PPAR Research
Volume 2020 (2020), Article ID 6633022, 7 pages
https://doi.org/10.1155/2020/6633022

Research Article

MicroRNA-21 Contributes to Acute Liver Injury in LPS-Induced Sepsis Mice by Inhibiting PPARα Expression

Xianjin Du,¹ Miao Wu,² Dan Tian,² Jianlin Zhou,³ Lu Wang,¹ and Liying Zhan¹

¹Department of Critical Care Medicine, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuchang, Wuhan, Hubei 430060, China
²Department of Emergency, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuchang, Wuhan, Hubei 430060, China
³Department of Orthopedics, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuchang, Wuhan, Hubei 430060, China

Correspondence should be addressed to Xianjin Du; duxianjin@whu.edu.cn, Jianlin Zhou; zhoujianlin2005@sina.com, and Liying Zhan; zhanliying@whu.edu.cn

Received 13 October 2020; Accepted 11 December 2020; Published 22 December 2020

Academic Editor: Xiao-Jie Lu

Copyright © 2020 Xianjin Du et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The severity of sepsis may be associated with excessive inflammation, thus leading to acute liver injury. MicroRNA-21 is highly expressed in the liver of a variety of inflammation-related diseases, and PPARα is also proved to participate in regulating inflammation. In the present study, the LPS-induced sepsis model was established. We found that microRNA-21 expression was upregulated in the liver of sepsis mice, and microRNA-21 inhibition significantly reduced the liver injury. The expression of liver injury markers, inflammation cytokines, and PPARα in the septic mice was higher than in antagomir-21 treated septic mice. In addition, we also found that PPARα is the target gene of microRNA-21; PPARα antagonist GW6471 could reverse the effect of antagomir-21. In conclusion, our study illustrated that microRNA-21 exacerbate acute liver injury in sepsis mice by inhibiting PPARα expression.

1. Introduction

Sepsis is a common cause of death in intensive care units [1]. It is a public health problem worldwide, and approximately 19 million people suffer from sepsis yearly [2]. Sepsis was defined as a “life-threatening organ dysfunction caused by a deregulated host response to infection” at the Sepsis-3 conference [3]. The acute liver injury occurs at any stage of sepsis; the dysregulation of hepatocyte function may be related to cytokine storm [4]. Liver injury can not only aggravate the development of the disease but also lead to death [5]. Despite the urgent need for effective therapeutic options, many new therapies have not improved the survival rate [6]. Therefore, understanding the pathogenesis of sepsis is very important for the treatment of sepsis.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to a nuclear hormone receptor superfamily [7]. PPARα is an isoform of peroxisome proliferator-activated receptors, which regulates adipocyte differentiation, fatty acid oxidation, and glucose metabolism [8]. More recently, emerging evidence revealed that PPARα activation could reduce the inflammatory response by promoting NF-κB inactivation [9]. In addition, the liver PPARα expression was found to be disturbed during sepsis. In a murine model of sepsis, liver PPARα expression was significantly associated with survival [10].

MicroRNA is a class of noncoding RNA, 19-22 nucleotides in length [11], which regulates gene expression at the posttranscriptional level by degrading message RNA or inhibiting its transcription [12]. MicroRNA microarray analysis showed the upregulation of microRNA-21 in sepsis patients [13]. Nevertheless, the role of microRNA-21 in sepsis-induced liver injury has not been fully elucidated.

In this study, we aim to explore the role of miR-21 and PPARα in the pathogenesis of sepsis-induced liver injury. For this purpose, we used antagomir-21 to inhibit miR-21 expression in murine sepsis model and detected the levels of proinflammatory cytokines (TNF-α, IL-1β, and IL-6), liver injury markers (AST, ALT), and PPARα expression.

2. Materials and Methods

2.1. Animal Models

All male C57BL/6 mice were obtained from Hubei Provincial Center for Disease Control and Prevention. The mice were housed in a pathogen-free facility with standard laboratory diet and water. At the age of 8 weeks, the mice were received three retroorbital intravenous injections of antagomir-21 (5’-UCAACAUCAGUCUGAUAAGCUA-3’; 16 mg/kg; ), antagomir control (5’-AAGGCAAGCUGACCCUGAAGUU-3’; 16 mg/kg; ), antagomir-21+GW6471 (16 mg/kg; 30 mg/kg; ), phosphate saline (PBS; ). Six PBS-treated mice were used as controls; the other mice received intraperitoneal injection of 5 mg/kg lipopolysaccharide to induce sepsis. At 24 h after LPS injection, all mice were sacrificed.

All experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health (2011).

2.2. Cell Culture

Mouse Biliary Duct Epithelial Cells and 293T cells were obtained from Newgainbio (Wuxi, China) and cultured in DM/F12 medium or Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin under 5% CO2 at 37°C.

2.3. Luciferase Reporter Assay

MiR-21 mimics (5’-UAGCUUAUCAGACUGAUGUUGA-3’) and NC-mimics (5’-UUCUCCGAACGUGUCACGUTT-3’) were purchased from Ribobio (Guangzhou, China). The pGL3 Luciferase Reporter Vectors (Promega) containing the PPARα-MT (5’-AAAAAAUCUGUUAGAUAAGCUA-3’) and PPARα-MuT (5’-AAUUAUAGUCAUACUAUUCGAA-3’) sequences were cotransfected with miR-21 mimics or NC-mimics (50 nM) into 293T and MBDEC cells. All transfections were performed using Lipofectamine 3000 (Invitrogen, USA). After 24 hours of incubation, 1×PLB was used to lyse the cells, and the luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega).

2.4. RNA Isolation and qRT-PCR

Total RNA was extracted from snap frozen mouse liver samples using TRIzol reagent (Invitrogen, USA). MiR-21 and mRNA levels were quantified by qRT-PCR assay. For miRNA, U6 was applied as endogenous control. For mRNA, GAPDH was used as endogenous control. All reactions were run on the ABI 7500Real-Time PCR System (Life Technologies, USA). The relative expression was calculated using 2-ΔΔCT method. All primers used in this study are as follows.

MiR-21 (stem-loop RT primer): 5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAACA-3’

MiR-21-F: 5’-GTGCAGGGTCCGAGGT-3’

MiR-21-R: 5’-GCCGCTAGCTTATCAGACTGATGT-3’

U6-F: 5’-AGCCCGCACTCAGAACATC-3’

U6-R: R: 5’-GCCACCAAGACAATCATCC-3’

GAPDH-F: 5’-CGTCCCGTAGACAAA ATGGTGAA-3’

GAPDH-R: 5’-GCCGTGAGTGGAGTCATACTGGAA CA-3’

PPARα-F: 5’-AACCTGAGGAAGCCGTTCTGTGACAT-3’

PPARα-R: 5’-GACCAGCTGCCGAAGGTCCACCAT-3’.

2.5. ELISA

The TNF-α, IL-1β, and IL-6 levels in liver or cultured supernatant were quantified using the Mouse TNF-α Precoated Elisa kit, Mouse IL-1β Precoated Elisa kit, and Mouse IL-6 Precoated ELISA kit, respectively.

2.6. Metabolic Analyses

Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined using the VITROS350 chemistry system (Johnson & Johnson, USA).

2.7. Histological Analysis of Liver

Liver samples from mice were fixed in 4% paraformaldehyde, then stained with hematoxylin and eosin. The liver damage photos were observed and recorded under light microscopy.

2.8. Western Blot

The nuclear protein of liver samples was extracted using NE-PER™ Nuclear Extraction Reagents (Thermo Fisher, USA). Protein was quantified using BCA Protein Assay (Thermo Fisher, USA). Total protein (50 μg) was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Membranes were incubated with the primary antibodies (1 : 1000) overnight at 4°C. After HRP-conjugated secondary antibodies (1 : 5000) incubation; the protein bands were visualized using the Clarity Western ECL Substrate (Bio-Rad, USA).

2.9. Statistical Analyses

Student’s test and one-way analysis of variance were used to analyze the significance between groups. was considered statistically significant. Data analysis was performed using SPSS 22.0 (IBM, Chicago, USA), and figures were designed using GraphPad Prism 8.3.0.

3. Results

3.1. Expression of miR-21 Is Increased in the Liver of Sepsis Mice

The animal experiments were performed according to the design (Figure 1(a)). After these mice were sacrificed, we collected the serum of the mice and isolated the livers. Subsequently, the expression of miR-21 in the liver was detected using qRT-PCR (Figure 1(b)). The results showed that miR-21 expression was significantly upregulated in septic mice; nevertheless, the injection of antagomir-21 totally blocked the upregulation of miR-21, suggesting that the sepsis mouse models with miR-21 inhibition were successfully established.

(a)

(b)

Figure 1: MiR-21 expression was increased in the liver of sepsis mice. (a) Mouse study design. (b) The miR-21 expression in the liver of the wide-type mice treated with PBS (WT) and the LPS-induced mice treated with PBS, antagomir-21 control (NC), antagomir-21 (A21), antagomir-21, and PPARα antagonist GW6471 (A21+G). QRT-PCR assay was used to detect the expression of miR-21. .

3.2. Antagomir-21 Reduces Liver Injury and Inflammation in Sepsis Mice

To further investigate the function of miR-21 in sepsis-induced liver injury, we next evaluated the pathological damage of livers and the levels of proinflammatory cytokines. As shown in Figures 2(a) and 2(b), the degree of liver damage in septic mice was significantly increased. Antagomir-21 administration alleviated the liver injury; however, in the antagomir-21 and GW6471 coadministration group, the degree of liver injury showed no changes. As expected in these mice of sepsis, the expression of liver injury markers (serum AST and serum ALT; Figures 2(c) and 2(d)) and proinflammatory cytokines (TNF-α, IL-1β, and IL-6; Figures 2(e)–2(j)) was notably reduced by antagomir-21. Nevertheless, GW6471 restored the levels of these cytokines again. To sum up above results, it demonstrated that miR-21 inhibition strongly decreased the liver injury and inflammation in septic mice. But the effects produced by miR-21 inhibition were reversed by PPARα antagonist GW6471.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

Figure 2: Antagomir-21 reduces liver injury and inflammation. (a, b) Histological analysis of livers. Tissue damages were scored in a scale of 0–4, with 0, 1, 2, 3, and 4 corresponding to 0%, <25%, 26%–50%, 51%–75%, and ≥76% of liver injury, respectively. (c, d) The serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. (e–g) ELISA assay was used to determine the levels of TNF-α, IL-1β, and IL-6 in livers. (h–i) ELISA assay was used to determine the levels of TNF-α, IL-1β, and IL-6 in serum. , .

3.3. MiR-21 Directly Interacts with PPARα

It is well known that miRNAs participate in a variety of physiological activities by regulating gene expression at the posttranscriptional level. To investigate the molecular mechanisms of miR-21 in sepsis, we used TargetScan to identify the potential genes, and PPARα was found to be one of the most relevant genes to sepsis. Starbase (http://starbase.sysu.edu.cn) was used to predict the putative binding sites between PPARα and miR-21 (Figure 3(a)). Luciferase report assay was performed to test whether miR-21 can directly bind to PPARα. As shown in Figure 3(b), in the 293T and MBDEC cells that cotransfected with PPARα WT and miR-21 mimics, the luciferase activities were significantly reduced, but there were no changes in the PPARα-MuT group. The results indicated that PPARα is a target gene of miR-21.

(a)

(b)

Figure 3: MiR-21 directly interacts with PPARα. (a) The putative binding sites between PPARA WT and miR-21. (b) Luciferase activity was detected in luciferase reporter vectors harboring PPARA WT/MuT sequences and miR-21 mimics cotransfected 293T and MBDEC cells. .

3.4. Antagomir-21 Reduces Liver Injury and Inflammation by Restoring PPARα Expression

To further verify whether miR-21 regulates the expression of PPARα in the liver. We detected the PPARα expression in the liver of five groups; three liver samples was randomly selected from each groups (Figures 4(a) and 4(b)). The total RNA was extracted from liver tissues, and the protein was extracted from the nucleus. The results showed that PPARα expression in PBS and AC groups was markedly decreased compared with the WT group. In addition, antagomir-21 treatment increased the PPARα expression in the liver, but GW6471 inhibited the level of PPARα nuclear protein. Aggregating all the results, we found that antagomir-21 alleviated liver injury and inflammation by restoring PPARα expression.

(a)

(b)

Figure 4: Antagomir-21 reduced liver injury and inflammation by restoring PPARα expression. (a) The expression of PPARα mRNA in livers was detected by qRT-PCR. (b) The expression of PPARα nuclear protein in livers was detected using western blot. .

4. Discussion

This study illustrated that miR-21 suppression attenuated liver injury in LPS-induced sepsis mice, by potentiating PPARα expression, which suggested a contribution of miR-21 in the pathogenesis of sepsis-induced liver injury. In addition, antagomiR-21 and PPARα represented anti-inflammatory activities in septic mice. These findings demonstrated that the miR-21/PPARα pathway might serve as a potential target in sepsis therapy.

A proinflammatory status is the key feature of sepsis, and the liver plays an important role in inflammation [14]. According to reports, miR-21 is upregulated in various inflammatory diseases, including myocardial injury [15], nonalcoholic steatohepatitis [16], and osteoarthritis [17]. However, the role of miR-21 in sepsis has not been fully elucidated. At first, we confirmed that miR-21 expression was elevated in the liver of LPS-induced sepsis mice. Then, to investigate the molecular mechanism of miR-21 in sepsis, we screened out some genes with miR-21 binding sites. Previous reports have shown that PPARα expression is reduced in the liver of NASH patients, and PPARα activation inhibits liver fibrosis in mice [18, 19]. These researches promoted us to verify the connection between miR-21 and PPARα. Subsequently, we found that in the liver of sepsis mice model, PPARα expression was decreased when miR-21 expression was increased. In addition, antagomir-21 restored PPARα expression and attenuated sepsis-induced liver injury, whereas PPARα antagonist GW6471 blocked the inhibitory effect of antagomir-21 on liver injury. Altogether, our study indicated that miR-21 regulated liver inflammation through PPARα inhibition.

Nevertheless, there are several limitations in our study. Recent studies reported that miR-21 was primarily expressed in biliary and inflammatory cells in the liver, rather than in hepatocytes. In this study, the cellular source of miR-21 was not elucidated; the function of miR-21/PPARα axis needs to be further explored.

In addition, miR-21 and PPAR agonists are considered druggable targets [20, 21]. This study showed that the mir-21/PPARα pathway might be an interesting new strategy for sepsis treatment. Some PPAR agonists, however, show side effects that cause them to be discontinued [22], and mir-21 antagonism treatment may also have side effects. Therefore, rigorous study design and safety monitoring are essential.

In conclusion, in LPS-induced sepsis mice model, we demonstrated that miR-21 contributed sepsis-induced liver injury and inflammation by inhibiting PPARα expression. Therefore, this study may provide an attractive potential target for the treatment of sepsis.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors confirm that there are no conflicts of interest.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 81601670) and Hubei Province Natural Science Fund (2014CFB302). We sincerely appreciate all lab members.

References

J. Cohen, J.-L. Vincent, N. K. J. Adhikari et al., “Sepsis: a roadmap for future research,” The Lancet. Infectious diseases, vol. 15, no. 5, pp. 581–614, 2015.

L. Van Wyngene, T. Vanderhaeghen, S. Timmermans et al., “Hepatic PPARα function and lipid metabolic pathways are dysregulated in polymicrobial sepsis,” EMBO Molecular Medicine, vol. 12, no. 2, article e11319, 2020.

M. Singer, C. S. Deutschman, C. W. Seymour et al., “The third international consensus definitions for sepsis and septic shock (Sepsis-3),” JAMA, vol. 315, no. 8, pp. 801–810, 2016.

N. Nesseler, Y. Launey, C. Aninat, F. Morel, Y. Mallédant, and P. Seguin, “Clinical review: the liver in sepsis,” Critical Care, vol. 16, no. 5, p. 235, 2012.

J. Yan, S. Li, and S. Li, “The role of the liver in sepsis,” International Reviews of Immunology, vol. 33, no. 6, pp. 498–510, 2014.

T. Evans, “Diagnosis and management of sepsis,” Clinical Medicine, vol. 18, no. 2, pp. 146–149, 2018.

I. Issemann and S. Green, “Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators,” Nature, vol. 347, no. 6294, pp. 645–650, 1990.

B. Tunctan, S. P. Kucukkavruk, M. Temiz-Resitoglu, D. S. Guden, A. N. Sari, and S. Sahan-Firat, “Bexarotene, a selective RXRα agonist, reverses hypotension associated with inflammation and tissue injury in a rat model of septic shock,” Inflammation, vol. 41, no. 1, pp. 337–355, 2018.

J. Korbecki, R. Bobinski, and M. Dutka, “Self-regulation of the inflammatory response by peroxisome proliferator-activated receptors,” Inflammation research: official journal of the European Histamine Research Society, vol. 68, pp. 443–458, 2019.

S. W. Standage, C. C. Caldwell, B. Zingarelli, and H. R. Wong, “Reduced peroxisome proliferator-activated receptor alpha expression is associated with decreased survival and increased tissue bacterial load in sepsis,” Shock, vol. 37, no. 2, pp. 164–169, 2012.

D. Portius, C. Sobolewski, and M. Foti, “MicroRNAs-dependent regulation of PPARs in metabolic diseases and cancers,” PPAR Research, vol. 2017, Article ID 7058424, 2017.

T. X. Lu, A. Munitz, and M. E. Rothenberg, “MicroRNA-21 is up-regulated in allergic airway inflammation and regulates IL-12p35 expression,” Journal of Immunology, vol. 182, no. 8, pp. 4994–5002, 2009.

Z. Xue, Q. Xi, H. Liu et al., “miR-21 promotes NLRP3 inflammasome activation to mediate pyroptosis and endotoxic shock,” Cell death & disease, vol. 10, p. 461, 2019.

P. Strnad, F. Tacke, A. Koch, and C. Trautwein, “Liver - guardian, modifier and target of sepsis,” Nature Reviews. Gastroenterology & Hepatology, vol. 14, no. 1, pp. 55–66, 2017.

R. Ji, Y. Cheng, J. Yue et al., “MicroRNA expression signature and antisense-mediated depletion reveal an essential role of MicroRNA in vascular neointimal lesion formation,” Circulation Research, vol. 100, no. 11, pp. 1579–1588, 2007.

X. Loyer, V. Paradis, C. Hénique et al., “Liver microRNA-21 is overexpressed in non-alcoholic steatohepatitis and contributes to the disease in experimental models by inhibiting PPARalpha expression,” Gut, vol. 65, no. 11, pp. 1882–1894, 2016.

D. Iliopoulos, K. N. Malizos, P. Oikonomou, and A. Tsezou, “Integrative microRNA and proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks,” PLoS One, vol. 3, no. 11, article e3740, 2008.

S. Francque, A. Verrijken, S. Caron et al., “PPARalpha gene expression correlates with severity and histological treatment response in patients with non-alcoholic steatohepatitis,” Journal of Hepatology, vol. 63, no. 1, pp. 164–173, 2015.

M. Pawlak, E. Baugé, W. Bourguet et al., “The transrepressive activity of peroxisome proliferator-activated receptor alpha is necessary and sufficient to prevent liver fibrosis in mice,” Hepatology, vol. 60, no. 5, pp. 1593–1606, 2014.

X. W. Wang, N. H. Heegaard, and H. Orum, “MicroRNAs in liver disease,” Gastroenterology, vol. 142, no. 7, pp. 1431–1443, 2012.

J. M. Hesselink and T. A. Hekker, “Therapeutic utility of palmitoylethanolamide in the treatment of neuropathic pain associated with various pathological conditions: a case series,” Journal of Pain Research, vol. 5, pp. 437–442, 2012.

J. C. Fruchart, “Selective peroxisome proliferator-activated receptor alpha modulators (SPPARMalpha): the next generation of peroxisome proliferator-activated receptor alpha-agonists,” Cardiovascular diabetology, vol. 12, p. 82, 2013.