Letter to the Editor: Lianhe Xiaozhi ointment for metabolic dysfunction-associated steatotic liver disease - a gut-liver-brain axis perspective

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly nonalcoholic fatty liver disease, is a growing global health burden with limited approved therapies. In a recent article published in the World Journal of Gastroenterology, Nie et al reported that Lianhe Xiaozhi ointment (LXO)—a patented traditional Chinese medicine (TCM) formulation derived from Huanglian Wendan decoction—markedly ameliorates MASLD in high-fat diet models through activation of the peroxisome proliferator-activated receptor alpha (PPARα) pathway. This letter commends the comprehensive multi-omics and in vivo approach of Nie et al, emphasizes its concordance with prior findings on PPARα’s central role in hepatic lipid metabolism, and discusses how LXO’s gut microbiota-PPARα-inflammation axis introduces a novel therapeutic dimension for MASLD. We provide a comparative analysis of LXO’s effects vs established MASLD interventions, noting concordant improvements in weight gain, metabolic indices, liver injury, and inflammation. We also offer a constructive critique of the study’s limitations, including the need for causal validation (e.g., Ppara knockout models) and translational investigation (bioactive compound pharmacokinetics and human trials). Finally, we expand on mechanistic hypotheses suggested by the authors, proposing exploration of the gut-liver-brain axis and associated neuroimmune outcomes in MASLD and outlining future research directions such as PPARα-targeted validation, serum pharmacochemistry, and neuroinflammatory profiling. This commentary affirms the hypothesis-defining and field-advancing significance of Nie et al’s work, demonstrating how integrating TCM insights with cutting-edge mechanistic studies can drive innovative MASLD therapies.

Key Words: Lianhe Xiaozhi ointment; Traditional Chinese medicine; Metabolic dysfunction-associated steatotic liver disease; Gut-liver-brain axis; Peroxisome proliferator-activated receptor alpha; Gut microbiota

Core Tip: Lianhe Xiaozhi ointment (LXO), a multi-herb formulation, profoundly improved experimental metabolic dysfunction-associated steatotic liver disease (MASLD) through a unique mechanism linking gut microbiota modulation to peroxisome proliferator-activated receptor alpha (PPARα) activation in the liver. This letter commends the study’s comprehensive strategy (network pharmacology, omics, in vivo or in vitro validation) and positions the findings in the context of emerging MASLD therapies. By comparing LXO’s outcomes to prior interventions and proposing next-step studies (e.g., Ppara knockout validation, metabolite profiling, brain inflammation assays), we underscore LXO as a concept-proof therapy that opens new avenues via the gut-PPARα-liver axis for metabolic liver disease.

TO THE EDITOR

Metabolic dysfunction-associated steatotic liver disease (MASLD) is now recognized as the leading cause of chronic liver disease worldwide, affecting roughly one-third of adults and projected to afflict over half the global population by 2040[1]. This surge, driven by the obesity and type 2 diabetes pandemic, underscores an urgent need for effective treatments[1]. MASLD [previously non-alcoholic fatty liver disease (NAFLD)] encompasses a spectrum from simple steatosis to steatohepatitis [metabolic dysfunction-associated steatohepatitis (MASH)] and cirrhosis, and is tightly linked to the metabolic syndrome. However, its pathogenesis is multifactorial and incompletely understood, involving complex crosstalk among hepatic lipid metabolism, chronic inflammation, and extrahepatic systems[2]. Notably, the gut-liver axis has emerged as a key player: Dysbiotic intestinal microbiota and a leaky gut can drive hepatic fat accumulation and inflammation via endotoxins and altered metabolites[2]. Likewise, MASLD’s impact is not confined to the liver-systemic and neuroimmune effects which are increasingly appreciated. Heightened chronic inflammation in MASLD is implicated in distant organ damage, including promoting neuroinflammation and cognitive decline[3]. Despite this systemic reach, no pharmacotherapy has yet been approved specifically for MASLD/MASH, making it a critical frontier in hepatology.

Within this context, Nie et al’s study[4] published in World Journal of Gastroenterology is timely and important. They investigated Lianhe Xiaozhi ointment (LXO), a novel traditional Chinese medicine (TCM)-based therapy, as a multi-target intervention for MASLD. Traditional herbal formulations like LXO, which is derived from a classic decoction (Huanglian Wendan) and has accumulated empirical success in metabolic disorders, offer an intriguing therapeutic avenue. Yet scientific validation of their active components and mechanisms is essential for wider acceptance. Nie et al[4] addressed this by integrating ultra-high-performance liquid chromatography-tandem mass spectrometry, network pharmacology (a systems-level, database- and computation-assisted approach that links herbal constituents to putative molecular targets and pathways), hepatic transcriptomics, and in vivo and in vitro models. The result is a rich dataset linking LXO to specific molecular pathways-notably peroxisome proliferator-activated receptor alpha (PPARα) activation-that could explain its MASLD benefits. In the sections below, we elaborate on the key insights and strengths of their findings, relate them to prior high-impact studies, and discuss how this work propels the field forward.

Key insights and methodological strengths

Nie et al[4] present a comprehensive, multi-platform analysis of LXO in MASLD, which is commendable for its depth and rigor. First, through phytochemical profiling and network pharmacology, they identified six pivotal bioactive compounds in LXO-including flavonoids (hesperetin, quercetin-3-O-glucuronide) and alkaloids (palmatine, armepavine, coclaurine, arvenin I) that likely mediate its effects. Notably, these compounds showed high predicted affinity for PPARα in docking analyses, immediately suggesting a unifying mechanism. The authors then demonstrated in MASLD mice that LXO treatment produced multi-faceted benefits: It retarded weight gain, improved insulin resistance (lowered fasting glucose), corrected dyslipidemia (reduced triglycerides), and lowered liver enzymes, indicating reduced steatotic liver injury. Histologically, LXO alleviated hepatic steatosis and inflammation, with treated mice showing markedly less fat deposition and inflammatory cytokine expression than untreated high-fat diet controls. These outcomes address core facets of MASLD’s clinical phenotype and underscore LXO’s potential as a holistic therapy.

Crucially, Nie et al[4] probed mechanistic pathways underlying these benefits. Unbiased transcriptomic profiling highlighted PPAR signaling as a top pathway upregulated by LXO. Zooming in, they confirmed that LXO robustly activated PPARα in the liver, increasing hepatic PPARα protein abundance together with Ppara mRNA expression, alongside canonical Cpt1a, Acox1, and Hmgcs2 transcripts that promote fatty-acid β-oxidation and ketogenesis. Consistently, LXO-treated livers showed increased β-hydroxybutyrate (ketone) levels and reduced triglyceride content, reflecting a metabolic shift toward fat catabolism. Importantly, LXO also blunted activation of NF-κB inflammatory signaling and lowered hepatic interleukin (IL)-1β and IL-18 cytokines-an effect likely linked to PPARα’s known anti-inflammatory function via NF-κB transrepression. The study’s in vitro arm further strengthens causality: LXO treatment in fatty acid-loaded hepatocytes reduced intracellular lipid accumulation comparable to fenofibrate (a known PPARα agonist), whereas co-treatment with a PPARα antagonist (GW6471) markedly attenuated LXO’s lipid-lowering and gene-induction effects. This pharmacological validation firmly implicates PPARα activation as a necessary mechanism for LXO’s action.

Another innovative aspect is the link established between LXO, gut microbiota, and PPARα. The authors found that LXO partially normalized gut microbiome dysbiosis caused by a high-fat diet, for instance restoring a healthier Bacteroidetes/Firmicutes ratio and increasing probiotic genera like Akkermansia and Butyricicoccus while suppressing potentially harmful taxa. Strikingly, one bacterium enriched by LXO (Parabacteroides distasonis) is known to produce fatty acids that activate PPARα. Correspondingly, LXO-treated mice showed higher hepatic levels of endogenous PPARα ligands (e.g., docosahexaenoic acid, linolenic acid). These findings propose a compelling feed-forward loop: LXO alters gut flora to favor PPARα-activating metabolites, which then augment hepatic PPARα signaling a multi-system mechanism rarely demonstrated so clearly in prior MASLD studies. The investigators encapsulate this concept in their proposed model[5]. Their integrated approach, spanning chemical characterization, omics, microbiome analysis, and functional tests, exemplifies a methodological strength, lending credibility to LXO’s therapeutic effects and mechanistic claims.

Comparison with prior literature

Nie et al’s findings[4] resonate with and extend prior knowledge in several ways. Notably, PPARα activation emerges as a common denominator. PPARα has long been recognized as a central regulator of hepatic lipid homeostasis and inflammation. Patients with non-alcoholic steatohepatitis (NASH) have reduced hepatic PPARα expression correlated with more severe insulin resistance and histologic activity, whereas interventions that ameliorate steatohepatitis (weight loss, bariatric surgery, etc.) tend to restore PPARα levels. Consistently, Ppara-deficient mice/models are predisposed to worse diet-induced fatty liver, underscoring PPARα’s protective role. Therapeutically, synthetic PPARα agonists such as fibrates have shown lipid-lowering and anti-steatotic effects. For example, fenofibrate improved hepatic steatosis in preclinical NASH by enhancing autophagic lipid clearance (lipophagy)[6]. However, fibrates alone have yielded only modest histological benefit in clinical NASH trials, possibly because MASLD’s multifactorial nature demands multi-target approaches. Here, LXO’s advantage is its multi-component synergy: It not only activates PPARα akin to a fibrate, but also modulates parallel pathways (gut microbiome, inflammatory signaling) that single agents may not address. Intriguingly, a recent study identified hyodeoxycholic acid (HDCA), a bile acid derivative, as an endogenous modulator of PPARα: HDCA supplementation ameliorated NAFLD in mice by facilitating PPARα nuclear localization, an effect abolished in Ppara knockout models[7]. This report highlights the therapeutic potential of targeting PPARα “from the inside” via metabolic intermediates. LXO appears to achieve a similar end through herbal metabolites and gut microbiota changes, positioning it at the nexus of Western (nuclear receptor) and Eastern (holistic gut-organ) paradigms.

Another area of comparison is the gut microbiome’s role in MASLD interventions. Growing evidence links dysbiosis to NAFLD progression by promoting endotoxemia and hepatic inflammation. Approaches like probiotics, prebiotics, and fecal microbiota transplant have been explored to restore gut-liver homeostasis in NAFLD[2]. Notably, a recent study showed that HDCA ameliorated NAFLD by facilitating hepatic PPARα nuclear localization, reinforcing the therapeutic potential of targeting endogenous PPARα modulators[7]. In the realm of herbal medicine, other TCM formulations have also exhibited this dual gut-liver modulation. For instance, Qushi Huayu decoction improved NAFLD outcomes in rodents by regulating the gut microbiome and activating nuclear receptors (like PXR) in the liver[8]. More importantly, a recent multicenter randomized trial in humans demonstrated that Qushi Huayu significantly reduced liver fat and alanine aminotransferase (ALT) levels in NAFLD patients, outperforming a comparator medicine, while also correcting gut microbiota imbalances[9]. This high-quality trial validates the real-world potential of TCM formulas. The LXO study by Nie et al[4] complements these findings: It provides a mechanistic blueprint (via PPARα and specific fatty acids) for how herbal interventions can confer metabolic and anti-inflammatory benefits. LXO’s effect on raising beneficial microbes like Akkermansia and Butyricicoccus (producers of short-chain fatty acids) and lowering pathogenic genera is particularly noteworthy. These microbial shifts mirror those seen with healthy lifestyle changes and certain pharmaceuticals (e.g., GLP-1 agonists have been reported to increase Akkermansia as well).

In terms of metabolic outcomes, LXO’s broad efficacy aligns with the multi-system improvements observed with aggressive interventions like weight loss and pan-PPAR agonists. Weight reduction through diet or bariatric surgery remains the most effective treatment for MASLD, as it simultaneously improves glycemic control, liver fat, and inflammation (often with PPARα upregulation as a downstream effect). Pharmacologically, a new class of pan-PPAR agonist (e.g., lanifibranor, targeting α/δ/γ) has shown promise in NASH by tackling dyslipidemia, insulin resistance, and inflammation together[10]. MASLD and type 2 diabetes are “two sides of the same coin,” suggesting that multi-faceted agents will be needed to address the intertwined metabolic and hepatic derangements. LXO functions as a “poly-pill” in this sense its components likely engage PPARα (fat oxidation), PPARδ (perhaps influencing cholesterol and inflammation[11]), and even PPARγ pathways indirectly (through improved adipose function or adipokines), although PPARγ was not the focus in the paper. Furthermore, LXO’s demonstrated improvements in ALT/aspartate aminotransferase (AST) and cytokines echo those reported with newer agents like obeticholic acid (an FXR agonist) or semaglutide (GLP-1 RA) in trials, therapies which mainly target metabolic and inflammatory pathways. While direct comparisons are difficult, LXO’s ability to favorably modulate multiple MASLD hallmarks places it in line with modern combination approaches.

Limitations and constructive critique

While the work of Nie et al[4] significantly advances our understanding of LXO, certain limitations temper the interpretation and offer directions for further validation. First, the evidence for PPARα’s role, although strongly supported by antagonist experiments, would be more definitive with genetic loss-of-function models. The authors acknowledge the absence of experiments in Ppara knockout mice. Demonstrating that LXO fails to ameliorate MASLD in hepatocyte-specific Ppara knockout mice (or conversely, that overexpression of Ppara enhances LXO’s effects) would cement the causal link between LXO and PPARα activation. This is especially important given PPARα’s broad involvement in metabolism—confirming LXO’s dependence on this pathway guards against the possibility of off-target effects driving the benefits. Second, the study’s insightful network pharmacology and docking results remain putative; the binding of LXO’s compounds to PPARα was predicted in silico but not directly measured. Follow-up biophysical assays (e.g., surface plasmon resonance or PPARα transactivation reporter assays) could rank the most potent LXO constituents and guide semi-synthesis of optimized analogues.

Third, the gut microbiota findings, while intriguing, are descriptive in nature. Changes in microbiome composition were catalogued, but the study did not assess functional metabolites (such as quantifying short-chain fatty acids, bile acids, or other microbial products in serum). As the authors note, these limit understanding of how microbiota alterations translated to host metabolic effects. Future metabolomic profiling and perhaps fecal microbiota transfer experiments (transplanting gut flora from LXO-treated mice to germ-free or MASLD mice) could clarify the gut-mediated contribution to LXO’s efficacy. Fourth, an important translational gap is the lack of serum pharmacochemistry, i.e., systematic identification of absorbable prototype constituents and their circulating metabolites after dosing, to define in vivo exposure and plausible bioactive entities. Herbal formulations undergo extensive biotransformation; thus, the true bioactive species at the target (liver) might differ from the original constituents. A “TCM modern research” approach integrating serum metabolite profiling (as in emerging network pharmacology + serum pharmacochemistry strategies) would strengthen confidence that the identified compounds (hesperetin, palmatine, etc.) are relevant in vivo and could serve as quality control markers or leads for drug development.

Finally, it must be emphasized that all data in Nie et al’s study[4] are preclinical-derived from mice and hepatocyte models. While these models were well-chosen (diet-induced MASLD mice mimic human metabolic syndrome), rodent results do not always translate to humans. The lack of any clinical or human tissue validation is a cautionary point. For instance, the doses of LXO used in mice and their human equivalent dose need consideration for safety and feasibility. We encourage the authors and others to pursue early-phase clinical trials, perhaps starting with MASLD patients with metabolic syndrome, to test LXO’s efficacy and tolerability in humans. Learning from prior NAFLD trials, suitable noninvasive endpoints [magnetic resonance imaging-derived proton density fat fraction (MRI-PDFF) for liver fat, serum ALT, enhanced liver fibrosis (ELF)/FibroScan for fibrosis, etc.] could be employed. In summary, these limitations—absence of genetic causality proof, limited functional microbiome analysis, unknown pharmacokinetics, and no human data—are not faults per se (many are beyond the scope of an initial study) but are opportunities to elevate LXO research from an exciting finding to a validated therapeutic approach.

Mechanistic expansion: Gut-liver-brain axis and neuroimmune links

One particularly forward-looking aspect that warrants discussion is the possible involvement of the gut-liver-brain axis in LXO’s effects. Nie et al[4] understandably focused on liver outcomes, but given MASLD’s systemic nature, it is intriguing to hypothesize how LXO’s metabolic and anti-inflammatory benefits might extend beyond the liver. Chronic MASLD is associated not only with cardiovascular complications but also with cognitive impairment and neuroinflammation, driven by peripheral inflammation and perhaps gut-derived signals crossing the blood-brain barrier[3,12]. The improvement in pro-inflammatory cytokines (IL-1β, IL-18) seen with LXO could conceivably ameliorate the chronic low-grade inflammation affecting the brain. Furthermore, Akkermansia (significantly increased by LXO) has been linked to improved gut barrier integrity and reduced endotoxemia, which might lower the influx of inflammatory mediators to both liver and brain[12].

We propose exploring whether LXO can favorably impact neuroinflammatory markers or behavior in MASLD models. For example, in mice with MASLD, tests of cognitive function (novel object recognition, maze tests) and measurement of microglial activation in the hippocampus could be conducted with/without LXO treatment. Such experiments would inform whether LXO’s impact is confined to metabolic organs or if it holistically improves the “brain fog” and fatigue often reported in NAFLD patients. Mechanistically, this dovetails with the emerging concept that treating liver disease may relieve associated neurological burden-essentially targeting a liver-brain inflammatory reflex. If LXO shows benefits in this realm, it would position the therapy not just as liver-centric, but as a modulator of neuroimmune crosstalk—a truly integrative outcome aligning with TCM’s whole-body perspective. Additionally, certain herbs in LXO (e.g., Coptis chinensis containing berberine, or Poria cocos) have reported anxiolytic or anti-neuroinflammatory properties in other contexts, which could contribute to such effects. While speculative, raising these possibilities broadens the horizon of MASLD therapy to encompass multi-organ health, something future studies of LXO could capitalize on.

Future research directions

Building on Nie et al’s foundation[4], we outline several concrete avenues for future LXO research.

PPARα pathway validation: As noted, using hepatocyte-specific Ppara knockout models will be critical. If LXO fails to improve MASLD in Ppara-deficient mice, this would provide definitive evidence that Ppara is indispensable for the proposed mechanism. Conversely, testing LXO alongside a known PPARα agonist (e.g., fibrate) for additive or synergistic effects could reveal if LXO has PPARα-independent benefits (e.g., additional PPARδ/γ or other pathway activation).

Active compound isolation and pharmacokinetics: Guided by the six key compounds identified, researchers should isolate or synthesize these constituents to test them individually and in combination. Determining which compounds (or their metabolites) reach effective concentrations in plasma and liver is vital. A serum pharmacochemistry workflow—profiling circulating LXO-derived prototypes and metabolites after administration—can clarify the truly bioavailable ingredients. For instance, quercetin-3-O-glucuronide might be extensively metabolized; its presence or absence in plasma will tell us if it directly drives PPARα activation or if its downstream metabolites do. Such studies also facilitate standardization of LXO (ensuring batch-to-batch consistency based on active markers) for clinical use.

Expanded multi-omics (metabolomics & proteomics): Integrating untargeted metabolomics in both the serum and liver of LXO-treated vs control MASLD models could identify pathways not initially considered. This may reveal changes in bile acid profiles, amino acid derivatives, or eicosanoids that contribute to LXO’s effects. Similarly, liver proteomics could complement transcriptomics to ensure key pathway proteins (not just mRNAs) are modulated. Multi-omics correlation with gut microbiome data (i.e., metabolite changes linked to specific microbiota shifts) would strengthen the gut-liver mechanism hypothesis.

Gut microbiome causality tests: To directly test the contribution of the microbiome, experiments using antibiotic-treated (germ-depleted) mice or germ-free mice colonized with MASLD microbiota could be employed. If LXO loses efficacy when gut microbes are wiped out, that underscores microbiota mediation. Alternatively, transferring fecal microbiota from LXO-treated mice to MASLD mice (fecal transplant) might partially reproduce the metabolic benefits, indicating microbial metabolites carry some of LXO’s therapeutic signal. Metagenomic analysis of the microbial genes/pathways upregulated by LXO would also identify which metabolic functions (e.g., short-chain fatty acid synthesis, bile acid transformation) are altered.

Neuroinflammatory and systemic endpoints: As discussed, future studies should include endpoints beyond the liver. Measuring circulating inflammatory markers (IL-6, tumor necrosis factor-alpha, etc.), gut barrier integrity (plasma lipopolysaccharide levels, intestinal tight junction protein expression), and markers of brain inflammation (microglial markers or neurotrophic factors in brain tissue) in LXO-treated MASLD models would determine if LXO confers systemic anti-inflammatory benefits. If feasible, behavioral assays for cognitive function or mood in rodent models could add a translational perspective, given NAFLD’s association with reduced quality of life and depression.

Early-phase clinical translation and mechanism-linked endpoints: Before large-scale trials, standardized manufacturing and safety pharmacology of LXO should be established, including batch-to-batch quality control and acute/chronic toxicity profiling. To make the translational pathway more concrete, we suggest an early-phase, randomized, double-blind, placebo-controlled study in adults with imaging-defined MASLD (e.g., elevated MRI-PDFF) and concomitant metabolic risk (overweight/obesity and/or insulin resistance), while excluding significant alcohol intake, viral hepatitis, decompensated cirrhosis, and major confounders of lipid metabolism. A 12-16-week proof-of-concept window would allow assessment of both efficacy and target engagement. Key efficacy endpoints may include change in MRI-PDFF (primary), serum ALT/AST and noninvasive fibrosis readouts (e.g., transient elastography and ELF; secondary), alongside metabolic indices (homeostasis model assessment of insulin resistance, triglycerides). Importantly, mechanism-aligned pharmacodynamic measures could be incorporated, such as circulating β-hydroxybutyrate and hepatic lipid-oxidation signatures as surrogates of PPARα activation, coupled with plasma fatty acid/bile acid profiling and longitudinal gut microbiome readouts to test the proposed microbiota-metabolite-PPARα axis. Exploratory outcomes may include patient-reported fatigue/cognitive complaints or brief neurocognitive testing to preliminarily evaluate the gut-liver-brain hypothesis without overextending trial complexity.

Taken together, these future directions aim to fill the gaps in mechanistic understanding and bridge the preclinical-clinical divide. They also represent hypothesis-generating experiments that could reveal whether LXO’s benefits extend to related metabolic diseases (e.g., obesity, type 2 diabetes, or even ALD-alcoholic liver disease, where gut microbiota and PPARα are also relevant).

CONCLUSION

Nie et al’s study[4] on LXO represents a meaningful step at the interface of traditional medicine and contemporary hepatology. By linking LXO to PPARα activation—potentially reinforced by gut microbiota-derived metabolites—the work provides a plausible mechanistic rationale for its multi-pronged benefits in experimental MASLD. Beyond reinforcing LXO’s therapeutic promise, our letter offers a distinct conceptual advance: We propose that evaluating multi-herb therapies for systemic metabolic diseases should move from a liver-centric, single-pathway view toward a gut-liver-brain axis framework that captures inter-organ immunometabolic crosstalk. Framed in this way, LXO is not merely a candidate that improves hepatic steatosis, but a test case for a systems-level evaluation strategy in which herbal interventions are assessed by: (1) A mechanistic chain that connects microbiota remodeling to host ligands and nuclear-receptor signaling (e.g., microbiota - metabolites - PPARα); and (2) Downstream systemic consequences that extend to gut barrier function and neuroimmune phenotypes. This paradigm shift is particularly relevant for MASLD, where chronic low-grade inflammation and metabolite signaling link the liver to extrahepatic complications, including cognitive and neuroinflammatory features. In closing, we commend Nie et al[4] for an elegant and thorough investigation that not only substantiates LXO’s benefits but also stimulates new questions. It positions LXO as a field-advancing prototype: A blend of ancient wisdom and modern evidence, urging us to think beyond single-target drugs toward integrated solutions for complex diseases like MASLD. As research builds on these findings—validating mechanisms, ensuring safety, and testing efficacy in humans—we may witness LXO (or derivatives thereof) evolving from bench to bedside. Such a development would embody the concept of “East meets West” in medicine, ultimately benefiting the millions of individuals worldwide at risk of steatotic liver disease.

ACKNOWLEDGEMENTS

During the preparation of this work, the authors used ChatGPT 5 in order to correct grammatical errors and improve the language and readability of the manuscript.