Machine Learning and Experimental Validation Ubiquitination-Related Key Genes in Sepsis: The Perspective of Chinese-Western Pharmaceutical Therapeutic Strategies

Summary

Keywords

Sepsis; Ubiquitination modification; Machine learning; Chinese medicine monomers; Small molecule drugs

Introduction

Sepsis is a life-threatening organ dysfunction syndrome caused by a dysregulated host response to infection. Its core pathological mechanisms include immune imbalance and organ damage. It can occur at any age, with high incidence and mortality rates. Approximately 48.9 million new cases of sepsis and 11 million sepsis-related deaths occur annually, making it a major global public health concern

^1,

^2,

^3,

⁴

. In China, recent surveys have shown that there are 4.8 to 6.1 million hospitalized sepsis cases annually, and the standardized sepsis-related mortality rate is 66.7 per 100,000 people

^5,

⁶

. However, early diagnosis and precise treatment of sepsis still face significant challenges. In recent years, the rise of molecularly targeted drugs has provided a new direction for sepsis treatment. Researchers have developed various molecularly targeted drugs aimed at specific inflammatory mediators or immune pathways

^7,

^8,

^9,

^10,

¹¹

. By precisely intervening in key inflammatory pathways, the immune homeostasis of the body can be restored. However, most of the existing targeted drugs focus on a single pathway, and the molecular mechanisms of sepsis have not been fully elucidated. In particular, the relevant immune mechanisms are extremely complex, so their clinical application and effectiveness still have significant limitations. Failure to precisely regulate the excessive inflammatory response and immunosuppressive state in sepsis patients will be unfavorable for treatment outcomes

¹²

. This indicates the importance of developing diagnostic biomarkers for sepsis to achieve individualized and precise treatment. With the development of high-throughput sequencing technology

¹³

, in the face of the current challenges in molecular targeted therapy for sepsis, mining key regulatory genes and signaling pathways of sepsis through bioinformatics methods has become an important strategy for exploring its pathogenesis and potential therapeutic targets.

Ubiquitination is a process in which ubiquitin molecules specifically modify target proteins under the action of enzymes (such as ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, ubiquitin ligases, and deubiquitinating enzymes). It participates in almost all life activities by regulating protein localization, metabolism, function, and degradation

¹⁴

. Ubiquitination plays a crucial role in the development of various inflammatory diseases and tumorigenesis

¹⁵

. The ubiquitination system plays an important role in the pathological process of sepsis by regulating inflammatory signaling pathways, cell death, etc.

^16,

^17,

¹⁸

. For example, E3 ligases regulate the activation of the NLRP3 inflammasome by catalyzing the ubiquitination of the NLRP3 inflammasome or other components of the inflammasomes, inducing apoptosis of immune cells, and thus exacerbating the development of sepsis

^19,

²⁰

. In contrast, YTHDF1 promotes NLRP3 ubiquitination by upregulating WWP1, thereby inhibiting caspase-1-dependent pyroptosis and alleviating sepsis

²¹

. In recent years, based on the core role of ubiquitination in sepsis, potential molecular targets have been provided for the intervention and treatment of sepsis. Researchers have developed various targeted treatment strategies for sepsis, such as natural compound regulation, proteasome inhibitors, and nanotechnology delivery systems. For instance, Gramine improves sepsis-induced myocardial dysfunction by binding to NF-κB p105 and inhibiting its ubiquitination

²²

. The ubiquitin-activating enzyme E1 inhibitor PYR-41 can reduce lung injury in septic mice

²³

. Intravenous administration of Skp2 mRNA-encapsulated lipid nanoparticles (LNP) can inhibit ferroptosis in lung epithelium and alleviate lung injury in septic mice

²⁴

. Although these strategies show potential in experimental studies, their clinical application still has a long way to go. Meanwhile, there is currently a lack of reliable indicators to evaluate the changes in the ubiquitination status in patients with sepsis, making it impossible to accurately identify patient subgroups that may benefit from specific targeted therapies. The research on ubiquitination-related genes (UbRGs) in sepsis, their dynamic regulatory networks, and their clinical translation value urgently need in-depth exploration.

Due to the current lack of approved specific drugs for sepsis, many researchers have also turned their attention to Traditional Chinese Medicines (TCMs) research. TCM has a long-standing history in preventing and treating various diseases. Xuebijing (XBJ) is a TCM compound injection approved by the State Food and Drug Administration (SFDA) of China for the clinical treatment of sepsis. Studies have confirmed that XBJ injection is effective in treating sepsis by integrating and regulating multiple pathways such as immunity, metabolism, and coagulation through multiple components and multiple targets

²⁵

. It can also effectively treat disseminated intravascular coagulation (DIC) caused by severe sepsis

²⁶

and significantly reduce the mortality rate of sepsis patients

²⁷

.Not only TCM compounds but also various small-molecule compounds isolated from TCM have shown potential in treating sepsis. Research indicates that many small-molecule compounds provide precise targeted intervention and treatment for sepsis by regulating multiple signaling pathways, such as NF-κB, TLR4, MAPK, NLRP3, AMPK, and PI3KAKT

²⁸

. For example, Berbamine (BA, BBM) dihydrochloride, isolated from TCMs like Berberis, Coptis chinensis, and Cortex Phellodendri, plays a protective role in sepsis by targeting Notch1 to regulate the inflammatory response of macrophages, inhibiting inflammation and promoting the degradation of Notch1, significantly improving the survival rate of septic mice and reducing lung inflammation

²⁹

.Moreover, small-molecule compounds can also affect the pathological process of sepsis by regulating the ubiquitination pathway. For instance, the small-molecule PSSM0332 dissociates the CRL4A DCAF8 E3 ligase complex, reduces the ubiquitination of NcoR1, and inhibits the inflammatory response in a mouse model of sepsis-induced myocardial dysfunction

³⁰

.Therefore, TCM and small-molecule drugs have significant value in the treatment of sepsis. Exploring the synergistic treatment of TCM, its active ingredients, and small-molecule drugs, evaluating the biological network mechanism between drugs and diseases through a multi-component, multi-target regulatory network, especially focusing on ubiquitination-related targets, and achieving precise targeted treatment according to the disease phenotypes and genotypes of patients can more effectively promote patient recovery and safeguard patient life and health, providing new potential for sepsis treatment.

However, how to integrate the ubiquitination regulatory network with changes in the immune microenvironment remains a challenge in the study of the molecular mechanism of sepsis. Traditional studies often screen differentially expressed genes based on a single dataset, but batch effects and technical heterogeneity may lead to result bias

³¹

. Integrative analysis of multi-cohort data can improve the reliability of gene screening. In addition, machine learning algorithms (e.g., LASSO regression, Random Forest) have shown unique advantages in feature gene screening and disease prediction model construction, capable of identifying key molecules with biological significance and diagnostic potential from high-dimensional data

^32,

^33,

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. Recent studies have indicated that the immune cell infiltration patterns (e.g., macrophage polarization, T-cell exhaustion) in sepsis patients are closely associated with disease progression

^35,

^36,

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. However, there is still a lack of systematic evidence on how UbRGs affect the outcome of sepsis by regulating immune cell functions.

Against the above background, we explored the differentially expressed ubiquitination-related genes and their regulatory pathways in sepsis by integrating multi-omics data. Three machine learning algorithms, namely LASSO regression, random forest (RF), and XGBoost

³⁸

, were employed to screen key feature genes. Combined with CIBERSORT immune infiltration analysis and ROC curve analysis based on the current dataset, we exploratorily assessed the preliminary discriminative ability of candidate genes. We further validated the transcriptional expression levels of these candidates in a sepsis macrophage model using RT‑qPCR. Finally, we screened small‑molecule drugs and traditional Chinese medicine compounds that may target ubiquitination‑related processes in sepsis, and verified their binding affinities via molecular docking. Overall, this study aims to provide new evidence for the identification of candidate biomarkers and the development of targeted therapeutic strategies grounded in the ubiquitination regulatory network.

Materials and Methods

Results

Discussion

In this study, four datasets (GSE32707, GSE54514, GSE69528, GSE137340) from the GEO database were standardized and analyzed, and 3,679 differentially expressed genes (DEGs) in sepsis were identified. Among them, 1,656 were upregulated and 2,023 were downregulated in the sepsis group. This extensive change in gene expression highlights the complex molecular landscape of sepsis. A total of 1,838 UbRGs were screened from the GeneCards database, and by intersecting with sepsis DEGs, 448 sepsis-related UbRGs were obtained. Functional enrichment analysis of these UbRGs indicated that they are involved in various biological processes, molecular functions, and cellular components, suggesting that ubiquitination may be a key mechanism regulating the immune response and inflammatory pathways during sepsis. Pathway analysis showed that these UbRGs were significantly enriched in 15 key pathways, such as the NF-κB signaling pathway, apoptosis, and ubiquitin-mediated proteolysis, which is consistent with recent research findings. For example, tumor necrosis factor receptor-associated factor 6 (TRAF6), with ubiquitin E3 ligase activity, activates the NF-κB signaling pathway through K63-site ubiquitination, while K48-site polyubiquitination negatively regulates the signal feedback between RANKL and IFN-γ

⁴¹

. In contrast, the deubiquitinase, TNFα-induced protein 3 (TNFAIP3), terminates NF-κB and MAP kinase signaling by removing K-63-linked polyubiquitin chains from TRAF2, TRAF6, and NEMO/IKKγ, thereby inhibiting tissue inflammation

⁴²

. TNFAIP3 was found to exert an anti-inflammatory effect in the retinal vasculature by inhibiting NF-κB activation

⁴³

. Downregulation of TNFAIP3 expression may lead to excessive activation of NF-κB signaling, thereby exacerbating the systemic inflammatory response

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.

Using machine learning models (LASSO, XGBoost, and RF), we identified 11 characteristic ubiquitination‑related genes (UbRGs) and evaluated their discriminative ability between normal and sepsis groups based on differential expression analysis and ROC curves within the current merged dataset. The statistically significant expression differences of these genes between the normal and sepsis groups suggested their potential as candidate biomarkers. Subsequently, we focused on four characteristic DE‑UbRGs (FBXO7, SIAH1, SUMO2, and UBE2C). FBXO7, SIAH1, and SUMO2 were significantly downregulated in the sepsis group (P < 0.05), while UBE2C was significantly upregulated (P < 0.05). In subsequent experiments using LPS‑treated THP‑1 cells, RT‑qPCR was performed to preliminarily validate the transcriptional expression of these four UbRGs, which showed trends consistent with the bioinformatics results, thereby providing initial experimental support for their candidacy as biomarkers. Nevertheless, it should be noted that RT‑qPCR only reflects mRNA abundance and does not directly demonstrate changes at the protein level or ubiquitination activity. However, the functional differences of FBXO7 between THP-1 cells and RAW264.7 cells may be mainly attributed to three aspects: first, inherent species differences, namely distinct gene regulation and signaling pathway dynamics between human and mouse; second, differences in cell origin and differentiation status: THP-1 is an induced immature human monocyte line, whereas RAW264.7 is a terminally differentiated mouse macrophage line; third, the multifunctional nature of FBXO7 itself and its cell‑type dependence. Therefore, these differences reflect the species specificity and cellular context dependence of FBXO7 function, and caution should be exercised when extrapolating the results to other species or cell models. We conducted immune infiltration analysis and GSEA analysis on the 4 UbRGs, indicating that they may play important roles in the inflammatory response and the maintenance of cellular homeostasis.

The pathological process of sepsis is closely associated with the dynamic changes in the infiltration pattern of immune cells. Recent studies have suggested that ubiquitination genes are involved in the pathological process of sepsis by mediating protein degradation and regulating signal transduction pathways, thereby influencing the function and activity of immune cells

⁴⁵

. FBXO7 (F-box protein 7), a key component of the SKP1-CUL1-F-box (SCF) E3 ligase complex, can specifically recognize various substrate proteins and regulate the stability and function of target proteins through the ubiquitin-proteasome system (UPS). FBXO7 participates in the regulation of proteasome complexes, mitophagy, the cell cycle, cell proliferation, and germ cell differentiation

⁴⁶

. In this study, the immune infiltration analysis of FBXO7 showed a significant correlation with multiple immune cells. Positive correlations were observed with naive B cells, memory B cells, two mast cell states, and neutrophils, especially the strongest correlation with neutrophils. There were more negatively correlated cell types, including plasma cells, several T cell types, NK cells, all macrophage subtypes, etc., with the strongest negative correlation with M0 macrophages. These results imply that FBXO7 may inhibit the function of myeloid immune cells (such as macrophages and monocytes) while promoting the activities of certain B cells and neutrophils. Considering that the excessive activation of macrophages in sepsis exacerbates inflammation, the low expression of FBXO7 may weaken the inhibition of macrophages, that is, promote myeloid cell infiltration, leading to uncontrolled inflammation. In the GSEA analysis, the positively correlated pathways were concentrated in cellular stress response and metabolic regulation, such as acid secretion, mitophagy, malaria-related pathways, etc. These may reflect the adaptive mechanisms of cells under sepsis stress. The negatively correlated pathways were mainly related to energy metabolism and immune activation, such as oxidative phosphorylation, RIG-I signaling, proteasome, etc. This indicates that when FBXO7 is lowly expressed, cellular energy production is hindered, and the antiviral response ability decreases, which is consistent with the characteristics of the immune paralysis stage of sepsis. It may inhibit metabolic pathways and drive disease deterioration, making it a research target with translational value, but the dynamic changes in the course of human sepsis still need to be verified. Fbxo7 negatively regulates NF-κB signaling through the ubiquitination of TRAF2 and cIAP1

⁴⁷

. FBXO7 mediates the proteasomal degradation of UXT-V2, resulting in the inhibition of the NF-κB signaling pathway

⁴⁸

. Additionally, FBXO7 plays a role in mitophagy

⁴⁹

, and the chemical inhibition of FBXO7 reduces inflammation and confers neuroprotection by stabilizing the mitochondrial kinase PINK1

⁵⁰

. Overall, FBXO7 can inhibit the inflammatory response through the NF-κB signaling pathway and maintain mitochondrial homeostasis to suppress excessive inflammation. The mechanism research and targeted therapy of FBXO7 still face challenges, but it has shown significant clinical translational potential. A study has shown that ginsenoside Rg1 treats sepsis-induced acute lung injury by regulating FBXO3 (a homologous protein of FBXO7), providing direct evidence for targeting F-box proteins. This study found that Rg1 reduces the stability of FBXO3 in an m6A-dependent manner, thereby activating the PGC-1α/Nrf2 signaling pathway and alleviating oxidative stress injury

⁵¹

. Considering the structural and functional similarities between FBXO7 and FBXO3, this study provides strong theoretical support for targeting FBXO7 in the treatment of sepsis. The main challenges in targeted therapy of FBXO7 include differences in tissue-specific functions, sepsis heterogeneity, and drug delivery obstacles. Future research should focus on verifying the regulatory mechanism of FBXO7 in sepsis, optimizing the characteristics of lead compounds, and developing personalized treatment strategies. Combining the strategies of repurposing old drugs and combination therapy, targeting FBXO7 is expected to provide new treatment options for this complex syndrome of sepsis, compensating for the limitations of the current mainly supportive treatment.

SIAH1 (Seven in Absentia Homolog 1) is a RING-type E3 ubiquitin ligase that participates in the regulation of key biological processes such as cell apoptosis, autophagy, metabolic stress response, and tumorigenesis by mediating the ubiquitination and degradation of specific substrate proteins. Its function is significantly "environment-dependent". Previous studies have shown that SIAH1 regulates cell signaling pathways by promoting the ubiquitination and degradation of substrates. For example, SIAH1 can promote the ubiquitination of RECK, thereby affecting cell migration and invasion related to matrix metalloproteinase 9 (MMP9)

⁵²

. In non-alcoholic fatty liver disease (NAFLD), SIAH1 regulates lipid metabolism by interacting with Sterol Carrier Protein 2 (Scp2) and promoting its ubiquitination and degradation

⁵³

. These mechanisms indicate that SIAH1 not only plays a role in the occurrence and development of cancer but is also an important regulator of metabolic diseases. However, there is a lack of systematic literature reports on the relationship between the function of SIAH1 in the ubiquitination and deubiquitination processes and sepsis. This study found that low expression of SIAH1 may indicate immune dysregulation and metabolic defects in sepsis patients, and its expression level may be related to the severity of the disease. Low expression in sepsis causes dual damage through "attenuated autophagy + immune quiescence": on the one hand, it weakens cell stress protection, and on the other hand, it hinders immune activation, jointly promoting the progression of sepsis. Temporal-specific regulation targeting this gene may be a potential therapeutic strategy. SIAH1 is positively correlated with the infiltration of innate immune cells (monocytes, resting NK cells) but significantly negatively correlated with the markers of adaptive immune activation (T/B cell activation, dendritic cells, regulatory T cells), especially the strong negative correlation with NK cell activation (r = -0.27), suggesting that SIAH1 may promote the immunosuppressive state by inhibiting the acquired immune response. A spinal cord injury model revealed a new mechanism of SIAH1 in the inflammatory response: after lipopolysaccharide (LPS) stimulation, the formation of the GAPDH-SIAH1 complex in microglia increases, activating the NF-κB pathway and releasing pro-inflammatory factors such as IL-6 and TNF-α. Nrf2 can alleviate inflammation by inhibiting this complex - overexpression of Nrf2 reduces SIAH1 expression by 50% and decreases the microglia proliferation rate by 35%

⁵⁴

. In the GSEA analysis, high expression of SIAH1 is significantly enriched in cell autophagy (Autophagy/Mitophagy), ferroptosis, and FoxO signaling, indicating that it may affect the sepsis process by regulating cell metabolic stress and survival pathways. At the same time, it significantly inhibits pathways related to primary immunodeficiency, DNA replication, and autoimmunity, confirming its negative regulatory effect on immune activation, which is consistent with the results of immune infiltration. In summary, in sepsis, SIAH1 has dual or even opposite effects in different cell types and disease stages. The actual impact of low SIAH1 expression on the immune system is cell-type-specific and contradictory: in lymphoid cells, low SIAH1 expression leads to excessive apoptosis of T/B cells and abnormal activation of NK cells due to c-Myb accumulation; while in myeloid cells, its low expression alleviates inflammatory damage by blocking the GAPDH-SIAH1 complex

^7,

^55,

^56,

⁵⁷

. That is, in the early inflammatory stage of sepsis, high expression of SIAH1 in myeloid cells (such as macrophages/microglia) may activate the NF-κB pathway by forming the GAPDH-SIAH1 complex, promote the release of IL-6/TNF-α, and at the same time degrade key autophagy proteins to weaken the cell metabolic protection ability and cause abnormal activation of NK cells, jointly exacerbating inflammatory damage; inhibiting SIAH1 at this time can alleviate the pathological response. In the late immunosuppressive stage, low expression of SIAH1 in lymphoid cells (T/B cells) accelerates immune cell exhaustion and worsens immune paralysis due to the inability to effectively degrade pro-apoptotic proteins such as caspase-3. At this time, enhancing SIAH1 activity is needed to maintain lymphocyte homeostasis.

SUMO2 (small ubiquitin-like modifier 2), as an important member of the SUMO family, regulates the stability, localization, and function of target proteins through SUMOylation modification and plays a crucial role in inflammatory responses and immune regulation

⁵⁸

. Rachael Barry et al. showed that the E3 SUMO ligase MUL1 mediates the SUMOylation of NLRP3 in the form of SUMO2/3 and inhibits the activation of the NLRP3 inflammasome

⁵⁹

. However, Ying Qin et al. found that the E3 SUMO ligase TRIM28 specifically binds to NLRP3, promotes its SUMO1, SUMO2, and SUMO3 modifications, thereby inhibiting the ubiquitination and protein degradation of NLRP3 and promoting NLRP3 expression and inflammasome activation. TRIM28 and MUL1 may mediate NLRP3 SUMOylation at different lysine sites and regulate NLRP3 activity through different mechanisms

⁶⁰

. In sepsis, Xinyong Liu et al. found that ginkgolic acid may promote inflammation and macrophage apoptosis in sepsis by inhibiting the SUMOylation process and increasing the activity of NF-κB p65

⁶¹

. From the immune infiltration analysis of SUMO2 in this study, it is significantly correlated with various immune cells: positively correlated with pro-inflammatory cells (monocytes, M0/M1 macrophages), but negatively correlated with some anti-inflammatory cells (resting mast cells, activated NK cells). GSEA results for SUMO2 show that the top-ranked pathways are neurodegenerative diseases (Huntington's, Parkinson's), which appear unrelated to sepsis but are essentially linked to dysregulated mitochondrial quality control and are closely associated with immunometabolic reprogramming. Sepsis is a metabolically complex disease. During sepsis, cellular energy metabolism shifts from oxidative phosphorylation (OXPHOS) to glycolysis (the Warburg effect)—a transition that occurs in nearly all cell types. Glycolysis can enhance immune activation, but it is also a cause of immunosuppression; excessive glycolysis instead leads to immune suppression. Mitochondrial dysfunction and metabolic disturbances are key drivers of sepsis mortality. Mitochondrial damage leads to metabolic reprogramming, which results in energy depletion and ROS burst, followed by exacerbated inflammation, which in turn causes further mitochondrial damage. This cycle ultimately leads to organ exhaustion, progressing to septic shock and multiple organ failure. Therefore, mitochondrial dysfunction is the root of metabolic reprogramming, and restoring OXPHOS depends on maintaining mitochondrial structure and function. In sepsis, SUMO2 may influence mitochondrial function, thereby regulating glycolysis and the inflammatory response, affecting cellular metabolic reprogramming and the progression of sepsis. Meanwhile, the activation of autoimmune pathways such as "graft-versus-host disease" and "autoimmune thyroiditis" indicates that there may be a mechanism similar to autoimmunity in sepsis. The low expression of SUMO2 in sepsis may participate in disease progression by regulating myeloid cell infiltration (pro-inflammatory), adaptive immune suppression (B/NK cells), and metabolic reprogramming (OXPHOS/glycolysis). The enriched neurodegenerative and autoimmune pathways provide a new perspective for understanding the multi-organ damage in sepsis. The "pro-inflammatory" and "immune suppression" caused by SUMO2 may be the molecular basis of the dynamic process of "early hyperinflammation - late immune paralysis" in sepsis. Therapeutic strategies based on SUMO2 need to strictly consider spatiotemporal specificity. Targeting the SUMO2 regulatory network (such as enhancing its myeloid-specific expression or supplementing SUMOylation substrates) may simultaneously improve excessive inflammation and immune paralysis, providing new ideas for treatment.

UBE2C (Ubiquitin-conjugating enzyme E2 C) is a core component of the ubiquitin-proteasome system (UPS). As a member of the ubiquitin-conjugating enzyme E2 family, it is responsible for transferring ubiquitin molecules to specific substrates and mainly mediates lysine 11-linked polyubiquitination (K11-linked polyubiquitination). Its core functions include cell cycle regulation, maintenance of genomic stability, and cross-regulation of NF-κB signaling. UBE2C is a specific E2 enzyme of the anaphase-promoting complex/cyclosome (APC/C), ensuring the normal exit from mitosis by degrading mitotic cyclins (e.g., Cyclin B1, Securin). In studies related to inflammatory diseases, UBE2C is considered a potential prognostic marker and therapeutic target

^62,

⁶³

. In this study, we found that UBE2C may drive immune imbalance through a dual mechanism of "promoting macrophage polarization and inhibiting T cell function" while activating metabolic stress and neurodegeneration-related pathways to exacerbate multi-organ damage. Further research should focus on verifying its specific role in macrophage metabolic reprogramming and T cell exhaustion and evaluating its potential as a staging diagnostic marker or immunotherapeutic target for sepsis. Immune infiltration analysis of UBE2C suggests a significant correlation with various immune cells, particularly a prominent positive correlation with macrophage subtypes. The correlation coefficients of M0, M1, and M2 macrophages range from 0.19 to 0.29, and the p-values are all highly significant. This may imply that UBE2C is involved in macrophage polarization or functional regulation. Some studies have also confirmed this. High expression of UBE2C is significantly positively correlated with M1 macrophage infiltration in sepsis patients (r = 0.29, P < 1e - 09), suggesting that it may exacerbate the pathological process by promoting the activation of pro-inflammatory signaling pathways (e.g., IL - 6/JAK/STAT3)

⁶⁴

. The most obvious negative correlations are with CD8+ T cells and naive CD4+ T cells, with correlation coefficients of -0.13 and -0.2, respectively. Positive correlations are concentrated in innate immune cells, while negative correlations tend to be with adaptive immune cells. UBE2C-mediated protein degradation may consume key proteins in immune cells (e.g., T cells), leading to immune exhaustion.Looking at the GSEA results, the top positively enriched pathways are neurodegenerative disease pathways, with Huntington's disease, Parkinson's disease, and prion disease ranking at the forefront, and all NES values are above 2.2. These pathways are involved in oxidative stress and mitochondrial function, potentially implicating UBE2C in cellular stress responses, possibly through the Warburg effect and regulation of mitochondrial dynamics, thereby influencing the progression of sepsis. The most strongly enriched signaling pathways are ribosome, cell cycle, and oxidative phosphorylation, suggesting that this gene may influence basic cell metabolism and proliferation. The most significantly negatively enriched pathways are related to transplant rejection and autoimmunity, especially the Th1/Th2 cell differentiation pathway with an NES value of -2.32, which corresponds to the negative correlation with T cells in immune infiltration.

To develop individualized treatment strategies targeting four characteristic UbRGs, we screened potential small-molecule drugs, TCMs, and their active ingredients. Based on the molecular docking results, Sirolimus showed high binding affinity to all four DE-UbRGs. Sirolimus is a well-known autophagy inducer and mTOR inhibitor

^65,

⁶⁶

. mTOR inhibition activates overall protein degradation through the ubiquitin-proteasome system and autophagy

⁶⁶

. The mTOR signaling pathway plays a crucial role in the pathological process of sepsis

^68,

^69,

⁷⁰

. Ubiquitination is involved in post-translational protein modification and plays an important role in the immune response, being closely related to the occurrence and progression of diseases

^19,

⁷¹

. Some studies have shown that the role of basic fibroblast growth factor (bFGF) in myocardial ischemia/reperfusion (I/R) recovery is related to the inhibition of excessive autophagy and the increase in ubiquitinated protein clearance rate by activating the PI3K/Akt/mTOR signaling pathway

⁷²

. Additionally, in a sepsis mouse model, Sirolimus alleviates lung injury by inhibiting the differentiation of Th17 cells through RORγt and STAT3 dysfunction

⁷³

. Therefore, considering the existing research results, Sirolimus has known immunomodulatory properties and good safety. By inhibiting mTORC1, it may act through multiple coordinated pathways such as stabilizing mitochondrial function in a ubiquitination-dependent manner, regulating the inflammatory response, and enhancing antioxidant defense, making it a promising candidate drug for targeted ubiquitination therapy of sepsis. Subsequently, a low-dose regimen can be verified through sepsis models to avoid excessive immunosuppression. Lysine, as an essential amino acid involved in protein synthesis, can be used as a nutritional supplement for adjuvant therapy. Temozolomide plays a crucial role in the treatment of gliomas

⁷⁴

. Downregulating UBE2C expression inhibits aerobic glycolysis by regulating p53 expression, thereby enhancing the sensitivity of glioma cells to Temozolomide

⁷⁵

. However, there are case reports of a patient with glioblastoma receiving Temozolomide and radiotherapy being diagnosed with sepsis due to phlegmonous gastritis

⁷⁶

, and another patient with high-grade neuroendocrine tumors developing neutropenic sepsis after receiving PRRT combined with capecitabine or Temozolomide

⁷⁷

. The specific mechanisms need further exploration. Pyrrolidonecarboxylic acid, as a safe molecule, also warrants further exploration for its adjuvant treatment value. p-Chloroamphetamine and Cycloheximide, due to potential toxicity risks, are more suitable as tools for mechanism research rather than therapeutic drugs

^78,

⁷⁹

. Alachlor

⁸⁰

and Iproplatin

⁸¹

are not suitable for clinical research due to toxicity or efficacy issues, but their mechanism research still promotes the development of related fields. Therefore, some of the screened small-molecule compounds exhibit significant cytotoxicity or carcinogenic risk. They are recommended only for mechanistic studies and are not suitable as candidate drugs for sepsis treatment. Additionally, the immunosuppressant sirolimus and certain chemotherapeutic agents may increase the risk of secondary infections, and thus require structural optimization as well as systematic safety evaluation under rigorous standards. From the TCMs screening results, we obtained TCMs corresponding to four characteristic UbRGs, namely FBXO7 (Oenanthe javanica), SIAH (semen arecae and Areca Catechu L.), SUMO2 (Brassica rapa L.), UBE2C (Fructus Arctii lappae), along with the corresponding active ingredients of the above TCMs. However, TCMs contain numerous components, and some known components are toxic to the human body. Most of the time, purification of active ingredients is required. Therefore, this study only explores components with medicinal value. Through literature review and database retrieval, it was found that Oenanthe javanica has many active ingredients and has anti - inflammatory applications in traditional medicine

⁸²

, but there are few studies directly targeting sepsis. Previous studies have shown that FBXO7 inhibits the NF - κB pathway, regulates mitochondrial autophagy, and modulates the inflammatory response

^47,

^48,

^49,

⁵⁰

. FBXO7 has the highest docking score with Kaempferol (-6.5 kcal/mol). Some studies have shown that Kaempferol reverses acute kidney injury in sepsis models by inhibiting macrophage infiltration in septic mice and suppressing the NF - κB/AKT signaling pathway

^83,

⁸⁴

. Secondly, FBXO7 also has relatively stable binding ability with Isorhamnetin (-6.0 kcal/mol) and Quercetin (-6.1 kcal/mol). Yang JH et al. isolated Isorhamnetin from Oenanthe javanica. Studies on rats and lipopolysaccharide (LPS) - activated macrophages confirmed that Isorhamnetin prevents acute inflammation by blocking NF - κB activation

⁸⁵

. Xue Y et al. found that Quercetin inhibits the activation of the NLRP3 inflammasome in epithelial cells triggered by Escherichia coli O157:H7, partly by preventing ROS production and activating autophagy

⁸⁶

. Aming Sang et al. demonstrated that Quercetin inhibits oxidative stress - mediated endoplasmic reticulum stress by activating the SIRT1/AMPK pathway, alleviating sepsis - induced acute lung injury

⁸⁷

. Therefore, the active ingredients of Oenanthe javanica (especially flavonoids) have multi - target anti - inflammatory and antioxidant properties, which meet the pathological needs of sepsis. Oenanthe javanica and its active ingredients may regulate the inflammatory response in sepsis and reduce organ damage (especially in the kidneys and lungs) by regulating the ubiquitination - related target FBXO7, inhibiting the NF - κB pathway, and maintaining mitochondrial homeostasis. However, there is currently no experimental evidence for the direct regulation of FBXO7 or ubiquitination by Oenanthe javanica, and further experiments are needed for verification.

We employed the same method to investigate the TCMs Arecae Semen and Arecae Pericarpium (Areca Catechu L.) in the SIAH mapping. Low - dose Arecae Semen, its extracts, and compounds exhibit pharmacological effects such as anthelmintic, antibacterial, anti - inflammatory, analgesic, antioxidant activities, and improvement of gastrointestinal function. The important mechanisms involved include reducing ROS production, inhibiting the activation of NADPH oxidase, increasing the activity of antioxidant enzymes, and influencing MAPK, AKT, TLR, NF - κB, Nrf - 2, PI3K, and STAT3 signaling pathways

^88,

^89,

^90,

⁹¹

. Studies have shown that patients with septic shock and secondary organ dysfunction have reduced antioxidant status and increased lipid peroxidation

⁹²

. X Terra et al. found that proanthocyanidins reduce LPS - induced ROS production and exert anti - inflammatory effects by inhibiting the activities of ERK1/2 and IKKb. Procyanidin B1 shows excellent binding ability with SIAH1 in molecular docking results, with a vina score of - 9.3 kcal/mol. Jing Xing et al. demonstrated that Procyanidin B1 binds to the TLR4 - MD - 2 heterodimer, inhibits the downstream activation of MAPK and NF - κB signaling pathways, and exerts anti - inflammatory effects on LPS - treated THP1 cells

⁹³

. In sepsis, Procyanidin B1 may bind to SIAH1, inhibit the ubiquitination and degradation of pro - inflammatory factors, block the excessive activation of NF - κB, and alleviate the inflammatory storm in sepsis. Its potential is clear, but the specific mechanism still needs to be clearly verified. It is worth noting that Procyanidin B1 has MW>500, NorO>10, NHorOH>5, violating the Lipinski's rule of five for oral bioavailability. However, many drugs have exceeded the Lipinski's rules

⁹⁴

. Procyanidin B1 shows a high affinity for SIAH1 (- 9.3 kcal/mol). As a natural polyphenolic compound, it can be metabolized and decomposed by gut microbiota into a series of low - molecular - weight polyphenol metabolites, which can then be absorbed and enter the systemic circulation

^95,

⁹⁶

. Therefore, it still has high research value. The other two active ingredients, Catechin and ent - Epicatechin, have obvious biological activities such as antioxidant, anticancer, anti - inflammatory, antimutagenic, anti - aging effects, and improvement of liver function. Studies by Jung A. J., Joseph Shay et al. showed that they mainly exert anti - inflammatory and protective effects through ROS scavenging and Nrf2 antioxidant stress response

^97,

^98,

^99,

¹⁰⁰

. Their specific roles in sepsis need further exploration. In general, the active ingredients of Arecae Semen may target the SIAH1 - ubiquitination pathway, providing a new strategy for sepsis inflammation regulation. In particular, the anti - inflammatory mechanism of Procyanidin B1 shows significant potential. However, since Arecae Semen has been classified as a Group 1 carcinogen by the WHO, and its fibrogenic effect is clear

^101,

¹⁰²

. Therefore, Arecae Semen raw materials cannot be directly used for treatment. Carcinogenic risks need to be avoided through ingredient purification/structural modification, and its efficacy and mechanism in sepsis models need to be further verified.

As mentioned previously, SUMOylation is closely related to sepsis

^58,

⁶¹

. SUMO2 and the potential TCM turnip (Brassica rapa L.), a traditional medicinal plant, have active components that regulate inflammation, oxidative stress, and immune responses through multiple targets

^103,

^104,

^105,

¹⁰⁶

. However, there are no reports in the existing literature about its direct regulation of SUMOylation. The results of drug screening and molecular docking suggest a potential new mechanism. Among the five components in this study, Liquiritin deserves the most attention, with a high oral bioavailability (OB, 65.69%) and drug-likeness (DL, 0.74), and the optimal docking score (-6.5 kcal/mol). Studies by Shijiao Zheng

¹⁰⁷

, Shan-Qi Mou

¹⁰⁸

, Hongling Zhou

¹⁰⁹

, et al. have shown that Liquiritin can alleviate sepsis-related inflammation and organ damage in the heart, lungs, kidneys, etc. stimulated by lipopolysaccharide (LPS) by activating the Nrf2 signaling pathway, AMP-activated protein kinase-dependent signaling pathway, and inhibiting the JNK/Nur77/c-Jun pathway. SUMO2 regulation may be a new target for the treatment of sepsis, but this is only a prediction and requires further experimental verification.In addition, Liquiritin and Quercetin are also active components of turnip. As mentioned before, Isorhamnetin can prevent acute inflammation by blocking NF-κB activation

⁸⁵

; Quercetin can inhibit the activation of the NLRP3 inflammasome

⁸⁶

, activate the SIRT1/AMPK pathway to inhibit endoplasmic reticulum stress mediated by oxidative stress, and alleviate sepsis-induced acute lung injury

⁸⁷

. Isorhamnetin and Quercetin may reduce the inflammatory response in sepsis by inhibiting NF-κB and reducing the SUMOylation of the NLRP3 inflammasome.β-sitosterol should not be ignored either. It can interfere with multiple cellular signaling pathways, including those related to the cell cycle, cell death, proliferation, survival, invasion, angiogenesis, metastasis, anti-inflammation, anti-cancer, liver protection, anti-oxidation, and cardiac protection. It shows no obvious toxicity in pharmacological screening, and its pharmacokinetic characteristics have been extensively studied

¹¹⁰

. β-sitosterol promotes the activation of antioxidant enzymes (such as superoxide dismutase, catalase, and glutathione) and inhibits lipid peroxidation by activating the Nrf2 and Nrf2/heme oxygenase-1 (HO-1) signaling pathways. It can also inhibit the expression of proteins involved in cellular signaling pathways (such as JAK3/STAT3 and NF-kB) and plays a key role in regulating the inflammatory/immune response

^111,

¹¹²

. For example, it can affect microglial polarization by inhibiting the TLR4/NF-κB signaling pathway, thereby reducing neuroinflammation and alleviating neuropathic pain

¹¹³

.The anti-oxidative and anti-inflammatory effects of the active components in turnip (Brassica rapa L.) are highly relevant to the key pathophysiological processes in sepsis, such as oxidative stress and uncontrolled inflammation. The research suggests that these effects may be achieved through the regulation of SUMO2-mediated protein SUMOylation modification.

As a member of the ubiquitin-conjugating enzyme E2 family, UBE2C participates in the regulation of the cell cycle and inflammation. UBE2C is mapped to Fructus arctii, which is the fruit of Arctium lappa L. It contains many chemical constituents and has anti-cancer, anti-viral, anti-inflammatory, and anti-oxidant effects

^114,

^115,

¹¹⁶

. The study by Weifang Zhang et al. showed that Fructus arctii inhibits microglial activation and reduces the levels of inflammatory factors through the Let-7E-regulated Toll-like receptor (TLR) signaling pathway

¹¹⁷

. Arctigenin, an important component of Fructus arctii, has potent anti-inflammatory, anti-oxidant, and anti-viral effects

^118,

¹¹⁹

. Studies by Supriya R Hyam et al.

¹²⁰

, Xianjuan Kou et al.

¹²¹

, Xiang Xu et al.

¹²²

, and Xianbao Shi et al.

¹²³

found that arctigenin inhibits key upstream pathways of NF-κB, such as PI3K/AKT, JAK-STAT, TLR4, and TNFR1, through multi-pathway crosstalk, and polarizes M1 macrophages into M2-like macrophages, ultimately alleviating various inflammations, such as in acute lung injury and cranial neuroinflammation, highlighting its broad-spectrum anti-inflammatory potential. Its mechanism of action is highly synergistic, providing a theoretical basis for the development of multi-target anti-inflammatory drugs. Arctigenin may block the ubiquitination modification of key inflammatory regulatory proteins such as IκBα and STAT3 by targeting the catalytic center of UBE2C; or it may reduce the expression of UBE2C by inhibiting the NF-κB/STAT3 pathway, disrupting the positive feedback loop of inflammation-ubiquitination. It has significant therapeutic potential in inflammation-related diseases, offering new ideas for the development of dual-target drugs for "ubiquitination-immune inflammation". It is worth noting that the OB value of arctigenin is only 8.05%, and pharmacokinetic studies have shown

¹²⁴

that the significant first-pass metabolism of arctigenin hinders its in vivo and clinical efficacy after oral administration. However, due to its medicinal value, it was still included in this study, and the molecular docking results showed that it stably binds to UBE2C (-7.1 kcal/mol). It is recommended to administer it via a route other than oral. As another important active component of Fructus arctii, arctiin also has important immune-enhancing and anti-inflammatory effects. Arctiin improves glomerulonephritis by inhibiting NF-kappaB activation and nuclear translocation and reducing the levels of these pro-inflammatory cytokines

¹²⁵

. The study by Bo Zhou et al.

¹²⁶

showed that arctiin prevents LPS-induced acute lung injury by inhibiting the PI3K/AKT signaling pathway in mice. This suggests that it may target UBE2C for the treatment of sepsis complicated by acute kidney injury and acute lung injury, protecting renal tubular epithelial cells through a dual mechanism of anti-oxidation/anti-ubiquitination disorder. Additionally, C Ribeiro Nogueira et al. found that septic patients showed significant deficiencies in retinol and beta-carotene

¹²⁷

. The study by Farouk K El-Baz et al.

¹²⁸

showed that oral administration of beta-carotene can reduce cyclophosphamide (CP)-induced acute lung injury and inflammation. The molecular docking in this study showed that the vina score of beta-carotene with UBE2C is -7.5 kcal/mol, indicating stable binding. Perhaps beta-carotene can be used as an adjuvant treatment for sepsis-related conditions, reducing the inflammatory response and supplementing nutrition. Fructus arctii and its active components may regulate protein ubiquitination by targeting UBE2C, blocking the core inflammatory pathways of sepsis from the upstream and hitting the core mechanism of uncontrolled inflammation in sepsis, providing an innovative strategy for the development of a new generation of anti-sepsis drugs.

In this study, by integrating multi-omics data and employing machine learning algorithms, we optimized the screening and identification process for sepsis-related biomarkers. Through differential expression analysis, functional enrichment analysis, and integration of gene expression with clinical information, we performed extensive preliminary screening and further focused on four characteristic ubiquitination-related genes (UbRGs). The expression changes of these genes were validated using independent-sample PCR experiments, enhancing the credibility of the findings. Subsequently, we focused on immune infiltration analysis, GSEA, and drug screening for the four characteristic UbRGs, which revealed the critical roles of ubiquitination‑related genes (UbRGs) in sepsis and helped identify novel potential intervention targets and therapeutic strategies. However, all findings from this study are exploratory and hypothesis‑generating, and all gene–drug associations and mechanistic inferences require further validation through protein‑level and functional experiments.

Nevertheless, this study has the following limitations: (1) Although public databases provided a relatively large sample size, detailed clinical information—such as the type of infectious pathogen and organ failure scores—was lacking, which may affect the clinical relevance and translational value of the findings. The 11 characteristic DE‑UbRGs demonstrated some discriminative ability for distinguishing sepsis from control samples in internal validation within the current merged dataset; however, the AUC estimates were derived from internal data and have not been validated in independent external cohorts. Therefore, they should not be interpreted as confirmed clinical diagnostic performance. Prospective, multi‑center clinical cohorts are still needed to confirm the true generalizability of these 11 gene markers for sepsis discrimination. (2) The mechanistic validation is insufficient. The RT‑qPCR experiments in the in vitro cell model only verified changes in target gene expression at the mRNA level and did not provide direct evidence regarding protein expression levels, E3 ligase activity, ubiquitination modification status, or causal relationships. Ubiquitination is a complex post‑translational modification process involving a multi‑enzyme cascade, and mRNA abundance does not simply correspond to protein function. Consequently, all mechanistic discussions in this study are hypotheses based on bioinformatic predictions and require further verification through experiments such as western blotting, co‑immunoprecipitation (Co‑IP), ubiquitination assays, gene knockout/overexpression, and animal models. (3) Safety concerns regarding candidate drugs: Some of the screened small-molecule compounds (e.g., Areca catechu, Areca catechu and Cycloheximide) exhibit significant cytotoxicity or carcinogenic risk. They are recommended only for mechanistic studies and are not suitable as candidate drugs for sepsis treatment. Additionally, the immunosuppressant Sirolimus and certain chemotherapeutic agents may increase the risk of secondary infections, and their safety needs to be systematically evaluated under rigorous standards. (4) Limitations of the traditional Chinese medicine (TCM) screening model: The “single component–single target” analysis model employed in this study does not fully capture the holistic characteristics of TCM, such as multi‑component synergy and multi‑pathway regulation. Additionally, the screening criteria (e.g., oral bioavailability, drug‑likeness) may exclude some potential active ingredients that require metabolic activation or can only be administered via parenteral routes. (5) Superficial validation of drug–target interactions: Current investigation of the drugs is limited to virtual screening and affinity calculations; the specific binding sites and regulatory mechanisms remain to be experimentally elucidated. Practical application of these drugs will require comprehensive and rigorous evaluations of pharmacodynamics, pharmacokinetics, toxicology, animal studies, and clinical trials, and their druggability may be further improved through formulations such as nanoformulations or sustained‑release tablets.

Subsequent research can employ single‑cell multi‑omics technologies to resolve the spatiotemporal expression characteristics of ubiquitination‑related genes across different stages of sepsis, further exploring the specific mechanisms of ubiquitination in each phase of sepsis, particularly in early intervention and treatment. In addition, studies should combine animal models and clinical samples to construct multi‑omics integrated network models and systematically investigate the mechanisms linking ubiquitination and sepsis. By validating target functions, integrating ubiquitination modification omics to decipher global regulatory networks, and conducting pharmacokinetic‑pharmacodynamic studies of active components along with in vitro and in vivo functional validation, the potential of the characteristic UbRGs as subtyping markers for sepsis and as a basis for personalized medicine can be further confirmed, thereby enhancing their clinical applicability.

Conclusion

Using machine learning, this study identified ubiquitination-related candidate genes linked to sepsis pathology—involving inflammation and immune microenvironment changes—and also preliminarily explored potential TCM monomers and small-molecule drugs. Of note, all findings are exploratory and hypothesis-generating. The proposed molecular mechanisms and drug–target interactions require subsequent validation at the protein level, via ubiquitination functional studies, and in animal models. Thus, this work offers initial candidate targets and a conceptual basis for further research on ubiquitination in sepsis, but does not represent validated clinical translation protocols.

Data availability statement

1.Publicly available datasets were analyzed in this study. This data can be found here: Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) database, including GSE32707, GSE54514, GSE69528, and GSE137340.

2.The components of TCMs were mainly obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database (https://old.tcmsp-e.com/tcmsp.php), the HERB database (http://herb.ac.cn/).

Funding

This work was supported by grants from Guangxi Natural Science Foundation (No.2021GXNSFBA196017) and National Natural Science Foundation of China (No.82302461 and No.82360374).

Author contribution

Lixiong Zeng (First Author) : Writing-Original Draft, Writing-Review & Editing, Conceptualization, Data Curation, Formal Analysis, Visualization, Methodology, Software; Yongbing Yu (First Author) ：Writing - Original Draft, Investigation, Validation, Formal analysis, Visualization, Software, Methodology; Junzhi Yang: Formal analysis, Validation, Methodology; Houyu Gan: Data Curation, Investigation, Methodology; Qiuli Chen: Data Curation, Investigation, Methodology; Xueyi Chen: Data Curation, Investigation, Visualization Jihua Feng: Formal analysis, Validation, Software, Funding acquisition; Kanglai Wei (Corresponding Author):Conceptualization, Resources, Supervision, Validation, Writing-Original Draft, Writing-Review &Editing; Jianfeng Zhang (Corresponding Author)：Methodology, Writing - Review & Editing, Funding acquisition, Resources, Supervision, Project administration; Jie Yang (Corresponding Author)：Methodology, Writing - Review & Editing, Resources, Supervision, Project administration, Investigation, Conceptualization, Data Curation, Resources.

Author information

The authors wish to acknowledge that Lixiong Zeng, Yongbing Yu and Junzhi Yang contributed equally to this work and are considered co-first authors. Kanglai Wei, Jianfeng Zhang, and Jie Yang are co-corresponding authors.

Acknowledgements

The authors acknowledge the technical support provided by the Guangxi Key Laboratory of Intelligent Precision Medicine and Guangxi Anrenxin Biotechnology Co., Ltd.

Competing interests

The authors declare no competing interests.

Ethics declaration

Not applicable.

References