MK-2206 – Tamoxifen Chemical

Sulforaphane prevents chromium-induced lung injury in rats via activation of the Akt/GSK-3b/Fyn pathway

A B S T R A C T
Chromium (Cr) is an internationally recognized carcinogenic hazard that causes serious pulmonary toxicity. However, Cr-induced pulmonary toxicity lacks effective treatment to date. Sulforaphane (SFN), a well-known organosulfur compound, has gained increasing attention because of its unique biological function. This study investigates if SFN could decrease K2Cr2O7-induced pulmonary toxicity and a po- tential mechanism involved using a rat 35-day Cr-induced pulmonary toxicity model and the mouse alveolar type II epithelial cell line (MLE-12). The results showed that SFN prevented Cr-induced oxidative stress, histopathological lesions, inﬂammation, apoptosis, and changes in protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK-3b) levels in vivo and in vitro. However, SFN can not play the protective effect against K2Cr2O7-induced cell injury after treating by an Akt-speciﬁc inhibitor (MK-2206 2HCl) in MLE-12 cells. Furthermore, SFN increased the expression of nuclear factor-E2-related factor-2 (Nrf2) phase II detoxiﬁcation enzymes. Collectively, this study demonstrates that SFN prevents K2Cr2O7-induced lung toxicity in rats through enhancing Nrf2-mediated exogenous antioxidant defenses via activation of the Akt/GSK-3b/Fyn signaling pathway. SFN may be a novel natural substance to cure Cr- induced lung toxicity.

1.Introduction
Chromium (Cr) is a common environmental pollutant with high toxicity in natural water and other resources (Alaoui-Sosse´ et al., pentavalent Cr by antioxidants accompanied by reactive oxygen species (ROS), which destroy the balance of oxidation and anti- oxidation (Kova´ˇcik et al., 2013; Luo et al., 2016; Thatoi et al., 2014).It was reported that long-term exposure to Cr caused histo- pathological changes in the lung (Beaver et al., 2009). More and more attention has been paid to lung toxicity caused by various ways (Tan et al., 2018a). Pulmonary toxicity induced by Cr involves a variety of mechanisms including the production of free radicals, inﬂammation, and apoptosis (Salama et al., 2016). It is of great clinical signiﬁcance to study the potential mechanism of Cr- induced lung toxicity to alleviate the toxic effect of Cr.Nuclear factor-E2-related factor-2 (Nrf2), a redox-regulated gene, has an important effect against oxidative stress (Ali et al., 2018; Lv et al., 2020; Tan et al., 2018b). Recently, studies reported that Nrf2 translocates into the cell nucleus and activates ARE- dependent gene expression, including transcription of target genes including heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO-1) during oxidative stress (Leclercq et al., 2018; Ray et al., 2012; Taguchi et al., 2011). Cr exposure acceler- ates oxidative stress and ROS production. Therefore, oxidative Fig. 1. SFN protects against K2Cr2O7-induced changes of hematological indexes and oxidative stress in rats. (A and C) The number for WBC and (B and D) RBC in blood samples. (C and D) The doses of SFN and K2Cr2O7 were both 4 mg kg—1. (E and H) MDA level, (F and I) GSH concentration, and (G and J) SOD activity in the lung. (HeJ) The doses of SFN and K2Cr2O7 were both 4 mg kg—1. Values are mean ± SEM (n ¼ 7). *Signiﬁcantly different (p < 0.05) vs control group; #Signiﬁcantly different (p < 0.05) vs K2Cr2O7 group stress may be a main factor of Cr-induced lung toxicity, and a po- tential target of Cr protective medicines. Protein kinase B (Akt) is involved in the regulation of cell sur- vival, proliferation, and apoptosis (Baiyun et al., 2018; Chan et al., 2018; Wang et al., 2017). Glycogen synthase kinase 3 beta (GSK- 3b) is a kind of serine/threonine protein kinase, which regulates cell and tissue metabolism (Shang et al., 2015). Many studies demon- strated that Nrf2 is targeted by GSK-3b, which partially regulates and activates Fyn, a negative regulatory factor of Nrf2 (Bitar and Al- Mulla, 2011; Niture et al., 2011; Zhang et al., 2012). Furthermore, Akt is an upstream regulator of GSK-3b that controls its activity (Lee et al., 2014; Liu et al., 2018a). Akt activation contributes to the deactivation of GSK-3b by enhancing its phosphorylation at Ser9 of GSK-3b (Gong et al., 2012). Therefore, the Akt/GSK-3b/Fyn signaling pathway and the activation of Nrf2 may play a key role in Cr- induced lung toxicity.Natural substances have attracted attention for protecting or- ganisms against injury (Calabro´ et al., 2019; Lu et al., 2018; Prince et al., 2016; Prince et al., 2019; Yang et al., 2018a). Sulforaphane(SFN), a naturally-occurring isothiocyanate compound, is found in these cruciferous vegetables including cabbage, broccoli, and brussels sprouts (Teng et al., 2019; Xu et al., 2019), and has antioxidation, anti-inﬂammation, anti-bacterial, and anti-tumor effect (Atwell et al., 2015; Bergstro€m et al., 2011). SFN is an indi- rect antioxidant and can induce phase II detoxiﬁcation enzymes and antioxidant genes (Visalli et al., 2017). We established a rat 35-day Cr-induced poisoning model to discuss K2Cr2O7-induced pul- monary toxicity and the potential mechanism. In this study, we hypothesized that SFN alleviates lung toxicity in rats chronically exposed to hexavalent Cr, and explored potential signaling path- ways related to Akt/GSK-3b/Fyn to increase our understanding of the biological effect of SFN. 2.Materials and methods 2.1.Chemicals and reagents SFN was acquired from AbMole BioScience (USA), and dissolved in dimethyl sulfoxide (DMSO). K2Cr2O7 was acquired from Tianli Chemical Reagent Co., Ltd. (Tianjin, China). The terminal deoxy- nucleotidyl transferase dUTP nick end labeling (TUNEL) kit was obtained from KeyGen Biotech Co. Ltd (Nanjing, China). Superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) assay kits were acquired from Nanjing Jiancheng Bioengineering Fig. 2. SFN ameliorates K2Cr2O7-induced rat lung toxicity. (A) Parafﬁn sections of lung tissues from control, K2Cr2O7, K2Cr2O7 þ SFN, and SFN groups were stained with HE. (magniﬁcation 200×). Hemorrhage, red arrow; Inﬂammatory cell inﬁltration, black arrow. Bar ¼ 100 mm. (B) The relative protein levels of NF-kB and IL-1b in lung tissues and (C) values of quantitative analysis. (D) Representative TUNEL images of lung tissues, (E) the ratio of apoptosis, (F) the apoptosis-related protein levels, and (G) values of quantitative analysis. Values are mean ± SEM (n ¼ 4). *Signiﬁcantly different (p < 0.05) vs control group; #Signiﬁcantly different (p < 0.05) vs K2Cr2O7 group.Fig. 3. SFN protects against K2Cr2O7-induced changes of white cell counts and cytokines in BALF. (A) IL-1b concentration, (B) TNF-a concentration, (C) total white cell count, and (D) neutrophil count in BALF. Values are mean ± SEM (n ¼ 7). *Signiﬁcantly different (p < 0.05) vs control group; #Signiﬁcantly different (p < 0.05) vs K2Cr2O7 group.Institute (China). CCK-8, JC-1, and ROS detection kits were from Beyotime Institute of Biotechnology (Jiangsu, China). TNF-a and IL- 1b enzyme-linked immunosorbent assay (ELISA) kits were pur- chased from Abcam (Cambridge, UK). Trizol was purchased from Ambion (USA), cDNA synthesis kit and SYBR Green RT-qPCR SuperMix Kit were acquired from Vazyme Biotech Co., Ltd (USA), and 2 PCR Taq Plus Master Mix with dye was got from Applied Biological Materials (Vancouver, Canada). Rat ACTB endogenous reference genes primers were from Sangon Biotech Co. Ltd. (Shanghai, China) and DNA markers were from Tiangen Biotech Co. Ltd. (Beijing, China). Antibodies of Akt, p-Akt, GSK-3b, p-GSK-3b, Fyn, Nrf2, HO-1, NQO1, NF-kB, IL-1b, Bcl-2, Bax, Bcl-xl, and Histone 3 were obtained from Santa Cruz Biotechnology (Santa Cruz, USA). The antibody of GAPDH was acquired from Hangzhou Goodhere Biotechnology (Hangzhou, China). All secondary antibodies were from ZSGB-BIO (Beijing, China). Nuclear and Cytoplasmic Protein Extraction kit, BCA protein assay kit, Radio-Immunoprecipitation Assay, and phenylmethanesulfonyl ﬂuoride were from Beyotime Institute of Biotechnology (Shanghai, China). 2.2.Animal and treatments The animal protocol was approved (Grant Number: 20180309) by the Ethical Committee for Animal Experiments (Northeast Agricultural University, Harbin, China). Fifty-six male Wistar rats, 6e8 w old and weighing 160e180 g, were provided by the Harbin Veterinary Research Institute (Harbin, China), and domesticated for 1 w before the start of experiments. Light cycles were set on a 12/ 12 h light/dark cycle with a temperature of 21 ± 2 ◦C, room humidity of 55 ± 5%, and standard diet and water provided ad libitum. First, we induced the K2Cr2O7-induced lung toxicity model. Twenty-eight rats were randomly divided into four groups (n ¼ 7): control, K2Cr2O7 high-dose, K2Cr2O7 medium-dose, and K2Cr2O7 low-dose groups. The control group rats were intraperitoneally injected with 0.9% (w/v) saline. The K2Cr2O7 high-dose, medium- dose, and low-dose groups rats were intraperitoneally injected with K2Cr2O7 (6, 4, and 2 mg kg—1, respectively, dissolved in sterile saline) once daily. The test period was 35 d.Then, we tested interventions with SFN in the K2Cr2O7 lung toxicity rats. Twenty-eight rats were divided into four groups (n 7): control, SFN, K2Cr2O7, and K2Cr2O7 SFN groups. Rats in the control group were intraperitoneally and subcutaneously injected with sterile saline. Rats in the SFN group were subcuta- neously injected with 4 mg kg—1 SFN solution and intraperitoneally injected with normal saline every d. Rats in the K2Cr2O7 group were intraperitoneally injected with 4 mg kg—1 K2Cr2O7 solution and subcutaneously injected with sterile saline every d. Rats in the K2Cr2O7 SFN group were intraperitoneally injected with 4 mg kg—1 K2Cr2O7 solution and subcutaneously injected with 4 mg kg—1 SFN solution every d. The test period was 35 d and ether anesthesia was administered 24 h after the last treatment. Blood samples were used by blood testing. And bronchoalveolar lavage ﬂuid (BALF) were collected to assess total white cell count, neutrophil count, and cytokines. Lung tissue samples were quickly frozen in liquid nitrogen, and then stored at —80 ◦C. 2.3.Blood testing The amount of red blood cells (RBC) and white blood cells (WBC) in the blood of rats were measured by a Bayer ADVIA 120 Blood Cell Analyzer. The total white cell count in BALF of rats was measured by a hemocytometer. Neutrophil count was determined by counting 200 cells from slides prepared by BALF and stained by Wright- Giemsa. 2.4.Oxidative stress biomarker assay GSH Assay Kit (A006-2-1), MDA Assay Kit (A003-1-2), and SOD Assay Kit (A001-3-2) which were acquired from Nanjing Jiancheng Bioengineering Institute were selected to determine lung GSH and MDA content, and SOD activity. Assay was conducted according to the manufacturer’s instructions (Lu et al., 2019; Yan et al., 2019). 2.5.Histopathology Lung tissues of rats were ﬁxed in 4% formaldehyde at 4 ◦C overnight, embedded in parafﬁn, cut to a 3-mm thickness, and stained with hematoxylin and eosin (H&E). Morphology was observed under a light microscope (BX-FM: Olympus Corp, Tokyo, Japan). Fig. 5. SFN enhances pneumonocyte viability and migration ability, and attenuates ROS levels and mitochondrial membrane potential in MLE-12 cells treated by K2Cr2O7.(A) Cells viability, (B) ROS levels, (C) cells migration ability, and (D) values of quanti- tative analysis. Values are mean ± SEM (n ¼ 6). (E) The mitochondrial membrane potential. Values are mean ± SEM (n ¼ 4, magniﬁcation 200×). Different letter showed signiﬁcant difference (p < 0.05). 2.9. Quantitative real-time PCR assay Fig. 4. Upregulation of Nrf2 by SFN is achieved by stimulating the Akt/GSK-3b/Fyn pathway in the lung of rats treated by K2Cr2O7. The relative protein levels of (A) Akt and p-Akt, (B) GSK-3b and p-GSK-3b, (C) Nrf2 and Fyn in nucleus, (E) Nrf2, HO-1, and NQO1, and (D and F) values of quantitative analysis. Values are mean ± SEM (n ¼ 4). (G) The relative mRNA levels of Fyn, Nrf2, HO-1, and NQO1 in lung of rats were detected by qRT-PCR. Values are mean ± SEM (n ¼ 5). *Signiﬁcantly different (p < 0.05) vs control group; #Signiﬁcantly different (p < 0.05) vs K2Cr2O7 group. 2.6.Cr content Lungs from K2Cr2O7 and K2Cr2O7 SFN groups were washed, blotted dry and weighed. Tissue samples were digested with concentrated nitric acid in a Microwave Digestion System. Total Cr content was determined by atomic absorption spectrophotometry (atomic absorption spectrophotometer, Varian 840 with Zeeman correction with GTA 100 graphite furnace and automatic seeder) (Quinteros et al., 2007). 2.7.Enzyme-linked immunosorbent assay TNF-a and IL-1b concentration in BALF were determined by corresponding ELISA kits according to the instructions recom- mended by the manufacturers, respectively. 2.8TUNEL assay Pneumocyte apoptosis was detected by a TUNEL assay kit. The sections were observed under a ﬂuorescence microscope (Olympus BX41, Nikon, Tokyo, Japan).To determine the expressions of, Fyn, Nrf2, HO-1 and NQO1, quantitative real-time PCR (qRT-PCR) was performed. qRT-PCR analysis was performed as previously described (Han et al., 2019; Liu et al., 2019; Zhang et al., 2019). b-actin was used as an inter- nal reference for mRNA. The gene primer sequences are as follows, Fyn: forward-GAA GCA CGG ACA GAA GAT GAC CTG and reverse- TGC CTG GAT GGA GTC AAC TGG AG; Nrf2: forward-GCC TTC CTC TGC TGC CAT TAG TC and reverse-TCA TTG AAC TCC ACC GTG CCT TC; HO-1: forward-TAT CGT GCT CGC ATG AAC ACT CTG and reverse-GTT GAG CAG GAA GGC GGT CTT AG; NQO1: forward-AGA AGC GTC TGG AGA CTG TCT GG and reverse-GAT CTG GTT GTC GGC TGG AAT GG. Relative RNA levels were calculated by the 2^—DDCt method. 2.10.Cell culture and treatments The mouse alveolar type II epithelial cell line (MLE-12) was purchased from American Type Culture Collection (USA) and cultured in Dulbecco’s modiﬁed Eagle’s F12 Ham medium (DME/F- 12) supplemented with 2% FBS and 1% penicillin-streptomycin. 2.11.Cell viability assay MLE-12 cells were treated with 1 mg mL—1 K2Cr2O7 for 24 h after pretreating with 0.1 mM SFN for 30 min. Cell viability was deter- mined by the CCK-8 kit. 2.12.Cell migration analysis MLE-12 cells were pretreated with 0.1 mM SFN for 30 min. Before the beginning of the migration test, the cell layer was scraped using the tip of a pipette. Next, MLE-12 cells were added with 1 mg mL—1 K2Cr2O7 for 24 h. Time-lapse images were captured for cell morphology analysis.Fig. 6. Upregulation of Nrf2 by SFN is achieved by the stimulating Akt/GSK-3b/Fyn pathway in MLE-12 cells treated by K2Cr2O7. The relative protein levels of (A) Akt and p-Akt, (B) GSK-3b and p-GSK-3b, (C) Nrf2 and Fyn, (E) HO-1 and NQO1, and (D and F) values of quantitative analysis. Values are mean ± SEM (n ¼ 4). *Signiﬁcantly different (p < 0.05) vs control group; #Signiﬁcantly different (p < 0.05) vs K2Cr2O7 group; &Signiﬁcantly different (p < 0.05) vs K2Cr2O7 þ SFN group. 2.13. Measurement of ROS generation According to the manufacturer’s instructions, the ROS genera- tion was examined by a ROS Assay Kit from Beyotime Institute of Biotechnology which can be detected with a ﬂuorescent enzyme reader. MLE-12 cells were pretreated with 0.1 mM SFN for 30 min after treatment with 10 mM of the Akt-speciﬁc inhibitor, MK-2206 2HCl, for 1 h and then cocultured with 1 mg mL—1 K2Cr2O7 for 24 h. Then, MLE-12 cells were incubated with 10 mM DFCH-DA at 37 ◦C for 20 min. Fluorescence was determined using excitation and emission wavelengths of 485 and 525 nm, respectively, with a 96- point well scan on a SpectraMax iD3 (Molecular Devices, USA). 2.14. Measurement of mitochondrial membrane potential Mitochondrial membrane potential was examined by a assay kit with JC-1. MLE-12 cells were treated with 1 mg mL—1 K2Cr2O7 for 24 h after pretreating with 0.1 mM SFN for 30 min. After treatment, cells were incubated with 5 mg mL—1 JC-1 at 37 ◦C for 20 min. Then, stained cells were imaged immediately at 200 magniﬁcation using a ﬂuorescence microscope (Nikon Eclipse Ti-s, Japan). 2.15. Western blot analysis Immunoblot analysis was performed as previously described with use of the following antibodies: Akt, p-Akt, GSK-3b, p-GSK-3b, Fyn, Nrf2 in cytosolic and nucleus, HO-1, NQO1, NF-kB, IL-1b, Bcl-2, Bax, and Bcl-xl (Li et al., 2020; Zhang et al., 2017; Zhu et al., 2019). Histone 3 and GAPDH were used as an internal control for normalization of protein expression. 2.16. Proteineprotein interaction analysis Proteineprotein interaction (PPI) analysis of differentially expressed genes was ﬁnished by the STRING database (http:// string-db.org/). We built a network based on the target gene sequence of the selected species from the database. 2.17. Statistical analysis Data are presented as the mean ± SEM. Statistical analyses were performed with SPSS 19.0 software (USA). Statistical analyses were performed by one-way ANOVA and Tukey’s post hoc test to assess signiﬁcance. Values of p < 0.05 were considered statistically signiﬁcant. Fig. 7. PPI analysis. Protein network of proteins regulated between oxidative stress-related genes, apoptosis-related genes, and inﬂammation-related genes expressed in the lung of rats. 3.Results 3.1.SFN protects against K2Cr2O7-induced changes of hematological indexes and oxidative stress in rats Numbers of WBC increased in a dosage-dependent manner after treatment with K2Cr2O7, and the number of WBC was increased signiﬁcantly in the K2Cr2O7 high- and medium-dose groups. The number of RBC was decreased in a dosage-dependent manner in the K2Cr2O7 groups. SFN treatment reversed the K2Cr2O7-induced increases in WBC numbers and restored the RBC counts (Fig.1AeD). Lung GSH concentration was signiﬁcantly decreased in the K2Cr2O7 high- and medium-dose groups, and lung MDA level was signiﬁcantly increased in the K2Cr2O7 groups. Fig. 1G showed a signiﬁcant decrease in lung SOD activity in the K2Cr2O7 groups. SFN signiﬁcantly reversed the K2Cr2O7-induced increases of lung MDA level and restored lung GSH concentration and SOD activity(Fig. 1EeJ). 3.2.SFN ameliorates K2Cr2O7-induced rat lung toxicity HE staining of the control and SFN groups showed normal lung tissue structures. The K2Cr2O7 group had marked hemorrhage in the alveolus and inﬂammatory cell inﬁltration. However, SFN attenuated these K2Cr2O7-induced pathological changes (Fig. 2A). SFN signiﬁcantly attenuated Cr accumulation in the lung (Fig. 2B). The expression of NF-kB and IL-1b in the lung was increased signiﬁcantly in the K2Cr2O7 group and restored by SFN (Fig. 2CeD). SFN treatment reversed the signiﬁcantly higher number of TUNEL-positive cells in the K2Cr2O7 group, indicating that SFN restored K2Cr2O7-induced apoptosis. Bax protein levels were increased signiﬁcantly, and Bcl-2 and Bcl-xl protein levels were decreased signiﬁcantly in the K2Cr2O7 group. However, SFN reversed these changes in apoptosis-related proteins, including Bcl- 2, Bcl-xl, and Bax (Fig. 2EeH).SFN signiﬁcantly reversed the K2Cr2O7-induced increases of IL- 1b and TNF-a concentrations in BALF (Fig. 3AeB). And SFN signif- icantly inhibited the K2Cr2O7-induced increases of total white cell and neutrophil counts in BALF (Fig. 3CeD). 3.3.Upregulation of Nrf2 by SFN is achieved by stimulating the Akt/ GSK-3b/Fyn pathway in the lung of rats treated by K2Cr2O7 K2Cr2O7 treatment signiﬁcantly decreased the activation of Akt, Nrf2, HO-1, and NQO1, and increased GSK-3b activity. These results revealed that K2Cr2O7 signiﬁcantly increased Fyn accumulation and reduced Nrf2 in cell nucleus. However, SFN activated Akt and Nrf2, and inhibited GSK-3b activity and the nuclear translocation of Fyn (Fig. 4). 3.4.SFN enhances pneumonocyte viability and migration ability, and attenuates ROS levels and mitochondrial membrane potential in MLE-12 cells treated by K2Cr2O7.We found that K2Cr2O7 signiﬁcantly reduced cell viability (Fig. 5A) and migration ability (Fig. 5CeD), and signiﬁcantly increased ROS levels (Fig. 5B) in MLE-12 cells. However, SFN treatment protected against the K2Cr2O7-induced effect (Fig. 5). When MLE-12 cells was treated with an Akt-speciﬁc inhibitor (MK- 2206 2HCl), SFN, and K2Cr2O7, these results showed that the pro- tection of cell viability by SFN was inhibited, and the inhibition of ROS levels by SFN was abolished (Fig. 5AeB).Exposure to K2Cr2O7 increased green ﬂuorescence by JC-1 staining in MLE-12 cells, indicating mitochondrial depolarization was induced by K2Cr2O7. Pretreated with SFN prevented this change, demonstrating SFN protected mitochondria from K2Cr2O7-induced damage (Fig. 5E). 3.5.Upregulation of Nrf2 by SFN is achieved by the stimulating Akt/ GSK-3b/Fyn pathway in MLE-12 cells treated by K2Cr2O7 In vitro, these results showed that K2Cr2O7 treatment signiﬁ- cantly decreased the activation of Akt, Nrf2, HO-1, and NQO1, and increased GSK-3b activity. K2Cr2O7 signiﬁcantly increased Fyn accumulation and reduced Nrf2 in cell nucleus. Moreover, vitro results showed that SFN increased the activation of Akt and Nrf2, and inhibited GSK-3b activity and the nuclear translocation of Fyn in MLE-12 cells. However, treatment with an Akt-speciﬁc inhibitor (MK-2206 2HCl) signiﬁcantly reversed the effect of SFN (Fig. 6). 3.6.PPI analysis Functional interaction networks revealed a relationship be- tween oxidative stress and the Akt/GSK-3b/Fyn signaling pathway, as well as the regulation of apoptosis and inﬂammation (Fig. 7). 4.Discussion Cr can enter organisms by numerous methods, leading to the accumulation of Cr in tissues, including the liver, kidney, heart, and lung (García-Nin~o and Pedraza-Chaverri, 2014; Hemmati et al.,2008; Yin et al., 2019; Zhao et al., 2019). In our study, histopatho- logical analysis indicated that SFN reversed the pulmonary toxicity of K2Cr2O7. Our results revealed that SFN attenuated the accumu- lation of Cr in the lung. Because of the health-promoting effects of SFN (Briones-Herrera et al., 2018), we speculated that SFN might inhibit the entering of chromium to the cells or promote the removal of chromium, and the related molecular mechanism re- mains to be further studied. In addition, the accumulation of K2Cr2O7 induced hematological toxicity, which was prevented by SFN treatment. Therefore, SFN can be used as an effective thera- peutic drug to restrain the pulmonary toxicity of K2Cr2O7.SOD, GSH, and MDA are key indicators of oxidative stress in organisms (Chen et al., 2016; Katayama and Mine, 2007; Yang et al., 2018b; Zhang et al., 2018b). We found that oxidative stress partic- ipated in the pathophysiological process of K2Cr2O7-induced lung toxicity by increasing MDA level and reducing GSH concentration and SOD activity. Of note, SFN protected against K2Cr2O7-induced oxidative stress. In vitro, results demonstrated that SFN protected against K2Cr2O7-induced oxidative stress by measuring cell viability and ROS production. These demonstrate that SFN allevi- ates pulmonary oxidative stress induced by K2Cr2O7 by eliminating ROS and enhancing the antioxidant enzyme defense system. Oxidative stress activates NF-kB leading to excessive inﬂam- mation, which indicates that inﬂammation and oxidative stress are closely related during disease progression (Li et al., 2019a; Su et al., 2019; Turillazzi et al., 2016; Yang et al., 2017). The activated inﬂammation increases the production of cytokines and chemo- kines (Petriello et al., 2018). The transcription factor NF-kB is closely related to cytokine production and immune responses, which oc- curs in many proinﬂammatory and cell death pathways (Kaulmann et al., 2016; Morel et al., 2008; Wei et al., 2018). IL-1b is an important proinﬂammatory cytokine that is related to many auto- immune diseases and which has a key role in anti-infection effect (Ahn et al., 2018; Dong et al., 2019). In our study, results demon- strated that Cr exposure promoted the expression of NF-kB and IL- 1b, whereas SFN efﬁciently suppressed the effect of Cr. Hence, SFN alleviates pulmonary inﬂammation induced by K2Cr2O7 via decreasing proinﬂammatory cytokines levels.Hexavalent Cr causes oxidative stress, inﬂammation, and apoptosis (Lei et al., 2008). Furthermore, oxidative stress is a known signal of apoptotic initiation, and the release of inﬂammatory cy- tokines is also related to the mechanism of Cr-induced apoptosis (Malik et al., 2016). Excessive ROS can lead to lipid peroxidation in the mitochondrial membrane, decreasing mitochondrial mem- brane potential, and impairment of mitochondrial morphology further lead to the accumulation of ROS, resulting in a vicious circle between mitochondria and ROS (Wang et al., 2019). SFN prevents intrinsic apoptosis induced by oxidative stress in noncancer cells(Negrette-Guzma´n et al., 2013). Our results suggested that K2Cr2O7 resulted in oxidative injury of mitochondrial membrane, and caused the mitochondrial depolarization and activation of mitochondrial-dependent apoptotic pathway associated with Bax, Bcl-2, and Bcl-xl, whereas SFN reversed all of these function of K2Cr2O7. Thus, a possible explanation is that the protective effect of SFN against K2Cr2O7 is probably related to the regulation of pul- monary oxidative stress, which attenuates K2Cr2O7-induced pulmonary apoptosis. Nrf2 is a major redox regulator that regulates antioxidant genes and phase II detoxifying enzymes to maintain the intracellular redox homeostasis (Li et al., 2019c; Xin et al., 2018; Yang et al., 2016). Many studies have revealed that K2Cr2O7-induced lung toxicity decreased level of Nrf2, HO-1, and NQO1, which regulate redox ability and promote toxic excretion (O’Hara et al., 2006). SFN, a well-known activator of Nrf2, interrupted Nrf2-Keap1 protein- protein interaction to promote Nrf2 phosphorylation and trans- location to the nucleus, and decreased Nrf2 degradation, thus initiating the transcription of downstream antioxidant genes (Hu et al., 2011). Our results suggested that SFN alleviates K2Cr2O7- induced lung toxicity through increasing the expression and effect of Nrf2. Furthermore, our results demonstrated that SFN restores the decreased expression of Akt induced by K2Cr2O7. Akt is an up- stream regulator of GSK-3b and can inhibit GSK-3b activity through promoting the phosphorylation of GSK-3b, and Akt upregulates Nrf2 and promotes Nrf2 translocation to the nucleus (Liu et al., 2018b; Wang et al., 2008). Evidences showed that Fyn phosphor- ylates Nrf2 favoring its nuclear export, and inhibition of the GSK- 3b/Fyn pathway impedes the nuclear export of Nrf2, then holding Nrf2 transcriptional action in the nucleus (Jain and Jaiswal, 2007). In this study, we found that the downregulation of Nrf2 by K2Cr2O7 is accompanied by inhibition of Akt, activation of GSK-3b, and accumulation of Fyn in the nucleus, whereas SFN suppressed the effect of K2Cr2O7. Our study demonstrated that the regulation of SFN on Nrf2 may be mediated by the combined action of Nrf2 and Akt, and the effect of Akt may be linked to the SFN-induced acti- vation of Nrf2 through the Akt/GSK-3b/Fyn signaling pathway. To conﬁrm this, we treated MLE-12 cells with an Akt-speciﬁc inhibitor (MK-2206 2HCl) and SFN, and demonstrated that activation of Nrf2 and its downstream genes NQO1 and HO-1 induced by SFN was attenuated, and the GSK-3b/Fyn pathway was activated. Together, SFN prevents K2Cr2O7-induced lung toxicity in rats through acti- vating Nrf2 partially via activation of the Akt/GSK-3b/Fyn signaling pathway.

5.Conclusion
In conclusion, our study demonstrates that SFN prevents K2Cr2O7-induced lung toxicity in rats via activation of the Akt/GSK- 3b/Fyn signaling pathway. Dietary intake of SFN may offer a novel and safe therapeutic approach to Cr-induced lung toxicity.