Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide, affecting about 25% of the adult population, of which 10% - 30% may progress to non-alcoholic steatohepatitis (NASH) and hepatic fibrosis, ultimately leading to cirrhosis or hepatocellular carcinoma [1]. NAFLD is characterized by the rapid progression of abnormal accumulation of hepatic lipids, hepatocellular damage, necroinflammation, and fibrosis [2]. In the United States, the prevalence of NAFLD continues to climb with the obesity and metabolic syndrome epidemics and has become the second leading cause of liver transplantation [3]. Therefore, early identification of hepatic steatosis and liver fibrosis is critical to intervene in disease progression [4], but current clinical reliance on liver biopsy carries invasive risks. Vibration-controlled transient elastography (VCTE) is being increasingly used clinically as a non-invasive alternative. Recent observational studies have shown that VCTE has high accuracy in assessing hepatic steatosis grading and liver fibrosis staging [5][6].

The C-reactive protein-albumin-lymphocyte (CALLY) index is a novel inflammatory biomarker designed based on CRP, albumin, and lymphocyte counts [7]. In existing studies, CALLY has been widely used for the prognosis of various malignant tumors, including colorectal cancer [8], esophageal cancer [9], gastric cancer [10], oral cancer [11], breast cancer [12], and non-small cell lung cancer [13]. In addition, the CALLY index has a high predictive value in cardiovascular diseases [14]-[16].

Inflammation is a hallmark of NAFLD progression [17], and the CALLY index plays a role in predicting hepatic steatosis and hepatic fibrosis due to the association between inflammation, immunity, and nutrition. However, currently, no studies have focused on the association between the CALLY index and hepatic steatosis and hepatic fibrosis. To address this research gap, we utilized the National Health and Nutrition Examination Survey (NHANES) to investigate the potential relationship between the CALLY Index and hepatic steatosis and liver fibrosis in adults.

In this study, 6226 adults were included based on inclusion and exclusion criteria, and the mean age of the participants was 47.668 years ± 17.297 years. Of these participants, 44.67% were male, 55.33% were female, 12.42% were Mexican American, 34.13% were non-Hispanic white, 24.99% were non-Hispanic black, and 28.46% were from other races. The mean (SD) for CAP was 263.351 dB/m ± 62.846 dB/m, for LSM was 5.757 kPa ± 4.601 kPa, and for CALLY was 1.530 ± 1.176. Table 1 lists all the clinical characteristics of the CAP participants as column-stratified variables. Compared to the non-NAFLD group, the severe steatosis group was more likely to have participants in the lowest quartile. Compared to the non-NAFLD group, the participants in the highest quartile were more likely to be older males, Mexican Americans, and non-Hispanic whites. They also exhibited good education, higher rates of smoking, rates of diabetes and hypertension, and elevated levels of ALT, AST, TC, TG, and LDL, compared with lower levels of HDL. Of particular note, individuals with severe hepatic steatosis had significantly lower ln CALLY values compared with those without NAFLD. In addition, a lower proportion of patients with severe steatosis was found in the highest ln CALLY quartiles (Q3 and Q4), which was significantly different (P < 0.001) compared with the non-NAFLD group.

Table 1. Weighted characteristics of the study population based on the controlled attenuated parameter (CAP).

Mean ± SD for continuous variables: P-value was calculated by a weighted linear regression model. % for categorical variables: P-value was calculated by the weighted chi-square test. LDL-Cholesterol: Low-Density Lipoprotein Cholesterol; HDL-Cholesterol: High-Density Lipoprotein Cholesterol; ALT: Alanine Transaminase; AST: Aspartate Aminotransferase.

Table 2 lists all the clinical characteristics of LSM individuals as column-stratified variables. Compared to the normal group, the cirrhotic group was more likely to be well-educated males exhibiting a higher prevalence of hypertension and diabetes mellitus. As well as elevated ALT, AST, and TG levels, in contrast, they had lower TC and HDL levels. The cirrhosis group had significantly lower ln CALLY than individuals without liver fibrosis, with lower proportions found in the highest ln CALLY quartiles (Q3, Q4, P < 0.001).

This study provides a comprehensive analysis of the association between the CALLY Index and hepatic steatosis and liver fibrosis in US adults using NHANES data from 2017 to 2020. Analysis of data from 6226 participants showed that the CALLY index was negatively associated with hepatic steatosis and hepatic fibrosis in US adults, even after adjusting for potential confounders through multivariate linear regression. A higher CALLY index was associated with a lower likelihood of angina. Notably, there was an inverted U-shaped (inflection point −0.59) relationship between the ln CALLY index and hepatic steatosis. In addition, there was an L-shaped relationship between the ln CALLY index and hepatic fibrosis, with an inflection point of 2.59. When the ln CALLY index was below the inflection point, an increase in the ln CALLY index was able to reduce the prevalence of hepatic fibrosis, which had a protective effect. To the best of our knowledge, this is the first study to report the relationship between the CALLY index and liver fibrosis and steatosis.

The CALLY index is a multidimensional assessment system integrating inflammatory response, nutritional status, and immune function constructed by Lida’s team, and its clinical value has been validated in the prognostic prediction of hepatocellular carcinoma (HCC) [7]. Among them, C-reactive protein (CRP), a classical inflammatory marker, has been shown to be closely associated with the severity of hepatic steatosis. A cohort study that included 393 patients showed that each elevated CRP level was positively associated with hepatic steatosis and fibrosis [19]. A study showed that CRP is an independent risk factor for the development of NAFLD [20]. Albumin is a medium-sized housekeeping protein with various functions such as osmoregulation, antioxidant, and anti-inflammatory, which accounts for more than half of the total serum composition of the human body. Diseases such as cirrhosis are not only associated with decreased albumin synthesis, but also with specific changes in its structure and function [2]. A study of 5.7 million Chinese adults showed that reduced Alb was positively associated with ≥F2, ≥F3, and F4 fibrosis [21]. In addition, existing studies have shown that lymphocytes play a unique role in the process of liver fibrosis and steatosis [22]-[24]. Distinguishing from traditional single biomarkers, the innovation of the CALLY index lies in the systematic integration of multidimensional pathophysiological processes. For example, although CRP reflects the intensity of hepatic inflammation, it is unable to assess the synthetic function or immunoregulatory status; whereas a decrease in albumin, although suggestive of impaired hepatic function, may mask the underlying inflammatory activity. By combining the three, the CALLY index can more accurately identify people at high risk for the transition from hepatic steatosis to fibrosis, enabling early intervention and improved outcomes. This provides new perspectives and strong evidence for exploring its underlying mechanisms and clinical applications.

NAFLD comprises a disease continuum ranging from steatosis with or without mild inflammation (NAFLD) to non-alcoholic steatohepatitis (NASH), characterized by necroinflammation and a more rapid progression of fibrosis than in NAFLD [25]. However, the role that inflammation plays in the progression of NAFLD is unclear. Various factors, such as lipotoxicity and LPS, activate NLRP3 inflammatory vesicles through the TLR4/NF-κB pathway or a series of pathogen-associated molecular patterns (PAMP) and damage-associated molecular patterns (DAMP), which in turn produce large amounts of IL-1β and IL-18, leading to inflammatory responses and cellular pyropoiesis, thus exacerbating liver injury [26]. Meanwhile, activation of inflammatory signaling pathways such as JNK, IKK/NF-κB, and JAK/STAT can cause insulin resistance and promote further development of hepatic steatosis and fibrosis [27]. Activation of innate immunity also promotes infiltration and aggregation of inflammatory cells in the liver, exacerbating liver inflammation and injury. For example, in the early stages of liver injury, Kupffer cells (KC) rapidly acquire a pro-inflammatory phenotype through the secretion of tumor necrosis factor and chemokines that trigger the recruitment of circulating monocyte-derived macrophages, which in turn exacerbate the onset and progression of NASH and liver fibrosis [28]. Neutrophils, on the other hand, drive hepatic metabolic inflammation by forming neutrophil extracellular traps (NETs) that interact with other pro-inflammatory immune cells [29]. In addition, NK cells have antifibrotic properties by secreting interferon γ to kill activated HSC and help to clear senescent activated hepatic stellate cells. Several studies have shown that the increased number of NK cells in NASH patients may be associated with elevated levels of NK cell-activating cytokines (e.g., IL-2, IL-12) [30]. Natural killer T (NKT) cells have a dual effect in NASH; they not only play an antifibrotic role by killing activated HSC, but may also accumulate in progressive NASH and promote the fibrotic process [31].

This study ensured national representativeness and broad generalizability of the sample through the use of the NHANES database and the assessment of the CALLY Index as a new non-invasive biomarker, which may provide strong support for the assessment of risk of hepatic fibrosis and steatosis. The application of the CALLY Index can assist primary care physicians in identifying high-risk individuals and provides a practical and cost-effective tool. However, despite the clinical relevance demonstrated in this study, some limitations remain. For example, because this study was a retrospective cross-sectional analysis, causality could not be established, and it was susceptible to inherent bias, while the accuracy of the results may have been affected by the way the data were collected and selection bias. In particular, the study did not take into account the participants’ medication use, which is particularly relevant for patients with NAFLD. In addition, the assessment of hepatic steatosis and hepatic fibrosis relies on transient elastography, which, although highly accurate, still differs from biopsy results and may therefore influence the judgment of the extent of liver disease. Furthermore, BMI and waist circumference were not included as covariates due to collinearity with metabolic variables; however, residual confounding by adiposity cannot be ruled out.

Nonetheless, the negative correlation of the CALLY index with hepatic fibrosis and steatosis suggests that individualized therapeutic approaches focusing on reducing inflammation and improving nutritional status in patients with lower indices may be helpful in enhancing metabolic health and preventing the progression of hepatic fibrosis. To further validate the potential of the CALLY index in assessing hepatic fibrosis and hepatic steatosis, future studies still need to overcome the limitations in the current study, such as the effects of temporality and potential confounders, and incorporate other more precise diagnostic methods for in-depth analysis.

In conclusion, our study demonstrates a negative association between the CALLY index and hepatic fibrosis and steatosis, supported by a large, nationally representative sample. However, further studies are needed to validate these findings in prospective studies and explore potential mechanisms.

*Corresponding author.