Evaluation of hepatic sonic hedgehog protein expression in the diagnosis of metabolic dysfunction-associated steatohepatitis

Abstract

Metabolic dysfunction-associated steatohepatitis (MASH) is an advanced form of metabolic dysfunction-associated fatty liver disease, with diagnosis relying on liver biopsy. The identification of ballooned hepatocytes in routine hematoxylin-eosin staining is subjective and exhibits significant interobserver variability. Sonic hedgehog homolog (SHH) is specifically expressed in ballooned hepatocytes and holds potential as a positive immunohistochemical marker. This review evaluates the diagnostic value of SHH expression in MASH based on available evidence. Studies demonstrate that SHH staining improves the consistency of interpretation of ballooned hepatocytes and correlates with disease severity, serum biomarkers, and fibrosis staging. However, most evidence comes from single-center retrospective studies with a limited number of observers. The independent predictive value of SHH requires validation through prospective multicenter cohort studies. In summary, SHH immunohistochemistry is a promising adjunctive diagnostic tool for MASH, though its clinical application remains in the preliminary stages.

Key Words: Sonic hedgehog; Metabolic dysfunction-associated steatotic liver disease; Metabolic dysfunction-associated steatohepatitis; Ballooned hepatocytes; Liver fibrosis

Core Tip: Sonic hedgehog homolog (SHH) is specifically expressed in ballooned hepatocytes and can be detected by immunohistochemistry. SHH staining may improve the consistency of ballooned hepatocyte interpretation and is associated with the severity of metabolic dysfunction-associated steatohepatitis. However, current evidence primarily comes from single-center retrospective studies. SHH immunohistochemistry is a promising adjunctive diagnostic tool, but multicenter prospective validation is still required before routine clinical application.

INTRODUCTION

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously referred to as non-alcoholic fatty liver disease (NAFLD), refers to hepatic steatosis occurring in the presence of one or more cardiometabolic risk factors without harmful alcohol intake[1]. NAFLD encompasses a broad spectrum of diseases and can be further classified into non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), representing an increasingly severe global health burden that affects approximately one-quarter of the adult population worldwide[2,3].

Non-alcoholic steatohepatitis is an advanced form of NAFLD, characterized by hepatic steatosis, inflammation, hepatocyte injury, and varying degrees of fibrosis[4]. Metabolic dysfunction-associated steatohepatitis (MASH) is marked by inflammation and hepatocyte injury in the hepatic lobules and portal areas, presenting as ballooning degeneration, fibrosis, and steatosis. Some NASH patients may progress to cirrhosis and exhibit a higher risk of hepatocellular carcinoma[5,6]. Therefore, the diagnosis of MASH is crucial for risk stratification in MASLD patients. Currently, histological diagnosis remains the gold standard for differentiating NASH from NAFL[5,7,8]. Defining MASH requires liver biopsy, with diagnosis based on three key criteria: Hepatic steatosis ≥ 5%, lobular inflammation, and hepatocyte injury characterized by ballooning hepatocytes[9]. However, conventional staining techniques often fail to identify ballooning hepatocytes, leading to interobserver and intraobserver diagnostic discrepancies[10]. Several studies have reported that immunohistochemical staining with cytokeratin 8/18 improves the detection of ballooned hepatocytes[11,12]. Cytokeratin 8/18 exhibits diffuse cytoplasmic expression in normal hepatocytes but is not expressed in ballooned hepatocytes, and it can also label Mallory-Denk bodies, which may be present in degenerated hepatocytes[11].

Meanwhile, hepatocyte sonic hedgehog homolog (SHH) signaling proteins can also serve as potential biomarkers for identifying and quantifying ballooning changes in hepatocytes. Preliminary evidence suggests that SHH expression correlates with the histological severity of MASH[13,14] and may participate in the regulation of disease progression[15]. Guy et al[13] demonstrated that qualitative assessment of SHH expression in liver biopsy specimens via immunohistochemistry (IHC) was associated with diagnostic outcomes and treatment responses in NASH. Recent studies by Han et al[16] indicate that elevated SHH expression even at low levels may serve as an independent predictor of disease progression in NASH patients. Can SHH IHC become a reliable adjunctive tool for MASH diagnosis? To understand the diagnostic value of SHH as a biomarker, it is essential to first clarify its role in the pathogenesis of MASH. This article systematically evaluates existing diagnostic evidence based on the pathophysiological mechanisms of SHH and explores its translational prospects and limitations. The hypothesis regarding the role of SHH in the development mechanism of MASH is summarized in Figure 1.

Figure 1 Schematic hypothesis of sonic hedgehog homolog in the pathogenesis of metabolic dysfunction-associated steatohepatitis. CK8/18: Cytokeratin 8/18; SHH: Sonic hedgehog homolog; TGF-β1: Transforming growth factor-β1.

THE ROLE OF SHH IN THE PATHOGENESIS OF MASH

Overview of the SHH pathway

The hedgehog (HH) pathway is a conserved and highly complex signaling cascade involved in cell growth, survival, and fate determination. The HH pathway comprises three ligands: Sonic HH, Indian HH (Ihh), and desert HH, which exhibit distinct tissue localization and molecular functions. The HH pathway can be simplified into four fundamental components: HH ligands, receptor Patched, signal sensor smoothened, and effector transcription factor glioma-associated oncogene homolog (GLI)[17]. Under normal physiological conditions, the HH signaling pathway participates in embryonic development and tissue repair processes. In healthy adult livers, this pathway remains largely dormant and is reactivated only during liver injury or regeneration[18]. Mechanistically, the SHH signaling pathway regulates endoplasmic reticulum stress, oxidative stress, and hepatic lipotoxicity injury - cellular stress types that serve as core drivers of hepatocyte injury in MASH models[19-21].

To systematically evaluate the evidence of SHH in MASH diagnosis and pathogenesis, we extracted and organized data from the included original studies (Table 1). We searched for published meta-analysis articles in the following databases: Web of Science, PubMed, and China National Knowledge Infrastructure. Three sets of keywords were used: (1) Sonic HH; (2) NAFLD, NASH, MASLD, MASH; and (3) Liver, hepatic. The search time range covered from the establishment of the databases to March 30, 2026. Additionally, a manual search of reference lists was also performed to identify other eligible studies. Inclusion criteria were: (1) Original studies (not reviews, commentaries, or case reports); (2) Subjects involving humans, animals, or in vitro models; (3) Detection of SHH expression; and (4) Reporting data on SHH’s correlation with MAFLD/NAFLD/NASH/MASH diagnosis, severity, consistency, mechanisms, or clinical applications. Exclusion criteria included: (1) Studies unrelated to the liver; (2) Studies focusing solely on other Hh pathway members (e.g., Ihh, GLI) without SHH detection; (3) Lack of original data reporting SHH disease associations; and (4) Inability to obtain full-text access. Based on the above literature screening process, we ultimately included 19 original studies covering diagnostic research, clinical trial sub-studies, mechanistic studies (cellular/animal models), and in vitro models. To clearly present the core evidence of each study, we extracted key information including study type, sample size, major SHH-related findings, diagnostic performance and consistency, disease severity association, and limitations.

Table 1 Key characteristics and sonic hedgehog homolog-related findings of included original studies.

Main research methods	Sample	Key findings related to SHH	Limitations	Ref.
Diagnostic studies	Liver tissue from 100 cases of hepatocellular carcinoma postoperatively	SHH IHC assisted in identifying ballooned cells; combined with hematoxylin and eosin staining resulted in ballooned hepatocyte grading upgrade in 20 cases and downgrade in 0 cases	Single-center design; 2 observers	Kusano et al[41]
Cross-sectional study	69 cases of NAFLD	SHH expression was significantly associated with ballooning degeneration, fibrosis, and M65/M30; moreover, SHH positivity in diabetic patients independently predicted advanced fibrosis	Small sample size and mixed biopsy types	Estep et al[14]
Substudy of clinical trial	59 cases of NASH	Vitamin E treatment resulted in SHH-positive hepatocytes reduction, which was associated with decreased AST levels, reduced ballooning degeneration, and reduced fibrosis	Subgroup analysis sample size was small	Guy et al[38]
Mechanistic clinical and applications	7 cases of NASH and 3 cases of A1AT	Endoplasmic reticulum stress induces SHH production in hepatocytes; the number of SHH-positive cells correlates with fibrosis staging and degree of ballooning degeneration	Small sample size	Rangwala et al[20]
Mechanism, animal model and clinical	16 cases of NAFLD+ animals	HH pathway activation mediates epithelial-mesenchymal transition in ductal cells and promotes fibrosis; fibrosis exacerbates in Ptc+/- mice	Small clinical sample size	Syn et al[25]
Mechanism research	3 cases of NASH+ cells	Ballooned hepatocytes downregulate caspase-9 and induce SHH via c-Jun N-terminal kinase/activator protein-1, forming an autophagic survival pathway	Only 3 clinical samples were available	Kakisaka et al[21]
In vivo	Mouse model	As little as 2%-5% of hepatocytes expressing SHH can induce hepatic fibrosis; SHH promotes the transformation of hepatocellular adenoma into hepatocellular carcinoma	Mouse model; not validated with inhibitors	Chung et al[28]
In vitro model	Human primary hepatocytes + fibroblasts	The 3D cell sheet model successfully induced human ballooned hepatocytes, with SHH secretion increasing by 1.4-2.0 times and myofibroblast activation	Utilizes skin fibroblasts, which are non-physiological	Gao et al[42]
Fundamental research	Mouse + patient serum	SHH promotes NASH via the HSP90β-miR-28-5p axis; serum miR-28-5p can serve as a diagnostic biomarker	SHH tissue staining has not been evaluated	Zhang et al[37]
Clinical intervention	436 cases (subgroup: 120 cases)	Improvement of NASH after bariatric surgery with significant reduction in plasma SHH levels	Non-diagnostic studies	Cabré et al[39]
Clinical + basic	135 cases of NASH+ cells	SHH and TGF-β1 exhibit a positive correlation in NASH (r = 0.6) and are co-localized around ballooned hepatocytes of HSCs	No independent diagnostic data available	Zhou et al[15]
Mechanism research	Mouse cells	The HH signal induces glycolytic reprogramming through HIF1α, driving HSCs activation	SHH has not been validated in NASH models	Chen et al[22]
Mechanism research	db/db mice + cells	Leptin induces SHH expression via PI3K/Akt pathway, activates HH signaling, and promotes HSCs transdifferentiation	The leptin deficiency model cannot fully simulate human conditions	Choi et al[30]
Mechanism research	ob/ob/db/db mouse	Leptin deficiency leads to decreased Smo expression, attenuation of HH signaling, and impaired HSCs activation	Single-gene model, ineffective with exogenous leptin therapy	Xie et al[31]
Mechanism research	Transgenic mice	The HH signal induces HH-induced long non-coding RNA through Gli, which regulates PPARγ via IGF2BP2, thereby influencing hepatic lipid metabolism		Jiang et al[34]
Case-control	38 cases of liver cirrhosis (8 cases of NASH)	SHH levels are significantly elevated in liver cirrhosis without variation across different etiologies; yes-associated protein 1 nuclear translocation is limited	Only 8 cases in the NASH group	Mohagheghi et al[29]
Mechanism research	Mouse + LX-2 cells	The HIF-1α inhibitor AMSP-30m alleviates hepatic fibrosis by inhibiting the SHH pathway; cyclopamine exhibits synergistic effects with AMSP-30m	CCl4 model	Lu et al[36]
Mechanism research	CCl4 rats	Bromelain acid B alleviates hepatic fibrosis by inhibiting the expression of SHH, Ptch1, Smo, and Gli1	Only mRNA was detected, with no protein validation performed	Tao et al[40]
Retrospective cohort	190 cases of MASLD	SHH IHC was used as an adjunct diagnostic tool for MASH; SHH served as an independent predictor (OR = 7.15)	Single-center design, retrospective nature, and wide CI span	Han et al[16]

SHH: Sonic hedgehog homolog; IHC: Immunohistochemistry; NAFLD: Non-alcoholic fatty liver disease; NASH: Non-alcoholic steatohepatitis; AST: Aspartate aminotransferase; A1AT: Alpha-1 antitrypsin; HH: Hedgehog; Ptc+/-: Patched haploidy deficiency; TGF-β1: Transforming growth factor-β1; HSC: Hepatic stellate cell; HIF1α: Hypoxia-inducible factor 1-α; PI3K: Phosphatidylinositol 3-kinase; Akt: Protein kinase B; Gli: Glioma-associated oncogene homolog; PPARγ: Peroxisome proliferator-activated receptor γ; IGF2BP2: Insulin-like growth factor 2 mRNA-binding protein 2; CCl₄: Carbon tetrachloride; MASLD: Metabolic dysfunction-associated steatotic liver disease; OR: Odds ratio; CI: Confidence interval.

Ballooned hepatocytes are the primary source of SHH

The ballooning degeneration of hepatocytes, as a characteristic lesion of MASH, continuously undergoes intense endoplasmic reticulum stress and oxidative stress, becoming the primary source of SHH production[22]. Rangwala et al[20] demonstrated through in vitro experiments that tunicamycin endoplasmic reticulum stress could elevate SHH mRNA levels in primary mouse hepatocytes by 16.1-fold and Ihh mRNA levels by 5.4-fold. Additionally, conditioned medium from erythromycin-treated groups increased Gli reporter gene activity by 14.7-fold, with cyclopamine blocking this effect, thereby revealing for the first time the mechanism by which endoplasmic reticulum stress induces SHH production via the Hh pathway. Kakisaka et al[21] further elucidated the autophagic survival function of SHH under lipotoxic stress, finding significantly reduced caspase-9 expression in ballooned hepatocytes of NASH patients. In caspase-9-knockdown Huh-7 cells, lipotoxic stress induced SHH expression through the c-Jun N-terminal kinase/activator protein-1 pathway, forming an autophagic survival signaling pathway, while inhibition of the SHH pathway triggered apoptosis. Studies also showed that when hepatocyte injury was prevented (e.g., cysteine protease-2 deficiency), cells became resistant to free fatty acid-induced apoptosis, and SHH expression was not induced[23]. These findings suggest that SHH expression is closely correlated with the state of hepatocyte injury.

Association of SHH with disease severity and fibrosis in MASH

However, under pathological conditions, persistent abnormal activation of this pathway can induce pathological changes such as liver fibrosis[18]. Additionally, SHH expression is negatively correlated with prognosis in patients with NASH[24]. Clinical practice and multiple animal model studies on NAFLD have confirmed that SHH pathway activation is closely associated with the risk of hepatocyte injury/death, liver inflammation, severity of liver fibrosis, and incidence/mortality rates of liver-related diseases[13,25-28]. Rangwala et al[20] first reported that SHH protein is overexpressed in ballooned hepatocytes and acts as a pro-fibrotic factor in diseased livers; SHH IHC revealed positive expression of SHH in ballooned hepatocytes, with the number of SHH-positive cells correlating with fibrosis staging. Syn et al[25] systematically elucidated the mechanism of SHH in NASH fibrosis, finding that SHH treatment induces ductal cell expression of α-smooth muscle actin, downregulates bone morphogenetic protein 7 and E-cadherin, and promotes epithelial-mesenchymal transition. In Patched haploidy deficiency (Ptc+/-) mice, enhanced Hh pathway activity was observed, and Ptc+/- mice exhibited more pronounced fibrosis compared to wild-type mice after methionine-choline-deficient diet-induced NASH. Clinical sample analysis showed progressive increases in SHH-positive cells, Gli2-positive cells, and S100A4-positive cells from NAFL to NASH and then to cirrhosis, with S100A4-positive cells in cirrhotic patients being ≥ 6 times higher than those in NAFL. Chung et al[28] established a liver-specific SHH-overexpressing transgenic mouse model using hydrodynamic transfection technology, directly validating the pathogenic role of SHH in hepatic fibrosis. The study found that only 2%-5% of hepatocytes expressed SHH, and significant hepatic fibrosis emerged in mice after 6 months, accompanied by activation of hepatic stellate cells (HSCs), upregulation of pro-fibrotic genes, and increased hepatocyte apoptosis. Although SHH overexpression alone for 13 months did not spontaneously induce hepatocellular tumors, co-expression with P53R172H and KRASG12D significantly promoted the transformation of hepatocellular adenomas into hepatocellular carcinoma, suggesting that SHH indirectly promotes hepatocellular carcinoma development by creating a pro-oncogenic microenvironment (fibrosis, cellular injury). This study was the first to demonstrate in animal models the pathogenic threshold effect of SHH - overexpression in only 2%-5% of hepatocytes could induce fibrosis, indicating that low-level SHH is pathogenic. However, the hydrodynamic transfection used in this study involved acute overexpression rather than chronic endogenous activation, differing from the gradual progression observed in NASH. Current research suggests that SHH may participate in the pathological process of MASH through multiple mechanisms. Chen et al[22] further elucidated the role of HH signaling in hepatic fibrosis from a metabolic regulation perspective, finding that during the transdifferentiation of quiescent HSCs into myofibroblasts, glycolysis-related genes were upregulated 6-25-fold, while gluconeogenesis-related genes decreased by 90%. Mechanistic studies indicate that HH signaling mediates this metabolic reprogramming through hypoxia-inducible factor 1-α (HIF-1α) activation, with Gli1/Gli2 directly binding to the HIF-1α promoter. In myofibroblast-specific Smo knockout mice, hepatic fibrosis was significantly alleviated, providing causal evidence for the pathogenic effects of SHH.

Crosstalk between SHH and other signaling pathways

In studies investigating the interactions between SHH and other signaling pathways, Zhou et al[15] found a positive correlation between SHH and transforming growth factor-β1 (TGF-β1) expression in NASH (r = 0.6), with both co-localizing in HSCs surrounding ballooned hepatocytes, suggesting synergistic regulation of NASH progression. Mohagheghi et al[29] demonstrated significantly elevated SHH levels across various etiologies of cirrhosis without inter-etiological differences, while yes-associated protein 1 primarily functions through cytoplasmic accumulation with limited nuclear translocation. These findings indicate that SHH and TGF-β1 constitute a shared fibrotic pathway and may continue to play a dominant role in fibrosis progression, whereas yes-associated protein 1’s function may be more focused on regeneration rather than fibrosis. Notably, leptin-deficient ob/ob mice exhibited obesity but reduced susceptibility to steatohepatitis and hepatic fibrosis. One possible mechanism involves leptin-mediated SHH activation, as decreased SHH activity in leptin-deficient maternal mice may suppress inflammation and fibrosis[30,31].

Beyond its role in liver repair, multiple studies have demonstrated HH signaling’s involvement in lipid metabolism regulation of adipocytes[32,33]. Recent hepatocyte studies further elucidated this mechanism[34]. SHH regulates long non-coding RNA (HH-induced long non-coding RNA) expression, thereby participating in high-fat diet-induced hepatic lipid metabolism regulation[35]. By inducing osteopontin expression to promote hepatic inflammatory cell infiltration and altering glucose metabolism in HSCs to facilitate fibrosis[22,34], SHH ligands secreted by damaged hepatocytes can activate TGF-β1, subsequently promoting HSCs transformation, with both mechanisms jointly regulating the disease progression of NASH[15]. Notably, intervention targeting the SHH pathway with inhibitors improves NASH outcomes in human and murine models, further supporting the critical role of SHH in the pathophysiology of MASH[22]. Recent studies targeting the SHH pathway have further enriched our understanding of its functions. A recent study by Lu et al[36] revealed that the HIF-1α inhibitor AMSP-30m significantly reduced the expression of SHH pathway-related proteins in carbon tetrachloride-induced hepatic fibrosis models and alleviated fibrosis severity. In vitro experiments further confirmed that the SHH inhibitor cyclopamine inhibits HIF-1α-induced HSCs activation. Related studies demonstrated no correlation between circulating serum SHH signaling and hepatic SHH signaling[14], suggesting potential abnormalities in post-translational modifications of SHH in ballooned hepatocytes, which may involve the processing of full-length SHH into N-terminal and C-terminal fragments (e.g., cholesterol modification) or the proteasomal degradation of SHH C-terminal fragments. Zhang et al[37] revealed a novel mechanism of SHH in NASH, demonstrating that SHH promotes NASH progression through the HSP90β-miR-28-5p axis. In hepatocytes, SHH reduces ubiquitination of HSP90β via the deubiquitinase USP31, thereby preventing its degradation and facilitating hepatic lipid synthesis. HSP90β levels are significantly elevated in NASH mouse models, inducing secretion of miR-28-5p-rich exosomes. miR-28-5p directly targets and downregulates Rap1b levels, subsequently enhancing nuclear factor kappa B transcriptional activity in macrophages and stimulating inflammatory cytokine expression. The study also found markedly elevated serum miR-28-5p levels in NASH patients, suggesting its potential as a diagnostic biomarker for NASH. This discovery provides a new mechanistic perspective on SHH’s role in NASH-SHH not only exerts paracrine effects on HSCs but also influences immune cell function through exosome-mediated intercellular communication. However, the study did not directly evaluate the correlation between SHH tissue staining and serum biomarkers, and the specific role of exosomes in SHH signaling requires further validation. Future research should assess the synergistic value of serum miR-28-5p combined with liver tissue SHH staining in MASH diagnosis and explore the feasibility of SHH-positive exosomes as non-invasive diagnostic markers. This finding reminds us that upregulated SHH expression may merely be a node in a complex signaling network, and its independent predictive value in diagnosis should be interpreted within a broader context of signal interactions, avoiding its interpretation as a single “driving” biomarker.

However, these findings also highlight the potential of SHH as a therapeutic response biomarker. Guy et al[38] first demonstrated in the PIVENS clinical trial sub-study that the vitamin E group exhibited more significant reduction in SHH-positive hepatocytes compared to the placebo group (P < 0.05), and that SHH-positive hepatocytes decreased more markedly in treatment responders than non-responders (P = 0.007). Cabré et al[39] observed that postoperative NASH improvement was associated with a significant decline in plasma SHH levels in patients undergoing bariatric surgery. These findings suggest that SHH reduction may serve as an effective histological marker for NASH treatment efficacy. Additionally, Tao et al[40] found that salvianolic acid B alleviated hepatic fibrosis by inhibiting SHH, Ptch1, Smo, and Gli1 expression, while Lu et al[36] reported that the HIF-1α inhibitor AMSP-30m also exerted anti-fibrotic effects through SHH pathway inhibition. These preclinical studies provide supportive evidence for the role of the SHH pathway in fibrosis, but their primary value lies in indicating potential therapeutic intervention rather than establishing causal evidence in human MASH.

The aforementioned mechanism studies provide a theoretical foundation for SHH as a histological marker of MASH. Since SHH is specifically highly expressed in ballooned hepatocytes and correlates with disease severity, immunohistochemical detection of SHH may assist in pathological diagnosis. The following section will systematically evaluate current research evidence regarding SHH IHC in MASH diagnosis.

RESEARCH EVIDENCE OF SHH IHC IN THE DIAGNOSIS OF MASH

Multiple studies have demonstrated that SHH IHC significantly improves the detection accuracy of ballooned hepatocytes and may enhance inter-pathologist interpretation consistency. Kusano et al[41] evaluated the value of SHH and keratin 8/18 IHC for ballooned cell detection in postoperative liver tissues from 100 hepatocellular carcinoma cases. They found that when using hematoxylin and eosin staining alone, the weighted kappa coefficient for ballooned degeneration scoring by two pathologists was 0.710, whereas the kappa coefficient increased to 0.806 after combined SHH IHC, with background liver disease diagnosis altered in 15% of patients. These findings suggest that SHH IHC can effectively reduce interobserver variability and improve the reliability of MASH pathological diagnosis. However, the study had limitations including single-center design and involvement of two observers from the same department, which may overestimate real-world consistency levels. Furthermore, the study primarily focused on changes in ballooned hepatocytes scores rather than the MASH diagnosis itself. Although a “15% change in background liver disease diagnosis” was reported, the diagnostic basis was the NAFLD activity score (requiring simultaneous presence of steatosis, inflammation, and ballooning), not merely SHH positivity. Additionally, only the following data were reported: “20 cases showed upgrading of ballooned hepatocytes scores after SHH staining, with no cases of downgrading”, and “15 cases of background liver disease diagnosis changes”. These data can roughly suggest that SHH staining “detected” more ballooned hepatocytes, but they did not provide sensitivity or specificity under the “true diagnostic gold standard”. For example, no assessment was made on the proportion of SHH-positive cells that were genuine ballooned hepatocytes (positive predictive value), nor was there evaluation of whether SHH-negative cells contained missed ballooned hepatocytes (false-negative rate).

Estep et al[14] conducted quantitative analysis of SHH expression in liver biopsy tissues from 69 NAFLD patients using computer-aided morphometry, revealing extremely high interobserver correlation (rho = 0.939, P < 0.0001), indicating that computer-assisted morphometry quantification techniques can effectively eliminate subjective interpretation differences. The study also identified SHH as an independent predictor of advanced fibrosis (odds ratio = 1.986), though the small sample size and mixed use of puncture biopsy and wedge biopsy specimens may introduce sampling variability. Furthermore, the study did not establish a “diagnostic threshold” for SHH nor calculate the sensitivity/specificity using histological NASH as the gold standard. The text mentions that “approximately half of the patients had extremely low or negative SHH levels”, suggesting a high false-negative rate in SHH staining, but this was not quantified.

Han et al[16] further validated this finding in 190 patients with MASLD, demonstrating that SHH IHC improved the interpretation consistency kappa value from 0.65 to 0.85, and SHH was an independent predictor of MASH (odds ratio = 7.15). Although the study had a large sample size, it may have been subject to selection bias due to its single-center retrospective design. Additionally, the odds ratio value exhibited a wide 95% confidence interval range (1.43-35.85), raising concerns about the precision of point estimates. Moreover, the odds ratio value reflects association strength rather than diagnostic accuracy.

Although these studies consistently demonstrate that SHH IHC enhances diagnostic consistency for MASH and correlates with disease severity, the strength of evidence warrants critical evaluation. The diagnosis of MASH remains complex, yet current research only establishes associations between SHH expression and MASH diagnosis through retrospective studies. Moreover, SHH expression may only improve consistency in single histological features, and staining interpretation criteria require further quantification. Further exploration and more robust evidence are still needed.

CONCLUSION

Although the evidence reviewed in this article indicates an undeniable coexistence of increased SHH levels in the MASH cohort, evidence for causality remains weak. Similar to multiple studies on non-alcoholic fatty liver disease, observational and retrospective studies dominate the literature, which inherently carry biases. However, this reflects the challenges encountered in real-world clinical practice and accurately mirrors the diagnostic dilemmas associated with MASH. Regrettably, there is currently no conclusive evidence supporting the inclusion of SHH in diagnostic criteria. Given the nearly universally reproducible detection methods, SHH emerges as an attractive biomarker. Undoubtedly, given the significant challenges in MASH diagnosis and prognosis, efforts should be made to maximize the potential role of SHH.