Mitochondrial transplantation and platelet rich plasma for the treatment of non-alcoholic fatty liver disease

Abstract

Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent global health concern, contributing to the development of insulin resistance, diabetes, cardiovascular disease, cirrhosis, and hepatocellular carcinoma. Since no approved drugs for the treatment of NAFLD exist, there is an urgent need for novel therapeutic strategies. Two such strategies are mitochondrial transplantation and platelet rich plasma (PRP) therapy. In preclinical NAFLD, mitochondrial transplantation alleviates steatosis by improving the hepatic imbalance between fatty acid utilization and synthesis. Moreover, it reduces excessive reactive oxygen species production and lipid peroxidation, thereby reducing inflammation and fibrosis. In contrast, PRP therapy ameliorates hepatic damage induced by xenobiotics by deactivating stellate cells, reducing fibrosis and apoptosis, and decreasing inflammation via NF-κB inhibition, while enhancing antioxidant defenses. These effects may be related to the improvement of NAFLD observed in a preclinical study. We propose that a combination of mitochondrial transplantation and PRP therapy may represent a novel approach for treating NAFLD by targeting different aspects of NAFLD in a complementary manner. We discuss the limitations of these therapies, as preclinical studies addressing NAFLD with these therapies are scarce, and there are no clinical trials in humans.

Key Words: Non-alcoholic fatty liver disease; Steatohepatitis; Inflammation; Cirrhosis; Oxidative stress; Electron transport chain; Tissue regeneration; Hepatocyte; Steatosis; Mitochondria

Core Tip: Liver diseases driven by mitochondrial dysfunction and oxidative stress have not yet been effectively treated, creating a need for novel therapeutic solutions. Mitochondrial transplantation restores energy metabolism, while platelet rich plasma (PRP) therapy reduces fibrosis via growth factors. This is the first review to propose the combination of mitochondrial transplantation and PRP as a novel therapeutic strategy for non-alcoholic fatty liver disease. The strategy addresses mitochondrial dysfunction, oxidative stress, and fibrosis while promoting liver regeneration, offering a promising alternative where pharmacological treatments are limited.

INTRODUCTION

Liver diseases are becoming increasingly prevalent worldwide. Several potential causes of liver disease exist. These factors include a sedentary lifestyle, unhealthy eating habits, obesity, diabetes, dyslipidemia, insulin resistance, and metabolic syndrome. These conditions can lead to non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC)[1,2]. A significant proportion of patients with NAFLD progress to NASH or HCC. Between 12% and 40% of individuals develop NASH at 8-13 years. Approximately 15% of the patients with NASH or early fibrosis progress to cirrhosis within the same period. Additionally, approximately 7% of patients with cirrhosis develop HCC within 10 years, highlighting the progressive and potentially serious nature of this condition[3]. An estimated 58417006 cases of cirrhosis and other chronic liver diseases were reported worldwide in 2021, with 1425142 deaths attributed to these conditions in the same year[4]. However, the outlook is even more concerning, as these numbers are projected to increase significantly by 2030[5]. One factor contributing to this unfavorable projection is the difficulty of accurately and promptly diagnosing these diseases. The lack of early symptoms, combined with limited sensitivity of noninvasive tests for early-stage detection, complicates diagnosis[6].

However, a standard treatment for NAFLD has not yet been established[7]. Some drugs currently used in the management of NAFLD were not initially developed for its specific treatment but to target metabolic abnormalities commonly associated with the disease[8]. Some of these include antibiotics, GLP-1 analogs, metformin, insulin, pioglitazone, statins, and sodium-glucose cotransporter type 2 inhibitors. However, the efficacy of drug treatments for NAFLD varies depending on the stage of the disease and patient profile; thus, there is no established therapy for NAFLD. Furthermore, these treatments should complement lifestyle changes; however, sustaining these changes over time is challenging[9].

Mitochondrial dysfunction is widely recognized as a critical factor in the development and progression of NAFLD into NASH and fibrosis. Therefore, the liver mitochondria have been suggested as potential targets for NAFLD treatment[10,11]. The role of mitochondria in the progression of NAFLD has been confirmed by the effects of resmetirom, a drug approved by the FDA in 2024 to treat moderate-to-advanced liver fibrosis. Resmetirom reduces hepatic steatosis by improving mitochondrial functions like β-oxidation of fatty acids, mitophagy, and mitochondrial biogenesis. Additionally, resmetirom addresses fibrosis by inhibiting lipogenesis and fibrogenesis through the suppression of transforming growth factor-β (TGF-β) signaling[12,13]. This finding is consistent with the idea that combination therapies targeting several pathways may enhance the effectiveness of NAFLD treatment, as demonstrated by the fact that less than 50% of patients treated with single therapies experienced a positive response[7].

Given the fundamental role of mitochondrial dysfunction and oxidative stress in the onset and progression of NAFLD and the emerging potential of combination therapies targeting multiple mechanisms, this study reviews preclinical evidence supporting the therapeutic efficacy of mitochondrial transplantation and platelet rich plasma (PRP) therapy in NAFLD treatment. First, we provided a context on the role of mitochondrial dysfunction and oxidative stress in the onset and progression of NAFLD. Next, we discussed the rationale for mitochondrial transplantation and PRP therapy in NAFLD by critically reviewing the existing preclinical evidence for their efficacy. Finally, we discussed the challenges that must be addressed to ensure the safety of these therapies. Our goal is to encourage further discussion on combining these therapies to treat NAFLD, design human clinical trials that establish the safety of these protocols, and optimize the dosage of mitochondrial transplantation and PRP for clinical use.

MITOCHONDRIAL DYSFUNCTION AND OXIDATIVE STRESS AS COMMON PATHOLOGICAL MECHANISMS IN LIVER DISEASES

Under normal physiological conditions, the mitochondrial electron transport chain (ETC) converts the chemical energy stored in reduced coenzymes (NADH or FADH₂) into a mitochondrial membrane potential (Δψ) by translocating protons across the inner mitochondrial membrane. This process uses the oxidation energies of NADH, FADH₂, and the electron transport intermediates[14]. The energy of Δψ is central to various processes fundamental to cell survival. For instance, Δψ drives ATP synthesis in mitochondria and ion transport, which helps modulate cellular energy levels and ionic homeostasis[15]. Additionally, Δψ acts as a physiological signal that modulates programmed cell death and quality control processes, such as mitophagy and autophagy. Moreover, it influences the formation of mitochondrial networks through mitochondrial fusion/fragmentation cycles, also known as mitochondrial dynamics[16]. Furthermore, ETC complexes are a primary source of reactive oxygen species (ROS), which can activate processes, such as inflammation or fibrosis, that contribute to the progression of diseases[17].

Alterations in these processes contribute to the progression of NAFLD. In obesity, excess nutrients and lipolysis in adipocytes increase the circulating free fatty acid levels[18]. Hepatocytes take up these fatty acids, which increase mitochondrial fatty acid oxidation and ETC activity. However, oversaturation of these processes eventually decreases the activity of the ETC complexes[19,20]. This has been partly attributed to a decrease in the cardiolipin content. Cardiolipin is a phospholipid unique to mitochondria and is involved in maintaining the structure and function of ETC complexes, as well as their interaction with the inner mitochondrial membrane[21]. Moreover, hepatic steatosis decreases Δψ, which can lead to mitochondrial fission, cytochrome c release, and liver damage via apoptosis induction[22,23]. Decreased mitophagy has been observed in NAFLD, contributing to the propagation of fissured mitochondria with impaired function and damaged mtDNA[24]. Decreased ETC activity worsens hepatic lipid accumulation by inhibiting the β-oxidation of fatty acids in mitochondria due to decreased regeneration of NAD⁺ and FAD²⁺ in complexes I and II of the ETC[25].

In contrast, decreased ETC complex activity increases ROS production[26], which promotes inflammation and the progression from steatosis to NASH[27]. This occurs via an increase in lipid peroxidation, which activates the nuclear factor-kappa B (NF-κB) pathway, resulting in the increased expression of inflammatory mediators such as tumor necrosis factor α (TNF-α), and interleukins (ILs) like IL-1β, IL-6, and IL-8. Furthermore, mtDNA released from damaged hepatocytes can activate Toll-like receptor 9, initiating innate immune responses that exacerbate hepatic inflammation[11,28].

As liver damage progresses, mitochondrial dysfunction and oxidative stress persist, contributing to the development of fibrosis through alterations in the urea cycle and mutations in the subunits of ETC complexes[29,30]. In addition, liver damage and inflammation activate hepatic stellate cells, which are responsible for the secretion and deposition of the extracellular matrix (ECM)[31]. These events promote fibrogenesis and extensive tissue remodeling, ultimately driving the progression[32,33].

In later stages, mitochondrial dysfunction is a hallmark of HCC, the most common primary liver cancer. In HCC, the mitochondria display excessive ROS production, hypoxia-induced stress, impaired unfolded protein responses, dysregulated dynamics, and mitophagy. Additionally, mtDNA damage and disruption of oxidative phosphorylation amplify mitochondrial dysfunction and contribute to oncogenesis[34,35]. A key metabolic hallmark of hepatic cancer cells is the shift toward aerobic glycolysis, known as the Warburg effect, which allows rapid ATP generation even under oxygen-rich conditions. Mitochondrial defects in tumor cells compromise aerobic respiration, promote inefficient electron transport, and generate excessive ROS. This results in oxidative damage to lipids, proteins, and DNA, which exacerbates mitochondrial failure and supports tumor progression[36,37].

The key pathological features associated with mitochondrial dysfunction and oxidative stress in various liver diseases such as steatosis, inflammation, fibrosis, metabolic dysregulation, uncontrolled cell proliferation, and somatic mutations[38-43] are summarized in Table 1, which highlights their roles in disease progression and severity.

Table 1 Mitochondrial dysfunction and oxidative stress in liver diseases.

Liver disease	Main pathological alterations	Role of oxidative stress	Role of mitochondrial dysfunction	Ref.
NAFLD	Hepatic lipid accumulation, mild inflammation, metabolic dysregulation, lipid accumulation, altered autophagy and mitochondrial quality control	Increased ROS production promotes lipid peroxidation and mild inflammatory response, oxidative stress contributes to progression toward NASH	Early mitochondrial damage affects lipid metabolism and redox balance, drives lipid dysregulation, impairs energy homeostasis, and alters mitochondrial turnover	Zheng et al[38], Dabravolski et al[39], and Trinchese et al[40]
NASH	Steatosis, inflammation, hepatocyte injury and fibrosis	ROS and RNS overproduction activate NF-κB and upregulate TNF-α, IL-1β, IL-6, IL-8	mtDNA release activates TLR9-mediated inflammation; mitochondrial damage enhances lipotoxicity and fibrosis	Simões et al[11], Rodrigues et al[28], and Carter-Kent et al[41]
HCC	Cell proliferation, somatic mutations, altered mitochondrial dynamics (fusion/fission, mitophagy)	ROS promote DNA damage and tumor progression, oncogenic signaling, and resistance to apoptosis	Mitochondrial dysfunction enhances Warburg effect	Chen et al[42] and Su et al[43]

NAFLD: Non-alcoholic fatty liver disease; NASH: Non-alcoholic steatohepatitis; HCC: Hepatocellular carcinoma; ROS: Reactive oxygen species; RNS: Reactive nitrogen species; IL: Interleukin; TNF-α: Tumor necrosis factor α; NF-kB: Nuclear factor-kappa B.

MITOCHONDRIAL TRANSPLANTATION IN NAFLD

In mitochondrial transplantation, mitochondria are isolated from a cell source and released postnatally into individuals to produce a therapeutic response[44]. Mitochondrial transplantation has the potential to be an effective therapy against NAFLD progression, as experimental evidence from preclinical models of NAFLD and other diseases suggests that various aspects of mitochondrial dysfunction involved in the pathophysiology of NAFLD can be improved using this approach.

Biological basis of mitochondrial transplantation

The concept of mitochondrial transplantation originated from pioneering in vitro observations of the transfer of the mitochondrial genome from isolated mitochondria to yeast spheroplasts, which are incapable of respiration[45]. Subsequently, it was confirmed that respiration-impaired mammalian cells can incorporate mitochondria incubated in their culture medium and regain respiratory function[46,47]. Further evidence that mitochondrial transfer occurs in vivo comes from studies in which mitochondrial transfer was observed by administering free mitochondrial cells to living organisms and improving certain pathological conditions, such as acute lung injury[48], spinal cord injury[49], or cardiac damage due to ischemia-reperfusion[50]. Another significant finding was the detection of intact, freely circulating mitochondria with functional respiration in the bloodstream of healthy individuals, as well as their transfer between different organs[51]. Mitochondria are transferred between donor and recipient cells via tunneling nanotubes[52], extracellular vesicles[53,54], cell adhesion[40], or the release of free mitochondria by donor cells[55]. These breakthroughs have encouraged the study of mitochondrial transplantation from healthy cells or tissues into diseased organisms as a novel approach to treating various diseases, including NAFLD.

Current research on mitochondrial transplantation for NAFLD

Few studies have addressed the effectiveness of mitochondrial transplantation as a therapeutic strategy for treating NAFLD. The first study to evaluate mitochondrial transplantation in C57BL/6J mice with diet-induced NAFLD[56]. Mitochondria were isolated from HepG2 cells using standard differential centrifugation. Percoll or sucrose gradients were not used to increase the purity. The mitochondria were administered via intravenous injection at a dose of 0.5 mg/kg body weight three times at 3 days intervals or six times to evaluate the dose-dependent nature of the treatment. The outcomes were reduced blood levels of alanine transaminase (ALT) and aspartate transaminase (AST) and decreased hepatic lipid levels. At the mitochondrial level, restoration of mitochondrial ultrastructure and increased activity of complex IV at the ETC and ATP levels were observed. Increased glutathione (GSH) and superoxide dismutase (SOD) levels and decreased ROS and lipid peroxidation levels were observed.

Another study examined the transfer of mitochondria in C57BL/6 mice via xenotransplantation of human bone marrow-derived mesenchymal stromal cells (MSCs) that had differentiated into the hepatocyte lineage into the liver[57]. NASH was induced using a methionine-choline-deficient diet[58]. MSCs were reported to prevent several impairments induced by the NASH diet, such as decreased expression of proteins from the ETC and fatty acid β-oxidation, as well as increased expression of proteins involved in hepatic lipid synthesis. Furthermore, transplantation of MSCs prevents a decrease in proteins involved in triglyceride secretion via very low density lipoprotein (VLDL) and reduces the levels of 4-hydroxynonenal (4-HNE), a product of lipid peroxidation[57].

A third study[59] evaluated mitochondrial transplantation in an NAFLD model induced by a high-fat diet and streptozotocin to induce diabetes. Mitochondria were isolated from the muscles of healthy rats using standard differential centrifugation without density gradients. Mitochondria were then administered intravenously at a dose of 0.4 mg/kg. Outcomes were evaluated at 24 hours and 48 hours after transplantation. Decreased blood levels of ALT and AST and cell hypertrophy were observed. The decrease in the activity of all ETC complexes was reversed, with a modest effect on complexes I and III. With regard to oxidative stress, the decrease in SOD, catalase, and GSH activities was reversed, and lipid peroxidation levels decreased. Mitochondrial administration decreased IL-6 and NF-κB mRNA levels; however, there was no histological evidence of decreased inflammation. Likewise, an increase in PGC-1α/AMPK/AKT/PI3K was reported, but there was no functional evidence of increased mitochondrial biogenesis or oxidative phosphorylation. Additionally, mitochondrial administration has a hypoglycemic effect, improving blood lipid levels and blood pressure.

Potential benefits of mitochondrial transplantation

Although evidence regarding the therapeutic potential of mitochondrial transplantation for treating NAFLD comes from a limited number of preclinical studies[56-59], it can be suggested that mitochondrial transplantation may efficiently interfere with NAFLD development by targeting several key processes involved in its pathogenesis. According to the multiple-hit theory of NAFLD development (Figure 1), hepatic lipid accumulation results from an imbalance between the lipid synthesis and utilization pathways. Insulin resistance increases de novo lipogenesis in the liver and lipolysis in adipose tissue. These processes increase plasma free fatty acid levels, their uptake in the liver, and their incorporation into triglycerides (TG). Excess dietary lipids promote hepatic triglyceride accumulation. Additionally, mitochondrial fatty acid β-oxidation decreases, as does the export of TG from the liver via VLDL secretion. The resulting lipid accumulation causes steatosis, the first hit, and lipotoxicity, which affects several processes, including insulin receptor signaling. Lipotoxicity causes mitochondrial dysfunction, which inhibits ETC function and oxidative phosphorylation while increasing ROS generation, the second hit. Excess ROS can oxidize the accumulated lipids from steatosis, generating reactive aldehydes such as 4-HNE and malondialdehyde (MDA). These molecules and ROS can trigger liver inflammation (the third hit) by activating the production of proinflammatory molecules. These molecules contribute to the development of fibrosis by promoting collagen synthesis via the activation of Kupffer and stellate cells, constituting the fourth hit. Additional factors include increased intestinal permeability to bacteria and exposure to pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharides. This further increases inflammation and liver fibrosis[60].

Figure 1 Multiple hit progression of non-alcoholic fatty liver disease. LPS: Lipopolysaccharides; ROS: Reactive oxygen species; ETC: Electron transport chain; DAG: Diacyl glycerol; CR: Ceramides; MDA: Malondialdehyde; 4-HNE: 4-hydroxynonenal; DNL: De novo lipogenesis; AC5L5: Long-chain fatty acyl-CoA ligase; Fabp1: Liver-specific fatty acid-binding protein 1; IL: Interleukin; TNF-α: Tumor necrosis factor α; NF-kβ: Nuclear factor-kappa B; VLDL: Very low density lipoprotein. Image provided by Servier Medical Art (https://smart.servier.com/), licensed under CC BY 4.0 (https://creativecommons.org/Licenses/by/4.0/).

The results of mitochondrial transplantation studies[56-59] suggest a hypothetical mechanism of action involving improvements in several targets considered in the multiple-hits theory (marked with green marks in Figure 2). Mitochondrial transplantation increases the expression of genes involved in mitochondrial biogenesis. This increase may be related to enhanced ETC activity and ATP synthesis, as well as lower mitochondrial ROS production. Additionally, there was a decrease in the expression of genes involved in de novo lipid synthesis, as well as an increase in the expression of genes involved in mitochondrial fatty acid β-oxidation and VLDL secretion. These changes may mediate the decrease in steatosis. Additionally, mitochondrial transplantation increased antioxidant systems, consistent with decreased ROS, lipid peroxidation, and 4-HNE levels. This may mediate a decrease in inflammation and fibrosis. These effects on the liver appear to influence the development of metabolic syndrome, as improved blood pressure, hyperglycemia, and dyslipidemia have been observed[59].

Figure 2 Role of mitochondrial dysfunction in the progression of different stages of non-alcoholic fatty liver disease. Δψ: Mitochondrial membrane potential; ROS: Reactive oxygen species; FFA: Free fatty acids; ETC: Electron transport chain; Cyt c: Cytochrome c; TG: Triglycerides; ECM: Extracellular matrix; IL: Interleukin; TNF-α: Tumor necrosis factor α; NF-κB: Nuclear factor-kappa B. Image provided by Servier Medical Art (https://smart.servier.com/), licensed under CC BY 4.0 (https://creativecommons.org/Licenses/by/4.0/).

Challenges and limitations of mitochondrial transplantation

Despite these encouraging results, further investigation is necessary to determine the effects of mitochondrial transplantation on other factors that contribute to NAFLD development (marked with blue crosses). It is unclear whether mitochondrial transplantation modulates intestinal permeability, PAMP-induced inflammation and fibrosis, lipolysis in the adipose tissue, the release of free fatty acids into the bloodstream, or their uptake by the liver. The effect of mitochondrial transplantation on hepatic insulin resistance and the levels of lipids that stimulate this phenomenon, including ceramides and diacylglycerols, is unknown. It is unclear whether the mechanism by which mitochondrial transplantation reduces fibrosis involves the reduced activation of Kupffer and stellate cells. Furthermore, the impact of mitochondrial transplantation on the progression of NAFLD to HCC and its capacity to resolve cirrhosis remains uncertain.

Other issues to be resolved include identification of the least immunogenic and most efficacious mitochondrial transplantation technique with the fewest side effects. Preclinical studies conducted thus far have evaluated the effects of mitochondrial transplantation for periods no longer than one month. Therefore, it is unclear whether the effects of transplantation are long-lasting or whether chronic regimens of mitochondrial administration are necessary. Furthermore, two studies[56,59] involved injecting isolated mitochondria for mitochondrial transplantation. Since mitochondria are isolated by differential centrifugation in the absence of density gradients, contamination with other organelles, particularly the endoplasmic reticulum, which is physiologically associated with the outer mitochondrial membrane, is probable. Additionally, the injection of isolated mitochondria may result in their transfer to organs other than the liver, which could have harmful effects. For instance, accidental transfer of mitochondria to neoplastic cells with mitochondrial dysfunction can increase their metastatic potential[61]. Furthermore, mitochondrial transplantation via MSC xenotransplantation in immunocompromised mice, as reported by Hsu et al[57], could limit the translatability of this approach to immunocompetent humans. Another important concern is the development of autoimmunity, adaptive immune responses, or worsening of NAFLD or other comorbidities due to a mismatch between the mtDNA of the donated mitochondria and the nuclear DNA of recipient cells[62].

In summary, mitochondrial transplantation has the potential to be an effective therapy for NAFLD and certain comorbidities of metabolic syndrome through a mechanism involving a reduction in steatosis, improvement in mitochondrial function, and a decrease in oxidative stress, inflammation, and fibrosis. Nevertheless, these results are backed up by a small number of studies that other research groups should replicate. In addition, there are several gaps regarding safety and dosage that require further studies, including more research on the direct targeting of mitochondria to the liver, ensuring the purity of mitochondrial preparations without contamination by other organelles, which could be ensured through the adherence to good manufacturing practices, and the characterization of the recipient and donor genomes to reduce the risk of immunogenicity. Similarly, the dosage that considers the chronic nature of this disease and the risk of genotoxicity due to the probable integration of mtDNA into the recipient DNA should be investigated. Additionally, the dosage should be based on a rationale that considers adjustments to body weight and metabolic rate.

PRP: CHARACTERISTICS AND APPLICATIONS

PRP is a biological product derived from blood obtained through centrifugation, which results in the concentration of platelets at levels that are 2-5 times higher than those found in whole blood. The preparation of PRP is subject to variation, with single or double centrifugation being the most common method. However, a novel approach has been developed that incorporates water evaporation post-centrifugation, resulting in a volume reduction by half and doubling of platelet content[63-65].

To guarantee clinical efficacy, PRP should contain approximately 1000 × 10³/μL of non-activated platelets[66]. Some components of PRP that may contribute to its therapeutic effects include insulin-like growth factor 1, TGF-β, and hepatocyte growth factor[67], which plays an important role in liver regeneration[68]. Other proteins found in PRP include platelet-derived growth factor and vascular endothelial growth factor, as well as cell adhesion proteins such as fibrin, fibronectin, and vitronectin, which promote cell migration, attachment, and tissue repair[64,69].

Many of these bioactive molecules have regenerative properties that enhance cell proliferation, angiogenesis, and bone regeneration in musculoskeletal disorders such as osteoarthritis[63]. Preclinical and clinical studies have explored the efficacy of PRP in various diseases, highlighting the diverse mechanisms that drive regeneration (Table 2)[66,70-75]. However, information regarding the efficacy of PRP in treating liver diseases such as NAFLD is extremely limited (Table 2). The following sections briefly review the effects of PRP on oxidative stress, inflammation and hepatic diseases to further support its therapeutic application in NAFLD.

Table 2 Regenerative mechanisms and applications of platelet rich plasma in preclinical and clinical studies.

Model/disease	Outcomes and mechanism	Ref.
Porcine and human cartilage cells/osteoarthritis	Stimulated chondrocyte proliferation, cartilage matrix production, suppressed inflammation via NF-κB inhibition	Monteiro et al[66]
Equine tenocytes, tendinopathies	Protected against hydrogen peroxide damage, reduced protein and lipid peroxidation, prevented cell death, increased nuclear Nrf2 levels, induced antioxidant enzyme expression	Tognoloni et al[71]
Human anterior cruciate ligamentocytes, ligament injury	Increased DNA content and metabolic activity (cell proliferation), no significant extracellular matrix production	Krismer et al[72]
Rabbits, osteoarthritis	Enhanced cartilage repair, increased glycosaminoglycan production	Monteiro et al[66]
Horses, osteoarthritis	Alleviated lameness	Monteiro et al[66]
Mice, cirrhosis	PRPEV reduced liver fibrosis, promoted hepatocyte proliferation, increased anti-inflammatory M2 macrophages	Maeda et al[73]
Rats, chronic liver disease	Improved liver enzymes, lipid profile, oxidative stress markers	Mansour et al[74]
Rats, bile duct ligation-induced cirrhosis	ADMSCs + PRP reduced inflammation, hepatocyte damage, collagen deposition	Shivaramu et al[75]
Humans (30 patients), knee osteoarthritis	Reduced pain by 33.4%	Monteiro et al[66] and Kwon et al[70]

Nrf2: Nuclear factor erythroid 2-related factor 2; PRPEV: Platelet-rich plasma-derived extracellular vesicles; ADMSC: Adipose-derived mesenchymal stem cell; PRP: Platelet rich plasma.

Antioxidant and anti-inflammatory properties of PRP

In an in vitro model of osteoarthritis, PRP stimulated the proliferation of cartilage cells and the production of cartilage matrix in both porcine and human samples, while simultaneously suppressing inflammation in osteoarthritic cartilage cells via NF-kB inhibition[66]. In vivo, PRP has been demonstrated to enhance cartilage repair and glycosaminoglycan production in rabbit osteoarthritis models[66,70]. In tendinopathies prevalent in equine and human athletes due to oxidative stress, PRP demonstrates antioxidant properties in vitro by protecting equine tenocytes from hydrogen peroxide-induced damage, reducing protein and lipid peroxidation, preventing cell death, increasing the nuclear levels of the redox-dependent nuclear factor erythroid 2-related factor 2 (Nrf2), and inducing antioxidant enzyme expression to support tendon homeostasis and regeneration[71]. Similarly, in an in vitro model of the human anterior cruciate ligament, the application of 2.5% PRP resulted in a significant increase in both DNA content and metabolic activity, suggesting enhanced cell proliferation. Importantly, PRP does not substantially increase ECM production[72].

Regarding inflammation, leukocyte-rich PRP reduced inflammation by inhibiting proinflammatory cytokines, such as IL-1, IL-6, and TNF-α, promoting macrophage polarization to an anti-inflammatory M2 phenotype, and suppressing NF-κB activity. Additionally, it enhanced angiogenesis by stimulating the formation of micro vessels in pathological tissues[76].

Use of PRP in liver disease models

In preclinical models of liver disease, PRP-derived extracellular vesicles reduced liver fibrosis, promoted hepatocyte proliferation, and increased anti-inflammatory M2 macrophages in a mouse cirrhosis model[77], and late PRP treatment improved liver enzymes, lipid profiles, and oxidative stress markers in a rat model of thioacetamide-induced chronic liver disease, highlighting the therapeutic potential of PRP components in hepatic regeneration[74]. Moreover, the combination of adipose-derived mesenchymal stem cells with PRP enhanced hepatoprotection in a rat model of bile duct ligation-induced cirrhosis, reducing inflammation, hepatocyte damage, and collagen deposition[78].

Platelets promote liver regeneration through anti-fibrotic and anti-apoptotic effects by deactivating stellate cells and activating the Akt pathway[79]. PRP has a protective effect against carbon tetrachloride-induced hepatotoxicity by reducing oxidative stress through an increase in GSH levels and a decrease in lipid peroxidation. No PRP-induced damage was detected in healthy animals in this study[80]. Moreover, an increase in the enzymatic activity of NADPH-quinone oxidoreductase-1 (NQO1) was detected in rats with thioacetamide-induced liver damage[77]. This further supports the role of Nrf2 in the effects of PRP since NQO1 gene expression is modulated by Nrf2[81].

From the above studies, the antioxidant, anti-inflammatory, and anti-fibrotic effects of PRP on liver damage induced by xenobiotics or experimental cirrhosis suggest a therapeutic role for PRP in NAFLD. This is consistent with a previous report in which PRP application reduced liver damage in a murine model of NASH, decreased ALT and AST blood levels, and improved hepatic steatosis and dyslipidemia[78]. However, this study did not describe the conditions under which the PRP was obtained or its platelet or leukocyte content. Therefore, it is difficult to infer whether the results of this study have potential clinical efficacy, given that it is unknown whether the PRP used meets the requirement of containing 1000 × 10³/μL of non-activated platelets to guarantee its clinical efficacy, as previously suggested[66].

Interplay between mitochondrial transplant and PRP therapy

It can be inferred that there is crosstalk between mitochondrial transplantation and PRP therapy, as both therapies share some targets in the development of NAFLD (Figure 1). Mitochondrial transplantation activates AMPK, which, in turn, phosphorylates Nrf2 at Ser550, inducing Nrf2 phosphorylation and the expression of antioxidant enzymes, such as SOD and catalase[82]. PRP also activates Nrf2-regulated antioxidant enzymes synthesis like NQO1[77]. Thus, the antioxidant effects of both therapies would reinforce each other, and therefore their anti-inflammatory effect, since MDA and 4-HNE levels can decrease with PRP[71,74] and mitochondrial transplantation[57], and these end products of lipid peroxidation function as inflammation-inducing molecules.

In terms of their anti-inflammatory and antifibrotic effects, mitochondrial transplantation decreases inflammatory molecules such as IL-6 and NF-κB, thereby mitigating fibrosis by attenuating the activation of stellate cells and macrophages[49,50]. In turn, PRP inhibits NF-κB-driven inflammation in macrophages and reduces fibrosis in stellate cells via growth factors such as TGF-β and PDGF, as well as extracellular vesicles[77]. Their coordinated action at the tissue level may amplify the anti-inflammatory and anti-fibrotic effects. The synergistic effects of both therapies are not trivial, as this would have the potential to reduce the doses of mitochondrial transplantation and PRP required to obtain a therapeutic effect and could consequently decrease the possibility of adverse effects.

CHALLENGES AND FUTURE DIRECTIONS IN PRP

Although the therapeutic potential of PRP and mitochondrial transplantation in liver disease is promising, several challenges remain. Despite these promising findings, the variability in PRP formulations, due to differences in preparation methods and composition, complicates reproducibility, precludes cross-study comparisons, and hinders optimization for specific clinical conditions, necessitating standardized protocols to enhance efficacy and evaluate cost-effectiveness[79,83,84]. For example, in a rabbit model of major hepatectomy, post-resection PRP injection showed no significant effect on liver regeneration and was associated with high mortality[80]. Platelet hyperactivation is a critical consideration in PRP therapy, especially for conditions such as preeclampsia and pregnancy-induced hypertension. In these conditions, a disrupted balance of thromboxane A2 and prostacyclin may exacerbate thrombo-inflammation[85]. In musculoskeletal applications, underlying inflammatory or autoimmune diseases may trigger adverse effects due to proinflammatory platelet phenotypes[86]. Therefore, careful patient selection should also be essential in patients with NAFLD to mitigate these risks.

Future research should prioritize the validation of the safety and efficacy of combined PRP and mitochondrial transplantation in NAFLD and cirrhosis. Therefore, efforts are needed to develop targeted delivery systems and synergistic protocols that leverage the regenerative and bioenergetic benefits of these therapies.

CONCLUSION

Mitochondrial transplantation is the transfer of mitochondria to a patient from a healthy donor or from the patient's own cells to produce a therapeutic response by restoring mitochondrial function. PRP therapy is a treatment that uses the individual's own blood growth factors to accelerate and improve the repair of damaged tissues. Both therapies have the potential to be a treatment for NAFLD by addressing multiple targets responsible for the development of this disease. However, there are several issues that have not been studied regarding the use of these novel therapies related to their manufacture, safety, dosage, and efficacy. The combination of PRP and mitochondrial transplantation offers a promising therapeutic approach for the treatment of liver diseases, including NAFLD, NASH, cirrhosis, and HCC. PRP has been shown to promote tissue regeneration, reduce inflammation, and mitigate fibrosis through the release of growth factors and extracellular vesicles. Mitochondrial transplantation enhances these effects by restoring hepatocyte bioenergetics and mitigating oxidative stress, thereby addressing mitochondrial dysfunction, which is critical to the pathogenesis of metabolic liver diseases, such as NAFLD. The combination of the regenerative and anti-inflammatory properties of PRP with the ability of mitochondrial transplantation to enhance cellular energy metabolism and resilience provides a multifaceted approach for addressing the intricate pathophysiology of NAFLD, NASH, cirrhosis, and HCC. This approach provides a promising avenue for the development of novel therapeutic strategies.