MyD88 signaling pathway

MyD88 signaling pathway

Pharmacological Research 111 (2016) 509–522 Contents lists available at ScienceDirect Pharmacological Research journal homepage: www.elsevier.com/lo...

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Pharmacological Research 111 (2016) 509–522

Contents lists available at ScienceDirect

Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs

Dioscin alleviates lipopolysaccharide-induced inflammatory kidney injury via the microRNA let-7i/TLR4/MyD88 signaling pathway Meng Qi, Lianhong Yin, Lina Xu, Xufeng Tao, Yan Qi, Xu Han, Changyuan Wang, Youwei Xu, Huijun Sun, Kexin Liu, Jinyong Peng ∗ College of Pharmacy, Dalian Medical University, Western 9 Lvshunnan Road, Dalian 116044, China

a r t i c l e

i n f o

Article history: Received 14 April 2016 Received in revised form 15 June 2016 Accepted 14 July 2016 Available online 16 July 2016 Keywords: Lipopolysaccharide Inflammatory acute kidney injury Micro let7i TLR4 Dioscin MyD88

a b s t r a c t We previously reported the potent effect of dioscin against renal ischemia/reperfusion injury, but little is known about the role of dioscin in lipopolysaccharide (LPS)-induced inflammatory kidney injury. The present work aimed to investigate the effects and potential mechanisms of dioscin in preventing LPS-induced kidney injury. In vivo injury was induced in rats and mice with an intraperitoneal injection of LPS (10 mg/kg), and in vitro studies were performed on NRK-52E and HK-2 cells challenged with LPS (0.5 ␮g/ml). Our results indicated that dioscin significantly protected against renal damage by decreasing blood urea nitrogen and creatinine levels and reversing oxidative stress. Mechanistic studies demonstrated that dioscin markedly up- regulated the level of the microRNA let-7i, resulting in significant inhibition of TLR4 expression. Dioscin significantly down-regulated the levels of MyD88, NOX1 and cleaved caspase-8/3; inhibited the nuclear translocation of NF-␬B; inhibited PI3K and Akt phosphorylation; increased the levels of SOD2; and decreased the mRNA levels of IL-1␤, IL-6, MIP-1␣, Fas and FasL. In vitro, transfection of microRNA let-7i inhibitor and TLR4 DNA were applied, and the results further confirmed the nephroprotective effect of dioscin in suppressing TLR4/MyD88 signaling and subsequently inhibiting inflammation, oxidative stress and apoptosis. Furthermore, the abrogation of cellular MyD88 expression by ST2825 eliminated the inhibitory effect of dioscin on the levels of nuclear NF-␬B, cleaved caspase-3, SOD2 and ROS. These data indicated that dioscin exerted a nephroprotective effect against LPS-induced inflammatory renal injury by adjusting the microRNA let-7i/TLR4/MyD88 signaling pathway, which provided novel insights into the mechanisms of this therapeutic candidate for the treatment of inflammatory kidney injury. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction

Abbreviations: LPS, lipopolysaccharide; MDA, malondialdehyde; NO, nitric oxide; ROS, reactive oxygen species; GSH, Px glutathione peroxidase; CAT, catalase; TLR4, toll like receptor 4; MyD88, myeloid differentiation primary response gene; NOX1, NADPH oxidase 1; NF-␬B, nuclear factor kappa B; PI3K, phosphatidylinositol 3-kinases; Akt, serine/threonine kinase; SOD2, superoxide dismutase; IL-1␤, interleukin-1␤; IL-6, interleukin-6; MIP-1␣, macrophage inflammatory protein1␣; Fas, factor associated with suicide; FasL, factor associated with suicide ligand; AKI, acute kidney injury; ICU, intensive care unit; ATN, acute tubular necrosis; miRs, microRNAs; TLRs, toll-like receptors; TUNEL, Terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling; FADD, factor associated with suicideassociated death domain-containing protein; DMSO, dissolving with dimethyl sulfoxide; CMC-Na, sodium carboxylmethyl-cellulose; DMEM, Dulbecco’s minimum essential medium; FBS, Fetal bovine serum. ∗ Corresponding author. E-mail address: [email protected] (J. Peng). http://dx.doi.org/10.1016/j.phrs.2016.07.016 1043-6618/© 2016 Elsevier Ltd. All rights reserved.

Acute kidney injury (AKI), a critical care syndrome, is characterized by a rapid decrease in renal function, which is responsible for substantial resource utilization and mortality in intensive care unit (ICU) patients [1]. The clinical conditions leading to AKI are caused by various factors, including sepsis-induced infection [2], cardiac surgery [3], liver or kidney transplantation [4], and contrast media-induced nephropathy [5]. Endotoxic (lipopolysaccharide, LPS) shock, the secondary outcome of systemic infections, remains the most common trigger for AKI. Sepsis-induced AKI results in up to 50% of mortality in ICU patients [6]. Therefore, it is necessary to seek effective therapeutic methods or pharmaceuticals to reduce the mortality of AKI in the clinic. Acute tubular necrosis (ATN) is usually caused by ischemia/reperfusion injury and LPS-mediated inflammatory responses [7]. LPS is derived from the cell wall of Gram-negative bacilli [8] and stimulates a renal inflammatory cascade resulting

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from cytokine-chemokine responses that can result in kidney end-organ damage. The factors involved in LPS-induced AKI include renal hemodynamic changes, endothelial dysfunction, renal interstitial inflammatory infiltration, microthrombus in the glomeruli and renal tubular congestion, among others [9]. Some inflammatory cytokines that are released from renal cells play critical roles in the pathogenesis of AKI [10]. Although sepsis is the most important cause of AKI, the underlying mechanisms are not completely understood. Numerous basic and clinical experiments have demonstrated that inflammation plays a vital role in the primary and secondary injury phases of septic AKI [11,12]. LPS activates a large number of pro-inflammatory cytokines, which can exacerbate the production of reactive oxygen species (ROS) and trigger tubular cell death. Therefore, the development of a novel and effective anti-inflammatory drug may be an efficient method to reverse LPS-induced AKI [13]. MicroRNAs (miRs) are characterized by the inhibition of mRNA transcription or the promotion of mRNA degradation in the occurrence and development of various diseases [14]. Many studies have reported that miR-let-7i, a member of the let-7 family of miRs, targets the Toll-like receptor 4 (TLR4) gene and inhibits its expression [15,16]. Toll-like receptors (TLRs), a family of 13 identified members in mammals, are evolutionarily conserved, widely expressed and belong to type I transmembrane proteins [17]. TLR4, a leading receptor for LPS, can regulate innate and adaptive immune responses, and may have a pathophysiological role in inflammation [18]. Upon stimulation with LPS, TLR4 can activate two classic signaling pathways: the myeloid differentiation factor 88 (MyD88)-dependent and -independent pathways [19]. LPS induces the formation of a signaling complex between MyD88 and phosphatidylinositol 3-kinase (PI3K). Serine/threonine kinase (Akt), the downstream target of PI3K, triggers inflammatory cytokine release and stimulates nuclear factor kappa B (NF-␬B) translocation [20]. NADPH oxidase 1 (NOX1) is adjusted by LPS through MyD88-dependent inflammatory signaling, which leads to ROS generation and a reduction in antioxidant enzymes [21]. In addition, MyD88 transforms pro- caspase-8 into cleaved caspase-8 through a series of downstream signals, followed by an increase in the levels of cleaved caspase-3 and the expression of several apoptosis-related genes [22]. Therefore, inhibiting the TLR4/MyD88 signaling pathway via up-regulating miR-let-7i to suppress inflammation-mediated oxidative stress and apoptosis represents a potential nephroprotective treatment strategy. Traditional Chinese medicines have been used in China to prevent and treat diseases for thousands of years, and some active natural products, including alpinetin from Alpinia katsumadai Hayata [23], leonurine from Leonurus cardiaca [24] and an extract from Cordyceps sobolifera [25], have been applied to treat LPS-mediated AKI. Thus, natural products from medicinal herbs are promising for the treatment of LPS- induced AKI. Dioscin (Dio, shown in Supplemental Fig. 1), a natural steroid saponin, is isolated from various herbs [26]. Pharmacological investigations have shown that dioscin has anti-tumor, antihyperlipidemic and anti-fungal activities [27–29]. Our previous studies have indicated that dioscin has potent effects against carbon tetrachloride (CCl4 )-and paracetamol-induced acute liver damage [30,31] and non-alcoholic fatty liver disease (NAFLD) [32] and anti-inflammatory activities against hepatic ischemia/reperfusion damage [33], cerebral injury [34], renal injury [35] and alcoholic liver fibrosis [36]. However, no studies have reported the effects and molecular mechanisms of dioscin in preventing LPS-induced AKI. Therefore, we explored the effects and possible mechanisms of dioscin in preventing LPS-induced inflammatory kidney injury.

2. Materials and methods 2.1. Chemicals and reagents Dioscin with the purity of over 98% was isolated from Dioscorea nipponica Makino in our laboratory [26,37,38]. Dioscin with a purity >99% was purchased from Shanghai Tauto Biochemical Technology Co., Ltd. (Shanghai, China). The dioscin was added to the serum-free medium after it was dissolved in a final concentration of dimethyl sulfoxide (DMSO) of less than 0.1% for the in vitro experiments or suspended in 0.5% sodium carboxylmethyl-cellulose (CMC-Na) for the in vivo experiments. The BUN, Cr, MDA, NO, GSH-Px, and CAT kits were obtained from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). The tissue protein extraction kit was obtained from Keygen Biotech. Co., Ltd. (Nanjing, China). The bicinchoninic acid (BCA) protein assay kit and Nuclear and Cytoplasmic Protein Extraction Kit were purchased from the Beyotime Institute of Biotechnology (Jiangsu, China). 4 ,6 -Diamidino-2-phenylindole (DAPI), tris (hydroxymethyl) aminomethane (Tris), sodium dodecyl sulfate (SDS) and CMC-Na were purchased from Sigma-Aldrich (St. Louis, MO, USA). Penicillin and streptomycin were obtained from Hyclone Laboratories, Inc. (MA, USA). Dulbecco’s minimum essential medium (DMEM), fetal bovine serum (FBS) and trypsin were purchased from Gibco (CA, USA). TRIZOL, a PrimeScript® RT Reagent Kit with gDNA Eraser (Perfect Real Time), and SYBR® Premix Ex TaqTM II (Tli RNase H Plus) were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). The SanPrep Column MicroRNA Mini-Prep Kit, MicroRNA First Strand cDNA Synthesis Kit and MicroRNA Quantitation PCR Kit were purchased from Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The miR-let-7i inhibitor and Lipofectamine 2000 were purchased from RiboBio Co., Ltd. (Guangzhou, China). LPS was purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Cell culture The NRK-52E normal rat kidney epithelial cell line and HK-2 human kidney tubular epithelial cell line were purchased from the Institute of Biochemistry Cell Biology (Shanghai, China) and maintained in DMEM supplemented with 10% FBS and antibiotics (100 IU/ml penicillin and 100 mg/ml streptomycin) in a humidified atmosphere of 5% CO2 and 95% O2 at 37 ◦ C. 2.3. Toxicity assay The NRK-52E and HK-2 cells were plated in 96-well plates at a density of 5 × 104 cells/ml per well for 24 h and treated with various concentrations of dioscin (0, 50, 100, 200, 400, 800 or 1000 ng/ml) for an additional 24 h. The cells were then analyzed according to the MTT method. The absorbance of the samples was quantified at 490 nm using a spectrophotometer (Thermo Fisher Scientific, MA, USA). 2.4. LPS-induced cell injury LPS was prepared in ultrapure water and used to make a series of working dilutions of 0.0625, 0.125, 0.25, 0.5, 1.0 and 2.0 ␮g/ml in serum-free DMEM for direct application to the cell cultures [39]. The NRK-52E and HK-2 cells were plated in 96-well plates at a density of 5 × 104 cells/ml per well for 24 h before they were challenged with various concentrations of LPS and then incubated for 24 h. Cell viability was assessed with the MTT assay. Based on the MTT results, the concentration of LPS that was sufficient to induce the cell injury was optimized.

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2.5. Cell viability assay

2.9. Western blotting assay

The NRK-52E and HK-2 cells were seeded in 96-well plates at a density of 5 × 104 cells/ml per well for 24 h and pretreated with various concentrations of dioscin (50, 100 or 200 ng/ml) for 6, 12 and 24 h before they were challenged with LPS (0.5 ␮g/ml) for 24 h. The model groups were cultured without the dioscin pretreatment, as described above, and the control groups were cultured in serumfree DMEM under normal conditions during the entire experiment. The MTT assay was used to assess cell viability.

The total, nuclear and cytoplasmic protein samples from the cells and kidney tissues were homogenized using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitor cocktail 2. The protein concentrations of the samples were determined using a BCA Protein Assay kit (Bio-Rad, Hercules, CA, USA) and standards. The proteins (50 ␮g) were denatured with sodium dodecyl sulfate (SDS) sample buffer, separated on 10% SDS-polyacrylamide gel electrophoresis (PAGE) gels, and then transferred to PVDF membranes (Millipore, Massachusetts, USA). After being blocked with 5% skim milk for 3 h at room temperature, the membranes were incubated with primary antibodies (listed in Supplemental Table 1) overnight at 4 ◦ C. After the addition of the anti-rabbit or antimouse secondary antibody for 2 h at room temperature, the protein bands on the membranes were detected using an enhanced chemiluminescence system and a Bio-Spectrum Gel Imaging System, respectively (UVP, California, USA). The intensity values of the bands were adjusted relative to the expression of the internal reference (IOD value of the target protein versus the IOD value of the corresponding internal reference).

2.6. LPS-induced acute kidney injury in vivo Male SD rats weighing 200–250 g (n = 60) and male C57BL/6J mice weighing 18–22 g (n = 60) were obtained from the Experimental Animal Center at Dalian Medical University (Dalian, China) (SCXK: 2013-0003). All experimental procedures were performed in strict accordance with PR China Legislation Regarding the Use and Care of Laboratory Animals, and all experiments were approved by the Animal Care and Use Committee of Dalian Medical University. After adapting to the new environment for one week, the rats were randomly divided into five groups (n = 12 per group) as follows: control, LPS-treated, and dioscin-treated (15, 30 or 60 mg/kg once daily for 7 days) groups, including LPS + Dio 60, LPS + Dio 30, and LPS + Dio 15. The mice were randomly divided into five groups (n = 12 per group) as follows: control, LPS-treated, and dioscin-treated (20, 40 or 80 mg/kg once daily for 7 days) groups, including LPS + Dio 80, LPS + Dio 40, and LPS + Dio 20. The animals in the control and model groups were administered 0.5% CMC-Na. The animals in the model groups and dioscin-treated groups were intraperitoneally injected with LPS (Sigma-Aldrich L3129, 10 mg/kg) at 6 h before sacrifice [40,41]. Blood samples were obtained from the rats and mice and centrifuged to produce serum. Then, the blood samples were stored at −20 ◦ C and the kidney samples were stored at −80 ◦ C for further analysis.

2.10. Quantitative real-time PCR assay The total RNA samples were extracted from the cells and kidney tissues using TRIZOL reagent (TaKaRa Biotechnology Co., Ltd., China) according to the manufacturer’s protocol. Then, a reverse transcription polymerase chain reaction (RT-PCR) was performed using a Prime-Script RT reagent kit (TaKaRa Biotechnology Co., Ltd., China) and a TC-512 PCR system (TECHNE, UK) after the concentration of the extracted RNA was measured. The forward (F) and reverse (R) primers for the tested genes are provided in Supplemental Table 2. Relative quantitation was performed using the Ct method for recurrent versus primary expression, with GAPDH as an endogenous control, and the fold changes were calculated for each gene. Eventually, the amount of the unknown template in our study was calculated using a standard curve.

2.7. Assessments of biochemical parameters

2.11. Quantification and silencing of miR-let-7i

The serum BUN and Cr levels were detected using specific kits according to the manufacturer’s instructions. The levels of MDA, NO, GSH-Px and CAT in the tissues were determined using commercial kits according to the manufacturers’ recommendations. All assays were performed in triplicate.

The total microRNA samples were isolated using the SanPrep Column MicroRNA Mini-Prep Kit (Sangon Biological Engineering Technology & Services Co., Ltd., China). Reverse transcription was performed using a MicroRNA First Strand cDNA Synthesis Kit, and the levels of the mature miRNA were quantified using real-time PCR with a MicroRNAs Quantitation PCR Kit (Sangon Biological Engineering Technology & Services Co., Ltd., China) and an ABI 7500 real-time PCR system (Applied Biosystems, USA) (the primers for miR-let-7i are listed in Supplemental Table 3). The U6 small nucleolar RNA (Sangon Biological Engineering Technology & Services Co., Ltd., China) was used for normalization. The NRK-52E and HK-2 cells were seeded in 6-well plates (2 × 105 cells/well) in serum-free medium and transfected with the inhibitor (50 nM) or negative controls mixed with Lipofectamine 2000 (RiboBio Co., Ltd., China) according to the manufacturer’s instructions (the sequence of the miR-let-7i inhibitor is listed in Supplemental Table 3). The negative controls consisting of random sequences had no detectable effects on the cell lines. The level of the TLR4 protein was detected to confirm whether the transfection was successful. Twenty-four hours after transfection, the cells were subjected to serum deprivation for 24 h in the presence or absence of the dioscin pretreatment (200 ng/ml) before they were challenged with LPS (0.5 ␮g/ml) for an additional 24 h. Then, the levels of the TLR4, cleaved caspase-3, SOD2 and nuclear p65 proteins were assayed. In addition, the ROS levels were determined

2.8. Histological and immunofluorescence assays The kidney tissues were fixed in 10% formalin, embedded in paraffin, and sectioned into 5-␮m slices. The slices were stained with hematoxylin-eosin (H&E) and photographed using a light microscope (Nikon Eclipse TE2000-U, NIKON, Japan) at 400 × magnification. For the immunofluorescence staining of TLR4, p-PI3K and NF-␬B, the tissue slices or formalin-fixed cells were incubated with anti-TLR4, anti-p-PI3K or anti-NF-␬B antibodies (listed in Supplemental Table 1) and an Alexa fluor-labeled secondary antibody, followed by DAPI (5 ␮g/ml), according to the manufacturer’s instructions. TUNEL staining was performed with an In Situ Cell Death Detection Kit (TMR Red; Roche, NJ, USA) according to the manufacturer’s instructions. The immunostained images were captured using a fluorescent microscope (Olympus BX63, Japan) at 200 × magnification. Immunofluorescence detection of NF-␬B was performed using a laser scanning confocal microscope (Leica, TCS SP5, Germany).

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Fig. 1. Effects of dioscin in protecting NRK-52E and HK-2 cells against LPS-induced injury. (A) Survival rates of NRK-52E and HK-2 cells treated with LPS (0.0625, 0.125, 0.25, 0.5, 1.0 or 2.0 ␮g/ml) for 24 h. (B) Effects of dioscin on the proliferation of cells challenged with LPS. NRK-52E and HK-2 cells were seeded in 96-well culture plates. After a 1-day incubation, the cells were pretreated with different concentrations of dioscin (50, 100 or 200 ng/ml) and challenged with LPS (0.5 ␮g/ml) for 24 h and then subjected to MTT assay. (C) Effects of dioscin on cell viability. NRK-52E and HK-2 cells were seeded in 96-well culture plates. After a 1-day incubation, the cells were treated with various concentrations of dioscin and then subjected to MTT assay after 6, 12 and 24 h incubation. (D) Effects of dioscin (50, 100 or 200 ng/ml) on the cellular morphology of NRK-52E and HK-2 cells for 12 h treatment under bright image (100 × magnification) observations. Data are presented as the mean ± SD (n = 5). * P < 0.05 and ** P < 0.01 compared with model groups.

and the number of apoptotic cells was determined using the TUNEL assay.

determined and the number of apoptotic cells were determined using TUNEL assay.

2.12. TLR4 overexpression DNA transfection 2.13. MyD88 inhibitor treatments in cells Transfection was performed when the NRK-52E and HK-2 cells were cultured to 70–80% confluence in six-well or 96-well plates. The cells were transfected with the TLR4 gene (pEX) or a nonbinding control gene using Lipofectamine 2000 reagent according to the manufacturer’s protocol. Twenty-four hours after transfection, the cells were subjected to serum deprivation for 24 h in the presence or absence of the dioscin pretreatment (200 ng/ml) before they were challenged with LPS (0.5 ␮g/ml) for an additional 24 h. Then, the protein levels of TLR4, MyD88, cleaved caspase-3, SOD2 and nuclear p65 were assayed. In addition, the ROS levels were

The NRK-52E and HK-2 cells were plated in 6-well plates (2 × 105 cells/well), and then the cells were exposed to ST2825 (20 ␮M) for 2 h [42]. After incubation, the cells were subjected to serum deprivation for 24 h before they were challenged with LPS (0.5 ␮g/ml) in the presence or absence of dioscin (200 ng/ml) for an additional 24 h. Then, the levels of the MyD88, cleaved caspase3, SOD2 and nuclear p65 proteins were assayed. In addition, the ROS levels were detected and the number of apoptotic cells was determined using the TUNEL assay.

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Fig. 2. Effects of dioscin on LPS-induced acute kidney injury in rats and mice. (A) Effects of dioscin on the serum Cr and BUN levels in rats and mice. (B) Acute kidney injury assessed by H&E staining (400 × original magnification) in rats and mice. (C) Effects of dioscin on the MDA, NO, GSH-Px, and CAT levels in rats and mice. Data are presented as the mean ± SD (n = 12). * P < 0.05 and ** P < 0.01 compared with model groups.

2.14. Statistical analyses

3. Results

All data were analyzed using the statistical software SPSS 18.0 and presented as the mean and standard deviation (SD). Statistically significant differences were determined using one-way ANOVA followed by the Newman-Keuls test. Comparisons between two groups were performed using an unpaired Student t-test. The results were considered to be statistically significant at P < 0.05.

3.1. Dioscin rehabilitates LPS-induced cell injury Exposure to LPS for 24 h induced a dose-dependent decrease in the cell viability of NRK-52E and HK-2 cells (Fig. 1A). Compared with the control groups (without LPS, set at 100%), the viability rates of the NRK-52E and HK-2 cells decreased from 71.0% and 73.3% to 24.3% and 23.0%, respectively, suggesting that LPS injured the NRK-52E and HK-2 cells. The optimal concentration of LPS

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Fig. 3. Dioscin up-regulates the expression levels of miR-let-7i and inhibits the levels of TLR4 and MyD88 in vitro and in vivo. (A) Effects of dioscin on the miR-let-7i levels in vitro and in vivo using real-time PCR assay. (B) Effects of dioscin on the TLR4 levels in vitro and in vivo using immunofluorescence staining (200 × magnification). (C) Effects of dioscin on the protein levels TLR4 and MyD88 in vitro and in vivo by western blotting assay. Cropped gels are shown, and the full-length gels are presented in Supplemental Fig. 6. Data are presented as the mean ± SD (n = 3). * P < 0.05 and ** P < 0.01 compared with model groups.

that induced cell injury in our tests was 0.5 ␮g/ml. As shown in Fig. 1B, dioscin (50, 100, 200 and 400 ng/ml) significantly protected the NRK-52E and HK-2 cells against LPS-induced injury in a doseand time-dependent manner. Compared with the LPS groups, a 12 h treatment with dioscin (50, 100 and 200 ng/ml) significantly increased NRK-52E and HK-2 cell viability by 1.21-, 1.41-, 1.58, and 1.35-fold and by 1.14-, 1.32-, 1.49-, and 1.3-fold, respectively. The

NRK-52E and HK-2 cells were pretreated with the compound at concentrations of 50, 100 and 200 ng/ml for 12 h, which was optimized for the subsequent experiments. Importantly, the selected concentrations of dioscin were not toxic to the cells (Fig. 1C). In addition, the cell morphological changes of the cells in model groups including unclear cell border, partial of cells dissolution and death, reduction after LPS stimulation were found, which were

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Fig. 4. Dioscin down-regulates PI3K/Akt inflammatory signaling in vivo and in vitro. (A) Effects of dioscin on the p-PI3K levels in vitro and in vivo using immunofluores-cence staining (200 × magnification). (B) Effects of dioscin on the protein levels of phosphorylated PI3K, Akt, and the nuclear translocation of NF-␬Bp65 in vitro and in vivo by western blotting assay. Cropped gels are shown, and the full-length gels are presented in Supplemental Fig. 7. (C) Effects of dioscin on the mRNA levels of MIP-1␣, IL-1␤ and IL-6 in vitro and in vivo using real-time PCR assay. Data are presented as the mean ± SD (n = 3). * P < 0.05 and ** P < 0.01 compared with model groups.

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Fig. 5. Dioscin inhibits LPS-induced apoptosis and oxidative stress in vivo and in vitro. (A) Representative micrographs of TUNEL-stained kidney sections and the cells (200 × magnification). (B) Effects of dioscin on the protein levels of cleaved caspase-8 and cleaved caspase-3 in vitro and in vivo by western blotting assay. (C) Effects of dioscin on the mRNA levels of Fas and FasL in vitro and in vivo by real-time PCR assay. (D) Effects of dioscin on the protein levels of NOX1 and SOD2 in vitro and in vivo by western blotting assay. Cropped gels are shown, and the full-length gels are presented in Supplemental Fig. 8. (E) Effects of dioscin on the ROS levels in vitro and in vivo. Data are presented as the mean ± SD (n = 3). * P < 0.05 and ** P < 0.01 compared with model groups.

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Fig. 6. The miR-let-7i inhibitor reverses the nephroprotective effects of dioscin. (A) Effects of dioscin on the protein levels of TLR4, cleaved caspase-3, SOD2 and nuclear NF-␬Bp65 in NRK-52E and HK-2 cells after transfection of miR-let-7i inhibitor by western blotting assay. Cropped gels are shown, and the full-length gels are presented in Supplemental Fig. 9 . (B) Effects of dioscin on the nuclear translocation of NF-␬B and the TUNEL-positive cell numbers transfected with miR-let-7i inhibitor and pretreated with or without dioscin using immunofluorescence staining (1200 × magnification) and TUNEL staining (200 × magnification), respectively. (C) Effects of dioscin on the ROS levels in NRK-52E and HK-2 cells with or without transfection of miR-let-7i inhibitor. (D) Effects of dioscin on the mRNA levels of IL-1␤, IL-6, MIP-1␣, Fas and FasL in NRK-52E and HK-2 cells transfected with miR-let-7i inhibitor using real-time PCR assay. Data are presented as the mean ± SD (n = 3). * P < 0.05 and ** P < 0.01 compared with model groups.

significantly prevented by dioscin in a dose-dependent manner (Fig. 1D). 3.2. Dioscin alleviates LPS-induced renal injury in vivo As shown in Fig. 2A, the serum BUN and Cr levels were markedly increased in the model groups and were significantly decreased by dioscin. Compared with the model groups, the blood urea nitrogen (BUN) and creatinine (Cr) levels in the rats treated with dioscin (60 mg/kg) were significantly reduced, by 50.3% and 48.9%, respectively, and the levels in mice treated with dioscin (80 mg/kg) were significantly reduced by 56.8% and 53.6%, respectively. As shown in Fig. 2B, the histopathological changes in the kidneys of the model groups included partial renal tubular epithelial vacuole degeneration or hyaline degeneration, swelling in the renal tubular epithelial cells, a narrowed lumen, interstitial angiectasis

hyperemia, and a large number of infiltrated inflammatory cells, which were markedly reversed by dioscin. As shown in Fig. 2C, dioscin clearly decreased the malondialdehyde (MDA) and nitric oxide (NO) levels and significantly increased the glutathione peroxidase (GSH-Px) and catalase (CAT) levels compared with the LPS-induced animals. 3.3. Dioscin up-regulates the expression levels of miR-let-7i in vivo and in vitro As shown in Fig. 3A, the expression levels of miR-let-7i were markedly decreased by LPS in vivo and in vitro compared with the control groups and were significantly up-regulated by dioscin in the high-dose-treated NRK-52E cells, HK-2 cells, rat and mouse kidney tissues by 1.86-, 1.61-, 0.98- and 1.26-fold, respectively, compared with the model groups.

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3.4. Dioscin decreases the levels of TLR4 and MyD88 in vivo and in vitro Based on the results of the immunofluorescence staining and western blotting (Fig. 3B–C), the expression levels of TLR4 and MyD88 in model groups were markedly increased compared with the control groups in vivo and in vitro and were significantly downregulated by dioscin. The results of the statistical analysis are provided in Supplemental Fig. 2A. 3.5. Dioscin down-regulates PI3K/Akt inflammatory signaling in vivo and in vitro As shown in Fig. 4A and B, high levels of PI3K were obvious in the model groups, which were significantly decreased by dioscin, based on the immunofluorescence assay. In addition, the levels of phosphorylated PI3K and Akt and the nuclear translocation of NF␬Bp65 were down-regulated by the compound (the results of the statistical analysis are provided in Supplemental Fig. 2B). As shown in Fig. 4C, the mRNA levels of pro-inflammatory factors, including macrophage inflammatory protein-1␣ (MIP-1␣), interleukin-1␤ (IL-1␤) and interleukin-6 (IL-6), were significantly increased in the model groups compared with the control groups and were all clearly restored by dioscin. 3.6. Dioscin inhibits LPS-induced apoptosis in vivo and in vitro As shown in Fig. 5A, more terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL)-positive cells displaying green fluorescence were observed in the model groups than in the dioscin-treated groups in vivo and in vitro. As shown in Fig. 5B–C, dioscin also significantly decreased the levels of apoptosis-related proteins or genes, including cleaved caspase8, cleaved caspase-3, factor associated with suicide (Fas) and factor associated with suicide ligand (FasL), in the LPS-induced cells and kidney tissues (the results of the statistical analysis are provided in Supplemental Fig. 2C). 3.7. Dioscin attenuates LPS-induced oxidative stress in vivo and in vitro As shown in Fig. 5D, dioscin notably down-regulated the levels of the NADPH oxidase 1 (NOX1) protein and up-regulated the expression levels of superoxide dismutase 2 (SOD2) in vivo and in vitro compared with the model groups (the results of the statistical analysis are provided in Supplemental Fig. 2D). In addition, the ROS levels in the high-dose dioscin-treated NRK-52E cells, HK2 cells, rat and mouse kidney tissues were significantly reduced by 37.1%, 35.8%, 32.0% and 40.1%, respectively, compared with the model groups (Fig. 5E). 3.8. MiR-let-7i inhibitor reverses the nephroprotective effects of dioscin To analyze the dioscin-mediated inhibition of the TLR4mediated inflammatory signaling pathway, a transfection approach using a miR-let-7i inhibitor was used to further elucidate the cellular effects of dioscin on miR-let-7i. As shown in Fig. 6A–B, transfection of the miR-let-7i inhibitor weakened the dioscininduced inhibitory effects of TLR4 and its downstream signaling intermediates. However, dioscin still down-regulated the expression levels of cleaved caspase-3, up-regulated the expression levels of SOD2, inhibited the nuclear translocation of NF-␬Bp65 and decreased the number TUNEL-positive cells after transfection (the bright images of the miR-let-7i inhibitor-transfected cells are provided in Supplemental Fig. 3A, and the statistical analysis of the

bands is provided in Supplemental Fig. 3B). Similar results were also found for the ROS levels, the expression levels of the inflammatory cytokines and the number of apoptotic cells following transfection with the miR-let-7i inhibitor (Fig. 6C–D). These results showed that the dioscin-induced suppression of the TLR4 signaling pathway might be mediated by the up-regulation of miR-let-7i. 3.9. TLR4 DNA abrogates the nephroprotective effects of dioscin To investigate the mechanisms of attenuating inflammation, we hypothesized that the anti-inflammatory capability of dioscin might result from down-regulating TLR4 signaling pathway. As shown in Fig. 7A–B, transfection of TLR4 DNA attenuated dioscininduced inhibitory effects to MyD88 and its downstream signaling intermediates. However, dioscin still decreased the protein levels of TLR4, MyD88, cleaved caspase-3, increased the protein levels of SOD2, inhibited the nuclear translocation of NF-␬Bp65, and decreased TUNEL-positive cell numbers after transfection (the bright images of the TLR4 DNA-transfected cells are provided in Supplemental Fig. 4A, and the statistical assays of the bands are provided in Supplemental Fig. 4B). Similar results were also found for ROS levels, the expression levels of inflammatory cytokines and the apoptotic cell numbers following transfection with TLR4 DNA (Fig. 7C–D). These findings suggested that TLR4 DNA transfection reversed the down-regulation effects of dioscin on the TLR4 signaling pathway after LPS stimulation. 3.10. MyD88 mediates the dioscin-induced inhibition of LPS-induced injury NRK-52E and HK-2 cells were incubated with dioscin to evaluate whether the modulation of miR-let-7i/TLR4 by dioscin had a vital relationship with MyD88. As shown in Fig. 8A, a 2 h challenge of the NRK-52E and HK-2 cells with ST2825 (20 ␮M) significantly inhibited MyD88 expression, cleaved caspase-3 expression and the nuclear translocation of NF-␬Bp65, and the increased the levels of SOD2 (the bright images after MyD88 inhibition are provided in Supplemental Fig. 5A, and the statistical analysis of the bands is provided in Supplemental Fig. 5B). The LPS-induced nuclear translocation of NF-␬Bp65, number of apoptotic cells, ROS levels and the expression levels of inflammatory cytokines and apoptosisrelated genes were partially abolished by ST2825 (Fig. 8B–D). These results showed that the dioscin- induced suppression of the TLR4 signaling pathway might be associated with the MyD88-dependent pathway. 4. Discussion AKI, characterized by a sudden loss of kidney function, increases the risk of end-stage renal disease and results in a profound impact on patients’ morbidity, mortality and medical expenses [43]. AKI prolongs the duration of the clinical course, which is mainly caused by acute hypoxia, trauma, toxic or septic insults to the renal parenchyma [44]. The LPS insult, which is involved in the pathogenesis of AKI, may lead to kidney failure and even multi-organ damage. Recently, numerous studies have examined LPS-induced AKI, but the pathogenesis of human AKI is complex and only partially understood [45]. Therefore, it is important to explore novel and effective pharmacological therapeutics to reverse LPS-induced AKI. Dioscin, a natural product, has potent effects on renal ischemia/reperfusion injury by adjusting the TLR4/MyD88 signaling pathway, as shown in our previous study [34]. In the present work, dioscin significantly protected against LPS-induced cell injury, as evidenced by its ability to improve NRK-52E and

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Fig. 7. TLR4 DNA abrogates the nephroprotective effects of dioscin. (A) Effects of dioscin on the protein levels of TLR4, MyD88, cleaved caspase-3, SOD2 and nuclear NF-␬Bp65 in NRK-52E and HK-2 cells after transfection of TLR4 DNA by western blotting assay. Cropped gels are shown, and the full-length gels are presented in Supplemental Fig. 10. (B) Effects of dioscin on the nuclear translocation of NF-␬B and the TUNEL-positive cell numbers transfected with TLR4 DNA and pretreated with or without dioscin using immunofluorescence staining (1200 × magnification) and TUNEL staining (200 × magnification), respectively. (C) Effects of dioscin on the ROS levels in NRK-52E and HK-2 cells with or without transfection of TLR4 DNA. (D) Effects of dioscin on the mRNA levels of IL-1␤, IL-6, MIP-1␣, Fas and FasL in NRK-52E and HK-2 cells transfected with TLR4 DNA using real-time PCR assay. Data are presented as the mean ± SD (n = 3). * P < 0.05 and ** P < 0.01 compared with model groups.

HK-2 cell viability and cellular morphology. In vivo, we demonstrated that dioscin significantly decreased the BUN and Cr levels and alleviated the histopathological changes. These data showed that dioscin had protective effect to inhibit LPS-induced AKI. LPS, a specific exogenous ligand for the TLR4 receptor, causes a substantial inflammatory response characterized by the recruitment of activated inflammatory cytokines and the induction of endothelial damage [46]. Evidence has demonstrated that the immune and inflammatory responses to microbial infection are mediated by TLR4, which also participates in the pathogenesis of LPS-induced AKI [47,48]. We further elucidated the molecular mechanisms involved in the dioscin-mediated protection against LPS-induced inflammatory kidney injury.

MiRs play important roles in many biological processes through mRNA degradation or translational suppression [14]. Let-7i, a member of the let-7 family of miRs, directly targets the TLR4 mRNA to down-regulate its expression [15,16]. In the present work, we found that the levels of miR-let-7i were markedly increased by dioscin in vitro and in vivo, accompanied by decreased levels of the TLR4 protein. Thus, miR-let-7i represented a potential therapeutic target for dioscin in the treatment of LPS-induced inflammatory kidney injury. Numerous signaling pathways are downstream of TLR4. One important pathway, the endogenous PI3K/Akt pathway, regulates negative feedback in response to LPS stimuli [49,50]. LPS activates the TLR4/MyD88-dependent signaling pathway, which results in the phosphorylation of PI3K and Akt, followed by the nuclear

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Fig. 8. MyD88 mediates the inhibitory effects of dioscin on LPS-induced injury. (A) Effects of dioscin on the protein levels of MyD88, cleaved caspase-3, SOD2 and nuclear NF-␬Bp65 in NRK-52E and HK-2 cells after inhibition of MyD88 by western blotting assay. Cropped gels are shown, and the full-length gels are presented in Supplemental Fig. 11 . (B) Effects of dioscin on the nuclear translocation of NF-␬B and the TUNEL-positive cell numbers pretreated with or without dioscin and MyD88 inhibitor using immunofluorescence staining (1200 × magnification) and TUNEL staining (200 × magnification), respectively. (C) Effects of dioscin on the ROS levels in NRK-52E and HK-2 cells with or without the inhibition of MyD88. (D) Effects of dioscin on the mRNA levels of IL-1␤, IL-6, MIP-1␣, Fas and FasL in NRK-52E and HK-2 cells after treatment with the MyD88 inhibitor using real-time PCR assay. Data are presented as the mean ± SD (n = 3). * P < 0.05 and ** P < 0.01 compared with model groups.

translocation of NF-␬B. The expression levels of inflammatory cytokines, including MIP-1␣, IL-1␤ and IL-6, are regulated by various transcription factors, one of the most important of which is NF-␬B [51]. Our results demonstrated that dioscin significantly decreased the levels of MyD88, MIP-1␣, IL-1␤ and IL-6; the levels of phosphorylated PI3K and Akt; and the nuclear translocation of NF-␬Bp65 in vitro and in vivo. Therefore, we hypothesized that the anti-inflammatory effects of dioscin might primarily result from the down-regulation of the MyD88-dependent TLR4-PI3K-Akt-NF-␬B pathway. Several studies have reported that the ROS generated by NADPH oxidase play a crucial role in the physiological process of sepsis [52,53]. During LPS-induced shock, the excessive release of ROS may trigger a hyper-activation of innate immune cells, overexpression of cytokines, and even end-stage organ injury [54]. TLR4 signaling can enhance NOX1 expression, followed by a series of

oxidative stress responses, including increased levels of ROS, NO, and MDA and reduced levels of antioxidant enzymes, including SOD2, CAT and GSH-Px, among others. High levels of SOD, CAT, and GSH-Px can protect acute kidney injury. In addition, excessive production of oxygen-free radicals can increase the level of MDA, which is one production of lipid peroxidation and a wellknown indicator of ROS [55]. In the present study, we found that the increased levels of MDA and NO, and the decreased levels of GSH-Px and CAT were all markedly reversed by dioscin, suggesting that dioscin showed prominent anti-oxidant activity against LPSinduced oxidative-nitrative kidney damage in vivo. Then, through western blotting assay, we found that the increased levels of NOX1, and the decreased levels of SOD2 were all significantly suppressed by dioscin in vitro and in vivo. These results demonstrated that TLR4/NOX1/ROS may be involved in the dioscin-mediated protection against LPS-induced inflammatory kidney injury.

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In addition, LPS can induce apoptosis via the MyD88-dependent TLR4 inflammatory signaling pathway [22]. Subsequently, MyD88 binds with Fas- associated death domain-containing protein (FADD), which promotes caspase activation and the expression of molecules including cleaved caspase-8, cleaved caspase-3, Fas, FasL, and others [56]. In the present work, we found that dioscin significantly reduced the levels of the cleaved caspase-8/3 proteins and the levels of the Fas and FasL mRNAs in vitro and in vivo. These results indicated that dioscin blocked LPS-induced apoptosis through the MyD88-dependent TLR4 signaling pathway. The objective of this study was to evaluate whether the inhibitory effect of dioscin during LPS-induced AKI was related to the MyD88-dependent TLR4 signaling pathway via the up-regulation miR-let-7i. To further elucidate the molecular mechanism, the methods of miR-let-7i inhibitor, TLR4 DNA overexpression and MyD88 inhibitor were used, and the levels of SOD2, ROS, cleaved caspase-3, nuclear p65, and inflammatory factors, as well as the numbers of apoptotic cells were measured. The results suggested that dioscin-induced alterations in inflammatory injury may be mediated by inhibiting the TLR4 signaling pathway through up- regulating miR-let-7i. In addition, we demonstrated that the levels of the proteins and mRNAs downstream of the MyD88 pathway were slightly increased in the LPS groups with and without MyD88 inhibition, which were not significantly different compared with the control groups. Importantly, the present study indicated that MyD88 served as a molecular link between inflammatory signaling and LPS-induced kidney injury. Thus, MyD88 was involved in the dioscin-mediated inhibition of LPS-induced renal injury in vivo and in vitro. In summary, based on our investigation, a simplified pathway to describe the possible involvement of miR-let-7i/TLR4/MyD88 signaling in dioscin-induced suppression of LPS-induced renal injury was investigated. Dioscin alleviated LPS- induced renal inflammatory injury, and it should be developed as a new potential candidate for clinical therapy. Competing financial interests The authors declare no competing financial interests. Acknowledgement This work was financially supported by the Program for Liaoning Innovative Research Team in University (LT2013019). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phrs.2016.07. 016. References [1] S.T. Vaara, V. Pettilä, K.M. Kaukonen, S. Bendel, A.M. Korhonen, R. Bellomo, M. Reinikainen, Finnish acute kidney injury study group, the attributable mortality of acute kidney injury: a sequentially matched analysis, Crit. Care Med. 42 (2014) 878–885. [2] M. Plataki, K. Kashani, J. Cabello-Garza, F. Maldonado, R. Kashyap, D.J. Kor, O. Gajic, R. Cartin-Ceba, Predictors of acute kidney injury in septic shock patients: an observational cohort study, Clin. J. Am. Soc. Nephrol. 6 (2011) 1744–1751. [3] H.T. Robert, M.I. James, H.R. Mitchel, AKI associated with cardiac surgery, Clin J. Am. Soc. Nephrol. 10 (2015) 500–514. [4] P.K. Li, E.A. Burdmann, R.L. Mehta, Acute kidney injury: acute kidney injury-global health alert, Nat. Rev. Nephrol. 9 (2013) 133–135. [5] N. Lameire, J.A. Kellum, KDIGO AKI guideline work group contrast-induced acute kidney injury and renal support for acute kidney injury: a KDIGO summary (Part 2), Crit. Care 17 (2013) 205.

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