Targeting Mitochondrial Dysfunction for the Prevention and Treatment of Non-Alcoholic Fatty Liver Disease

Abstract

Non-alcoholic fatty liver disease (NAFLD) has emerged as the most prevalent chronic liver disorder worldwide, with its prevalence closely linked to metabolic comorbidities. Mitochondrial dysfunction, a central mechanism underlying metabolic dysregulation, is recognized as a critical driver of NAFLD pathogenesis. This review systematically delineates the molecular mechanisms of mitochondrial dysfunction in NAFLD, encompassing multidimensional networks such as mitochondrial dynamics imbalance, oxidative stress, aberrant metabolic signaling, and inter-organelle crosstalk. Furthermore, we propose novel mitochondrial-targeted therapeutic strategies for NAFLD, including restoration of energy metabolism and mitigation of oxidative damage through antioxidant interventions, modulation of mitochondrial dynamics, and activation of autophagy. Finally, we summarize current research limitations and future directions in targeting mitochondrial dysfunction. By integrating existing evidence, this review establishes a robust theoretical framework for mitochondrial-targeted therapies in NAFLD, offering innovative clinical approaches and a transformative paradigm for addressing the global burden of metabolic liver diseases.

Introduction

Non-alcoholic Fatty Liver Disease (NAFLD) is one of the most prevalent chronic liver diseases globally, affecting approximately 24% of the adult population. Its pathological characteristic is the abnormal accumulation of lipids in the liver, which is closely associated with obesity, insulin resistance, and metabolic syndrome. As the disease progresses, NAFLD can evolve into non-alcoholic steatohepatitis (NASH), liver fibrosis, and even cirrhosis and liver cancer, posing a significant health burden among metabolic-related diseases. ¹ In recent years, NAFLD has been redefined as Metabolic Dysfunction- Associated Steatotic Liver Disease (MASLD), further highlighting the central role of metabolic abnormalities in its pathogenesis.^2,3

Mitochondrial dysfunction is a key pathological mechanism in the occurrence and development of NAFLD. In hepatocytes, mitochondria are responsible for fatty acid β-oxidation, regulation of reactive oxygen species (ROS), and maintenance of energy metabolic balance. Abnormal mitochondrial function directly leads to lipid metabolic disorders, enhanced oxidative stress, and activation of the inflammatory response. ⁴ Research has shown that in the hepatocytes of NAFLD patients, the ability of mitochondrial fatty acid oxidation decreases, and excessive production of ROS triggers lipotoxicity, mitochondrial DNA damage, and imbalance of mitophagy. These processes, in turn, drive hepatocyte injury, inflammatory infiltration, and fibrosis. ⁵ In addition, abnormal mitochondrial dynamics such as imbalance of fusion/fission and bioenergetic defects further exacerbate insulin resistance and systemic metabolic disorders, forming a vicious cycle. ⁶ Mitochondria are the energy hubs of cells, responsible for generating ATP and regulating key metabolic processes, including oxidative phosphorylation and fatty acid oxidation. ⁷ This central role makes them critical nodes for maintaining cellular homeostasis, and any dysfunction can trigger a chain reaction that impacts other organelles. For example, mitochondrial dysfunction can lead to insufficient energy supply or the accumulation of reactive oxygen species, which indirectly affects the calcium homeostasis and protein folding processes of the endoplasmic reticulum, as well as the lipid metabolism functions of peroxisomes. Therefore, mitochondrial dysregulation serves as a catalyst for various diseases, such as NAFLD. ⁸

Targeting mitochondrial dysfunction offers unique advantages for the treatment of NAFLD. Firstly, mitochondria serve as the central hub of metabolic regulation. Intervening in their function can simultaneously improve multiple pathological processes such as lipid metabolism, oxidative stress, and inflammation. ⁹ For instance, mitochondria-targeted antioxidants and mitochondrial quality regulators have demonstrated the potential to alleviate hepatic steatosis and fibrosis in animal models. ¹⁰ Secondly, personalized treatment strategies based on mitochondrial biomarkers are being developed, which are expected to enhance efficacy prediction and precise intervention. ¹¹ In addition, plant-derived mitochondrial regulators and metabolic intervention measures provide new directions for safe and multi-effective treatment options by improving mitochondrial function. ¹² Compared with traditional single-target therapies, targeting mitochondria not only addresses the metabolic root cause of NAFLD but also can delay disease progression through the synergistic action of multiple pathways, showing significant prospects for clinical translation. ¹³

In this paper, we systematically review the molecular mechanisms of mitochondrial dysfunction in NAFLD, propose novel intervention strategies for NAFLD targeting mitochondria, and summarize the current limitations and future directions of research on targeting mitochondrial dysfunction. This work establishes a solid theoretical foundation for mitochondrial-targeted therapies for NAFLD and provides new directions for the clinical treatment of NAFLD. Through interdisciplinary collaboration and technological innovation, mitochondrial-targeted therapy is expected to become a significant breakthrough in precision medicine for NAFLD, offering a new paradigm for the prevention and control of the global burden of metabolic liver diseases.

The Central Position of Mitochondrial Function in Metabolic Diseases

The pathogenesis of metabolic diseases involves metabolic disorders in multiple organs and abnormalities at the cellular and molecular levels. As the central regulator of energy metabolism and oxidative stress, mitochondrial dysfunction plays a crucial role in their pathological processes, see Figure 1.

Figure 1.

The central position of mitochondrial function in metabolic diseases. NAFLD, UPRmt unfolded protein response of mitochondria, Drp1 dynamin-related protein 1, Mfn 1/2 mitochondrial fusion protein, Mst1 macrophage stimulating 1, PINK1 PTEN induced putative kinase 1, AMPK adenosine 5'-monophosphate-activated protein kinase, ACC acetyl-CoA carboxylase, ATP adenosine triphosphate, PGC-1α peroxisome proliferator-activated receptor-gamma coactivator 1-α，ATF5 activating transcription factor 5, CHOP C/EBP-homologous protein, IRS-1 insulin receptor substrate 1, JUK jun N-terminal kinase, SREBP-1c sterol regulatory element-binding protein 1c ， ROS reactive oxygen species, IDH2 isocitrate dehydrogenase.

Metabolic hub Functions of the Mitochondria

Mitochondria, as the core of cellular energy metabolism, regulate whole-body energy homeostasis through oxidative phosphorylation (OxPhos) and fatty acid β-oxidation (FAO). Studies have shown that a decline in mitochondrial oxidative capacity is closely associated with obesity, insulin resistance, and non-alcoholic fatty liver disease (NAFLD). For instance, in the TALLYHO/Jng mouse model, the phenotypes of obesity and type 2 diabetes are directly related to reduced mitochondrial biogenesis and decreased OxPhos activity, mainly manifested as a decline in ATP production. ¹⁴ Additionally, the maintenance of mitochondrial membrane potential (ΔΨm) is crucial for fatty acid metabolism. In the hepatocytes of NAFLD patients, a decrease in mitochondrial membrane potential leads to the activation of the mitochondrial unfolded protein response, exacerbating lipid peroxidation. ¹⁵

Mitochondrial Dynamics and Metabolic Adaptation

Mitochondria regulate metabolic flexibility through the dynamic balance between fusion (mediated by Mfn1/2) and fission (mediated by Drp1). In MASLD, the mitochondrial network is finely balanced by fusion (MFN1/2, OPA1) and fission (DRP1, FIS1, MFF) to maintain cristae structure, electron transport, and lipid oxidation. ¹⁶ When metabolic and lipotoxic stress drive the system toward an “excessive fission/restricted fusion” shift, mitochondrial fragmentation reduces oxidative phosphorylation efficiency and promotes abnormal lipid droplet-mitochondria coupling, accompanied by leakage of ROS and oxidized mtDNA that activates the NLRP3 and cGAS-STING innate immune pathways. ¹⁷ Concurrently, endoplasmic reticulum stress triggers cristae collapse, apoptosis, and inflammatory amplification via the OMA1-OPA1 axis. Studies have shown that the expression of Mfn2, a mitochondrial fusion protein, is significantly reduced in patients with NAFLD and in mouse models. The loss of its function leads to fragmentation of the mitochondrial network, which in turn inhibits fatty acid oxidation capacity and promotes lipid accumulation. ¹⁸ This imbalance exhibits distinct cell-type specificity: hepatocytes require “moderate fission” to maintain metabolic flexibility, and excessive inhibition of DRP1 can conversely trigger the OMA1-integrated stress pathway to exacerbate injury, ¹⁹ whereas in Kupffer cells/infiltrating mononuclear macrophages, the fission shift and glycolytic inflammatory phenotype mutually reinforce to drive persistent inflammation. ²⁰ In obesity models, enhanced mitochondrial fission leads to functional fragmentation, which is associated with impaired insulin signaling pathways and reduced glucose uptake capacity. ²¹ Conversely, overexpression of the mitochondrial fusion protein Mfn2 can restore the integrity of the mitochondrial network in skeletal muscle, improve insulin sensitivity, and increase the glucose uptake rate. ²² Notably, mitochondrial dynamics also regulate the clearance of damaged mitochondria through mitophagy. In non-alcoholic fatty liver disease (NAFLD), upregulation of Mst1 inhibits Parkin-mediated mitophagy, with a 40% increase in caspase-3 activity, resulting in increased hepatocyte apoptosis. ²³

Mitochondria-Nucleus Signaling Interaction

Mitochondria regulate nuclear gene expression through retrograde signaling, which involves the unfolded protein response of mitochondria and the AMP - activated protein kinase (AMPK) pathway. The UPRmt responds to oxidative stress by activating the transcription factors ATF5 and CHOP, which upregulate the expression of mitochondrial chaperone proteins such as HSP60. ¹⁵ AMPK, acting as an energy sensor, promotes fatty acid oxidation in muscle tissue by phosphorylating acetyl-CoA carboxylase (ACC), thereby increasing the rate of β-oxidation. ²⁴ In addition, peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α), a key co-activator of mitochondrial biogenesis, shows a significant association between its reduced expression and age-related mitochondrial dysfunction. This mainly results in decreased ATP production and insulin resistance. ²⁵ PGC-1α is a key regulator of mitochondrial function. In NAFLD, PGC-1α expression is epigenetically suppressed, primarily through DNA hypermethylation in its promoter region. ²⁶ In a high-fat/high-sucrose diet NAFLD rat model, a conserved CpG site in the PGC-1α promoter was found to be hypermethylated, which directly led to a significant decrease in PGC-1α mRNA and protein expression levels. ²⁷ This hypermethylation is associated with the abnormal expression of metabolic genes, promoting hepatic lipid accumulation and insulin resistance. Interestingly, hypomethylation can increase PGC-1α expression and improve metabolic outcomes, and exercise and dietary interventions can lead to the hypomethylation of PGC-1α. This suggests that exercise and diet may serve as potential therapeutic strategies for NAFLD. ²⁸ However, patients generally have low adherence to long-term lifestyle changes, which limits the effectiveness of treatment. This requires the support of a multidisciplinary team, such as nutritionists and exercise coaches, to improve the sustainability of lifestyle therapies. ²⁹ Furthermore, there is no consensus on the optimal dietary regimen and exercise intensity, and further exploration is needed.

Mitochondrial Metabolism and Disease Mechanisms

In metabolic syndrome, abnormal mitochondrial function initially presents as insulin resistance. In skeletal muscle, incomplete mitochondrial β-oxidation results in the accumulation of medium- and long- chain acylcarnitines, which inhibits the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1). ³⁰ Intriguingly, excessive ROS in hepatocyte mitochondria can activate the expression of lipid-synthesizing enzymes such as sterol regulatory element-binding protein 1c (SREBP-1c) via the JNK pathway, accelerating steatosis and thereby exacerbating the progression of non-alcoholic fatty liver disease.^31,32 Simultaneously, oxidative stress in liver cells leads to a decrease in the activity of mitochondrial NADPH-dependent isocitrate dehydrogenase (IDH2), weakening the antioxidant capacity and further damaging mitochondrial DNA.^33,34

As metabolic gatekeepers, mitochondria have a functional network that encompasses energy sensing, redox balance, and cell fate determination, offering multi-dimensional targets for the mechanistic analysis and treatment of metabolic diseases.

Rationale for Mitochondrial Targeting

As the energy-metabolic center of eukaryotic cells, mitochondria are closely associated with the occurrence and development of various diseases when their functions are abnormal, such as lactate, cholesterol synthesis, lipid synthesis, glutamine. The physiological mechanisms of healthy and dysfunctional mitochondria in NAFLD see Figure 2. We systematically expound on the rationality of mitochondrial-targeted therapy from three aspects: the core physiological functions of mitochondria, the pathological mechanisms of mitochondrial dysfunction, and the biological basis of targeted intervention.

Figure 2.

The physiological mechanisms of healthy and dysfunctional mitochondria in NAFLD. Ⅰ electron transport chain Ⅰ, Ⅱ electron transport chain Ⅱ, Ⅲ electron transport chain Ⅲ, Ⅳ electron transport chain Ⅳ, ROS reactive oxygen species, FAO fatty acid β-oxidation.

The Core Physiological Function of the Mitochondria

Mitochondria generate adenosine triphosphate (ATP) through oxidative phosphorylation, accounting for over 90% of the cell's energy supply.^35,36 In addition, mitochondria are also involved in key physiological processes such as calcium homeostasis regulation, signal transduction, and programed cell death regulation.^37,38 Its double-membrane structure, consisting of an outer membrane and an inner membrane with dense cristae, provides a functional basis for the electron transport chain (ETC). However, it also becomes the main site for the production of ROS — the electron leakage from ETC complexes I and III can generate superoxide anions, which can then be converted into other highly reactive free radicals.^39,40 Notably, mitochondrial dynamic balance directly affects the efficiency of the ETC and the amount of ROS generated by regulating the cristae morphology and membrane potential. ⁴¹

Pathomechanism of Mitochondrial Dysfunction

In metabolic diseases and cardiovascular pathologies, mitochondrial dysfunction is characterized by an oxidative stress cascade reaction, dysregulation of dynamic equilibrium, and abnormal epigenetic modifications. In a diabetic cardiomyopathy model, excessive production of mitochondrial ROS leads to cardiomyocyte apoptosis and fibrosis. Experiments have shown that the mitochondria-targeted antioxidant Mito-TEMPO can significantly reduce the level of 8-hydroxydeoxyguanosine in cardiac tissue and improve left ventricular diastolic function. ⁴² In atherosclerosis, oxidative damage triggered by mitochondrial DNA (mtDNA) mutations promotes the formation of foam cells by activating the NLRP3 inflammasome. ⁴³ In studies of hypertension-related renal injury, Drp1-mediated excessive mitochondrial fission results in a 50% reduction in ATP synthesis in renal tubular epithelial cells, accompanied by an increase in ROS production. Inhibition of Drp1-GTPase activity can restore the integrity of the mitochondrial network and alleviate ischemia-reperfusion injury. ⁴⁴ In NAFLD, a decrease in the expression of the fusion protein OPA1 also causes fragmentation of hepatocyte mitochondria, exacerbating lipid peroxidation and the inflammatory response.^45,46 Moreover, in a hyperglycemic environment, abnormal methylation of mitochondrial DNA inhibits the expression of the NDUFB6 gene, hinders the assembly of complex I, and causes defects in the electron transport chain (ETC). This epigenetic change can be reversed by a mitochondria-targeted demethylase, restoring the insulin-secreting function of pancreatic β-cells.^47,48

Biological Basis for the Mitochondrial-Targeted Intervention

Targeting mitochondria is spatially specific. Lipophilic cationic compounds driven by the mitochondrial membrane potential, such as MitoQ and SS-31, can accumulate in the inner mitochondrial membrane, with their local concentrations significantly higher than those in the cytoplasm. Clinical trials have shown that MitoQ is significantly more effective than the traditional antioxidant vitamin E in improving vascular endothelial function in the elderly. ⁴⁹ Some studies have demonstrated that by regulating the oxidation state of cardiolipin, the stability of electron transport chain (ETC) complexes, mitochondrial autophagy, and apoptotic pathways can be simultaneously improved, restoring the ATP production of ischemic myocardium to the normal level. ⁵⁰ Additionally, by activating the mitochondrial PGC - 1α pathway, the density of retinal mitochondria in patients with diabetic retinopathy can be increased, and capillary leakage can be reduced.^51,52

These research advances not only validate the theoretical feasibility of mitochondrial - targeted therapy but also provide a molecular basis for the design of individualized treatment regimens in the era of precision medicine.

Molecular Mechanism of Mitochondrial Dysfunction in NAFLD

Imbalance in Mitochondrial Dynamics and Disorders of Lipid Metabolism

Imbalance in mitochondrial dynamics is a core characteristic of mitochondrial dysfunction in NAFLD. Multiple human liver tissue and transcriptome studies have shown that downregulation of MFN2 and restricted regulation of OPA1 are common in active MASH, while DRP1 and fission programs increase with the staging of inflammation and fibrosis, and are enriched in myeloid compartments such as Kupffer cells at the spatial/single-cell level. ⁵³ These “dynamic fingerprints” are positively correlated with pathological scores, fibrosis severity, and transcriptional inflammatory pathways, suggesting that fusion/fission imbalance is not merely a phenotypic accompaniment but one of the progression drivers. In addition, abnormal remodeling of phosphatidylglycerol (PG) disrupts mitochondrial membrane stability, triggers oxidative stress and mitochondrial DNA depletion, and ultimately exacerbates hepatocyte lipotoxicity. ⁵⁴ Noteworthy, excessive activation of Drp1, a mitochondrial fission protein, has been confirmed to be closely associated with hepatocyte insulin resistance and lipid peroxidation. It may affect lipid transport by regulating the mitochondrial-endoplasmic reticulum contact sites (MAMs). ⁵⁵ Clinically, baseline histology showing MFN2↓/DRP1↑ or corresponding transcriptional fingerprints often indicates a high-risk trajectory in the “inflammation-fibrosis pathway”. Combined with cf-mtDNA and standard non-invasive grading tools, it is expected to improve the accuracy of risk stratification and follow-up interpretation. ⁵⁶

Oxidative Stress and Mitochondrial Respiratory Chain Damage

Abnormalities in the mitochondrial oxidative phosphorylation (OXPHOS) system are key drivers in the occurrence and development of NAFLD. Clinical studies and animal models have shown that in the hepatocytes of NAFLD patients, the activities of mitochondrial complexes I, III, and IV are significantly reduced. This reduction leads to an increased electron leak in the electron transport chain (ETC), resulting in the production of ROS such as superoxide anions and hydrogen peroxide.^57,58 It is worth noting that the core feature of NAFLD is the imbalance in oxidative stress caused by excessive ROS production and reduced antioxidant capacity. ⁵⁹ During acute stress, only slight increases in AST and ROS are observed, but the antioxidant system can still compensate. ⁶⁰ In contrast, chronic stress leads to the persistent accumulation of ROS, collapse of the antioxidant system, and significant increases in ALT, AST, and ROS. ⁶¹ In the ammonia-induced liver injury model, increased ammonia concentration dose-dependently induces elevated serum ALT/AST levels, ROS accumulation, and a decrease in antioxidant enzyme activity. ⁶² This state of oxidative stress exacerbates liver injury. Firstly, it initiates a lipid peroxidation chain reaction. ROS attack polyunsaturated fatty acids, generating malondialdehyde (MDA) and 4-hydroxynonenal. These products directly damage mitochondrial DNA and proteins, thus forming a vicious cycle.^63,64 Secondly, it activates inflammatory signals. ROS promote the release of pro-inflammatory factors such as TNF-α and IL-6 through the NF-κB and NLRP3 inflammasome pathways, accelerating the activation of hepatic stellate cells and the fibrosis process.^57,65 Last but not least, there is an imbalance in the NAD⁺/NADH ratio. Damage to the mitochondrial respiratory chain leads to the depletion of NAD⁺, which inhibits the SIRT1/SIRT3-dependent deacetylation, further weakening the mitochondrial adaptive stress response.^66,67

Mitochondrial-Insulin Signaling Interaction

Mitochondrial dysfunction induces hepatic insulin resistance through multiple mechanisms, including the accumulation of diacylglycerol, abnormalities in mitochondrial-derived metabolites, and dysregulation of mitochondrial calcium homeostasis. Impaired mitochondrial β-oxidation leads to the accumulation of acyl-CoA intermediates, which activates the protein kinase C signaling pathway and inhibits the tyrosine phosphorylation of insulin receptor substrate.^58,68 Simultaneously, the accumulation of succinate and acetyl-CoA regulates the expression of gluconeogenic genes such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase through histone acetylation modification, thereby promoting hepatic glucose output. ⁶⁷ Abnormal function of mitochondria-associated membranes also results in the disruption of endoplasmic reticulum-mitochondrial calcium signaling, inhibiting the activity of the pyruvate dehydrogenase complex and thus exacerbating the impairment of glucose utilization.^55,69

Mitochondrial Autophagy Disorder

The autophagic mechanism for the selective clearance of damaged mitochondria is significantly impaired in NAFLD. Studies have found that a high-fat environment induces persistent depolarization of the mitochondrial membrane potential. However, the decreased stability of PTEN-induced putative kinase 1 (PINK1) results in the failure of Parkin recruitment, preventing the initiation of autophagosome formation.^55,70 The overexpression of perilipin 2, a lipid droplet-associated protein, hinders the binding of the autophagy receptor optineurin to damaged mitochondria, leading to the accumulation of dysfunctional mitochondria. ⁷¹ Mitochondrial ROS inhibit the nuclear translocation of transcription factor EB, reducing the activity of lysosomal enzymes such as cathepsin B and impeding the degradation of autophagosomes. ⁷⁰

Mitochondrial-ER Stress Coordination Mechanism

Endoplasmic reticulum stress and mitochondrial dysfunction form a positive feedback loop, influencing the progression of NAFLD. After the activation of the unfolded protein response, inositol-requiring enzyme 1α promotes Drp1-mediated mitochondrial fission through JNK phosphorylation, while simultaneously inhibiting peroxisome proliferator-activated receptor-γ coactivator-1α-dependent mitochondrial biogenesis. ⁶⁴ Phosphorylated eukaryotic initiation factor 2α upregulates the expression of activating transcription factor 4, leading to the overexpression of mitochondrial uncoupling protein 2, which exacerbates proton leak and energy dissipation.^72,73 Finally, there is the cross-action of lipotoxic metabolites. For example, the cholesterol oxidation product 27-hydroxycholesterol promotes the opening of the mitochondrial permeability transition pore via the mitochondrial membrane sterol transport protein steroidogenic acute regulatory protein, inducing apoptosis.^50,63

Imbalance in Energy Metabolism and Biological Clock Regulation

The core components of the biological clock directly regulate mitochondria-related genes. CLOCK/BMAL1, as core circadian transcription factors, control mitochondrial energy metabolism pathways by binding to the promoter of target genes, such as directly regulating the activity of key metabolic enzymes in processes like the tricarboxylic acid cycle and oxidative phosphorylation. ⁷⁴ In addition, factors such as chronic light exposure disruption and other circadian rhythm disturbances can lead to an abnormal increase in the activity of the mitochondrial fission protein DRP1, disrupting mitochondrial dynamics. This, in turn, lowers mitochondrial membrane potential, increases ROS production, and ultimately induces mitochondrial dysfunction.^75,76 Energy metabolism abnormalities mainly affect circadian rhythm precision through negative feedback, thus leading to mitochondrial dysfunction. Specifically, energy metabolism imbalance disrupts the expression rhythm of circadian clock genes such as Per and Cry. ⁷⁷ In skeletal muscles, high-intensity exercise can lead to the loss of the circadian oscillation of the Bmal1 gene, disrupting the activity of mitochondrial respiratory chain complexes and the rhythm of ATP synthesis. ⁷⁸ SIRT1 is a key molecule linking the biological clock to mitochondrial function. Its dysregulation weakens the control of mitochondrial biogenesis and antioxidant defense. Moreover, energy metabolism abnormalities alter NAD + levels, affecting the activity of the deacetylase SIRT1 and exacerbating mitochondrial damage. ⁷⁹

Mitochondrial dysfunction participates in the occurrence and development of NAFLD through a complex molecular network, involving multi-dimensional mechanisms such as imbalanced dynamics, oxidative damage, abnormal metabolic signaling, and interactions between organelles, see Figure 3. In-depth analysis of these pathways will provide a theoretical basis for the development of precise treatment strategies.

Figure 3.

Molecular mechanism of mitochondrial dysfunction in NAFLD. IRE1α inositol-requiring enzyme 1α ， JUK jun N-terminal kinase, PGC-1α peroxisome proliferator-activated receptor-gamma coactivator 1-α， Drp1 dynamin-related protein 1，Mfn2 mitochondrial fusion protein，eIF2α eukaryotic translation initiation factor 2α， ATF4 activating transcription factor, UCP2 uncoupling protein 2, ATP adenosine triphosphate, PG phosphatidylglycerol, ETC electron transport chain, MDA malondialdehyde, 4-HNE 4-hydroxynonenal, PUFA polyunsaturated fatty acid, ROS reactive oxygen species, TFEB transcription factor EB, NF-κB Nuclear factor kappa-B, IL-6 interleukin-6, TNF-α tumor necrosis factor-α ， OPTN osteopontin, PLIN2 perilipin 2, PINK1 PTEN induced putative kinase 1 ，DAG diacylglycerol, PKC protein kinase C, IRS insulin receptor substrate.

Therapeutic Strategies for the Targeting of Mitochondrial Dysfunction

Mitochondria, as the core of cellular energy metabolism, are closely associated with a variety of diseases when their function is impaired, including cardiovascular diseases, metabolic diseases, and neurodegenerative diseases.^36,80 Abnormal mitochondrial function is characterized by impaired oxidative phosphorylation, excessive production of ROS, a decrease in mitochondrial membrane potential, and imbalanced mitochondrial dynamics. ⁸¹ In recent years, therapeutic strategies targeting mitochondrial dysfunction have gradually become a research hotspot, encompassing multiple approaches such as antioxidant interventions, regulation of mitochondrial dynamics, gene therapy, and metabolic regulation.^82–84

Antioxidant Treatment Strategy

Mitochondria are the primary source of ROS. The excessive production of ROS can lead to oxidative stress-induced damage, accelerating cell apoptosis and organ function decline. In response to this, the development of mitochondria-targeted antioxidants has emerged as an important research direction. A mitochondria-targeted coenzyme Q analog can improve vascular endothelial function by inhibiting ROS accumulation. Clinical studies have shown that MitoQ can significantly enhance the endothelium-dependent vasodilation function (mediated by nitric oxide, NO) in the elderly population. ⁴² Targeting cardiolipin on the inner mitochondrial membrane can also reduce ROS generation and stabilize the mitochondrial membrane structure, demonstrating a protective effect in myocardial ischemia-reperfusion injury. ⁸⁵ Research reports indicate that N-acetylcysteine can alleviate mitochondrial oxidative stress and neuroinflammation in brain injury by increasing glutathione synthesis. ⁸⁶ Additionally, novel antioxidants such as Indole-TEMPO conjugates have shown potential for anti-inflammation and mitochondrial protection in animal models. ⁸⁷

Studies have reported that gut microbiota regulates mitochondrial bioenergetics and oxidative stress responses, influencing the progression of NAFLD. ⁸⁸ This is primarily achieved through metabolites such as short-chain fatty acids and bile acids, which interact with mitochondria and regulate bioenergetic balance, such as fatty acid oxidation and oxidative phosphorylation efficiency, thereby affecting hepatic lipid accumulation. ⁸⁹ Furthermore, harmful metabolites resulting from gut dysbiosis can induce excessive ROS release from mitochondria, triggering oxidative stress, which further damages mitochondrial DNA and respiratory chain complexes. Meanwhile, harmful metabolites activate pathways such as Toll-like receptors (TLRs), triggering liver inflammation and releasing pro-inflammatory factors such as TNF-α. ⁹⁰ These factors directly inhibit mitochondrial respiratory chain activity and induce mitochondrial permeability transition, leading to cell apoptosis. The above evidence suggests that modulating the gut microbiota is a key mechanism in influencing the mitochondrial oxidative stress state. ⁹¹

Regulation of the Mitochondrial Dynamics

The dynamic balance between mitochondrial fusion mediated by Mfn1/2 and OPA1 and mitochondrial fission mediated by Drp1 is crucial for maintaining mitochondrial function. Intervention strategies can be implemented by regulating mitochondrial dynamics. In obesity-related insulin resistance, Drp1 inhibitors such as Mdivi-1 can restore the integrity of the mitochondrial network and improve glucose metabolism in skeletal muscle. ⁹² Additionally, in diabetic nephropathy, activating the SIRT1/PGC - 1α pathway can enhance mitochondrial biogenesis and improve the function of renal tubular mitochondria. ⁸⁴ Targeting the mitochondrial permeability transition pore is a key approach for regulating mitochondrial dynamics. In ischemia-reperfusion injury, derivatives of cyclosporine A can inhibit the abnormal opening of mPTP, thereby reducing cardiomyocyte apoptosis. ⁹³ Although direct, selective, and clinically validated safe clinical-grade “fusion/fission-targeted drugs” remain lacking, multiple effective interventions have indirectly rebalanced the mitochondrial network or its downstream abnormalities, correlating with histological improvements. For instance, aerobic exercise inhibits excessive fission and enhances mitochondrial quality control via SIRT1-mediated DRP1 deacetylation. ⁹⁴ GLP-1 receptor agonists and FGF21-based agents have been shown in cellular, animal, and early human evidence to ameliorate lipotoxicity and enhance β-oxidation and mitophagy.^95,96 Meanwhile, the concept of direct DRP1 inhibition requires cautious consideration, as excessive inhibition at the hepatocyte level may be detrimental. This indicates that future strategies should emphasize cell-type selectivity, dosage and timing windows, as well as complementary combination with metabolic pathway drugs. ⁹⁷

Mitochondrial Autophagy Regulation

Mitochondrial DNA repair plays a crucial role in regulating mitophagy. For mitochondrial diseases caused by mtDNA mutations, gene-editing technologies such as CRISPR/Cas9 and heterologous mtDNA transplantation have shown potential. ⁹⁸ Activation of the PINK1/Parkin pathway can clear damaged mitochondria and enhance mitophagy. For instance, rapamycin promotes autophagy by inhibiting mTOR and improves mitochondrial function in non-alcoholic fatty liver disease.^99,100 Additionally, regulating the mitochondrial deacetylase SIRT3 can target mitochondria-nucleus communication to improve cardiac energy metabolism disorders and oxidative stress. ⁸¹

Metabolic Intervention is Associated with Energy Resuscitation

Mitochondrial energy metabolism remodeling stands as the core of treatment. Metformin enhances the efficiency of mitochondrial oxidative phosphorylation in diabetic cardiomyopathy by activating the AMPK/SIRT1 pathway. ¹⁰¹ Mitochondrial transplantation is a reliable approach for remodeling energy metabolism. In ischemic stroke, the transplantation of exogenous mitochondria can restore the energy supply to neurons and reduce the infarct volume. ¹⁰² Additionally, for sepsis-related mitochondrial dysfunction, supplementation with pyruvate dehydrogenase kinase inhibitors such as dichloroacetic acid can restore the activity of the tricarboxylic acid cycle, thereby reviving the energy metabolism pathway. ⁴⁷

Combination Therapy with Multiple-Target Intervention

Given the multifactorial nature of mitochondrial dysfunction, combination therapies hold greater promise. The combined action of antioxidant treatment and kinetic regulation yields remarkable results. For example, the combination of SS-31 and a Drp1 inhibitor synergistically improves mitochondrial structure and function in heart failure models. ⁸⁵ Meanwhile, targeting the SIRT1/PGC-1α pathway in combination with metformin can synergistically enhance mitochondrial biogenesis in diabetic nephropathy. ¹⁰³ Traditional Chinese medicines and natural compounds also demonstrate excellent efficacy in targeting mitochondrial disorders. For instance, astragaloside IV alleviates myocardial oxidative damage by inhibiting mitochondrial NOX4 and enhancing the activity of superoxide dismutase. ¹⁰⁴

Current Study Limitations

Insufficient Specificity of Mitochondrial Targeting Drugs

Mitochondria-targeted drugs are designed to act precisely on mitochondria for the treatment of related diseases. However, liver-targeted drugs typically face two challenges: how to ensure effective delivery of the drug to the target tissue and avoid toxic effects in other organs; and how to enhance the drug's bioavailability and stability, preventing premature degradation in the body.. In practical applications, it is difficult for drugs to be precisely enriched in mitochondria, and they often distribute widely in other tissues and organs of the body. This leads to unnecessary effects of the drugs at non-target sites, increasing the risk of systemic toxicity. For instance, some drugs may cause damage to vital organs such as the liver and kidneys, trigger adverse reactions, and affect the patients’ physical health. ¹⁰⁵ To address this problem, a preliminary idea is to modify the drugs so that they can recognize specific markers of mitochondria, thereby enhancing the drug's targeting ability. Additionally, novel drug delivery systems can also be explored to precisely transport drugs to mitochondria. ¹⁰⁶ For example, methods such as nanotechnology, liposomes, or antibody-drug conjugates (ADCs) can be utilized in combination with liver-specific targets for precise delivery, thereby increasing hepatic drug concentration and reducing systemic toxicity. Alternatively, surface modification, pegylation, or the addition of endogenous signaling molecules can enhance delivery efficiency, reduce clearance rate, and improve liver targeting.

Individual Differences and Challenges in Clinical Translation

There is significant heterogeneity within the NAFLD patient population, and individual differences such as age, gender, genetic background, metabolic status, and comorbidities may exert substantial impacts on treatment efficacy. ¹⁰⁷ The formulation of individualized treatment regimens needs to take these factors into account; however, there is currently a lack of comprehensive tools to fully assess patients’ treatment responses. The analysis and development of genomics, transcriptomics, metabolomics, as well as biomarkers including cf-mtDNA and liver function indicators, can provide personalized predictions of disease progression, treatment response, and drug resistance for each patient. Alternatively, by integrating big data and artificial intelligence technologies, precise drug treatment models for NAFLD patients can be established to explore the impacts of different genotypes, environmental factors, and other variables on drug responses. Through multivariate analysis, “optimal responder” patient populations can be identified, and corresponding clinical trial protocols can be designed.

A gap in Clinical Translational Outcomes

Currently, human clinical trials primarily focus on improving overall liver function using existing drugs or lifestyle interventions. For example, pioglitazone, as a PPARγ agonist, alleviates liver damage by regulating mitochondrial energy metabolism and oxidative stress. A retrospective, single-center trial explored the effect of pioglitazone on liver function improvement in patients with type 2 diabetes and alcoholic fatty liver disease. The results showed significant improvements in liver function markers in T2D patients with AFLD after 3 months of pioglitazone treatment. ¹⁰⁸ Studies have shown that high physical activity and cardiovascular health can indirectly regulate mitochondrial function by reducing hepatic lipid deposition and improving insulin sensitivity. ¹⁰⁹ However, specific therapies that directly target mitochondria remain at the preclinical research stage. Mitochondrial-targeted compounds face the challenge of complex metabolic network regulation. Clinical translation needs to address issues such as drug delivery, organ specificity, and long-term safety. ¹¹⁰ Additionally, the lack of standardized mitochondrial function testing methods hinders the objective assessment of therapeutic efficacy in clinical trials. ¹¹¹ Developing non-invasive mitochondrial function biomarkers, such as PBMCs respiratory function and mtDNA variations, to support clinical trial design, can advance the development of targeted therapies for mitochondrial oxidative stress, dynamics, and autophagy.

Challenges and Strategies for Pharmacokinetic (PK) Characteristics

NAFLD patients typically present with impaired liver function, which may affect drug metabolism and excretion; thus, pharmacokinetic (PK) profiles can vary significantly among different patient populations. The absorption, distribution, metabolism, and excretion (ADME) processes of drugs may be impacted by factors such as hepatic steatosis and reduced hepatic blood perfusion, leading to drug accumulation in the liver or suboptimal therapeutic efficacy. In preclinical studies, it is necessary to conduct PK research using small animal models that simulate different liver conditions, such as varying degrees of fat accumulation and liver fibrosis, and extrapolate the data to population models to further optimize clinical trial design. Additionally, by evaluating the PK characteristics of drugs across different clinical stages of NAFLD using in vitro and animal models, and integrating biomarkers and individual genomic information, personalized pharmacokinetic models can be constructed to predict drug performance in diverse patients.

Future Research Direction

Nanoparticles and liposomes, as precise targeted delivery systems, have demonstrated remarkable advantages in enhancing the mitochondrial enrichment of drugs. ¹¹² Nanoparticles possess unique physicochemical properties that enable them to precisely deliver drugs to mitochondria through either passive or active targeting. The principle lies in the fact that the particle size and surface properties of nanoparticles can be precisely regulated, allowing them to smoothly penetrate the cell membrane and mitochondrial membrane, thereby achieving efficient drug enrichment. ¹¹³ Liposomes, on the other hand, are vesicles composed of phospholipid bilayers, which exhibit excellent biocompatibility and membrane-fusion ability. They can encapsulate drugs and release them into the interior of mitochondria through fusion with the mitochondrial membrane. ¹¹⁴ Mitochondrial metabolites such as succinate and acylcarnitine have significant application value in disease staging. The level changes of these metabolites are closely related to the mitochondrial functional state and can reflect the occurrence and development process of diseases. By detecting the content of mitochondrial metabolites, important evidence can be provided for the early diagnosis, condition assessment, and prognosis prediction of diseases. ¹¹⁵ The combination of mitochondria-targeted drugs and anti-inflammatory/anti-fibrotic drugs has a unique mechanism of action and potential advantages. Mitochondrial dysfunction is often accompanied by inflammatory responses and fibrotic processes. Anti-inflammatory/anti-fibrotic drugs can alleviate these pathological changes, creating favorable conditions for the action of mitochondria-targeted drugs. ¹¹⁶ Developing stratified treatment strategies based on mitochondrial genomic polymorphisms has a solid basis and great significance. The mitochondrial genome exhibits rich polymorphisms, and the mitochondrial gene sequences may vary among different individuals, which can affect mitochondrial function and the response to drugs. By sequencing and analyzing the mitochondrial genomes of patients, their mitochondrial gene characteristics can be understood, and personalized treatment plans can be formulated. Based on this, network pharmacology and organ-on-a-chip technology play an important role in mitochondrial-targeted drug research.^117–119 Network pharmacology excels at identifying the action targets, regulatory pathways, and potential toxicity mechanisms of mitochondrial-targeted drugs at the system level, making it particularly suitable for studying complex systems. ¹²⁰ Organ-on-a-chip technology provides a highly physiologically relevant in vitro platform that can be used to directly assess the actual impact of mitochondrial-targeted drugs on mitochondrial function, organ-level efficacy, and toxicity in a simulated organ environment. ¹²¹ The integration of organ-on-a-chip technology with network pharmacology can more systematically and reliably advance mitochondrial-targeted drug research in the drug discovery and development process, overcoming the limitations of traditional models and improving the accuracy and efficiency of predictions.^122,123

Mitochondrial dysfunction is a central factor in the progression of many metabolic diseases. Targeting mitochondria has emerged as a new research direction in clinical studies, such as using peripheral blood mononuclear cells to assess mitochondrial respiratory function to monitor disease progression in NAFLD patients. ¹²⁴ Another example is the use of hydrogen sulfide (H₂S) to stimulate mitochondrial respiration, thereby improving age-related dysfunction and neurodegenerative diseases. ¹²⁵ Meanwhile, mitochondrial transplantation has shown potential in diseases such as NAFLD, but challenges remain in optimizing delivery methods, determining dosages, and sourcing donors. ¹²⁶ Based on this, mitochondrial-related indicators have been proposed as novel biomarkers for disease diagnosis. Traditional biomarkers include mitochondrial membrane potential, calcium ion levels, and ATP levels, while emerging biomarkers, such as cell-free mitochondria and blood cardiolipin levels, may more sensitively reflect mitochondrial responses to exposure. ¹²⁷ The lactate/pyruvate ratio (Lac-AA) is strongly correlated with mitochondrial dysfunction markers and can serve as a central node for integrating the assessment of metabolic disease severity. ¹²⁸ Mitochondrial-related microRNAs regulate mitochondrial gene expression and may also serve as novel biomarkers for mitochondrial-related diseases. ¹²⁹

NAFLD exhibits mitochondrial adaptive changes across different age groups. In children and adolescents, the basal metabolic rate is high, and mitochondrial compensatory capacity is strong, but obesity may accelerate functional depletion. ¹³⁰ In contrast, elderly patients experience a decline in mitochondrial autophagy and repair capabilities, reduced ROS clearance efficiency, and a higher risk of disease progression. Specifically, in the early stages of NAFLD, hepatocyte mitochondria respond to lipid overload by increasing fatty acid oxidation rates, forming a compensatory mechanism. ¹³¹ This adaptability may delay hepatic lipid accumulation, especially in the metabolically active groups of children and adolescents, whereas elderly patients are more likely to progress to NAFLD due to the decline in mitochondrial plasticity. Personalized treatment strategies are needed for NAFLD patients of different age groups. For example, children and adolescent patients can improve mitochondrial quality and fatty acid oxidation capacity through exercise and diet, significantly alleviating NAFLD progression. ¹³² In contrast, the decline in mitochondrial repair capacity is a core factor in the high progression risk in elderly patients, necessitating the use of pharmacological strategies such as antioxidants and autophagy enhancers. ¹³³

NAFLD involves various pathological mechanisms such as fat accumulation, inflammation, and fibrosis. Multi-target combination therapy is considered a rational choice because it improves treatment efficacy and safety by simultaneously targeting multiple pathways. ¹³⁴ Traditional Chinese medicine shows potential in the treatment of NAFLD due to its “multi-component, multi-pathway, and multi-target” characteristics. For example, Jiangzhi Decoction regulates lipid metabolism and inflammation through a multi-target mechanism. ¹³⁵ The combination of baicalin and acarbose not only improves liver histology and glucose tolerance but also reduces insulin resistance, confirming its efficacy. ¹³⁶ In clinical trials for NAFLD, combination therapies, where multiple drugs target different pathways, enable more patients to achieve primary endpoints compared to the placebo group, indicating that synergistic mechanisms can more effectively alleviate symptoms. ¹³⁷

Conclusion

NAFLD has emerged as the most prevalent chronic liver disease globally, and its prevalence is closely associated with metabolic disorders. Mitochondrial dysfunction, as a core mechanism of metabolic disturbances, exacerbates insulin resistance and inflammatory responses in hepatocytes through multiple pathways, including impaired oxidative phosphorylation, excessive production of ROS, imbalanced mitochondrial dynamics, and defective autophagy. Specifically, decreased expression of mitochondrial fusion proteins, over-activation of the fission protein Drp1, lipid peroxidation induced by oxidative stress, and imbalance of NAD⁺/NADH collectively drive the onset and progression of NAFLD. Intervention strategies targeting mitochondria, such as antioxidants, regulation of mitochondrial dynamics, activation of autophagy, and metabolic remodeling, have shown potential in improving mitochondrial function and alleviating NAFLD. However, the insufficient targeting of existing drugs and the heterogeneity between animal models and human diseases remain major challenges. Future research should combine precise delivery systems with multi-targeted combination therapies to promote the translation of mitochondrial-targeted strategies into clinical applications. This systematic review establishes a solid theoretical foundation for mitochondrial-targeted therapies in NAFLD and provides new directions for its clinical treatment. Through interdisciplinary collaboration and technological innovation, mitochondrial-targeted therapy is expected to become a significant breakthrough in precision medicine for NAFLD, offering a new paradigm for the prevention and control of the global burden of metabolic liver diseases.