CysLTR1 Blockage Ameliorates Liver Injury Caused by Aluminum

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CysLTR1 blockage ameliorates liver injury caused by aluminum-overload via PI3K/AKT/mTOR-mediated autophagy activation in vivo and in vitro Congli Hu, Junqing Yang, Qin He, Ying Luo, Zhihao Chen, Lu Yang, Honggang Yi, Huan Li, Hui Xia, Dongzhi Ran, Yang Yang, Jiahua Zhang, Yuke Li, and Hong Wang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00121 • Publication Date (Web): 10 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

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Molecular Pharmaceutics

Pharmacology

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

CysLTR1

blockage

aluminum-overload

ameliorates via

liver

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injury

PI3K/AKT/mTOR-mediated

caused

by

autophagy

activation in vivo and in vitro Congli Hu1* , Junqing Yang1*,Qin He2*, Ying Luo1, Zhihao Chen1, Lu Yang1, Honggang Yi1, Huan Li1, Hui Xia1, Dongzhi Ran1, Yang Yang1, Jiahua Zhang1, Yuke Li1, Hong Wang1#

AffiliationÖ 1

Department of Pharmacology, Chongqing Medical University, the Key Laboratory

of Biochemistry and Molecular Pharmacology, Chongqing 400016, China 2

Department of Hepatobiliary Surgery, 1st Affiliated Hospital, Chongqing Medical

University, Chongqing 400016, China

*co-first authors: Congli Hu, Junqing Yang, Qin He

#

Corresponding author: Hong Wang

Department of Pharmacology, Chongqing Medical University, Chongqing 400016, China; Tel: +86-23-68485161; Fax: +86-23-68485161; E-mail:


;

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Abstract Aluminum (Al) is a trivalent cation that can accumulate in animal organs, especially in the liver. We previously studies demonstrated that Al overload could induce liver morphologic aberrations and dysfunction. However, the molecular mechanism underlying liver injury caused by Al overload still remains unknown. In the present study, we investigated the relationship between leukotrienes receptors and PI3K / AKT / mTOR pathway in Al-induced liver injury in vivo and in vitro. We demonstrated that Al overload significantly increased the protein expression levels of CysLTR1, PI3K, AKT, mTOR and p62, while significantly decreased the LC3BII protein levels in rat liver, thus suggesting that autophagy process was inhibited in Al-overloaded rat liver. In addition, MK-571, an inhibitor of CysLTR1, effectively protected the human hepatocyte L02 cells against injury caused by Al exposure. Moreover,

CysLTR1

blockage

could

significantly

down-regulate

the

PI3K/AKT/mTOR pathway and activate autophagy. The effect of MK-571 on cell viability was abolished by the treatment with the autophagy inhibitor (wortmannin) but not with the autophagy agonist (rapamycin). Taken together, our results indicated that the blockage of leukotriene receptor of CysLTR1 promotes autophagy and further reduces hepatocyte death through PI3K/AKT/mTOR pathway inhibition. CysLTR1 thus could represent a potential target for the new drug development for chronic non-infective liver injury. Keywords: aluminum overload; liver injury; CysLTR1; PI3K/AKT/mTOR pathway; autophagy



Introduction Aluminum (Al) is a trivalent cation that can accumulate in animal organs, especially in the liver 1. Along with other studies, we previously demonstrated that Al overload could induce liver morphologic aberrations and dysfunction 2-4. However, the molecular mechanism involved in liver injury caused by Al overload still remains unknown. Inflammation plays a significant role in chronic liver injuries. Our previous studies suggested that Al exposure induced chronic liver injury probably associated to the oxidative stress and inflammation 5, 6. The chronic overload with AlCl3 increased the levels of the pro-inflammatory interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) 7. Moreover, rats exposed to AlCl3 increased the gene expression of TNF-α, interleukin-6 (IL-6) and IL-1β in the liver 8. A prolonged exposure of tissues or organs to high concentrations of inflammatory mediators can induce cells in high stress conditions, causing serious damages. Several studies have reported that cell damage caused by inflammation could be regulated by autophagy 9. Indeed, changes in autophagy levels represent a potential mechanism for the development of a large number of liver diseases, but the role of autophagy in liver injury relies on different causes and mechanisms 10, 11. Autophagy is a well-conserved catabolic process, responsible for the disposal of damaged organelles and metabolic macromolecules to maintain cellular homeostasis, and to supply a variety of substrates for cellular energy generation during harsh conditions, such as starvation 12. Additionally, autophagy has important regulatory


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effects on inflammation, affecting the pathological progression of inflammatory diseases. The crucial role of autophagy has been implicated in metabolic organs, such as liver, muscle, and adipose tissue, due to their unique metabolic macromolecule degrading activities 13. In our previous research we found that 5-lipoxygenase (5-LOX) contributed to liver injury elicited by Al overload 14. Meanwhile, caffeic acid, an inhibitor of 5-LOX, was reported to inhibit oxidative stress and reduce leukotrienes (LTs) levels 15. LTs are metabolites of arachidonic acid induced via the 5-LOX pathway. LTs include cysteinyl leukotriene (CysLTs; LTC4, LTD4, LTE4) and the dihydroxy leukotriene LTB4. Each LTs can bind to specific receptors, such as CysLTs to CysLTR1 and CysLTR2, LTB4 to LTB4R1 and LTB4R2 (or BLT1 and BLT2) respectively 16. LTs receptors belong to seven transmembrane domain G protein-coupled receptors and can bind ligands to mediate various signaling of inflammatory in cells. Recent studies showed that LTs were involved not only in inflammation, shock, allergic reactions and plasma extravasations, but also in different types of liver injury 17, 18. For example, LTD4 triggered a 3-fold transient increase in phosphatidylinositol 3-kinase (PI3K) phosphorylation 19, and also stimulated mouse embryonic stem (ES) cells proliferation and migration via PI3K/ AKT pathway 20. Indeed, LTD4 stimulation of a Gi/o coupled CysLT1R triggered the transactivation of epidemal growth factor receptor (EGF-R) through the intervention of PI3K and reactive oxygen species (ROS) 21. Endogenous LTB4 contributed to the activation of protein kinase C alpha (PKC-α), extracellular signal-regulated kinase (ERK 1/2) and PI3K, while endogenous CysLTs



to the activation of PKC-δ, p38 and PI3K 22. CysLT1R and LTB4R are thus involved in the activation of PI3K in some diseases. However, the molecular mechanism of CysLTs and LTB4R in Al-induced chronic liver injury remains unknown. Besides, the PI3K/AKT/mTOR pathway is widely recognized as a fundamental intracellular signaling pathway involved in both normal cell physiology and cancer pathology, and it is able to inhibit autophagy when activated 23. Since autophagy serves to maintain stability in the intracellular environment, LTs receptors are important response to the promotion of the inflammatory response. In this framework, we decided to investigate in vivo and in vitro whether the inhibition of LTs receptors could protect the liver from Al-induced injury by inhibiting PI3K/AKT/mTOR pathway.

Materials and methods Chemicals and reagents AlCl3·6H2O (Sinopharm Chemical Reagent, China), sodium gluconate (Chengdu Kelong Reagent, China) and maltolate (Sinopharm Chemical Reagent, China), they are all analytically pure. The antibodies of this experiment were as follows: CysLTR1,CysLTR2,LTB4R1 and LTB4R2 purchased from Bioss (Bioss, China); PI3 Kinase, p-AKT, p62, LC3I/II and Beclin-1 purchased from Cell Signaling Technology (CST, USA); AKT, p-mTOR purchased from Abcam (Abcam, UK); GAPDH, HRP-conjugated secondary antibodies purchased from Proteintech (Proteintech, Wuhan, China). The ELISA kits of this expriment were as follows: LTB4 and LTD4 purchased from Cusabio(Cusabio, Wuhan,China). LTC4 and LTD4


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purchased from mblio (Shanghai enzyme lian biotechnology, China). Animals Ten male Sprague Dawley rats (supplied from the Laboratory Animal Center of Chongqing Medical University), weighing 200± 20 g, were randomly and equally divided into two groups (5 rats/ group): control group and model group. The rats were housed in standard conditions (temperature = 25 ± 1 °C, humidity = 50 ± 2%, 12/12 h light/dark cycles, lights on from 8:00–20:00) with free access to water and standard pellet food. All procedures performed in studies involving animals were approved by the Animal Laboratory Administrative Center and the institutional Animal Ethics Committee at Chongqing Medical University [No. SYXK (Yu) 2012-0001]. Establishment of animal models After 3 days of acclimatization rats were treated intragastrically once a day, 5 days per week for 20 continuous weeks. The model group (5 rats) received 1 ml/100 g Al-gluconate solution (200 mg Al3+/Kg), and the control group (5 rats) treated with an equal volume of sodium gluconate solution 10. Al-gluconate solution (20 mg Al3+/ml) was produced by blending 17.9 g of AlCl3·6H2O and 9.9 g of sodium gluconate to 100 ml of double distilled water, then adjusted the pH to 6.0 10. Histopathological examination After the cessation of Al-gluconate or sodium gluconate administration, the rats (n=3 per group) were intraperitoneally anesthetized with 4 % chloral hydrate (1 ml/100 g) and transcardially perfused with 0.9% saline (100 ml) containing heparin followed by 4% paraformaldehyde (PH=7.2). And then the liver tissues were isolated



and blocked in 4% paraformaldehyde. After embedding, the livers were sliced into 4-5µm thick sections for Hematoxylin-Eosin (H&E). Hepatic pathological changes were examined by light microscopy. Liver function tests Peripheral blood was collected from each rat orbit (n=5 per group). The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were measured according to the manufacturer's manual of the kits (Jiancheng Bioengineering Ltd, Nanjing, China). Biochemistry test

Rat liver (n=5 per group) was dissected before perfusion with 4% paraformaldehyde. The content of malondialdehyde (MDA; Beyotime, China) and the activity of superoxide dismutase (SOD; Jiancheng Bioengineering Ltd, Nanjing, China) were measured by biochemistry assay kits. The protein content was measured by BCA protein assay kit (Beyotime, China). The levels of LTs (LTB4, LTC4, LTD4 and LTE4) in liver tissues were detected with ELISA kits. The detailed procedure performed according to the manufacturer's manual of the kits. Real-Time Quantitative PCR analysis

Total RNA was isolated from rat liver (n=4 per group) using Trizol (Takara, Japan) according to the manufacturer’ instructions and was reversed transcribed into cDNA using M-MULV (Beyotime, China). Realtime-qPCR was performed on the CFX Connect System (Bio-Rad Laboratories ,USA) and SYBR Green aPCR Master Mix (Bimake, USA) was used. The mRNA expressions of CysLTR1, CysLTR2,


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LTB4R1and LTB4R2 were quantified with the 2−∆∆Ct method. And the primers sequences were as follows: CysLTR1 forward, 5’-AAAACCTGCCAAGTCGAAGG-3’, and CysLTR1 reverse, 5’ACCACTGACTTCTGCATCCT- 3’; CysLTR2 forward, 5’-TTTTCATGCTCAACCTGGCC- 3’, and CysLTR2 reverse, 5’-CCAGGAAGCGCACAATACTC- 3’; LTB4R1 forward, 5’TCCTACACTTTCTGGCTCGG- 3’, and LTB4R1 reverse, 5’GCAGAAAAGACACCACCCAG- 3’; LTB4R2 forward, 5’ATGTCTGTCTGCTACCGTCC- 3’, and LTB4R2 reverse, 5’-ACAAGTGTGGCTGCTAGTGG-3’; GAPDH forward, 5’-GCAACTCCCATTCTTCCACC- 3’, and GAPDH reverse, 5’GCCTCTCTCTTGCTCTCAGT- 3’. Western blotting analysis The rat liver (n=4 per group) was weighed, then it was put into tissue lysate solution to extract. Tissue homogenate was centrifuged at 4 °C for 10 min with 12,000 × g. The content of protein was measured by a kit of BCA protein quantitation kit (Beyotime, China). A sample protein (10 µl) was isolated through sodium dodecyl sulphate polyacrylamide gel electrophoresis and transported to polyvinylidene difluoride membranes (Millipore, USA). The membranes were placed in blocking buffer (5% BSA) at room temperature for 1 h and then incubated with antibodies (CysLTR1, CysLTR2, LTB4R1, LTB4R1, p62, Beclin-1 and LC3B, dilution 1:500; PI3 Kinase, AKT, p-AKT, p-mTOR and GAPDH, dilution 1:1000) overnight at 4 °C.



Then, membranes were washed 3 times×10 min in TBS-Tween buffer, and incubated with HRP-conjugated secondary antibodies (1:3000) for 1 h at room temperature. Finally, washed with TBS-Tween buffer for 3 times×10min, immunoblot results were visualized by electrochemiluminescence (ECL), which was detected using a Bio-Rad imaging system (Bio-Rad, USA). Cell culture experiments Human hepatocytes of HL-7702 (L02) were purchased from BeNa Culture Collection (BNCC, Beijing, China). L02 cells were served as normal human hepatocytes in liver diseases 24, 25. The cells were cultured in RPMI-1640 medium (HyClone, USA) containing 10% fetal bovine serum (FBS) (Biotech, Germany) and in a humidified atmosphere (5% CO2, 37 °C). Cells were treated with Al-gluconate, Al(malt)3 and various inhibitors according to the experimental design. The Al-gluconate and Al(malt)3 was produced by mixing the solutions of AlCl3·6H2O and sodium gluconate or maltol which were respectively dissolved in distilled water and phosphate buffered saline (PBS). Afterwards, adjusted the pH to 7.0 by NaOH and filtered it with 0.22 µmol/L syringe filters. MTT assay L02 cells (n=6 per group) were seeded into 96-well plates at a density of 5.0 x103 cells in each well (100 µl). The cells were adherent growth and then drugs were added into them. After being incubated with various concentrations of drugs at the indicated time points, 20 µl of MTT solution (5 mg/ ml) (Genview, USA) was added in each well, keeping cells in culture(5% CO2, 37 °C) for 4 h. Then the original


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medium was removed, and DMSO (150 µl) (Sigma-Aldrich, USA) was added to each well to dissolve and mix the crystals with low-speed oscillation for 10 min. The absorbance (OD) of each well was measured at 490 nm using an Enzyme-linked immunoassay analyzer (Bio-Tech, USA). Cytotoxicity analysis Before the experiment, L02 cells (n=6 per group) were seeded into a 6-well plate at a density of 2.0 × 105 cells per well. The cells were adherent growth and then drugs were added into them. Incubating with various concentrations of drugs at the indicated time points, the LDH release into the medium was detected according to the Cytotoxicity Detection Kit (Beyotime, China). Flow cytometry analysis L02 cells (n=6 per group) were seeded into a 6-well plate at a density of 2.0 × 105 cells per well and cultured under standard conditions (37 °C with 5 % CO2). The cells were adherent growth and then drugs were added into them. Cells were collected after treating with drugs for 36 h. Cells were digested with EDTA-free trypsin for 1.0 min at 37 °C, collected, centrifugedÄ1000 r/min; 10 min) and the supernatant was discarded. Then cells were washed twice with cold PBS, in the end resuspended in 200 µl staining buffer. AnnexinV-FITC/PI staining was used in the dark, and cells were immediately analyzed on a flow cytometer. Electron microscopy analysis Collection of each group cells (n=3 per group) were centrifugedÄ1000 r/min; 10 min), and discarded the supernatant and fixed overnight in 2% glutaraldehyde (pH7.3)



in 0.1 M phosphate buffer saline at 4 °C. Images of autophagosomes were captured random from L02 cells sections using a transmission electron microscope. Statistical analysis Student’s t-test on GraphPad Prism 5 was used to analyze our experimental data, and P value of < 0.05 was considered significant. The results of western blotting were analyzed by Image J software.

Result The histopathology in rat liver treated with chronic Al overload We found significant abnormalities in the liver injury model induced by Al overloaded rats through hepatic histopathology staining. The Al-overload (model) group showed remarkable vacuolar degeneration (Fig 1B) and spotty necrosis (Fig 1C), compared with the control group (Fig 1A). The toxicity in rat liver treated with chronic Al overload The levels of Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly increased in the model of the chronic Al overloaded rats (Fig 2A and B, P< 0.01). Additionally, in the chronic Al overloaded group, the activity of SOD was remarkably decreased compared with the control group (Fig 2C, P< 0.05). However, it was significantly increased of the content of MDA in chronic Al-overloaded group (Fig 2D, P< 0.05). The levels of inflammation factors in rat liver treated with chronic Al overload The contents of LTs in chronic Al overloaded rat liver.. Compared with the


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control group, the levels of LTC4 (P< 0.01) and LTD4 (P< 0.05) were increased significantly in the Al overloaded group (Table 1). Changes in LTs receptors expression levels in rat liver treated with chronic Al overload For the evaluation of LTs receptor levels induced by Al overload treatment, we analyzed the mRNA and protein levels. Surprisingly, mRNA and protein of LTs receptor levels were not the same trend. We evaluated the main CysLTs receptor (CysLTR1, CysLTR2) and LTs receptors (LTB4R1, LTB4R2) in the rat liver exposed to chronic Al overload. The expression levels of CysLT1R, CysLT2R and LTB4R1 were significantly increased in chronic Al overloaded group except to LTB4R2 (Fig 3, P< 0.05). Meanwhile, the expression of CysLT1R mRNA was the most significant than others in chronic Al-overloaded group (Fig 3). In addition, the protein expression levels of CysLT1R and LTB4R1 in the model group were significantly higher than that in the control group (Fig 4, P< 0.05), while the expression levels of CysLT2R and LTB4R2 protein were equal to the control group. Obviously, the expression level of CysLT1R protein was the most significant (Fig 4). PI3K/AKT/mTOR expression in rat liver treated with chronic Al overload It is well known that PI3K/AKT/mTOR pathway plays an important role in regulating cell cycle, apoptosis and autophagy. And it is also a classical pathway in inflammation signaling pathway. It is obviously from Fig 5 (P

CysLTR1 Blockage Ameliorates Liver Injury Caused by Aluminum

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