Serum complement C3 as a diagnostic biomarker for metabolic dysfunction -associated steatotic liver disease in middle-aged and elderly adults: a cross-sectional study | BMC Gastroenterology

The present study set out with the objective of investigating the relationship between serum complement proteins (C1q, C3, C4, and total complement) and MASLD as well as SLF in middle-aged and elderly individuals. The study revealed that, in comparison with the control group, the MASLD group exhibited higher levels of C1q, C3, C4, and total complement. Further analysis demonstrated that elevated C3 levels were an moderate independent associated factor for MASLD in middle-aged and elderly individuals, with an inverted U-shaped nonlinear relationship observed. In addition, C3 exerted a direct influence on MASLD and an indirect influence through TG, accounting for 15.6% of the total effect. Furthermore, the diagnostic efficacy of C3 for MASLD in this population was found to be comparable to existing steatotic liver disease diagnostic models. However, in the context of middle-aged and elderly patients with MASLD complicated by SLF, complement proteins, including C3, did not demonstrate an independent association with SLF.

In recent years, the relationship between the complement system and MASLD has garnered increasing attention from the scientific community. The liver, as the primary site of complement protein synthesis, is particularly susceptible to complement-mediated injury, and dysregulation of the complement system can exacerbate hepatic inflammation and fibrosis [21]. The pathogenesis of MASLD is multifactorial in nature, involving chronic inflammation, insulin resistance, lipid deposition and oxidative stress, in which the complement system may play a crucial role [22, 23]. The complement system is a pivotal component of the innate immune system, which plays a critical role in host defence against pathogens, clearance of immune complexes, and lipid metabolism [24,25,26]. Proteomic studies have demonstrated significant upregulation of complement proteins in MASLD patients [27]. Mendelian randomisation analyses further support that elevated C3 levels are significantly associated with an increased risk of MASLD (OR = 1.65 [95% CI: 1.40–1.94]). A multitude of studies have reported a positive correlation between C3 levels and both the prevalence and severity of MASLD, with individuals in the highest quartile of C3 levels exhibiting a higher susceptibility to MASLD, suggesting that C3 serves as an independent associated factor for the disease [28,29,30,31]. Previous research on the association between complement and MASLD has primarily focused on the general adult population. In line with these findings, our study identified a positive correlation between elevated C3 levels and MASLD prevalence in middle-aged and elderly individuals, confirming C3 as an independent associated factor for MASLD in this demographic. Notably, the observed difference in C3 levels between MASLD patients and controls (132.0 mg/dL vs. 108.0 mg/dL) represents an increase of 22%. This magnitude of change is analogous to the differential observed in established inflammatory biomarkers during metabolic dysfunction and may reflect a significant burden of complement-mediated inflammation in MASLD patients [32].

The complement system is primarily activated through three pathways: the classical, lectin, and alternative pathways [21]. As the central component of the complement system, C3 serves as the convergence point for all three activation routes. Cleavage of C3 generates C3a and C3b, subsequently triggering C5 cleavage and amplifying the inflammatory cascade [33]. Notably, C3a, a key activation product and potent inflammatory effector, has been implicated in exacerbating TG metabolic dysregulation [34]. Moreover, acylation-stimulating protein, a metabolic derivative of C3, promotes TG synthesis and aggravates insulin resistance, thereby facilitating hepatic lipid accumulation [35]. A previous study demonstrated that TG mediates the association between C3 and MASLD, accounting for 14.0% of the total effect [28]. Consistent with this, our study also identified a partial mediation effect of TG in the relationship between C3 and MASLD among middle-aged and elderly individuals, with a mediation proportion of 15.6% (95% BootCI: 0.019–0.102). However, this mediation analysis must be interpreted with considerable caution. Given the cross-sectional design of our study, we cannot establish temporality or rule out unmeasured confounding between C3 and TG levels. It remains biologically plausible that the relationship could be reversed or subject to residual confounding. Therefore, these results should be viewed as hypothesis-generating, illustrating a potential pathway that requires validation through more robust methodological approaches such as longitudinal mediation frameworks or Mendelian randomization studies. These findings reinforce the hypothesis that C3 is associated with MASLD pathogenesis partly through dysregulation of lipid metabolism, offering mechanistic insights that may support the development of targeted therapeutic strategies.

This study is pioneering in its revelation of an inverted U-shaped nonlinear relationship between C3 levels and MASLD in middle-aged and elderly individuals. Within the range of 127–167 mg/dL, C3 was identified as an independent associated factor for MASLD. Conversely, when levels fell below 127 mg/L or exceeded 167 mg/L, the association with MASLD risk was attenuated. This nonlinear association may reflect complex underlying physiology; however, the observed reversal of association at higher levels should be interpreted with caution. Moderately elevated C3 levels (127–167 mg/L) are likely to promote hepatic inflammation and lipid accumulation through the aforementioned mechanisms. The apparent risk attenuation at lower and extremely high C3 levels may be influenced by statistical factors, unmeasured confounding variables, or yet unexplored biological pathways rather than necessarily representing a true protective effect. For instance, very high C3 levels could be associated with other compensatory metabolic or inflammatory states that complicate the relationship. While negative feedback mechanisms or anti-inflammatory compensation have been proposed in some contexts, direct evidence in MASLD is currently lacking, and these explanations remain hypothetical [36]. These findings may identify a critical range of C3 levels relevant for MASLD management. Animal studies support complement components as potential therapeutic targets, with research in rat models demonstrating that C3aR and C5aR antagonists significantly ameliorate high-fat diet-induced obesity, insulin resistance, and adipose tissue inflammation [37]. Future investigations should prioritize validating this nonlinear relationship and elucidating its underlying mechanisms. For individuals with moderately elevated C3, monitoring and risk assessment may be warranted. However, clinical strategies targeting C3 modulation would require substantial additional evidence from intervention studies. This finding provides novel insights into the complex relationship between complement activation and MASLD in ageing populations, but requires validation in independent cohorts and further mechanistic investigation.

The present study appraised the diagnostic performance of complement proteins and three non-invasive models for the identification of steatotic liver disease in middle-aged and elderly individuals with MASLD. The findings indicated that C3 demonstrated optimal diagnostic efficacy for MASLD (AUC = 0.80). At an optimal cut-off value of 116.50 mg/dL, C3 demonstrated a sensitivity of 80.45% and a specificity of 68.89% for the diagnosis of MASLD. The three non-invasive steatotic liver disease diagnostic models assessed were the FLD index, ZJU index, and TyG index. The FLD index was found to be a simple and efficient screening tool for MASLD, specifically developed for Chinese populations [38]. The ZJU index, developed by researchers from the First Affiliated Hospital of Zhejiang University, was established through a cross-sectional study of 9,602 participants and is suitable for MASLD screening in Chinese community populations [39]. A subsequent validation study involving 19,804 subjects confirmed the ZJU index’s diagnostic performance, demonstrating an AUC of 0.925 for MASLD detection, which was significantly superior to the FLI index [40]. The TyG index, derived from a cross-sectional study of 10,761 Chinese health examination participants aged ≥ 20 years, exhibited high sensitivity for MASLD detection when the index value exceeded 8.5 [41]. Validation of these three models in our study cohort revealed comparable diagnostic performance between the ZJU index (AUC = 0.80) and FLD index (AUC = 0.82) (P >0.05), both of which outperformed the TyG index (AUC = 0.75). Notably, C3 demonstrated diagnostic value equivalent to these established indices (FLD index: AUC = 0.82; ZJU index: AUC = 0.80; TyG index: AUC = 0.75). These findings carry important clinical implications. C3 level exceeding 116.50 mg/dL may indicate increased risk of steatotic liver disease, warranting further confirmation through ultrasound or CT imaging. However, as the measurement of C3 alone cannot provide a definitive diagnosis MASLD in middle-aged and elderly individuals, a comprehensive evaluation incorporating additional diagnostic parameters remains necessary. Future research should explore the potential value of combining C3 with other non-invasive diagnostic models to enhance MASLD detection in this population.

The present study revealed an inverse association between elevated C3 levels and the risk of SLF in middle-aged and elderly MASLD patients. Liver fibrosis staging is recognised as one of the most accurate predictors of mortality in MASLD patients, providing critical information about disease severity and prognosis [42]. The development of liver fibrosis in MASLD is associated with multiple associated factors, including genetic predisposition, metabolic disturbances (visceral obesity, insulin resistance, and unhealthy dietary patterns), and immune dysregulation [9]. Preliminary studies have indicated a potential role for complement activation in the promotion of hepatic fibrosis in MASLD. Complement activation products, specifically C3a and C5a, have been observed to interact with complement receptors present on the surfaces of macrophages, thereby modulating their inflammatory responses [43]. Both C3a and C5a have been shown to possess potent chemotactic and pro-inflammatory properties, with their receptors being widely expressed on various immune cells, particularly macrophage [44]. In the liver, Kupffer cells/macrophages activated by complement activation secrete inflammatory chemokines that recruit peripheral monocytes, thereby exacerbating hepatic inflammation in MASLD [45]. Beyond their pro-inflammatory effects, activated macrophages also promote fibrosis by modulating hepatic stellate cells [46]. Through the secretion of cytokines such as TGF-β, TNF-α, and IL-1β, activated Kupffer cells drive HSC differentiation into pro-fibrotic phenotypes that overexpress collagen and α-smooth muscle actin, thereby contributing to the development of liver fibrosis [46]. Most notably, a seminal study published in 2024 mechanistically linked the activation of complement C3 directly to the progression of MASH. This study showed that C3 potently stimulates the formation of neutrophil extracellular traps, which exacerbate hepatic inflammation and injury [47]. This finding extends the pro-fibrotic mechanisms of complement beyond the activation of macrophages in the liver.

It is important to note that the findings of this study do not contradict the established mechanisms. During advanced stages of fibrosis related to MASLD, C3 and C4 concentrations typically decline, primarily reflecting deteriorating hepatic synthetic function [48, 49]. In the multivariate logistic regression analysis conducted, elevated C3 levels were not identified as an independent protective factor against SLF in this population. However, this null finding must be interpreted with caution. Despite using age-adjusted FIB-4 cut-offs, residual confounding from unmeasured factors or the limited sample size of the SLF subgroup may have obscured a clinically relevant association. Therefore, we cannot conclusively rule out a relationship between complement levels and fibrosis progression. While an association was observed between C3 and risk reduction of SLF, the data does not support an independent protective role in fibrosis progression. These results underscore the intricate relationship between complement proteins and liver fibrosis in ageing MASLD patients. The findings underscore the necessity for large-scale prospective cohort studies to further elucidate the precise role of complement components in MASLD-associated fibrogenesis.

While this study provides insights into the relationship between C3 and MASLD in middle-aged and elderly populations, several limitations must be acknowledged. Firstly, the single-center design may limit the generalizability of our findings to broader populations. Secondly, although the identified association between C3 and MASLD is biologically plausible, the temporal sequence cannot be determined by cross-sectional design. We cannot rule out the possibility of reverse causation, whereby hepatic metabolic dysfunction and chronic inflammation in MASLD may lead to elevated complement synthesis, rather than C3 directly contributing to disease pathogenesis. Thirdly, while we adjusted for extensive covariates, residual confounding and the potential for misclassification bias-due to the reliance on ultrasonography for MASLD diagnosis and FIB-4 for SLF diagnosis-may influence the observed effects. Future studies employing multi-center longitudinal designs and more sensitive imaging are warranted. Fourthly, the exploratory analysis of multiple complement proteins necessitates caution in interpreting the results for analytes with weaker associations, as some findings may be susceptible to type I error due to multiple testing. Our mechanistic interpretations are also constrained by the lack of direct measurement of complement activation products and inflammatory cytokines. Finally, while the diagnostic utility of C3 is promising, its successful integration into clinical practice requires careful consideration of cost-effectiveness, availability across diverse healthcare settings, and seamless incorporation into existing diagnostic algorithms.

Future research should focus on validating these findings in larger, multicenter cohorts of middle-aged and elderly individuals to enhance generalizability. Prospective cohort studies with long-term follow-up would provide more robust evidence regarding causal relationships. More refined stratification analyses and stricter control of potential confounders would improve the reliability of future studies. The diagnostic accuracy could be further enhanced by developing multivariate models combining C3 with existing non-invasive steatotic liver disease diagnostic indices. At the basic research level, investigating the molecular mechanisms through which C3 is associated with to MASLD pathogenesis in aging populations – particularly its interactions with hepatocytes and inflammatory pathways – will help clarify the complement system’s precise role in age-related hepatic steatosis, inflammation and fibrosis. Such mechanistic insights could inform the development of complement-targeted precision therapies for elderly patients. Furthermore, evaluating complement inhibitors in aged MASLD animal models to assess their therapeutic effects on hepatic inflammation and fibrosis would provide critical experimental evidence for clinical translation in this specific population, while simultaneously revealing the complement system’s key mechanistic contributions to MASLD progression in aging individuals.