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. 2024 Jan 5;198(1):50–60. doi: 10.1093/toxsci/kfad132

Hepatocyte miR-21-5p-deficiency alleviates APAP-induced liver injury by inducing PPARγ and autophagy

Chao Xu 1,2,✉,#, Fang Yan 3,#, Yulan Zhao 4, Hartmut Jaeschke 5, Jianguo Wu 6,7, Li Fang 8,9, Lifang Zhao 10,11, Yuanfei Zhao 12,✉, Li Wang 13
PMCID: PMC11491925  PMID: 38180883

Abstract

Acetaminophen (APAP)-induced liver injury is one of the most frequent causes of acute liver failure worldwide. Significant increases in the levels of miRNA-21 in both liver tissues and plasma have been observed in APAP-overdosed animals and humans. However, the mechanistic effect of miRNA-21 on acute liver injury remains unknown. In this study, we generated a new hepatocyte-specific miRNA-21 knockout (miR-21-HKO) mouse line. miR-21-HKO and the background-matched sibling wild-type (WT) mice were treated with a toxic dose of APAP. Compared with WT mice, miR-21 HKO mice showed an increased survival, a reduction of necrotic hepatocytes, and an increased expression of light chain 3 beta, which suggested an autophagy activation. The expression of PPARγ was highly induced in the livers of miR-21-HKO mice after a 2-h APAP treatment, which preceded the activation of LC3B at the 12 h APAP treatment. miR-21 negatively regulated PPARγ protein expression by targeting its 3′-UTR. When PPARγ function was blocked by a potent antagonist GW9662 in miR-21-HKO mice, the autophage activation was significantly diminished, suggesting an indispensable role of PPARγ signaling pathway in miR-21-mediated hepatotoxicity. Taken together, hepatocyte-specific depletion of miRNA-21 alleviated APAP-induced hepatotoxicity by activating PPARγ and autophagy, demonstrating a crucial new regulatory role of miR-21 in APAP-mediated liver injury.

Keywords: drug-induced liver injury, microRNA, autophagy, PPARγ, LC3B

Acetaminophen (APAP) overdose causes the quintessential hepatotoxicity induced by the drug and is the most common single cause of liver injury in the United States and the United Kingdom (Mak et al., 2017). APAP-related adverse events continue to be a public health burden (Lee, 2004). APAP overdose is the leading cause for calls to Poison Control Centers (>100 000/year) and accounts for more than 50 000 emergency room visits, 2600 hospitalizations, and nearly 500 deaths per year as a result of APAP-associated acute liver failure (Major et al., 2016; Nourjah et al., 2006).

Noncoding RNAs (ncRNAs) are ribonucleic acid (RNA) molecules that are not translated into protein products (Zhao et al., 2017). Different classes of ncRNAs participate in different cellular processes, such as gene expression, RNA maturation, and protein synthesis (Gebert and MacRae, 2018). MicroRNAs (miRNAs, miRs) are small ncRNAs that regulate gene expression (Zhang et al., 2018) and have recently been the focus of studies in various liver diseases (Tran et al., 2017; Yang et al., 2015).

Expression of miR-21 has been found to be deregulated in almost all types of cancers and therefore was classified as an oncomiR. Recent studies have revealed important roles of miR-21 in liver injury. Using APAP overdose-induced liver injury as a mouse model, highly significant differences in the level of miR-21 in both liver tissues and in plasma between control and overdosed animals were observed (Wang et al., 2009). Similarly, miR-21 became dramatically elevated in the serum of APAP-overdosed patients, compared with the ischemic liver patients (Ward et al., 2014). Moreover, it has been demonstrated that patients with spontaneous recovery from acute liver failure showed significantly higher serum levels of miR-21 compared with nonrecovered patients. In liver biopsies, miR-21 expression was decreased in liver tissues of the spontaneous survivors (John et al., 2014). These observations suggest that miR-21 may exert an important role in APAP-induced hepatotoxicity.

Liver is composed of parenchymal hepatocytes and nonparenchymal cells including Kupffer cells, stellate cells, and liver sinusoidal endothelial cells. Hepatocytes perform most of the liver’s functions including metabolism, storage, digestion, and bile production, and their dysregulation contributes to various liver diseases (Lee et al., 2015; Smalling et al., 2013; Tsuchiya et al., 2015; Zhang et al., 2016). Despite recent advances on the miR-21 regulation of liver metabolic function, little is known about its hepatic cell-type specific effect during liver injury and the progression of liver diseases.

In the present study, we employed hepatocyte-specific miR-21 knockout (miR-21 HKO) mice to unravel the mechanistic function of miR-21 on the hepatotoxicity induced by APAP. Compared with the wild-type (WT) mice, miR-21 HKO mice exhibited a markedly low degree of liver damage and increased survival. This was contributed by the significant elevation of PPARγ as well as the activation of autophagy. We further showed that miR-21 played a negative regulatory role on PPARγ protein expression. Moreover, PPARγ antagonist GW9662 reversed the protective effect of loss of miR-21 on APAP-induced liver injury. These findings highlight a novel physio-pathological role for miR-21 in the regulation of APAP-induced hepatotoxicity mediated by PPARγ and suggest that targeting PPARγ signaling pathway may provide a crucial therapeutic approach in the treatment of APAP-induced hepatotoxicity involving miR-21.

Materials and methods

 

 

Animals

The embryonic stem cells for deletion of miR-21 were generated by the trans-NIH Knock-Out Mouse Project (KOMP) and obtained from the KOMP Repository (www.komp.org) (Lloyd, 2011). We contracted with KOMP to generate the germline-transmitted lacZ-neo-floxed mice. To generate hepatocyte-specific miR-21 deletion, we then crossed the floxed mice with act-FLPe/Alb-CRE lines to generate CRE+ (hepatocyte specific miR-21−/−, HKO), and their subline CRE− (WT) mice. Mice were fed a standard rodent chow diet (Harlan No. 2018) with free access to water and maintained in a 12-h light/dark cycle (light on 6 am to 6 pm), temperature-controlled (23°C), and pathogen-free facility. In vivo experiments were performed on male mice at the age of 6–8 weeks unless stated otherwise (n = 5–6 mice/group). All mice were sacrificed after overnight fasting unless otherwise indicated. For in vivo studies, mice were either given PBS (intraperitoneal; IP) or APAP (400 mg/kg, IP) after fasting for 12–15 h. GW9662 (in saline) were administered (2 mg/kg, IP) every 12 h for 3 times prior to the treatment of APAP. Protocols for animal use were approved by IACUC at the University of Connecticut.

Cell culture

HEK293T and Huh7 cells were purchased from ATCC and were made aliquots and stored in liquid nitrogen tank immediately after the first passage. The cell lines were passaged for less than 6 months when used for experiments. Cells were maintained in DMEM (high glucose) medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. All cell lines were maintained at 37°C in a humidified atmosphere of 5% CO2 in air.

Primary hepatocyte isolation

Primary hepatocytes were isolated by the 2-step collagenase perfusion described previously. The cells were plated in William E medium supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine and cultured in a humidified incubator at 37°C, 5% CO2. Cells were plated for 24 h before the appropriate treatments. Primary cultured hepatocytes were isolated and treated with APAP (5 μM).

Real-time PCR

RNA was isolated from the cells by TRIzol (Ambion) and cDNA synthesis was performed with High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher). And the quantitative PCR was performed using the Universal SYBR Green (Biorad). Relative quantification was calculated with normalization to HPRT1 (mouse). Specific primers are included in Supplementary Table 1.

Western blotting

The protein samples were extracted from liver tissue or cells. The protein concentration was measured using the bicinchoninic acid (BCA) method. Equal amounts of protein from different samples were separated in a 10% SDS-PAGE gel, then the samples were transferred into polyvinylidene difluoride membranes (Millipore) from the gel. After incubating for 1 h with 5% nonfat milk in TBST, the membranes were incubated overnight at 4°C with anti-p-JNK (1:1000, Cell Signaling), anti-CYP2E1 (1:1000, Cell Signaling), anti-LC3B (1:1000, Cell Signaling), anti-p62 (1:1000, Cell Signaling), anti-RIPK3 (1:1000, Cell Signaling), anti-MLKL (1:1000, Cell Signaling), anti-BIP (1:1000, Cell Signaling), anti-PARP (1:1000, Cell Signaling), anti-Caspase 8 (1:1000, Cell Signaling), anti- PPARγ (1:1000, Cell Signaling), anti-PGC1α (1:1000, Abcam), anti- PGC1β (1:1000, Novus), and anti-β-Actin (1:10 000, Sigma) antibodies. Following the primary antibodies, the membranes were incubated in correspondence secondary antibodies at a 1:3000 dilution for 1 h at room temperature. Immune complexes were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher). β-actin protein was evaluated as a loading control.

Antibodies

The antibodies were purchased from Sigma-Aldrich (β-Actin), Novus (PGC1β), Cell Signaling (p-JNK, CYP2E1, RIPK3, MLKL, BIP, PARP, Caspase 8, Beclin 1, p62, LC3B, mTOR, p-mTOR, AMPK, p-AMPK, PPARγ), and Abcam (PGC1α).

Transient transfection

Huh7 cells were transfected with miR-21 mimic or negative control using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) and plated in 6-well plates. Twenty-four hours later, the cells were harvested, and the mRNA and protein expression of PPARγ were detected.

Analysis of serum ALT and AST

Serum ALT and AST were detected by Infinity ALT/GPT kit (Thermo Fisher, TR71121) or Infinity AST/GOT (Thermo Fisher, TR70121) kit, respectively.

Plasma and hepatic APAP protein adducts measurement

Plasma and hepatic APAP adducts were measured by high-pressure liquid chromatography with electrochemical detection as described previously (Ni et al, 2012). Briefly, plasma samples and liver homogenates were dialyzed with a gel-filtration method to remove free APAP and APAP-GSH prior to digestion and analysis. A standard BCA assay was used to determine protein levels and adducts were normalized to total protein in the liver homogenates.

Histological analysis of liver sections

Freshly harvested mouse livers were fixed in formalin for 48 h and then embedded in paraffin. Paraffin sections at thickness of 4 µm were cut and subjected to xylene and ethanol rehydration prior to hematoxylin and eosin (H&E) and Masson trichrome staining. Eight fields of each slide were randomly taken under microscope and quantified by ImageJ software as described previously.

Microscopy for autophagy

Adenovirus-GFP-LC3 was kindly provided by Dr Wen-Xing Ding (Department of Pharmacology, Toxicology, and Therapeutics, The University of Kansas Medical Center) (Ni et al., 2012). To examine autophagy, primary hepatocytes were seeded in a 12 well-plate (2 × 105 in each well) and infected with adenovirus-GFP-LC3 (100 viral particles per cell) overnight. Cells were treated with APAP (5 µM). Fluorescence images were acquired under a Zeiss Axio fluorescence microscope.

Standard methods

RNA isolation, qPCR, cell culture, in vitro virus transduction and plasmid transfection, Western blot (WB), and confocal imaging were described previously. Transient transfection was described previously. For WB, equal amounts of proteins from 5 to 6 mouse livers in each group (n = 5–6/group) were pooled, and duplicate loading was used.

Statistical analysis

Data are shown as the mean ± standard error of the mean and are representative of at least 3 independent experiments. Statistical analysis was carried out using Student’s t test between 2 groups and 1-way ANOVA among multiple groups. p < .05 was considered statistically significant.

Results

 

Generation of hepatocyte-specific miR-21-deficient mice

Our preliminary analysis showed that the basal expression of miR-21 was approximately 2-fold higher in hepatocytes than in nonparenchymal cells (Figure 1A). Thus, we generated hepatocyte-specific miR-21 knockout mice to better understand its mechanistic role in APAP-induced liver injury. In brief, we produced miR-21 floxed mice and bred them with C57BL/6J mice expressing Cre recombinase under the control of albumin promoter to obtain heterozygotes. This progeny was then interbred to obtain hepatocyte-specific miR-21-deficient mice (HKO) (Figure 1B). qPCR analysis confirmed a specific depletion of miR-21 in HKO liver but not in other tissues (Figure 1C). A low level of miR-21 in HKO liver was likely contributed by its expression in hepatic nonparenchymal cells. Furthermore, we confirmed a complete deletion of miR-21 in primary hepatocytes isolated from HKO mice (Figure 1D). HKO mice did not show morphological or metabolic abnormality under normal physiological conditions.

Figure 1.

Figure 1.

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Generating hepatocyte specific miR-21 knockout mice. A, qPCR of miR-21 expression in hepatocytes and nonparenchymal cells isolated from mouse livers (n = 5/group). *p < .05. B, Schematic diagram for generating hepatocyte-specific miR-21 knockout mice (HKO). C, qPCR analysis of miR-21 expression in several tissues of miR-21 HKO mice and their WT littermates (n = 5 mice per group). D, qPCR analysis of miR-21 expression in isolated primary hepatocytes. *p < .05, **p < .01.

APAP-induced liver damage was alleviated in miR-21 HKO mice

To determine the effect of hepatocyte miR-21-deficiency on acute liver injury, miR-21 HKO and their WT littermates were administrated with APAP for 2, 6, and 12 h. H&E staining of liver sections revealed early sign of liver injury 2 h-post APAP administration, which was to a similar extent in both WT and miR-21 HKO (Figure 2A). The severity of liver injury worsened over an extended period of 12 h in WT mice, including inflammation, pericentral necrosis, and hemorrhage. However, the liver damage was alleviated in mR-21 HKO mice, as shown by the decreased necrotic areas and hepatocellular degeneration. Compared with control littermates, miR-21 HKO mice showed significantly lower levels of serum ALT and AST (Figure 2B), as well as increased survival rate during the prolonged exposure of APAP (Figure 2C).

Figure 2.

Figure 2.

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APAP-induced liver damage was alleviated in miR-21 HKO mice. miR-21 HKO and their WT littermates were administrated with APAP for 2, 6, and 12 h. Liver damage was detected by hematoxylin and eosin (H&E) staining of liver sections (A), serum levels of ALT and AST (B), survival rate analyzed by Kaplan-Meier method (C), mRNA levels of genes related to inflammation (D), and activation of JNK (E). *p < .05, **p < .01.

Next, we sought to compare the expression of inflammatory genes at the early response (2 h) and late response (12 h) stages. Hepatic mRNA expression of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), NACHT, LRR, and PYD domains-containing protein 3 (NALP3), tumor necrosis factor alpha (TNFα), and inducible nitric oxide synthase (iNOS) was markedly elevated in WT mice 12 h after APAP administration, whereas their induction was significantly blunted in miR-21 HKO (Figure 2D). APAP overdose-induced hepatic injury involved the early activation of c-Jun NH2-terminal kinase (JNK)-dependent (phosphorylated JNK) cell death pathway (Jaeschke et al., 2012). WB revealed a significant enhancement in the expression of phospho-JNK (p-JNK) in WT at 2 h post-APAP treatment while the activation of p-JNK was approximately 60% lower in miR-21 HKO versus WT (Figure 2E). By 12 h post-APAP treatment, p-JNK protein almost returned back to the baseline levels. Taken together, ablation of miR-21 in hepatocytes protected against APAP-induced hepatotoxicity.

The autophagy pathway was activated in APAP-treated miR-21 HKO mice

APAP-induced cell death mechanisms are initiated by the formation of the presumed reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which is generated mainly by the cytochrome P450 enzymes CYP2E1 in mice and humans (James et al., 2003). Glutathione (GSH) plays an important role in the detoxification of NAPQI (Jaeschke et al., 2012), and its depletion facilitates reactive oxygen species (ROS) production and liver injury (Supplementary Figure 1). It is known that adducts formation induced by APAP can be detected 2 h after APAP injection. Indeed, we detected plasma and liver protein adducts 2 h post-APAP administration, however, no significant differences were observed between WT and miR-21 HKO (Figure 3A). In addition, glutathione disulfide (GSSG) levels were too low to be detected and no significant differences of GSH levels between WT and miR-21 HKO 2 h after APAP-injection were observed (not shown). Consistent with this observation, Cyp2e1 mRNA expression was similarly increased in both WT and miR-21 HKO (Figure 3B). The results suggested that genetic depletion of miR-21 in hepatocytes did not affect the early response with regard to Cyp2e1 expression and activity to breakdown APAP.

Figure 3.

Figure 3.

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The autophagy pathway was activated in APAP-treated miR-21 HKO mice. A, miR-21 HKO and their WT littermates were administrated with APAP for 2 h. Plasma and liver APAP-adducts were detected. B, Cyp2e1 mRNA expression was determined at 2 and 12 h post-APAP administration. C, Cyp2e1 and multiple proteins in autophagy, apoptosis, endoplasmic reticulum stress, and necrosis pathways were examined at 12 h post-APAP administration. D, The mRNA levels of glutathione S-transferases (GSTs) in the liver were evaluated after APAP treatment for 12 h.

Because the significant protection of liver injury occurred 12 h post-APAP injection (Figure 2B), we next investigated the effect of APAP on hepatic CYP2E1 expression at this time point. Interestingly, Cyp2e1 mRNAs were markedly reduced 12 h post-APAP treatment in both WT and miR-21 HKO (Figure 3B). We also evaluated the mRNA levels of glutathione S-transferases (GSTs); a family of enzymes important to catalytic detoxification. Overall, the hepatic expression of Gstt2, Gstt3, Gstm2, and Gstm3 was markedly increased in miR-21 HKO versus WT under the conditions of phosphate-buffered saline (PBS) or APAP treatment (Figure 3D), and their increased expression may contribute to the decreased liver injury.

We further examined multiple proteins in apoptosis and autophagy pathways (Figure 3C). Interestingly, CYP2E1 protein was only slightly decreased in APAP versus PBS WT mice. It was approximately 30% decreased in miR-21 HKO versus WT by APAP treatment. Therefore, CYP2E1 protein showed a different pattern of change as compared to its mRNA levels (Figure 3B). The results suggest that Cyp2e1 mRNA and CYP2E1 protein expression underwent differential transcriptional regulation and post-translational modification under APAP-challenged condition, respectively.

Microtubule-associated protein 1 light chain 3 beta (LC3B) and sequestosome 1 (p62), the biomarker of autophagy, increased about 2-fold in miR-21 HKO mice after APAP treatment for 12 h, compared with control littermates. The protein levels of mixed lineage kinase domain like pseudokinase (MLKL), poly(ADP-ribose) polymerase 1 (PARP), and Caspase 8 were increased approximately 1.5-fold in miR-21 HKO versus WT mice. No significant differences in the expressions of receptor-interacting serine-threonine kinase 3 (RIPK3) and BIP in miR-21 HKO versus WT mice. Taken together, the protection from APAP toxicity in miR-21 HKO mice likely involves the activation of autophagy.

Transmission electron microscopy (TEM) is one of the best approaches to provide direct evidence for autophagy (Martinet et al., 2014). TEM analysis demonstrated a clear formation of autophagosome in livers of miR-21 HKO mice compared with WT mice (Figure 4A). Consistent with the in vivo studies, APAP treatment also increased GFP-LC3 puncta in primary cultured hepatocytes isolated from miR-21 HKO mice, indicating a significant accumulation of autophagosomes (Figure 4B). Interestingly, APAP treatment also significantly increased hepatic mRNA levels of the most autophagy-related genes in miR-21 HKO versus WT mice (Figure 4C). Mechanistic target of rapamycin kinase (mTOR) and AMP-activated protein kinase (AMPK) are 2 main regulators of autophagy (Kim et al., 2011). mTOR and p-mTOR proteins were increased approximately 2-fold and approximately 1.5-fold, respectively, in miR-21 HKO versus WT mice 12 h post-APAP treatment, whereas the levels of AMPK and p-AMPK showed no marked changes in KO versus WT mice (Supplementary Figure 2).

Figure 4.

Figure 4.

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The activation of autophagy in miR-21 HKO mice was induced upon APAP stimulation. A, Formation of autophagosome in vivo was detected using transmission electron microscopy at 6 h post-APAP administration. B, GFP-LC3 puncta in primary cultured mouse hepatocytes were evaluated after APAP treatment for 12 h. C, The mRNA levels of autophagy related genes in the liver at 2 and 12 h post-APAP administration. *p < .05, **p < .01.

miR-21 negatively regulates PPARγ expression by targeting its 3′-UTR

Mitochondrial injury is a critical alteration in APAP Toxicity (McGill et al., 2012). Notably, peroxisome proliferator activated receptor gamma (PPARγ) regulates mitochondrial biogenesis and plays an important role in mitochondria dynamics (Boland et al., 2013). Hepatic PPARγ mRNA levels were similar in WT and miR-21 HKO mice in control (PBS) and 2 h APAP treatment groups (Figure 5A). Interestingly, APAP treatment at 12 h time point dramatically decreased hepatic PPARγ mRNA levels in the WT mice but not in miR-21 HKO mice. However, peroxisome proliferative-activated receptor gamma coactivator 1 alpha (PGC1a) and beta (PGC1β) mRNA levels were not markedly different in WT and miR-21 HKO mice in PBS, 2 h and 12 APAP treatment groups, although PGC1β mRNA showed a significant reduction in both 12 h APAP-treated WT and miR-21 HKO mice.

Figure 5.

Figure 5.

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PPARγ proteins were significantly elevated in miR-21 HKO mice due to loss of miR-21 inhibition. Hepatic expression of genes involved in mitochondrial biogenesis and autophagy were determined in the mRNA levels (A) and protein levels (B). The expression of miR-21 (C) and PPARγ protein expression (D) in miR-21 mimic treated hepatocytes. E, The predicted miR-21 binding site located in the 3′-UTR of PPARγ. F, Cells were cotransfected with miR-21 mimic and luciferase reporter plasmids: empty vector only, synthetic consensus miR-21 binding sequences, wild-type PPARγ 3′-UTR and mutant of PPARγ 3′-UTR to assess the inhibition of miR-21 on PPARγ 3′-UTR activity. *p < .05, **p < .01.

Intriguingly, the basal hepatic protein levels of PPARγ were ∼ approximately 1.8-fold increased in miR-21 HKO versus WT mice in PBS groups (Figure 5B). A 2 h APAP treatment increased PPARγ protein levels approximately 4.5-fold in WT mice relative to its PBS controls. However, its induction at the same 2 h time point was approximately 13-fold higher in miR-21 HKO mice than in the WT mice. The transient induction PPARγ protein in WT mice by 2 h APAP may serve as an endogenous protective mechanism to react to APAP administration. At the 12 h time point after APAP treatment, PPARγ protein levels almost returned back to the basal levels as compared with PBS groups and mice developed liver injury. Because the basal levels of PPARγ protein was approximately 1.8-fold increased in HKO versus WT mice in PBS groups, it suggests a direct negative regulation of PPARγ protein by miR-21. In addition, LC3B activation (approximately 5-fold) occurred at 12 h post-APAP treatment in miR-21 HKO versus WT mice. In contrast, no significant changes were observed in PGC1a and PGC1β proteins in WT and miR-21 HKO mice. Taken together, the results suggest that the elevation of PPARγ proteins in miR-21 HKO mice are directly associated with the loss of miR-21, but are independent of its mRNA changes. The results also suggest that PPARγ activation precedes the activation of LC3B, which is indicative of autophagy.

MiRNAs are known to regulate protein expression by binding to 3′-UTR of its target. We next determined whether miR-21 negatively regulates PPARγ expression by targeting its 3′-UTR. We treated primary mouse hepatocytes with miR-21 mimics in vitro. As shown in Figures 5C and 5D, overexpression of miR-21 significantly decreased protein expression of PPARγ in hepatocytes relative to negative control treated cells. The human PPARγ mRNA (NM_138712.3) is predicted to be a potential target of miR-21 using bioinformatic databases (RNA22, TargetScan, and Pic Tar). A miR-21 binding site in the 3′-UTRs of its mRNA was identified (Figure 5E). To test whether miR-21 directly regulates PPARγ, we constructed a luciferase reporter construct containing the predicted miR-21 recognition sequence in the WT 3′-UTR of PPARγ inserted into pMir-GLO report. A construct harboring a mutant predicted miR-21 recognition sequence was also prepared and a construct harboring direct-match miR-21 binding site was used as a positive control. miR-21 decreased luciferase activities of the reporter constructs with miR-21 binding site and PPARγ 3′-UTR WT to 0.36 ± 0.04 and 0.48 ± 0.54 fold, respectively, in comparison with the negative control empty vector. Deletion of the predicted miR-21 binding site abolished the inhibitory effect of miR-21 (Figure 5F). These data indicate that miR-21 directly regulate PPARγ expression by targeting its 3′-UTR.

PPARγ played an indispensable role to protect against APAP-induced hepatotoxicity in miR-21 HKO mice

Based on the above findings, we hypothesize that PPARγ signaling pathway plays an important role to protect against APAP-induced hepatotoxicity in miR-21 HKO mice. To further address this question, we treated miR-21 HKO and WT mice with a potent PPARγ antagonist namely GW9662, prior to APAP treatment. GW9662 has been reported to affect PPARγ binding activity thus its function but not its mRNA expression. HE staining showed that the liver damage was similar between WT and miR-21 HKO mice at 2, 12, and 24 h after GW9662 and APAP treatment (Figure 6A). The levels of ALT and AST showed no marked differences between WT and miR-21 HKO mouse (Figure 5B). It is noted that the ALT values in WT GW+APAP group (Figure 6B) was lower than that in WT APAP group without GW (Figure 2B). This could be attributed to the reported anti-inflammatory property of GW9662 in diet-induced nonalcoholic fatty liver disease. With respect to the expression of genes related to inflammation, the levels of IL-1β, TNFα, and NLRP3 increased even higher in miR-21 HKO mice than that in WT mice (Figure 6C). The levels of IL-6, LY6G, MCP-1, and iNOS were similar between WT and miR-21 HKO mice. The pattern changes in hepatic PPARγ mRNAs in WT and miR-21 HKO mice with GW9662 treatment (Figure 6D) were similar in both types of mice without GW9662 (Figure 5A), suggesting no effect of GW9662 on PPARγ mRNA levels.

Figure 6.

Figure 6.

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Inhibiting PPARγ function blocked autophagy activation and reversed the projection against APAP-induced hepatotoxicity in miR-21 HKO mice. miR-21 HKO and their WT littermates were treated with GW9662, a potent antagonist of PPARγ prior to APAP treatment. Liver damage was detected by hematoxylin and eosin (H&E) staining of liver sections (B), serum levels of ALT and AST (B), and mRNA levels of genes related to inflammation (C). Hepatic expression of PPARγ was determined in the mRNA levels (D) and protein levels (E). Protein expression of phospho-JNK and LC3B were also determined. *p < .05, **p < .01.

We also examined the protein expression of several genes. p-JNK proteins were acutely and similarly induced at the 2 h GW+APAP groups in both the WT and miR-21 HKO mice, and the induction was significant diminished at the 12 h GW+APAP groups (Figure 6E). The results suggest that the JNK activation by APAP is not associated with loss of miR-21 and PPARγ. In contrast to the significant induction of LC3B proteins at 12 h APAP groups in miR-21 HKO mice versus WT mice (Figure 5B), GW9662 treatment abolished such induction at 12 h GW+APAP groups in miR-21 HKO mice versus WT mice (Figure 6E). In addition, the basal PPARγ protein levels were similar in WT and miR-21 HKO mice in the presence of GW9662. GW9662 treatment at 2 h GW+APAP groups (Figure 6E) abolished the elevation of PPARγ proteins in both WT and miR-21 HKO mice at 2 h APAP groups without GW9662 (Figure 4B). Taken together, the results further demonstrate that the induction of PPARγ by loss of miR-21 inhibition is responsible for the activation of autophagy in miR-21 HKO mice.

Discussion

In this study, we observed several new findings. First, deleting miR-21 specifically from hepatocytes in mice protected against APAP-induced hepatotoxicity. Second, this protective effect as a result of miR-21 ablation was positively associated with the activation of autophagy. Third, the elevation of PPARγ protein in miR-21 HKO preceded the activation of LC3B protein and autophagy. Fourth, miR-21 directly inhibited PPARγ mRNA 3′-UTR reporter activity as well as PPARγ protein expression. Fifth, miR-21 HKO mice lost protection against hepatotoxicity when PPARγ protein function was blocked by its antagonist GW9662. We conclude that the activation of PPARγ signaling pathway plays an indispensable role in miR-21 HKO mice to protect against APAP-induced liver injury (Figure 7). Our findings demonstrate a protective role for miR-21 in the induction of liver injury caused by APAP. Modulating miR-21 and PPARγ expression in hepatocytes may provide a potential strategy for the treatment of APAP-induced hepatotoxicity.

Figure 7.

Figure 7.

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Schematic diagram of the role of miR-21 in protecting against APAP-induced liver injury.

Mitochondrial dysfunction and oxidative stress are the central mediators in the pathogenesis of APAP-induced liver injury (Kon et al., 2004). Autophagy is usually activated as a survival mechanism in response to an adverse environment, which can eliminate damaged mitochondria and maintain mitochondrial homeostasis (ie, mitophagy) (Kim et al., 2007). Indeed, emerging evidence suggests that autophagic removal of damaged mitochondria may protect against APAP-induced liver injury. APAP overdose could induce autophagy. Pharmacological inhibition of autophagy by 3-methyladenine or chloroquine further exacerbated APAP-induced hepatotoxicity. In contrast, induction of autophagy by rapamycin inhibited APAP-induced hepatotoxicity. This protection could be mediated via the removal of damaged mitochondria and APAP protein adducts, as well as a reduction of ROS production (Ni et al., 2012, 2016; Tran et al., 2017). Meanwhile, hepatocyte-specific autophagy deficiency were found to be more susceptible to APAP-induced liver injury (Zhang et al., 2018). Similarly, knockdown of Parkin in mouse livers using adenovirus-shRNA significantly reduced mitophagy but increased JNK activation after APAP administration, which exacerbated APAP-induced liver injury(Yang et al., 2015). Moreover, activation of autophagy is proved a promising approach to attenuate APAP-induced liver injury. Lin et al. demonstrated the APAP-induced accumulation of adiponectin by AMPK-dependent activation of autophagy, which in turn removes damaged mitochondria, thereby ameliorating oxidative stress and necrosis (Lin et al., 2014). Metformin, a first-line drug to treat type 2 diabetes mellitus, protected against APAP acute hepatotoxicity in mice (Gebert and MacRae, 2018; Zhang et al., 2016). Metformin could attenuate the mitochondrial oxidant stress and mitochondrial dysfunction, which could be attributed to its ability of enhancing mitophagy (Zhao et al., 2017). In the present study, we firstly demonstrated that miR-21 ablation in the liver alleviated APAP-induced hepatotoxicity by the activation of autophagy and mitochondria biogenesis.

Mitochondria are highly dynamic organelles responding to cellular stress, whose integrity is central to efficient cellular energy production and cell survival (Tsuchiya et al., 2015). There is increasing evidence that key molecules modulate mitochondrial dynamics through important signaling pathways and are coordinated in response to pathophysiologic stresses such as hypoxia and nutrient deprivation (Boland et al., 2013). Mitochondrial biogenesis is induced by nutrient deprivation and in response to oxidative stress and requires the coordinated expression of nuclear and mitochondrial encoded genes that are co-regulated by transcription factors PPARγ and the key transcriptional co-factor, PGC-1α, β (Liangpunsakul et al., 2017; Tran et al., 2017). Defects in biogenesis are frequently lethal to cells and organisms. Mitophagy is a specialized form of autophagy in which mitochondria are targeted and engulfed by autophagosomes that fuse with lysosomes to degrade the encapsulated mitochondria (Novak, 2012). Mitophagy is promoted by a number of different mechanisms including Parkin-mediated pathway (Jaeschke et al., 2012). Our findings demonstrated that the protein expression of PPARγ were dramatically induced at 2 h after APAP treatment in miR-21 HKO mouse relative to WT mouse. In consideration of its regulation role in mitochondrial dynamics, miR-21 hepatocyte-specific knock out induced mitochondrial biogenesis, which promote ATP production and mitochondrial metabolism. Meanwhile, mitophagy was induced, which degrades the damaged mitochondria.

PPARγ is a key nuclear receptor that regulates glucose and lipid metabolism (Wang et al., 2014, 2016), which is known to play an active role in anti-inflammatory response (Uchimura et al., 2001; Wen et al., 2010). Activation of PPARγ signaling pathway has been proved to have significant hepatoprotective activity on APAP-induced hepatotoxicity (Gupta et al., 2014; Wang et al., 2017). Using Sprague Dawley rats, Gupta et al. demonstrated that pioglitazone, a PPARγ agonist, antagonized APAP-induced liver pathological damage, significantly reduced the elevated level of ALT/AST and also inhibits the free radical formation (Gupta et al., 2014). Similarly, Wang et al. showed that rosiglitazone, a synthetic PPARγ agonist, alleviated APAP-induced characteristic centrilobular necrosis in CD-1 mouse liver (Wang et al., 2017). In fact, activation of PPARγ played an important role in preventing acute liver injury. PPARγ agonist down-regulated hepatic inflammatory cytokines and adhesion molecules during ischemia/reperfusion-induced acute liver injury (Akahori et al., 2007; Kuboki et al., 2008). PPARγ agonist repressed hepatic inflammatory cytokines during cyclophosphamide-induced liver injury (El-Sheikh and Rifaai, 2014). However, the mechanism underlying the protective role of PPARγ on hepatotoxicity is still not fully elucidated. In this study, we demonstrated that miR-21 HKO mice were protected from APAP-induced hepatotoxicity, which may relate to the activation of autophagy and mitophagy. At the same time, PPARγ signaling pathway played an indispensable role in miR-21 HKO mice to protect APAP-induced hepatotoxicity. Several molecular pathways may be involved for the influence of PPARγ on mitochondrial function. A major pathway for injury-induced mitochondrial degradation is Parkin pathway that translocates to mitochondria and mediates mitochondrial degradation (Cai et al., 2012; Narendra et al., 2008). The adaptor protein p62 facilitates selective autophagy into the LC3B-regulated machinery for lysosomal degradation (Bjorkoy et al., 2005; Geisler et al., 2010). PPARγ agonist treatment has previously been shown to protect against mitochondrial dysfunction (Fuenzalida et al., 2007; Fujisawa et al., 2009). Therefore, activation of PPARγ could induce the degradation of defective mitochondria and upregulation of mitochondrial biogenesis to restore a healthy pool of functioning mitochondria, leading to mitochondrial homeostasis.

After our study is completed and we are in the process of submitting and publishing, we become aware of a new study (Huffman et al., 2023) which showed that loss of miR-21 protects against APAP-induced hepatotoxicity. However, the work from Huffman et al. does not diminish the significance of our work. First, the basic findings in our study are consistent with this newly published study. However, Huffman’s study did not provide mechanistic understanding as to why lacking miR-21 in mice protected against APAP-induced liver injury. Significantly, our study is distinguished from Huffman’s study in that we provided novel mechanistic insight and revealed a PPARγ-dependent signaling pathway in APAP-induced liver injury mediated by miR-21. We employed a newly developed hepatocyte-specific miR-21 HKO mice and found that the protective effect of loss of miR-21 on APAP-induced liver injury was contributed by the significant elevation of PPARγ as well as the activation of autophagy. Our findings suggest that targeting PPARγ signaling pathway may provide a crucial therapeutic approach in the treatment of APAP-induced hepatotoxicity involving miR-21. Therefore, the Huffman et al. study strengthens our findings and complements the conclusion of our study.

In summary, upon APAP intoxication, miR-21 ablation in the liver significantly induced the activation of PPARγ as early as 2 h due to loss of miR-21 inhibition, which in turn induced the upregulation of mitochondrial biogenesis and increased autophagy/mitophagy. Ultimately, APAP-induced hepatotoxicity was alleviated. These findings highlight a novel physiopathological role for miR-21 in the regulation of APAP-induced hepatotoxicity through PPARγ, and suggest a potential new approach in the treatment of miR-21-mediated hepatotoxicity induced by APAP by targeting the PPARγ signaling pathway.

Supplementary Material

kfad132_Supplementary_Data
kfad132_supplementary_data.docx (502.7KB, docx)

Acknowledgments

We sincerely thank Dr James L. Boyer from Yale Liver Center for assisting with the quantifications of area of necrosis.

Contributor Information

Chao Xu, Department of Endocrinology and Metabolism, Shandong Provincial Hospital affiliated to Shandong First Medical University, Jinan, Shandong, 250021, China; Shandong Provincial Key Laboratory of Endocrinology and Lipid Metabolism, Jinan, Shandong, 250021, China.

Fang Yan, Department of Pain Management, Shandong Provincial Hospital affiliated to Shandong First Medical University, Jinan, Shandong, 250021, China.

Yulan Zhao, Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310009, China.

Hartmut Jaeschke, Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.

Jianguo Wu, Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA; Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio 44195, USA.

Li Fang, Department of Endocrinology and Metabolism, Shandong Provincial Hospital affiliated to Shandong First Medical University, Jinan, Shandong, 250021, China; Shandong Provincial Key Laboratory of Endocrinology and Lipid Metabolism, Jinan, Shandong, 250021, China.

Lifang Zhao, Department of Endocrinology and Metabolism, Shandong Provincial Hospital affiliated to Shandong First Medical University, Jinan, Shandong, 250021, China; Shandong Provincial Key Laboratory of Endocrinology and Lipid Metabolism, Jinan, Shandong, 250021, China.

Yuanfei Zhao, Beijing Institute of Heart, Lung, and Blood Vessel Diseases, Beijing Anzhen Hospital Affiliated to Capital Medical University, Beijing, 100029, China.

Li Wang, Independent Researcher, Tucson, Arizona 85004, USA.

Supplementary data

Supplementary data are available at Toxicological Sciences online.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

National Natural Science Foundation (81974124), Academic Promotion Program of Shandong First Medical University (2019RC015), and the Natural Science Foundation of Shandong Province (ZR2021MH150).

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Supplementary Materials

kfad132_Supplementary_Data
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