Published online Oct 7, 2026. doi: 10.3748/wjg.121244
Revised: April 25, 2026
Accepted: June 30, 2026
Published online: October 7, 2026
Processing time: 165 Days and 17.1 Hours
Acute-on-chronic liver failure (ACLF) is a life-threatening condition characterized by acute hepatic decompensation and high short-term mortality. Although Wenyang Jiedu Huayu Formula (WYJD) has demonstrated therapeutic efficacy for alleviating liver injury and modulating immune inflammation, whether its hepatoprotective effects involve targeting succinate metabolism remains unclear.
To investigate whether WYJD exerts therapeutic effects in ACLF by targeting succinate metabolism and to elucidate the underlying molecular mechanisms.
ACLF rat model was prepared by sensitization using bovine serum albumin, followed by D-galactosamine/lipopolysaccharide challenge. Animals were assigned to normal control, Model, WYJD, and WYJD plus succinate dehydrogenase (SDH) inhibitor groups. Therapeutic efficacy was evaluated using hepatic histopathology and inflammatory cytokine profiling. Succinate metabolism-related molecular changes were systematically analyzed using targeted energy metabolomics, biochemical enzyme activity assays, western blotting, and reverse transcription quantitative polymerase chain reaction. Mitochondrial ultrastructure was examined using transmission electron microscopy. Intrahepatic macrophage infiltration was assessed using immunofluorescence staining.
WYJD treatment markedly attenuated hepatic necrosis and inflammatory cell infiltration in ACLF rat models. Metabolomic analysis revealed succinate accumulation and tricarboxylic acid cycle dysfunction in the ACLF liver tissues, which were effectively reversed by WYJD. Mechanistically, WYJD upregulated SDH activity and ex
WYJD ameliorated ACLF by acting partly through SDH to clear accumulated succinate and suppress hypoxia-inducible factor-1α-mediated inflammation, suggesting succinate metabolism as a potential therapeutic target.
Core Tip: This study demonstrated that Wenyang Jiedu Huayu Formula (WYJD) ameliorated acute-on-chronic liver failure by targeting succinate metabolism. WYJD acts partly through succinate dehydrogenase to clear accumulated succinate and suppress hypoxia-inducible factor-1α signaling, thereby correcting energy metabolic disturbances and attenuating macrophage-mediated inflammation. These findings suggested that succinate metabolism can be a potential therapeutic target in acute-on-chronic liver failure, and the study provided mechanistic insights supporting the clinical application of WYJD from a metabolic-immune perspective.
- Citation: Zhou J, Tian XL, Zhou YF, Zhou ZY, Tan NH, Chen B. Wenyang Jiedu Huayu Formula targets succinate metabolism to alleviate acute-on-chronic liver failure. World J Gastroenterol 2026; 32(37): 121244
- URL: https://www.wjgnet.com/1007-9327/full/v32/i37/121244.htm
- DOI: https://dx.doi.org/10.3748/wjg.121244
Acute-on-chronic liver failure (ACLF) is a severe clinical syndrome characterized by acute hepatic decompensation superimposed on chronic liver disease, and presents with high short-term mortality and poor prognosis[1,2]. Liver transplantation is the definitive treatment, but its clinical application is limited by donor shortage and high costs[1]. Therefore, elucidating the pathogenesis of ACLF and exploring safe and effective medical treatment strategies is of significant clinical importance.
The pathological mechanisms underlying ACLF involve intense systemic inflammation and immune-metabolic dysregulation[3,4]. To meet the immense energy demands of uncontrolled inflammation and immune cell activation, the body undergoes metabolic reprogramming, manifested as mitochondrial dysfunction, disruption of the tricarboxylic acid (TCA) cycle, and inhibition of oxidative phosphorylation (OXPHOS)[5-7]. This metabolic imbalance not only directly causes hepatocyte death and organ failure, but also reciprocally regulates immune cell activation and inflammatory responses by altering key metabolite levels, forming a detrimental metabolic-immunity cycle that accelerates disease progression[7,8]. Recent studies have identified succinate, an intermediate metabolite of TCA cycle, to be an important metabolic hub linking cellular metabolic status to inflammatory signaling[9,10]. Under stressful conditions such as hypoxia or inflammation, succinate dehydrogenase (SDH) activity is suppressed leading to aberrant succinate accumulation[9]. Accumulated succinate stabilizes hypoxia-inducible factor-1α (HIF-1α) by inhibiting prolyl hydroxylase activity, thereby driving glycolysis and promoting the expression of pro-inflammatory genes[11]. Therefore, targeting succinate metabolism to restore homeostasis may represent a novel strategy for ameliorating the metabolic disturbances and excessive inflammatory responses in ACLF.
Extensive clinical experience has been accumulated in traditional Chinese medicine for the prevention and treatment of liver failure. Based on the intertwined pathogenesis of ACLF characterized by “blood stasis”, “toxicity”, and “deficiency” in Chinese medicine theory, our research group established “resolving stasis, reinforcing deficiency, and promoting regeneration” as the core therapeutic principle, and accordingly developed the Wenyang Jiedu Huayu Formula (WYJD). Among the six herbal components, Chishao and Fuzi constituted the core herb pair and served as sovereign drugs for WYJD (Table 1). The long-term clinical application of WYJD in patients with ACLF has demonstrated favorable therapeutic efficacy, effectively reducing mortality rates, and the formula has been incorporated into relevant clinical guidelines[12,13].
| Chinese name | Scientific name | Common name | Weight (g) | Part used |
| Fu Pian | Aconitum carmichaelii Debeaux | Processed aconite lateral root | 10 | Processed lateral root |
| Bai Zhu | Atractylodes macrocephala Koidz | Largehead atractylodes rhizome | 30 | Rhizome |
| Yin Chen | Artemisia capillaris Thunb | Capillary wormwood herb | 30 | Aerial part |
| Dan Shen | Salvia miltiorrhiza Bunge | Salvia root | 30 | Root and rhizome |
| Chi Shao | Paeonia lactiflora Pall | Red peony root | 60 | Root |
| Yi Yi Ren | Coix lacryma-jobi L. var. ma-yuen (Roman.) Stapf | Coix seed | 30 | Seed |
Our previous studies on this core herb pair demonstrated that Chishao-Fuzi downregulates HIF-1α, inhibits glycolysis, and promotes macrophage polarization toward the anti-inflammatory M2 phenotype, thereby mitigating immune-inflammatory injury in ACLF[14,15]. These findings suggested that the active components within WYJD may similarly act upstream of HIF-1α regulation, with succinate as a key metabolic signaling molecule critical for HIF-1α stabilization serving as an important node connecting energy metabolism and immune inflammation. Based on this premise, the present study investigated whether WYJD ameliorated energy metabolic disorders and immune-inflammatory injury in ACLF by SDH activation, clearance of aberrantly accumulated succinate, and subsequent inhibition of HIF-1α signaling pathway from the perspective of succinate metabolic regulation.
Male Sprague-Dawley rats (specific pathogen-free, 110-150 g) were supplied by Hunan SJA Laboratory Animal Co., Ltd. Animals were kept under standardized conditions (22-26 °C, 50%-70% humidity, 12-hour light/dark cycle) with unrestricted access to food and water. All procedures were approved by the Ethics Committee of the First Affiliated Hospital of Hunan University of Chinese Medicine, No. ZYFY202503313.
Bovine serum albumin (BSA, AWB0157a) was obtained from Abiowell. Freund’s incomplete adjuvant (F5506), D-galactosamine (G0750), lipopolysaccharide (L2630), and dimethyl malonate (DMM, 136441) were purchased from Sigma-Aldrich.
WYJD consisted of six Chinese herbal medicines (Table 1) sourced from the First Affiliated Hospital of Hunan University of Chinese Medicine. The herbs were weighed according to the clinical prescription, soaked in distilled water for 30 minutes, and made into a decoction. Fu Pian was first decocted for 1 hour, after which the remaining five herbs were added, and the mixture was boiled for an additional 40 minutes. The decoction was filtered and the residue was re-boiled with distilled water for 30 minutes. The two filtrates were combined and concentrated to a final concentration of 1.71 g crude drug/mL for subsequent animal experiments. Chemical profiling was done using ultra-performance liquid chromatography-tandem mass spectrometry, as described previously[16].
The ACLF rat model was established as described previously (Figure 1)[14]. The rats were sensitized by subcutaneous multisite injections of BSA emulsion (0.5 mL containing 4 mg BSA) on days 1, 14, 24, and 34. From day 35, immunological challenge was performed via tail vein injections of BSA solution twice weekly for six consecutive weeks, with doses progressively increasing from 2 mg to 4 mg in 0.5 mg increments. On the day following the final tail vein injection, acute liver injury was induced by intraperitoneal administration of D-galactosamine (400 mg/kg) and lipopolysaccharide (100 μg/kg). Twelve hours later, the rats were anesthetized with 3% sodium pentobarbital (2 mL/kg, i.p.) and blood samples were collected from the abdominal aorta and liver tissues for subsequent analyses.
The rats were initially allocated to either a normal control group (NC, n = 6) or a model induction group (n = 34). During the induction period, 10 rats died. The remaining rats were assigned to the Model, WYJD, or WYIN (WYJD plus DMM) groups. Rats in the WYJD group received daily intragastric administration of WYJD decoction (17.1 g/kg), whereas those in the WYIN group received additional intraperitoneal injections of DMM (160 mg/kg)[17]. Equal volumes of saline were administered to the NC and Model groups. All interventions were continued until the acute challenge, and the final number of surviving rats in each group was used for analysis.
Fixed in 40 g/L formaldehyde, paraffin-embedded liver sections (5 μm) were stained with hematoxylin and eosin. The sections were examined and photographed under a light microscope.
Liver tissue sections were blocked with 5% BSA and incubated overnight at 4 °C with primary antibodies against CD68 (1:200, Servicebio, GB113109, China) and Ki67 (1:200, Abiowell, AWA10320, China). After washing, the sections were incubated with fluorophore-conjugated secondary antibodies (1:500) at 37 °C for 1 hour, counterstained with DAPI, and imaged using a fluorescence microscope (BA410T, Motic, China).
Liver specimens (1 mm³) were fixed in 2.5 g/L glutaraldehyde, post-fixed in 10 g/L osmium tetroxide, dehydrated, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined under a transmission electron microscope (JEM-1400FLASH, JEOL, Japan). Mitochondrial damage was semi-quantitatively assessed using the Flameng scoring system[18]. For each group, 20-30 randomly selected mitochondria from five randomly chosen fields per section were scored by two independent observers who were blinded to group allocation. The scoring criteria are listed in Table 2. The average score for each group was calculated and presented as mean ± SD.
| Score | Description |
| 0 | Normal mitochondrial morphology |
| 1 | Mild swelling, decreased matrix density, and separated cristae |
| 2 | Moderate swelling, translucent matrix, and intact cristae |
| 3 | Severe swelling, condensed matrix, and ruptured cristae |
| 4 | Severe swelling with ruptured cristae, complete loss of inner and outer membranes, and vacuolation |
Serum and liver homogenate levels of interleukin-1β (IL-1β, YJ037361), tumor necrosis factor-α (YJ002859), and IL-10 (YJ002813) were quantified using ELISA kits (Shanghai Enzyme-Linked Biotechnology, China) according to the manufacturer’s instructions.
Total RNA was isolated from the liver tissues using a TRIzol reagent, and complementary DNA was synthesized using a reverse transcription kit (CW2569, CWBIO, China). Quantitative polymerase chain reaction was performed on a PIKOREAL96 system (Thermo, United States) with SYBR Green Master Mix. The primer sequences are listed in Table 3. Relative expression was calculated by the 2-ΔΔCt method with β-actin as reference.
| Gene | Forward sequence (5′-3′) | Reverse sequence (5′-3′) | Size (bp) |
| SDHA | CTGTTGCCAAGGACCTAGCAT | AGCCTCTTCCTTCACGGAT | 70 |
| SDHB | CAAAACCTTCGCCATTTACCG | ATTAGAGCATCCAGCACCATCG | 32 |
| HIF-1α | ACGATTGTGAAGTTAATGCTCCC | AACCAACAGAAACGAAACCCC | 120 |
| β-actin | ACATCCGTAAAGACCTCTATGCC | TACTCCTGCTTGCTGATCCAC | 42 |
Liver lysates were prepared in RIPA buffer containing protease inhibitors, and protein concentrations were measured using bicinchoninic acid assay. Equal amounts of proteins were separated by sodium-dodecyl sulfate gel electrophoresis and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% skim milk and incubated overnight at 4 °C with primary antibodies against SDHA (1:1000, Proteintech, 14865-1-AP, China), SDHB (1:1000, Proteintech, 10620-1-AP, China), HIF-1α (1:1000, Abiowell, AWA13590, China), and β-actin (1:5000, Proteintech, 66009-1-Ig, China), then with HRP-conjugated secondary antibodies (1:5000). The bands were visualized using enhanced chemiluminescence and quantified using ImageJ.
Liver samples (50 mg) were homogenized in cold methanol, extracted with 80% methanol, incubated at -20 °C, and centrifuged. The supernatants were dried, reconstituted in methanol-water (1:1), and analyzed by liquid chromatography-tandem mass spectrometry (Shimadzu Nexera X2 LC-30AD, Japan; AB SCIEX QTRAP 6500+, United States) using a Kinetex F5 column (2.6 μm, 3.0 mm × 100 mm). Mobile phases were 10 mmol/L ammonium acetate (A) and acetonitrile (B) with gradient elution: 0-3 minutes 0% B, 3-10 minutes 0%-95% B, 10-12 minutes 95% B, 12-13 minutes 95%-0% B, 13-14 minutes 0% B. Electrospray ionization source parameters: Ion spray voltage ±5500/-4500 V, temperature 500 °C, gas1 55 psi, gas2 60 psi, curtain gas 35 psi. Multiple reaction monitoring mode was used to quantify TCA cycle intermediates.
Hepatic SDH activity (A022-1-1, Nanjing Jiancheng, China), ATP content (A095-1-1, Nanjing Jiancheng, China), NAD+/NADH ratio (S0176S, Beyotime, China), and succinate levels (E-BC-K902-M, Elabscience, China) were measured using commercial kits following standard protocols.
Data are presented as mean ± SD. GraphPad Prism 10 was used for statistical analysis and graphing. Normality was tested using Shapiro-Wilk and Q-Q plots. For multiple comparisons, one-way ANOVA with Tukey’s post-hoc test (normal distribution, equal variance) or Kruskal-Wallis with Dunn’s test (non-normal/unequal variance) was applied. P < 0.05 was considered significant.
To assess the therapeutic effect of WYJD on ACLF, we evaluated histopathological changes in liver tissues and systemic inflammatory markers. Hematoxylin and eosin staining revealed that compared with the NC group, the Model group exhibited disrupted hepatic architecture characterized by extensive hepatocyte necrosis, ballooning degeneration, and massive inflammatory cell infiltration. WYJD treatment significantly reduced necrosis and inflammatory infiltration, thereby improving liver tissue structure. The improvement in liver pathology was less pronounced in the WYIN group (WYJD combined with the SDH inhibitor DMM) than in the WYJD group (Figure 2A).
ELISA analysis showed that serum and hepatic levels of tumor necrosis factor-α and IL-1β were markedly elevated in the Model group, whereas IL-10 decreased. WYJD treatment significantly reversed these changes and restored the balance between pro- and anti-inflammatory mediators (Figure 2B and C). Co-administration with DMM partially abrogated the effects of WYJD on cytokine levels. These results indicated that WYJD alleviated liver injury and systemic inflammation in ACLF, potentially by modulating SDH activity.
Targeted metabolomics showed succinate accumulation and TCA cycle disturbances: To elucidate the metabolic basis of the action of WYJD in ACLF, we performed targeted energy metabolomics of liver tissues. Orthogonal partial least squares discriminant analysis score plot and permutation test results revealed a clear separation in the metabolic profiles among the four groups indicating no overfitting and confirming that ACLF modeling and subsequent drug intervention profoundly reshaped the hepatic metabolic landscape (Figure 3A and B). Heatmap analysis revealed a distinct clustering of key energy metabolites among the four groups (Figure 3C). Succinate levels were markedly elevated in the model group, indicating an aberrant accumulation under ACLF conditions. WYJD treatment significantly reduced succinate levels, an effect that was attenuated by SDH inhibition in the WYIN group. Other TCA cycle intermediates including fumarate, malate, and acetyl-CoA, decreased in the Model group and were restored by WYJD, and decreased in the WYIN group (Figure 3D). Quantitative analysis confirmed that succinate content was significantly increased and fumarate content was decreased in the Model group compared with the NC group (Figure 3E) suggesting metabolic blockade at the SDH-catalyzed step. WYJD treatment normalized these changes and this effect was abrogated by SDH inhibition. An independent biochemical assay for succinate content in liver homogenates corroborated these findings (Figure 3F). Notably, although the absolute succinate values differed between the two methods owing to their distinct analytical principles, both methods consistently demonstrated significant succinate accumulation in the Model group which was reversed by WYJD treatment, supporting the robustness of this finding.
Pathway enrichment analysis reveals restoration of TCA cycle and suppression of HIF-1α signaling: Kyoto Ency
WYJD improves cellular energy status and redox homeostasis: To assess the functional consequences of TCA cycle restoration, we measured hepatic energy parameters. Compared with the NC group, the Model group exhibited significantly reduced ATP content and NAD+/NADH ratio along with elevated lactate levels and lactate/pyruvate ratio, indicating enhanced glycolytic compensation. WYJD treatment restored ATP levels and the NAD+/NADH ratio while decreasing lactate levels and lactate/pyruvate ratio (Figure 3H and I). These results demonstrated that WYJD enhanced mitochondrial energy metabolism efficiency by restoring TCA cycle flux, thereby alleviating excessive glycolysis.
Based on metabolomics findings of aberrant succinate accumulation, we further validated SDH/succinate/HIF-1α signaling axis at the molecular level. SDH is a key enzyme involved in the TCA cycle and electron transport chain (ETC), and its functional status directly affects succinate metabolism and cellular energy homeostasis[19]. Given that the SDHA and SDHB subunits constitute the catalytic core of SDH, which is responsible for succinate oxidation and electron transfer[19,20], we focused on detecting their expression and SDH activity. Compared with the NC group, the Model group showed significantly reduced SDH activity in the liver tissue accompanied by downregulated mRNA and protein expression of SDHA and SDHB (Figure 4A-C). WYJD treatment reversed these changes by markedly enhancing SDH activity and upregulating SDHA/B expression. The effects of WYJD on SDH activity and expression were partially abrogated in the WYIN group.
Examination of downstream HIF-1α expression revealed that both protein and mRNA levels were significantly upregulated in the Model group, suppressed by WYJD treatment, and this suppression was reversed in the WYIN group (Figure 4D). These results demonstrated that WYJD promoted succinate clearance by upregulating SDH activity and expression, thereby inhibiting HIF-1α stabilization and expression.
Mitochondria are the primary sites of cellular aerobic respiration and energy conversion, and their structural integrity directly determines the functional efficiency of the TCA cycle and OXPHOS[21]. To validate the effect of WYJD on energy metabolism at the ultrastructural level, we examined mitochondrial morphology in hepatocytes using transmission electron microscopy. Hepatocytes in the NC group displayed normal mitochondrial morphology with distinct, dense cristae, and a uniform matrix. In contrast, mitochondria in the model group exhibited marked swelling, disrupted or absent cristae, and a vacuolated matrix, which are characteristic of severe mitochondrial dysfunction in ACLF. WYJD treatment attenuated mitochondrial swelling, partially restored the cristae structure, and increased matrix density. An improvement in mitochondrial function in the WYIN group was weaker than that in the WYJD group (Figure 5). These ultrastructural findings confirmed that WYJD improved mitochondrial morphology and function, an effect that is closely associated with SDH activity.
Macrophages are key effector cells in the hepatic inflammatory response during ACLF[22]. To clarify the dynamic changes in macrophages in liver tissue, we employed dual immunofluorescence staining with CD68 and Ki67. The results showed that, compared with the NC group, the Model group exhibited a significant increase in CD68+ macrophages with diffuse infiltration in the liver, whereas Ki67-positive signals showed no significant differences between the two groups (Figure 6). These findings indicated that the increased number of hepatic macrophages under ACLF conditions primarily originated from the recruitment of circulating monocytes rather than the local proliferation of Kupffer cells. WYJD intervention markedly reduced CD68+ macrophage infiltration, an effect that was partially reversed in the WYIN group treated with the SDH inhibitor (Figure 5). These results demonstrated that WYJD effectively suppressed macrophage recruitment to the liver in the ACLF model, an action partly dependent on SDH activity.
ACLF is associated with high short-term mortality due to immune-metabolic dysregulation[23]. Omics studies have revealed significant metabolic pathways remodeling and accumulation of key metabolites in patients with ACLF. These metabolic abnormalities directly modulate immune cell activation and inflammatory responses, creating a vicious cycle in which metabolic disorders and immune dysregulation reinforce each other[4,24]. Our previous clinical metabolomic analysis demonstrated that ACLF liver tissues exhibited characteristic metabolic reprogramming associated with aberrant hepatic macrophage activation[8]. The present study extends these findings by indicating that WYJD effectively eliminated abnormally accumulated succinate in the liver by upregulating SDH activity and expression. This is associated with suppression of HIF-1α stabilization and transcriptional activity, along with improvement in mitochondrial function, reversal of metabolic reprogramming, and attenuation of macrophage-mediated immune-inflammatory injury. These findings suggested a therapeutic mechanism for WYJD from a metabolic-immune perspective and provided experimental evidence supporting traditional Chinese medicine principles of resolving stasis, reinforcing deficiency, and promoting regeneration.
Recent studies have identified succinate, an intermediate metabolite of the TCA cycle, as an endogenous signaling molecule that accumulates under inflammatory conditions and directly participates in immune regulation[25,26]. In the ACLF model used in the present study, liver tissues exhibited significantly elevated succinate levels and reduced fumarate content, indicating SDH inhibition and the consequent blockade of succinate metabolism. Accumulated succinate promotes glycolysis and inflammation-related gene expression through HIF-1α stabilization and activates extracellular inflammatory signaling via the cell surface receptor SUCNR1[27-29]. This study systematically delineated a pathological cascade involving aberrant succinate metabolism, HIF-1α activation, pro-inflammatory cytokine release, and energy metabolic disturbances in an ACLF animal model. These findings advance our understanding of ACLF pathogenesis from inflammation to metabolic-immune dysregulation.
Given the pivotal role of succinate metabolism in ACLF progression, targeting this pathway can be a promising the
The present study demonstrated that WYJD specifically upregulated the expression of the catalytic subunits SDHA and SDHB and enhanced SDH enzymatic activity. This promoted conversion of succinate to fumarate, reduced succinate accumulation at its source, restored TCA cycle flux, enhanced OXPHOS efficiency, and attenuated glycolytic com
This study demonstrated that the regulatory effects of WYJD on energy metabolism extended to remodeling of the hepatic immune microenvironment. HIF-1α, a key transcription factor downstream of succinate, regulates diverse cellular responses including glycolysis and inflammation[31]. WYJD significantly downregulated HIF-1α expression while reducing hepatic succinate levels, providing an upstream transcriptional mechanism for its anti-inflammatory effects. Our previous work demonstrated that WYJD inhibits glycolysis via HIF-1α downregulation, thereby promoting macrophage polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype. We further demonstrated that WYJD modulated not only macrophage polarization, but also macrophage recruitment to the liver. WYJD treatment significantly reduced CD68+ macrophage infiltration to the liver in ACLF, which was partially abrogated by SDH inhibition, linking macrophage dynamics to succinate metabolism.
Our previous clinicopathological analysis similarly showed a marked increase in CD68+ cells in liver tissues from patients with ACLF, without a corresponding increase in Ki67 staining, suggesting that accumulated hepatic macrophages are derived primarily from recruited circulating monocytes rather than from local Kupffer cell proliferation[8]. HIF-1α is known to directly upregulate chemokines such as vascular endothelial growth factor and chemokine ligand 12, and adhesion molecules, thereby promoting monocyte and macrophage recruitment[32,33]. Based on our findings WYJD modulated the SDH-succinate-HIF-1α signaling pathway, we proposed that WYJD inhibition of macrophage infiltration occurred via SDH activation, reduction of succinate accumulation, and suppression of HIF-1α transcriptional activity, leading to downregulation of local hepatic chemotactic signals and ultimately preventing pathological recruitment of circulating monocytes. Preliminary data on chemokine expression from our previous studies supports this hypothesis[8]. Thus, this study not only demonstrated the inhibitory effect of WYJD on macrophage infiltration in an ACLF animal model but also provided experimental evidence on its regulation of macrophages recruitment through metabolic-immune crosstalk.
This study had several limitations. A DMM-only control group was not included in this study. However, a recent study demonstrated that DMM at the same dose (160 mg/kg, intraperitoneally) showed no direct hepatotoxicity in vitro or in vivo[17]. Therefore, the partial reversal of the protective effects of WYJD in the WYIN group might not be attributable to the nonspecific toxicity of DMM, although off-target effects cannot be formally excluded. SDH and HIF-1α expression were assessed in whole liver homogenates, which do not discriminate between hepatocyte-specific and macrophage-specific contributions. Whether WYJD primarily targets the SDH in hepatocytes or infiltrating macrophages remains to be determined.
We did not directly assess HIF-1α nuclear translocation or measure its downstream target genes. However, a previous study demonstrated that reduction in HIF-α protein levels correlates with decreased HIF transcriptional activity, as measured by luciferase reporter assays and downstream target gene expression[34]. Therefore, a significant reduction in HIF-1α protein levels observed after WYJD treatment strongly suggests decreased HIF-1α transcriptional activity.
Furthermore, as a multicomponent herbal formula, the identification of the specific active constituents of WYJD responsible for SDH modulation requires further investigation. The chemokine repertoire downstream of HIF-1α and its functional role in monocyte recruitment during ACLF also remains to be fully characterized. Finally, the present findings were obtained in a rat model of ACLF. Although this model recapitulates key features of human ACLF, the translational relevance of targeting succinate metabolism in patients requires further validation using human liver samples, patient-derived organoids, or clinical cohort studies.
This study systematically elucidated the metabolic-immune regulatory mechanism by which WYJD ameliorated ACLF. By acting partly through SDH, clearing aberrantly accumulated succinate, and suppressing HIF-1α signaling, WYJD effectively corrected energy disturbances, thereby attenuating mitochondrial damage and macrophage-mediated inflammatory responses. These findings provided a mechanistic basis for the clinical application of WYJD and suggested a framework for developing metabolic node-targeted therapeutic strategies involving metabolic-immune crosstalk for liver failure.
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