce the activity of antioxidant enzymes, and promote liver cell apoptosis. Fortunately, nano-Se treatment enhances the degree of hepatocyte resist oxidative damage or apoptosis that protect hepatocyte damage induced by sodium uoride. These results imply that uoride induced liver apoptosis, thereby causing liver damage in the mice. Nano-Se is capable to alleviate uoride-induced hepatocyte damage, that selenium can be preferred to prevent chronic uorosis-induced apoptosis.
Oxidative stress causes damage to cell components, especially mitochondria, which are the site of ROS production and also the target of oxidative stress-induced damage [8]; previous reports have shown that ROS accumulation induced the mitochondrial respiratory chain damage [4]. Excessive uorine intake increases the accumulation of ROS in the liver, suggesting that uorine destroys the mitochondrial respiratory chain, which is mainly characterized by the occurrence of oxidative stress and the decrease of ATP content [33]. It has been reported that sodium uoride exposure stimulates the production of ROS, causing oxidative stress [11; 25]. With the discovery and further understanding of mitochondrial role in apoptosis, the role of ROS in apoptosis has been paid more and more attention. Previous study showed that uoride exposure induced apoptosis has also been occurred in kidney and liver of carp, Cyprinus carpio [20]. Apoptosis and oxidation pathways are particularly important in the study of the cell-dependent molecular basis of liver and kidney damage induced by uoride [14]. Selenium may regulate the expression of apoptosis related factors, like Caspase-3, Caspase-9, Bcl-2 and Bax by enhancing the activity of Bcl-2 in cells, inhibit the activity of death regulating gene Bax, Caspase-3 and Caspase-9, for future delay the cell death regulating, thus protecting cells and extending cell life. Consistent with this result, the current study has been done in vivo concerning the apoptosis induced by uoride found that uoride has cytotoxic effects via affecting cell proliferation and even causing severe cell death. At the same time, our study also found that nano-Se can antagonize the apoptosis of hepatocytes in mice induced by uorine, and inhibit apoptosis signal transduction and antagonize apoptosis through the expression of apoptotic factors, such as Caspase-3, Caspase-9, Bax, P53, CYT-C and Bcl-2, as well as the activity of antioxidant enzymes.
Autophagy is best characterized as a survival response, it is a widespread mechanism of a mechanism of programmed cell death, and an important pathway of intracellular component degradation, which is crucial for cell activity and body health. Cells can remove damaged organelles through autophagy to maintain cell homeostasis and provide energy [22]. Most recent studies have reported that autophagy involves many biological events, such as cell proliferation and development, oxidative stress, cell survival, cell senescence, and cell death [49]. For a complete autophagy pathway, each step is regulated by autophagy-related genes, such as ATG5, P53, mTOR, LC3, Bcl-2 and Beclin1. As an important detoxifying organ, liver is vulnerable to damage by poisons, among which autophagy plays an important role in the process of hepatocyte damage [15]. LC3 is crucial for the formation of autophagosomes, and the level of LC3 can re ect the number of autophagosomes. LC3-I can be speci cally bound to the membrane of autophagosomes after transformation into LC3-II, so it is also considered as a marker protein for autophagy [24].
In our study, the expression of Beclin-1 was increased in uoride group, which was in agree with other report by Guo et al. who reported that 100 mg/L NaF increased the autophagy-related proteins in mice [12]. In particular, we observed that nano-Se could regulate the expression of Cyt-C and Beclin-1/Bcl-2 signaling pathway to target apoptosis and autophagy and mediate the liver damage affected by uorosis. Therefore, nano-Se has protective effects against uoride-induced hepatocytes and can play key role as a protective agent to target autophagy. Beclin-1 is the rst identi ed gene mediating autophagy in mammals and considered as a key regulator of autophagy. The autophagy precursor can be prevented by Beclin1 interacting proteins via regulating the activity of autophagy in cells [27]. Various autophagy regulatory proteins combine with Beclin1 to form protein complexes to regulate autophagy. The Bcl-2 interacts with Beclin1, resulting in preventing Beclin1-dependent autophagy leading to apoptosis. Therefore, Beclin1 can bind with autophagy-related proteins to regulate autophagy or apoptosis [18].
The current studies provide a novel evidence that uoride exposure induces apoptosis and autophagy, and results in hepatotoxicity. Particularly, we have demonstrated that the related regulatory mechanisms are mediated by Cyt-C and Beclin-1/Bcl-2 signaling pathways. In addition, nano-Se has protective effects against uoride-induced hepatocytes apoptosis, thereby reduce liver damage. The future studies should be investigated for nano-Se antidotal effect on uoride toxicity. Moreover, our ndings provide novel insights into the control mechanism of chronic uorosis hepatotoxicity, and further studies among the uoride, DNA damage and ROS are required to explore the uoride induced oxidative stress in hepatocytes.

Declarations
Ethics approval and consent to participate Availability of data and materials Yes, it will be provided by corresponding authors.

Competing interests
None of the authors has any con ict of interest.  Fluoride-induced apoptosis as revealed by evaluating the Cyt-C expression. (A&B) immunostaining of Cyt-C expression was increased in NaF treatment mice, and Nano-Se could alleviate uorosis-induced Cyt-C expression; (C) western blotting showed that the Cyt-C protein expression increased in the NaF treatment , while decreased in Nano-Se treatment; (D) qRT-PCR quanti cation revealed that the level of Cyt-C mRNA expression in the NaF group was signi cantly higher than control and Nano-Se groups, and Nano-Se decreasing impact observed on Cyt-C expression in uorosis-induced hepatocyte apoptosis.  All values are presented as mean ± SD.

Figure 5
Effects of nano-selenium on uoride exposure-induced hepatocytes autophagy. (A) expression levels of the autophagy marker genes (ATG-5, mTOR, and P62). (B) a quantitative analysis of the band density revealed that the level of ATG-5, mTOR, and P62 proteins in the uoride exposure-induced liver damage was highly notable, while nano-selenium partially alleviates the autophagy. All values are presented as mean ± standard deviation (M ± SD). *P < 0.05.