Melatonin  protects  against LPS-induced inflammation and oxidative stress in hepatocytes  by  enhancing mitophagy and mitochondrial biogenesis

doi:10.21203/rs.3.rs-207360/v1

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Melatonin protects against LPS-induced inflammation and oxidative stress in hepatocytes by enhancing mitophagy and mitochondrial biogenesis

https://doi.org/10.21203/rs.3.rs-207360/v1

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Background: Melatonin have a protective effect in the liver during sepsis by counteracting oxidative stress and reducing inflammatory responses. Melatonin also regulates mitochondrial biogenesis. This study explored the mechanisms by which melatonin protects against liver injury in experimental sepsis with a focus on mitophagy and mitochondrial biogenesis.

Methods and Results: An in vitro model of sepsis-induced hepatocyte injury was established using AML12 cells. Indicators of oxidative stress, inflammation, mitophagy and mitochondrial biogenesis were assessed. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 protein levels, intracellular reactive oxygen species (ROS) levels, lipid peroxidation (malondialdehyde [MDA] levels), and markers of mitophagy (PTEN-induced putative kinase 1 [PINK1] and Parkin) and mitochondrial biogenesis (peroxisome proliferator-activated receptor-gamma coactivator 1a [PGC-1α], nuclear respiratory factor 1 [NRF-1], mitochondrial transcription factor A [TFAM]) were significantly increased, while superoxide dismutase (SOD) activity and intracellular adenosine triphosphate (ATP) levels were significantly decreased in LPS-treated AML12 cells compared to controls. TNF-α and IL-6 protein levels and intracellular ROS and MDA levels were significantly decreased, while SOD activity, intracellular ATP levels, and markers of mitophagy and mitochondrial biogenesis were significantly increased by melatonin pre-treatment.

Conclusion: This study demonstrated that melatonin was involved in the maintenance of mitochondrial homeostasis in hepatocytes during sepsis. Mechanisms involved selective elimination of dysfunctional mitochondria through mitophagy and induction of mitochondrial biogenesis.

Molecular Biology

Melatonin

Sepsis

Hepatocyte

Mitochondria

Oxidative stress

inflammation

Sepsis is a global public health concern that is responsible for high mortality rates in intensive care units [1]. The Sepsis-3 task force defines sepsis as ‘a life-threatening organ dysfunction caused by a dysregulated host response to infection’ [2]. The liver has a crucial role in the defense against sepsis as it clears bacteria and mediates inflammatory responses. In critically ill patients, early hepatic dysfunction is a risk factor for poor prognosis and mortality [3], but the morbidity and mortality associated with sepsis can be decreased by reducing liver injury and restoring liver function [4]. Currently, there remains an unmet clinical need to fully elucidate the molecular mechanisms underlying liver dysfunction during sepsis.

Mitochondria are life-sustaining organelles that have a critical role in stress responses [5]. Ultrastructural damage and functional impairment are observed in mitochondria during sepsis, resulting in the depletion of adenosine triphosphate (ATP), the production of reactive oxygen species (ROS), cell damage induced by oxidative stress, and organ failure [6].

In mammals, mitochondrial quality control is regulated by various mechanisms that result in mitochondrial turnover [7]. During sepsis, defective mitochondrial quality control mechanisms amplify organ failure, while restoration of mitochondrial quality control mechanisms ameliorate organ failure [7]. In several in vivo and in vitro models of sepsis, augmented mitophagy and mitochondrial biogenesis increased the number of functional mitochondria and decreased mitochondrial ROS production, inflammation, and injury [8][9]. As mitochondrial damage and depletion are early manifestations of liver dysfunction during sepsis [10], mitophagy and mitochondrial biogenesis in hepatocytes represent potential therapeutic targets in sepsis.

Melatonin (N-acetyl-5-methoxytryptamine), a hormone produced by the pineal gland, contributes to numerous physiological functions, including stress, sleep-wake rhythm, body temperature cycles, and circadian rhythms [11]. Melatonin may have a protective effect in the liver during sepsis by directly scavenging ROS or upregulating antioxidant enzymes to counteract oxidative stress, reduce inflammatory responses, and increase ATP levels [12][13]. Melatonin may regulate mitophagy and mitochondrial biogenesis[14]. In a mouse model, melatonin induced mitophagy and reduced oxidative stress and inflammation in atherosclerotic lesions, and prevented atherosclerotic progression [15]. In HepG2 cells in vitro, melatonin enhanced mitochondrial biogenesis and had protective effects in cadmium-induced hepatotoxicity [16]. The protective effects of melatonin against sepsis-induced hepatocyte injury remain to be elucidated.

This study investigated the protective mechanisms of melatonin in an in vitro model of sepsis-induced hepatocyte injury, with a focus on mitochondrial quality control.

Cell culture

Mouse hepatocyte AML12 cells were cultured in DMEM/F12 medium (11330032, Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS; 10270106, Gibco), 100 U/mL penicillin, 100μg/mL streptomycin (15140122, Sigma-Aldrich; St. Louis, Mo, USA), insulin, transferrin, selenium (ITS) liquid media supplement (I3146, Sigma-Aldrich), and 40 ng/ml dexamethasone (D4902, Sigma-Aldrich) at 37°C and 5% CO2 in a humidified incubator.

Experimental Procedure

Melatonin (MEL) (M5250, Sigma-Aldrich) was dissolved in ethanol to a final concentration of 0.01%. When 80% confluence was reached, AML12 cells were divided into four groups: MEL, AML12 cells were treated with 1µM melatonin; LPS, AML12 cells were treated with 25μg/mL lipopolysaccharide (LPS from Escherichia coli O55:B5; L-2880, Sigma-Aldrich) for 24h; MEL+LPS, AML12 cells were pretreated with 1µM melatonin for 1h followed by 25μg/mL LPS for 24 h; and control, AML12 cells were treated with an equivalent volume of vehicle.

Measurement of intracellular ROS levels

Intracellular ROS levels were evaluated using a commercially available kit (S0033S, Beyotime Biotechnology, China), according to the manufacturer’s recommendations. Briefly, AML12 cells were trypsinized, centrifuged, and incubated with 2’,7’-dichlorofluorescein-diacetate (DCFH-DA) (10 μM in DMEM/F12). ROS was quantified as mean fluorescence intensity using a flow cytometer (BD Biosciences, San Jose, CA, USA) with 488 nm excitation and 525 nm emission.

Enzyme-linked immunosorbent assay (ELISA)

TNF-α and IL-6 protein levels were assessed in AML12 cell-free culture supernatants using a commercial ELISA kit (EMC102A, EMC004, Neobioscience Technology Company, China), according to the manufacturer’s instructions.

Biochemical analyses

Malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and intracellular ATP levels were assessed in AML12 cell cultures using appropriate kits (S0131S, S0101S, S0027, lipid peroxidation MDA assay kit, total SOD assay kit with WST-8, enhanced ATP assay kit [Beyotime Biotechnology, China]) according to the manufacturer’s instructions.

Western blot analysis

AML12 cells were homogenized in radioimmunoprecipitation assay (RIPA) buffer (P0013B, Beyotime Biotechnology, China) supplemented with a protease and phosphatase inhibitor cocktail (P1045, Beyotime Biotechnology, China). Total protein was extracted and analyzed using a BCA protein assay kit (P0012S, Beyotime Biotechnology). After separation on 10% SDS‑PAGE, proteins were transferred onto polyvinylidene difluoride membranes (Millipore), which were then blocked for 1 h at room temperature (5% BSA) and incubated with the following primary antibodies: PINK1 (1:1,000, 23274-1-AP, Proteintech), Parkin (1:1,000, 14060-1-AP, Proteintech), PGC-1α (1:1,000, 66369-1-Ig, Proteintech), NRF-1 (1:1,000, 12482-1-AP, Proteintech), TFAM (1:1,000, 19998-1-AP, Proteintech), and GAPDH (1:5,000, 60004-1-Ig, Proteintech) overnight at 4 ̊C. Subsequently, membranes were incubated with horse radish peroxidase (HRP)-conjugated secondary antibodies. Protein levels were quantified using densitometry and Image J software.

Statistical analysis

Statistical analysis was performed with SPSS 20.0. Data are presented as the mean ± SD of three individual experiments using GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA). Comparisons were performed using one-way analysis of variance (ANOVA) followed by a post hoc pairwise comparison (least significant difference [LSD]). P<0.05 was considered statistically significant.

Melatonin attenuated LPS-induced inflammatory response in hepatocytes

As proinflammatory cytokine levels are increased in sepsis, which may damage the liver, TNF-α and IL-6 protein levels were assessed in cell-free culture supernatants to investigate the effects of melatonin on the LPS-induced inflammatory response in hepatocytes. TNF-α and IL-6 protein levels were significantly increased in cell-free culture supernatants from LPS-treated AML12 cells compared to controls, and significantly decreased in cell-free culture supernatants from LPS-treated AML12 cells by melatonin pre-treatment (Fig.1).

Melatonin attenuated LPS-induced oxidative stress in hepatocytes

To investigate the redox status of hepatocytes during sepsis, intracellular ROS levels, lipid peroxidation (MDA level), and SOD activity were measured. Intracellular ROS and MDA levels were significantly increased in LPS-treated AML12 cells compared to controls, and significantly decreased in LPS-treated AML12 cells by melatonin pre-treatment. Conversely, SOD activity was significantly decreased in LPS-treated AML12 cells compared to controls, and significantly increased in LPS-treated AML12 cells by melatonin pre-treatment (Fig.2).

Melatonin increased intracellular ATP levels under septic conditions

To investigate mitochondrial function in hepatocytes during sepsis, intracellular ATP levels were assessed. Intracellular ATP levels were significantly decreased in LPS-treated AML12 cells compared to controls, and significantly increased in LPS-treated AML12 cells by melatonin pre-treatment (Fig.3).

Melatonin upregulated mitophagy-related protein levels

The PINK1-Parkin pathway is a main regulator of mitophagy. Western blot analysis showed that PINK1 and Parkin protein levels were significantly increased in LPS-treated AML12 cells compared to controls, and significantly increased in LPS-treated AML12 cells by melatonin pre-treatment (Fig.4).

Melatonin upregulated mitochondrial biogenesis-related protein levels

PGC-1α, NRF-1 and TFAM are the most relevant proteins in mitochondrial biogenesis. Western blot analysis showed that PGC-1α, NRF-1 and TFAM protein levels were significantly increased in LPS-treated AML12 cells compared to controls, and significantly increased in LPS-treated AML12 cells by melatonin pre-treatment (Fig.5).

This study explored the protective mechanisms of melatonin in an in vitro model of sepsis-induced hepatocyte injury with a focus on mechanisms involved in mitochondria quality control. Findings showed that melatonin reduced LPS-induced inflammation and oxidative stress and enhanced mitophagy and mitochondrial biogenesis in hepatocytes.

Patients with liver dysfunction often experience sepsis, with symptoms occurring in the early stages [4]. Melatonin has anti-inflammatory, anti-oxidative stress and anti-tumor effects, and may have a protective role in the pathogenesis of sepsis. A prior report showed that melatonin reduced sepsis-induced liver inflammation and oxidative stress, thereby alleviating liver damage and reducing liver dysfunction [17]. In the present study, LPS was used to establish an in vitro model of hepatocyte injury induced by sepsis. Consistent with previous results, melatonin pretreatment decreased the LPS-induced release of inflammatory cytokines from hepatocytes, implying that melatonin may reduce hepatocellular inflammation during sepsis.

Oxidative stress, defined as an imbalance between the production of ROS and their elimination by protective mechanisms, contributes to the pathogenesis of sepsis. Sepsis can cause metabolic imbalance, mitochondrial dysfunction, and the production of large quantities of ROS, which accumulate and damage hepatocytes [18]. Mitochondria produce and are damaged by ROS within cells. Mitochondrial ROS production mainly takes place in the electron transport chain during oxidative phosphorylation. Altered mitochondrial function can lead to ROS overproduction, which damages mitochondrial proteins and DNA, and produces a continuous cycle of ROS generation [19]. In the present study, intracellular ROS level was elevated in LPS-treated hepatocytes vs. controls, and this effect was reversed by pre-treatment with melatonin. MDA is formed during lipid peroxidation and is considered a marker of oxidative stress. SOD is a cellular antioxidant and responsible for the elimination of ROS [20]. In the present study, MDA levels were elevated and SOD activity was reduced in LPS-treated hepatocytes vs. controls, and these effects were reversed by pre-treatment with melatonin. These data suggest that melatonin may alleviate LPS-induced oxidative stress in hepatocytes.

Hepatocytes have developed a variety of mechanisms to maintain mitochondrial homeostasis, including mitophagy and mitochondrial biogenesis [7]. Mitophagy, or selective autophagy of mitochondria, can remove damaged mitochondria and prevent excessive production of ROS [6]. The PINK1-Parkin pathway is a main regulator of mitophagy. In sepsis, PINK1 accrues on the outer membrane of dysfunctional mitochondria, which induces the translocation of Parkin from the cytoplasm to the outer mitochondrial membrane and triggers mitophagy [7]. Accumulating evidence implies that mitophagy may have a protective effect on liver function during sepsis. Enhancing mitophagy reduced LPS-induced cardiomyocyte injury, oxidative stress, and inflammation [21], while Parkin-knockout mice exhibited worse cardiac function and constant degradation of mitochondrial metabolic functions compared to wild-type mice during sepsis [22]. In the present study, LPS induced mitophagy in hepatocytes vs. controls, and these effects were enhanced by melatonin pre-treatment, suggesting that melatonin may augment mitophagy in hepatocytes during sepsis.

Mitochondrial biogenesis, or the formation of new mitochondria, is mainly regulated by the PGC-1a-NRF1-TFAM pathway. PGC-1a is a transcriptional co-activator that promotes mitochondrial DNA replication and transcription by activating the transcription factor NRF1 and subsequently upregulating the expression of TFAM [7]. Mitochondrial biogenesis has a role in sepsis. In astrocytes, sepsis can stimulate mitochondrial biogenesis, increase the number of mitochondria in cells, and recover mitochondrial ultrastructure to meet the increased energy demands of the astrocytes under septic conditions [23][24]. One recent report showed that melatonin can enhance mitochondrial biogenesis in liver fibrosis and sustain mitochondria [14]. In the present study, PGC-1a, NRF1 and TFAM protein levels were elevated in LPS-treated hepatocytes vs. controls, and these effects were enhanced by melatonin pre-treatment. These results suggest that melatonin can promote mitochondrial biogenesis during sepsis.

Cellular ATP levels are a marker of mitochondrial function. In the present study, intracellular ATP levels were reduced in LPS-treated hepatocytes vs. controls, and this effect was reversed by melatonin pre-treatment. These results suggest that melatonin can improve mitochondrial function in hepatocytes during sepsis.

In conclusion, our study elucidated the role of melatonin in preserving mitochondrial homeostasis in hepatocytes during sepsis. Mechanisms involved selective elimination of dysfunctional mitochondria through mitophagy and induction of mitochondrial biogenesis. These data indicate melatonin has potential as a therapy for sepsis-induced liver injury.

Acknowledgements

Not applicable.

Funding

The authors did not receive support from any organization for the submitted work.

Availability of data and material

The datasets used during the present study are available from the corresponding author upon reasonable request.

Code availability

Not applicable.

Author contributions

Bin Hu, Zhijiang Chen, Lili Liang, Meiyu Zheng, Xinxin Chen and Yaxin Wang performed the experiments; Bin Hu and Zhijiang Chen analyzed the data; Bin Hu drafted the manuscript; Qiyi Zeng contributed to the interpretation of the results and critical revision of the manuscript. All authors had final approval of the submitted versions.

Competing interests

The authors declare that there have no competing interests.

Vincent J, Marshall JC, Ñamendys-Silva SA, François B, Martin-Loeches I, Lipman J, Reinhart K, Antonelli M, Pickkers P, Njimi H, Jimenez E, Sakr Y (2014) Assessment of the worldwide burden of critical illness: the Intensive Care Over Nations (ICON) audit. The Lancet Respiratory Medicine 2: 380-386. https://doi.org/10.1016/S2213-2600(14)70061-X
Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, Bellomo R, Bernard GR, Chiche J, Coopersmith CM, Hotchkiss RS, Levy MM, Marshall JC, Martin GS, Opal SM, Rubenfeld GD, van der Poll T, Vincent J, Angus DC (2016) The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 315: 801. https://doi.org/10.1001/jama.2016.0287
Kramer L, Jordan B, Druml W, Bauer P, Metnitz PGH (2007) Incidence and prognosis of early hepatic dysfunction in critically ill patients—A prospective multicenter study. CRIT CARE MED 35: 1097-1099. https://doi.org/10.1097/01.CCM.0000259462.97164.A0
Yan J, Li S, Li S (2014) The Role of the Liver in Sepsis. INT REV IMMUNOL 33: 498-510. https://doi.org/10.3109/08830185.2014.889129
Han D, Ybanez MD, Johnson HS, McDonald JN, Mesropyan L, Sancheti H, Martin G, Martin A, Lim AM, Dara L, Cadenas E, Tsukamoto H, Kaplowitz N (2012) Dynamic adaptation of liver mitochondria to chronic alcohol feeding in mice: biogenesis, remodeling, and functional alterations. J BIOL CHEM 287: 42165-42179. https://doi.org/10.1074/jbc.M112.377374
Garrabou G, Moren C, Lopez S, Tobias E, Cardellach F, Miro O, Casademont J (2012) The effects of sepsis on mitochondria. J INFECT DIS 205: 392-400. https://doi.org/10.1093/infdis/jir764
Wu Y, Yao YM, Lu ZQ (2019) Mitochondrial quality control mechanisms as potential therapeutic targets in sepsis-induced multiple organ failure. J Mol Med (Berl) 97: 451-462. https://doi.org/10.1007/s00109-019-01756-2
Mannam P, Shinn AS, Srivastava A, Neamu RF, Walker WE, Bohanon M, Merkel J, Kang MJ, Dela CC, Ahasic AM, Pisani MA, Trentalange M, West AP, Shadel GS, Elias JA, Lee PJ (2014) MKK3 regulates mitochondrial biogenesis and mitophagy in sepsis-induced lung injury. Am J Physiol Lung Cell Mol Physiol 306: L604-L619. https://doi.org/10.1152/ajplung.00272.2013
Piantadosi CA, Withers CM, Bartz RR, MacGarvey NC, Fu P, Sweeney TE, Welty-Wolf KE, Suliman HB (2011) Heme oxygenase-1 couples activation of mitochondrial biogenesis to anti-inflammatory cytokine expression. J BIOL CHEM 286: 16374-16385. https://doi.org/10.1074/jbc.M110.207738
Singer M (2014) The role of mitochondrial dysfunction in sepsis-induced multi-organ failure. VIRULENCE 5: 66-72. https://doi.org/10.4161/viru.26907
Tordjman S, Chokron S, Delorme R, Charrier A, Bellissant E, Jaafari N, Fougerou C (2017) Melatonin: Pharmacology, Functions and Therapeutic Benefits. CURR NEUROPHARMACOL 15: 434-443. https://doi.org/10.2174/1570159X14666161228122115
Kleber A, Kubulus D, Rossler D, Wolf B, Volk T, Speer T, Fink T (2014) Melatonin modifies cellular stress in the liver of septic mice by reducing reactive oxygen species and increasing the unfolded protein response. EXP MOL PATHOL 97: 565-571. https://doi.org/10.1016/j.yexmp.2014.10.009
Ozkok E, Yorulmaz H, Ates G, Aksu A, Balkis N, Sahin O, Tamer S (2016) Amelioration of energy metabolism by melatonin in skeletal muscle of rats with LPS induced endotoxemia. PHYSIOL RES 65: 833-842. https://doi.org/10.33549/physiolres.933282
Kang JW, Hong JM, Lee SM (2016) Melatonin enhances mitophagy and mitochondrial biogenesis in rats with carbon tetrachloride-induced liver fibrosis. J PINEAL RES 60: 383-393. https://doi.org/10.1111/jpi.12319
Ma S, Chen J, Feng J, Zhang R, Fan M, Han D, Li X, Li C, Ren J, Wang Y, Cao F (2018) Melatonin Ameliorates the Progression of Atherosclerosis via Mitophagy Activation and NLRP3 Inflammasome Inhibition. OXID MED CELL LONGEV 2018: 9286458. https://doi.org/10.1155/2018/9286458
Guo P, Pi H, Xu S, Zhang L, Li Y, Li M, Cao Z, Tian L, Xie J, Li R, He M, Lu Y, Liu C, Duan W, Yu Z, Zhou Z (2014) Melatonin Improves mitochondrial function by promoting MT1/SIRT1/PGC-1 alpha-dependent mitochondrial biogenesis in cadmium-induced hepatotoxicity in vitro. TOXICOL SCI 142: 182-195. https://doi.org/10.1093/toxsci/kfu164
Chen J, Xia H, Zhang L, Zhang H, Wang D, Tao X (2019) Protective effects of melatonin on sepsis-induced liver injury and dysregulation of gluconeogenesis in rats through activating SIRT1/STAT3 pathway. BIOMED PHARMACOTHER 117: 109150. https://doi.org/10.1016/j.biopha.2019.109150
Zhong W, Qian K, Xiong J, Ma K, Wang A, Zou Y (2016) Curcumin alleviates lipopolysaccharide induced sepsis and liver failure by suppression of oxidative stress-related inflammation via PI3K/AKT and NF-kappaB related signaling. BIOMED PHARMACOTHER 83: 302-313. https://doi.org/10.1016/j.biopha.2016.06.036
Quoilin C, Mouithys-Mickalad A, Lecart S, Fontaine-Aupart MP, Hoebeke M (2014) Evidence of oxidative stress and mitochondrial respiratory chain dysfunction in an in vitro model of sepsis-induced kidney injury. Biochim Biophys Acta 1837: 1790-1800. https://doi.org/10.1016/j.bbabio.2014.07.005
Ren Y, Yang X, Niu X, Liu S, Ren G (2011) Chemical characterization of the avenanthramide-rich extract from oat and its effect on D-galactose-induced oxidative stress in mice. J Agric Food Chem 59: 206-211. https://doi.org/10.1021/jf103938e
Li J, Shi W, Zhang J, Ren L (2019) To Explore the Protective Mechanism of PTEN-Induced Kinase 1 (PINK1)/Parkin Mitophagy-Mediated Extract of Periplaneta Americana on Lipopolysaccharide-Induced Cardiomyocyte Injury. Med Sci Monit 25: 1383-1391. https://doi.org/10.12659/MSM.912980
Piquereau J, Godin R, Deschenes S, Bessi VL, Mofarrahi M, Hussain SN, Burelle Y (2013) Protective role of PARK2/Parkin in sepsis-induced cardiac contractile and mitochondrial dysfunction. AUTOPHAGY 9: 1837-1851. https://doi.org/10.4161/auto.26502
Zhao YZ, Gao ZY, Ma LQ, Zhuang YY, Guan FL (2017) Research on biogenesis of mitochondria in astrocytes in sepsis-associated encephalopathy models. Eur Rev Med Pharmacol Sci 21: 3924-3934
Chen XL, Wang Y, Peng WW, Zheng YJ, Zhang TN, Wang PJ, Huang JD, Zeng QY (2018) Effects of interleukin-6 and IL-6/AMPK signaling pathway on mitochondrial biogenesis and astrocytes viability under experimental septic condition. INT IMMUNOPHARMACOL 59: 287-294. https://doi.org/10.1016/j.intimp.2018.04.020

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Melatonin protects against LPS-induced inflammation and oxidative stress in hepatocytes by enhancing mitophagy and mitochondrial biogenesis

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