Dietary luteolin protects against HgCl2-induced renal injury via activation of Nrf2-mediated signaling in rat

Accepted Manuscript Dietary luteolin protects against HgCl2-induced renal injury via activation of Nrf2-mediated signaling in rat

Xiao Tan, Biying Liu, Siyu Li, Jingjing Lu, Ruiqi Baiyun, Yueying Lv, Qian Lu, Zhigang Zhang PII: DOI: Reference:

S0162-0134(17)30602-5 doi:10.1016/j.jinorgbio.2017.11.010 JIB 10370

To appear in:

Journal of Inorganic Biochemistry

Received date: Revised date: Accepted date:

24 August 2017 4 November 2017 4 November 2017

Please cite this article as: Xiao Tan, Biying Liu, Siyu Li, Jingjing Lu, Ruiqi Baiyun, Yueying Lv, Qian Lu, Zhigang Zhang , Dietary luteolin protects against HgCl2-induced renal injury via activation of Nrf2-mediated signaling in rat. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jib(2017), doi:10.1016/j.jinorgbio.2017.11.010

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ACCEPTED MANUSCRIPT Dietary luteolin protects against HgCl2-induced renal injury via activation of Nrf2-mediated signaling in rat

Xiao Tana, 1, Biying Liua, 1, Siyu Lia, Jingjing Lua, Ruiqi Baiyuna, Yueying Lva,

College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street,

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a

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Qian Lua, Zhigang Zhanga,b,*

Harbin, China

Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine, 59

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Mucai Street, Harbin 150030, China

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b

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*Corresponding author:

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Professor Zhigang Zhang, College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Harbin 150030, China

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Phone: 86-0451-55190863

1

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E-mail: [email protected] These two authors contributed equally to this work.

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ACCEPTED MANUSCRIPT Abstract Luteolin (Lut) belongs to the flavonoid family with various beneficial bioactivities. Here, we investigated whether Lut attenuate mercuric chloride (HgCl2)-induced renal injury in rat. We found that oral gavage administration of Lut (80 mg/kg) alleviated

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anemia and renal histology upon HgCl2 treatment (80 mg/L). Lut also significantly

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reduced HgCl2-induced oxidative stress and inflammatory, presenting as the reduced

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malondialdehyde (MDA) formation, increased glutathione (GSH) level, and inhibited activation of nuclear factor kappa B (NF-κB). Moreover, Lut protected renal cells

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from HgCl2-induced apoptosis, as assessed by Terminal deoxynucleotidyl transferase

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dUNT nick end labeling (TUNEL) assay and the protein levels of B-cell lymphoma gene 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), Bcl-2-associated X protein

Lut

increased

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Furthermore,

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(Bax), and p53. Interestingly, Lut reduced renal mercuric accumulation in rat. nuclear

translocation

of

the

nuclear

factor-erythroid-2-related factor 2 (Nrf2), and subsequent protein expression of the

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antioxidant enzymes, heme oxygenase-1 (HO-1) and nicotinamide adenine

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dinucleotide phosphatase: quinone-acceptor 1 (NQO1). Our results suggest that Lut suppress HgCl2-induced renal injury via activation of Nrf2 signaling pathway.

Keywords: Luteolin; HgCl2; Renal injury; Nrf2; NF-κB; Apoptosis

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ACCEPTED MANUSCRIPT 1. Introduction Mercury, as one of the most toxic metallic contaminants, is released from natural emissions and anthropogenic pollutants [1]. Mercury included elemental, organic, and inorganic forms [2]. Excessive levels of Hg2+ can be found in vegetables, cereals, and

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seafood products [3,4]. Thus, mercury gradually accumulates in human and animal

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body corresponding to the onset of dietary exposure [5]. The Environmental

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Protection Agency in the United States sets the normal blood mercury concentrations in adults less 5.0 ug/L [6]. However, blood mercury concentration levels of adults

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were far beyond this value in many regions [7-9].

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Large amounts of Hg2+ accumulate in the kidney after exposure to elemental or inorganic forms of mercury, excess of which can induce renal injury [10]. Mercuric

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chloride (HgCl2) caused renal injury by inducing oxidative stress [11]. Oxidative

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stress has been implicated in mitochondrial injury and led to apoptosis. In addition, accumulating evidence suggested that oxidative stress plays a key role in modulating

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inflammation [12]. Therefore, antioxidant may have the potential protective effect on

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HgCl2-induced renal injury. Currently, mercury poisoning is primarily treated with metal chelating agents, such as meso-2, 3-dimercaptosuccinic acid and 2, 3-dimercaptopropanesulfonic acid, however, these agents can cause varying degrees of damage to the kidney [13]. Therefore, we sought to identify a drug with no side effects, believing that a foodborne natural product would have this characteristic. We previously reported remarkable results in prevention and treatment of arsenic poisoning using natural 3

ACCEPTED MANUSCRIPT products [14-16]. Luteolin (Lut, 3’,4’,5,7-tetrahydroxyflavone) is abundant in vegetables, fruits, and other natural plants, such as celery, green pepper, parsley, perilla leaf, and chamomile tea [17]. Lut was reported to have a wide variety of biological properties, including

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low toxicity, no mutagenicity, cell protection, sedating, anti-tumor, anti-oxidant,

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anti-inflammatory, and anti-apoptotic activities [17,18]. In recent years, Lut was

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shown to provide neuroprotection in models of traumatic brain injury via the nuclear factor-erythroid-2-related factor 2 (Nrf2) signaling pathway [19]. Lut also attenuated

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adipocyte-derived inflammatory responses via suppression of nuclear factor kappa B

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(NF-κB) signaling pathway [20]. In addition, Lut ameliorated cisplatin-induced renal apoptosis [21]. These findings suggested that Lut inhibited oxidative stress,

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inflammation, and apoptosis. However, the effects of Lut on HgCl2-induced renal

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injury have not been investigated so far. Consequently, based on the toxic effects of HgCl2 and the pharmacological

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properties of Lut, we investigated whether Lut would protect against HgCl2-induced

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renal injury and explored its detailed mechanism. 2. Materials and methods 2.1. Animals and treatments Twenty-eight 6–8-week-old healthy male Wistar rats, with a mean weight of 100–140 g, from the Experimental Animal Centre of Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences (Harbin, China) were housed in the Animal Quarters of the Northeast Agricultural University at 22 ± 2°C on a 12-h 4

ACCEPTED MANUSCRIPT light/dark cycle. They were allowed free access to standard rodent chow and tap water. After a week, twenty-eight rats were randomly divided into the following four groups: control, Lut, HgCl2, and HgCl2 + Lut groups (n = 7). The experimental treatments were administered for 56 d. The control group drank water; the Lut group drank water

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and was treated with Lut (80 mg/kg per day in 1% dimethylsulfoxide (DMSO)

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intragastrically) beginning on day 43; the HgCl2 group drank water with 80 mg/L

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HgCl2; the HgCl2 + Lut group drank water with 80 mg/L HgCl2 and was administered Lut (80 mg/kg per day in 1% DMSO intragastrically) beginning on day 43. Lut

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(purity, >98%) was from Xi’an Weiao (Shanxi, China). HgCl2 was from Beijing

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Chemical Factory (Beijing, China). The experiments that involved experimental animals were carried out in accordance to the Institutional Guidelines of the Northeast

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Agricultural University for the Care and Use of Experimental Animals and were

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approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University.

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2.2. Sample collection

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The rats were fasted after administration of the last treatment and placed in a metabolic cage for 24-h urine collection. Urine samples were obtained to measure total mercuric concentrations. After urine collection, all rats were anesthetized with ether on day 57. Blood samples were then collected by puncturing the retro-orbital venous sinus. Samples were centrifuged (4000 × g for 10 min) and serum was prepared for analysis. The kidneys were then harvested and each divided into 3 pieces. One was placed in a brown bottle for immersion in 10% formalin at 37°C for 24 h and 5

ACCEPTED MANUSCRIPT then used for histopathology and Terminal deoxynucleotidyl transferase dUNT nick end labeling (TUNEL) assay. The second piece was maintained at 4°C and malondialdehyde (MDA) and glutathione (GSH) levels of renal tissue were determined. The remaining renal tissue was stored at −80°C.

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2.3. Determination of blood index

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We used an automated Auto Hematology Analyzer BC-2600Vet (Mindray,

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Shenzhen, China) to determine the levels of white blood cells (WBC), red blood cell (RBC), and hemoglobin (HGB). Creatinine (CREA) and blood urea nitrogen (BUN)

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levels were assayed using UniCel DxC800 Synchron (Bekman, USA).

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2.4. Histopathological examination of the kidney

For histopathology, midsections of the kidney, each with a thickness of 1–2 mm,

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were prepared from each rat. They were fixed (10% formalin for 24 h, 37°C),

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dehydrated, embedded in paraffin and cut into 3 µm sections. The sections were then stained with hematoxylin and eosin (H&E) and examined under a light microscope

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(BX-FM; Olympus Corp, Tokyo, Japan).

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2.5. Measurements of MDA and GSH in the kidney MDA and GSH levels were measured using assay kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s protocols. 2.6. TUNEL assay Apoptotic cells were stained using a TUNEL detection kit (Keygen, Nanjing, China) using the manufacturer’s protocol. Briefly, sections were deparaffinized and rehydrated, then washed with PBS (3 × 3 min) followed by 1% Triton-100 at room 6

ACCEPTED MANUSCRIPT temperature for 15 min. After washing again with PBS (3 × 5 min), sections were treated with 3% H2O2-methanol for 15 min. Each was then washed with PBS (3 × 5 min) and treated with proteinase K (100 µL for 30 min) at 37°C. Sections were then incubated with 100 µL streptavidin-fluorescein isothiocyanate (FITC) working

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solution in the dark, in a humidified atmosphere, at 37°C for 1 h. Samples were then

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washed with PBS (3 × 5 min) and sections were incubated with 100 µL DAPI

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working solution and incubated in the dark for 5 min, then washed again with PBS (3 × 5 min). Finally, samples were observed by fluorescence microscopy, with apoptotic

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cells stained green and normal cells stained blue. The apoptosis rate = (total number

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of apoptotic cells / the total number of cells) × 100%. 2.7. Western blot analysis

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Nrf2, heme oxygenase-1 (HO-1), nicotinamide adenine dinucleotide phosphatase:

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quinone-acceptor 1 (NQO1), NF-κB, tumor necrosis factor alpha (TNF-α), B-cell lymphoma gene 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), Bcl-2-associated X

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protein (Bax), and p53 proteins were detected by western blotting. Primary antibodies

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against each protein were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies to GAPDH (Xianzhi, Hangzhou, China) were used as a standard control, while Histone H3 (Cell Signaling Technology, Beverly, MA, USA) was used as controls for nuclear Nrf2 and NF-κB respectively. Radio immunoprecipitation buffer (Beyotime Institute of Biotechnology, Jiangsu, China) and nuclear and cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology, Jiangsu, China) were used to protein extract from kidney tissue, respectively. Nuclear and cytoplasmic fractions 7

ACCEPTED MANUSCRIPT were used for Nrf2 and NF-κB immunoblotting. In total protein extract, protein expressions of Nrf2, HO-1, NQO1, NF-κB, TNF-α, Bcl-2, Bcl-xL, Bax, and p53 were determined. The protein concentration was estimated using BCA reagent (Beyotime Institute of Biotechnology, Jiangsu, China), and equal amounts of the proteins were

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separated on 12% SDS–PAGE and transferred to polyvinylidine difluoride membrane.

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Membranes were briefly washed in Tris-buffered saline (24.2 g Tris，80 g NaCl，1000

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mL distilled water) containing 0.05% Tween-20 (TBST) and blocked for 1 h in 5% nonfat dry milk (5 g nonfat dry milk, 100 mL TBST). Membranes were then

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incubated overnight at 4°C with appropriate antibodies diluted in 5% nonfat dry milk.

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They were then washed 4 times for 10 min in TBST and incubated with secondary antibody appropriately diluted in TBST for 1 h at room temperature. The membrane

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was then washed four times in TBST and bands detected using the Imager Amersham

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600 chemiluminescence system (General Electric Company, Fairfield, CT, USA). 2.8. Total mercuric analysis in the kidney and urine

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Briefly, approximately 0.5 g renal tissue and 1 mL urine were digested with a

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HNO3–H2SO4 solution, with heating. After samples were completely digested, they were diluted with deionized water. Mercuric concentrations were measured with a 930 System Atomic Fluorescence Spectrometer (Beijing Jitian Instrument Company, Beijing, China). Total mercuric concentrations in the kidney were expressed as mg/kg and total mercuric concentrations in the urine were expressed as mg/L. 2.9. Statistical analysis Data are presented as mean ± standard error of the mean (SEM). Statistical 8

ACCEPTED MANUSCRIPT analyses were performed with SPSS 19.0 software (SPSS, Chicago, IL, USA). Shapiro-Wilk was performed to assess the normality of the data, and Levene's Test for equality of variances was performed. One-way analysis of variance was used to determine differences among 4 groups. Tukey Test for post hoc multiple comparison

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was used to determine differences between means. A two-tailed p < 0.05 was

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considered as being significant.

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3. Results 3.1. Effects of Lut on blood index

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WBC level in the HgCl2 group was increased significantly compared with that in

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the control group (p < 0.05), but the WBC level in the HgCl2 + Lut group was significantly lower than in the HgCl2 group (p < 0.05, Fig. 1A). RBC (Fig. 1B) and

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HGB (Fig. 1C) levels in the HgCl2 group were reduced compared with those in the

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control group. However, Lut treatment caused a significant increase (p < 0.05) in the levels of RBC and HGB compared with those in the HgCl2 group.

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Serum CREA and BUN are useful markers and measuring their levels is a simple

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and convenient method to evaluate renal dysfunction. Therefore, we analyzed effects of HgCl2 and Lut on serum CREA (Fig. 1D) and BUN (Fig. 1E) levels. CREA and BUN levels were significantly higher in the HgCl2 group compared with in the control group (p < 0.05), but Lut significantly (p < 0.05) prevented the effects of HgCl2 on these markers. 3.2. Effects of Lut on renal histopathology Histopathological changes are direct indicators of renal injury. The H&E-stained 9

ACCEPTED MANUSCRIPT renal tissues from the control (Fig. 2A) and the Lut (Fig. 2B) groups appeared to have normal kidney tubules. In contrast, HgCl2-induced histopathological changes were evident in renal tissues, including tubular accumulation of protein material (protein cylindruria), destruction of tubular structures, necrosis and disorganization,

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inflammatory cell infiltration, interstitial fibrosis, dilation, and hyperemia of

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glomerular capillaries (Fig. 2C). Lut treatment significantly diminished these

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HgCl2-induced pathologies (Fig. 2D).

3.3. Effects of Lut on MDA and GSH levels in the kidney

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MDA and GSH levels in the kidney from all groups are shown in Fig. 3. In the

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HgCl2 group, the MDA (Fig. 3A) content in the kidney was far higher than in the control group (p < 0.05) and the GSH (Fig. 3B) content was lower (p < 0.05).

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However, Lut treatment markedly reversed these alterations (p < 0.05).

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3.4. Effects of Lut on the Nrf2-mediated signaling pathway To investigate the molecular basis of a protective effect of Lut against

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HgCl2-induced renal oxidative stress and inflammatory response, we examined

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whether Lut treatment affected renal expression of nuclear Nrf2 (Fig. 4A), total HO-1 (Fig. 4B), total NQO1 (Fig. 4C), nuclear NF-κB (Fig. 4D), cytosolic NF-κB (Fig. 4E), and total TNF-α (Fig. 4F). HgCl2 markedly reduced expression of nuclear Nrf2, total HO-1, total NQO1, and cytosolic NF-κB and increased expression of nuclear NF-κB and total TNF-α (p < 0.05). However, compared with the group receiving only HgCl2, Lut signifcantly reversed these effects (p < 0.05). 3.5. Effects of Lut on renal apoptosis, Bcl-2 family proteins, and p53 10

ACCEPTED MANUSCRIPT To determine whether the renoprotective effects of Lut were further detectable at the histopathological level, we employed TUNEL staining to examine apoptotic cells in the renal tissues (Fig. 5A). There were few TUNEL-positive cells in the control and Lut groups. HgCl2 alone induced an apoptotic rate of approximately 15.57%, while

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Lut treatment significantly decreased this HgCl2-induced apoptotic rate to 8.28% (p <

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0.05, Fig. 5B).

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We further investigated the molecular basis of a protective effect of Lut against HgCl2-induced renal apoptosis. Treatment of rats with HgCl2 decreased expression of

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anti-apoptotic Bcl-2 (Fig. 5C, p < 0.05) and Bcl-xL (Fig. 5D, p < 0.05). In

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HgCl2-treated rats, Lut administration significantly attenuated these decreases in expression of Bcl-2 and Bcl-xL (p < 0.05). We also evaluated expression of

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pro-apoptotic Bax and found it significantly increased after HgCl2 treatment (p <

(Fig. 5E, p < 0.05).

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0.05). However, added administration of Lut significantly reduced Bax expression

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In addition, p53 expression was also increased after HgCl2 treatment compared

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with in the control group (p < 0.05). Lut attenuated this HgCl2-induced increase of p53 expression (Fig. 5F, p < 0.05). 3.6. Effects of Lut on total mercuric concentrations in the kidney and urine To further examine how Lut prevented HgCl2-induced renal toxicity, mercuric retention in the kidney was measured. In the kidney (Fig. 6A), the total concentrations of mercury in the HgCl2 group were significantly higher than in the control group (p < 0.05). Treatment with Lut significantly attenuated mercuric retention (p < 0.05). Urine 11

ACCEPTED MANUSCRIPT is an important route of mercuric excretion. The total urine mercuric concentrations in the HgCl2 + Lut group were greater than in the HgCl2 group (Fig. 6B, p < 0.05). 4. Discussion Inorganic mercury is an important environmental pollutant that threatens human

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health. The most prominent effect of mercury is nephortoxicity [22]. The increased

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WBC count can be used as a biomarker of heavy metal intake [23]. Here, Lut

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effectively reduced the increased number of WBC caused by HgCl2, suggesting that Lut attenuated HgCl2-induced toxic effects partially through reducing total mercury

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accumulation in rats. Additionally, complete blood analysis indicated that HgCl2

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induced anemia in rats. Erythropoietin comes from the kidney and stimulates RBC and HGB production in adult mammals. Patients with renal injury are often anemic

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due at least to inadequate erythropoietin formation [24]. In our study, Lut alleviated

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the anemia induced by HgCl2 probably via restoring the production of erythropoietin. We speculate that Lut protected rats against HgCl2-induced renal injury through

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ameliorating anemia and decreasing WBC.

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The levels of serum CREA and BUN reflect the function of kidney [25]. Consistent with the renal histological observations, Lut effectively ameliorated HgCl2-induced renal injury. Hg2+ is characterized by higher affinity to thiol groups after entering the kidney, causing depletion of intracellular thiol groups and excessive production of reactive oxygen free radicals [26]. Measurements of lipid peroxidation production MDA and non-enzymatic antioxidant GSH indicated that oxidative stress involves in HgCl2-induced renal injury. The administration of Lut significantly 12

ACCEPTED MANUSCRIPT restored the levels of GSH and MDA induced by HgCl2. Therefore, the possible mechanism underlying the protective effect of Lutin HgCl2-induced renal injury is the inhibition of oxidative stress. Reactive oxygen species accumulation leads to mitochondrial dysfunction and

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DNA damage, which mediates and accelerates apoptosis [27-29]. The activation of

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p53, caused by DNA damage, serves as a major mediator of cellular stress [30]. Our

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result indicated that p53-mediated apoptosis occurred in HgCl2-induced renal injury. p53 modulates protein–protein interactions with members of the Bcl-2 family proteins, to

activation

of

Bax,

thereby

promotes

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leads

mitochondrial

membrane

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permeabilization and induces apoptosis [31]. In our study, Lut suppressed p53, accompanied by a decrease in the level of proapoptotic member Bax and increase in

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the levels of pro-survival members Bcl-2 and Bcl-xL. Furthermore, many studies have

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shown that NF-κB is activated by a wide range of biological stimuli, including oxidative stress [32], subsequently enhances transcriptional activation of target genes,

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including TNF-α. TNF-α have been found to promote inflammatory response [33].

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Lut inhibited renal inflammatory, as evidenced by the fact that Lut inhibited HgCl2-induced NF-κB nuclear translocation and the expression of TNF-α. Taken together, all these results indicated that Lut attenuated HgCl2-mediated inflammation and apoptosis caused by oxidative damage. It is also noteworthy that, some studies showed that NQO1 stabilized the tumor-suppressor p53 [34]. HO-1 could upregulate expression of Bcl-2 and Bcl-xL, and inhibite nuclear translocation of NF-κB [35]. They are both downstream proteins of Nrf2. A possible explanation is that Lut 13

ACCEPTED MANUSCRIPT protects kidney injury induced by HgCl2 via activating the Nrf2 pathway. Transcription factor Nrf2 is involved in protecting against oxidative damage [36,37]. Nrf2 translocates to the nucleus and forms heterodimers with other nuclear proteins and consequent activation of phase II detoxifying enzymes, such as HO-1

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and NQO1 [38,39], which thereby improves the antioxidant capacity of the body

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[40,41]. In our study, Lut treatment increased the Nrf2 nuclear translocation and

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enhanced expressions of NQO1 and HO-1. Thus, Lut plays a critical role in the

anti-apoptosis, and anti-inflammation.

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activation of Nrf2, which attenuates HgCl2-induced kidney toxicity by antioxidant,

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Interestingly, our results showed that Lut reduced mercuric accumulation in the kidney and significantly increased urinary excretion of Hg, which may play a key role

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in the protective effect of Lut against HgCl2-induced renal injury. Protection against

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mercury in the kidney has been shown to be associated with metallothionein (MT) [42], which contains a large number of cysteine residues and thiol groups. When Hg2+

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enter the kidney, some of the MT reacts with free radicals induced by HgCl2 and

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another portion forms Hg-MT complexes [42,43], which is excreted by urine [10]. This can promote detoxification to a certain extent. Therefore, we predicted that Lut might stimulate the kidney to produce more MT to increase the organ's capacity for binding with mercury ions and, eventually, increasing mercuric excretion in the urine. Future studies aim at further proof. 5. Conclusion

In conclusion, as shown in Fig. 7, our findings revealed that dietary luteolin 14

ACCEPTED MANUSCRIPT protects against HgCl2-induced renal injury via activation of Nrf2-mediated signaling pathway. This study provides beneficial evidence for the application of Lut supplementation may serve as an alternative treatment for renal injury. Conflict of interest

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None.

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Abbreviation

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Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma gene 2; Bcl-xL, B-cell lymphoma-extra large; BUN, blood urea nitrogen; CREA, creatinine; DMSO,

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dimethylsulfoxide; FITC, fluorescein isothiocyanate; GSH, glutathione; HGB,

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hemoglobin; HgCl2, mercuric chloride; HO-1, heme oxygenase-1; Lut, Luteolin; MDA, malondialdehyde; MT, metallothionein; NF-κB, nuclear factor kappa B; NQO1,

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nicotinamide adenine dinucleotide phosphatase: quinone-acceptor 1; Nrf2, nuclear

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factor-erythroid-2-related factor 2; RBC, red blood cell; TNF-α, tumor necrosis factor-α; TUNEL, Terminal deoxynucleotidyl transferase dUNT nick end labeling;

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WBC, white blood cell

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Acknowledgments

This work was funded by the National Science Foundation of China (31101868), Scientific Research Foundation for the Returned Overseas Chinese Scholars of Heilongjiang Province (LC2017007), Financial Assistance from Postdoctoral Scientific Research Developmental Fund of Heilongjiang Province (LBH-Q16013), Scientific Research Foundation for Excellent Returned Scholars of Heilongjiang Province, University Nursing Program for Young Scholar with Creative Talents in 15

ACCEPTED MANUSCRIPT Heilongjiang Province (UNPYSCT-2016012), and Academic Backbone Support

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NU

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Program (15XG17) approved by Northeast Agricultural University.

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Fig. 1. Effects of Lut on blood index. Samples were collected from control, Lut, HgCl2, and HgCl2 + Lut groups. (A) WBC, (B) RBC, (C) HGB, (D) CREA, and (E) BUN levels were measured. Data are the mean ± SEM, n = 7. * significantly different (p < 0.05) from control group and # significantly different (p < 0.05) from HgCl2 group.

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Fig. 2. Effects of Lut on renal histopathology. Paraffin sections of renal tissues

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were stained with hematoxylin and eosin (200 ×).

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Fig. 3. Effects of Lut on MDA and GSH levels in the kidney. Samples were collected from control, Lut, HgCl2, and HgCl2 + Lut groups. (A) MDA and (B) GSH

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Fig. 4. Effects of Lut on the Nrf2 signaling pathway and inflammatory. Western blot to evaluate the expression levels of (A) nuclear Nrf2, (B) total HO-1, (C) total NQO1, (D) nuclear NF-κB, (E) cytosolic NF-κB, and (F) total TNF-α in control, Lut, HgCl2, and HgCl2 + Lut group’s kidneys. GAPDH and Histone H3 were used as standard control. Data are the mean ± SEM, n = 7. * significantly different (p < 0.05) 26

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from control group and # significantly different (p < 0.05) from HgCl2 group.

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Fig. 5. Effects of Lut on renal apoptosis, Bcl-2 family proteins, and p53. Paraffin sections of renal tissues were treated with TUNEL staining and imaged by fluorescent

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TUNEL-positive cells to the total number of renal tissues. (A) Representative photographs of TUNEL staining in control, Lut, HgCl2, and HgCl2 + Lut groups.

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Scale bar = 20 µm. (B) Quantitative analysis of TUNEL-positive cells content in

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control, Lut, HgCl2, and HgCl2 + Lut groups. Data are the mean ± SEM, n = 7. * significantly different (p < 0.05) from control group and # significantly different (p <

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Western blot to evaluate the expression level of (C) Bcl-2, (D) Bcl-xL, (E) Bax,

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and (F) p53 in control, Lut, HgCl2, and HgCl2 + Lut group’s kidneys. GAPDH was used as a standard control. Data are the mean ± SEM, n = 7. * significantly different (p < 0.05) from control group and # significantly different (p < 0.05) from HgCl2 group.

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Fig. 6. Effects of Lut on total mercuric concentrations in the kidney and urine. Samples were collected from control, Lut, HgCl2, and HgCl2 + Lut groups. The total

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mercuric concentrations of (A) kidney and (B) urine were measured. Data are mean ±

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Fig. 7. The mechanism of Lut on protecting against HgCl2-induced renal injury.

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Lut protected against HgCl2-induced renal injury by alleviating oxidative stress and inflammation through regulating the Nrf2/NF-κB signaling pathway and inhibiting

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apoptosis by regulating Bcl-2 family proteins expression.

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Synopsis Potential mechanism of luteolin in HgCl2-induced renal injury. Luteolin protected against HgCl2-induced renal injury by alleviating oxidative stress and inflammation through regulating the nuclear factor-erythroid-2-related factor 2 (Nrf2) / nuclear factor kappa B (NF-κB) signaling pathway and inhibiting apoptosis by regulating B-cell lymphoma gene 2 (Bcl-2) family proteins expression.

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ACCEPTED MANUSCRIPT Highlights Oxidative stress is involved in HgCl2-induced renal injury.

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Dietary luteolin reduced renal mercuric accumulation.

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Dietary luteolin promoted mercuric excretion in the urine.

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