NAFLD is a major chronic liver disease closely associated with obesity, insulin resistance (IR), T2DM, dyslipidemia and other MetS components, and it is the hepatic manifestation of MetS. The pathogenesis of NAFLD is complex whereby the historical two-hit hypothesis is now replaced by a multiple parallel hits hypothesis also considering the impact of an altered adipokine secretory pattern, inflammation, gut microbiota, nutritional factors, genetic and epigenetic factors, and IR on the development and progression of NAFLD [20]. NAFLD and MetS are closely related in both phenotype and pathogenesis. Coexistence of NAFLD and MetS should not be expected in every patient, one may precede the other, and common therapeutic modalities are beneficial for both entities.

BMP9, a hepatic cytokine that regulates blood glucose and lipid metabolism, is closely related to the pathogenesis of NAFLD and has a certain protective effect on NAFLD. It can inhibit hepatic lipid deposition, transform white adipose tissue (WAT) to brown adipose tissue and play a leptin-like role such as increasing energy expenditure to exert lipid-lowering effect [21]. Furthermore, BMP9 can play a hypoglycemic role in regulating glucose metabolism by increasing insulin sensitivity and stimulating the synthesis and secretion of insulin release [8]. Kuo et al. found that BMP9 induced WAT browning, inhibited high-fat diet-induced obesity and improved obesity-mediated IR and NAFLD in mice [22]. Our previous study also found the expression of BMP9 was decreased in NAFLD mice and exogenous supplementation of BMP9 improved the phenotype of NAFLD [11]. However, the relationship between serum BMP9 and the severity of MetS, NAFLD has not been explored thoroughly so far.

In the present study, we observed a negative correlation between the concentration of BMP9 and several indicators of glycolipid metabolism, including TG, FPG, HbA1c and BMI, while it was positively associated with HDL. This was consistent with other studies. Xu et al. found in middle-aged and older populations [23] as well as patients with T2DM and NAFLD [10], circulating BMP9 levels positively correlated with HDL and negatively correlated with markers of adiposity such as waist-to-hip ratio (WHR) or BMI, parameters of glucose metabolism (FPG and HbA1c) and TG. Animal studies also showed that upregulation of BMP9 levels inhibited the expression of lipogenic genes, ameliorated triglyceride accumulation in liver, decreased serum TC and TG [24], improved glucose tolerance, decreased FPG and alleviate IR in mice [11].

NAFLD is closely associated with obesity, and BMI is currently the most widely used anthropometric measure for assessing obesity worldwide and the most critical risk factor for NAFLD [25]. In our study, we discovered the risk of developing NAFLD increased along with elevated serum BMP9 and BMI, which is consistent with Younossi et al [26]. They also found that the prevalence of NAFLD was positively correlated with an increase in BMI. It is noteworthy that there was a significantly higher risk of NAFLD observed in men compared to women in our study. This has been confirmed in other studies that the prevalence of NAFLD is higher in men compared to women, and in women, the prevalence tends to increase after menopause, potentially due to the possible protective role of estrogen [27].

NAFLD has insidious progression and lacks specific symptoms. Noninvasive assessment of the severity of NAFLD is a general trend. Serum transaminase is often used as an index to detect NASH clinically, however, it does not reflect the course and prognosis of NAFLD accurately [28]. Another frequently used biomarker for NASH is cytokeratin 18 (CK18), which exhibits an overall sensitivity of 66% and specificity of 82% [29]. Nevertheless, inflammatory markers such as ferritin, high-sensitivity C-reactive protein (CRP), tumor necrosis factor and interleukins were demonstrated poor accuracy and specificity in recognizing NASH [30]. FibroTouch is now widely used in clinical practice as a noninvasive assessment method with excellent diagnostic performance for hepatic steatosis and fibrosis. The sensitivity of CAP values in diagnosing hepatic steatosis at stages S1, S2 and S3 was 81.5%, 86.7% and 100.0%, respectively, and the specificity was 80.6%, 91.7% and 96.5%, respectively [31]. Additionally, LSM outperformed other noninvasive fibrosis indices, namely fibrosis 4 (FIB-4), aspartate aminotransferase-to-platelet ratio index (APRI) and γ-glutamyl transferase-to-platelet ratio index (GPRI), demonstrating significant superiority, albeit second to liver biopsy [32]. Therefore, we employed FibroTouch in place of liver puncture to measure the degree of steatosis and fibrosis. In our research, we observed an upward trend in transaminases, CAP and LSM in patients with NAFLD compared to the healthy population. Moreover, we identified significant differences in BMP9 concentration among different liver enzyme levels, CAP and LSM. The current study also showed serum BMP9 levels differentiated NASH at-risk (58.13 ± 2.82 ng/L) from the other groups: healthy control (70.32 ± 3.70) and NAFL (64.34 ± 4.76 ng/L, p < 0.0001). Notably, BMP9 is widely involved in the function of various receptors associated with liver fibrosis promotion, downstream signaling molecules and the expression of target genes in hepatocytes [33], thus making it a valuable serum diagnostic indicator [34]. All these findings suggest that BMP9 has the potential to serve as a biomarker in identifying at-risk NASH patients.

Uric acid levels may play a role in MetS, and high uric acid level is independently associated with all components of MetS such as hypertension, obesity, high triglycerides, low HDL and elevated FPG [23]. In addition, another meta-analysis of prospective studies concluded that there is an independent linear relationship between increased UA levels and the occurrence of MetS [35, 36]. Therefore, in our study we used UA instead of hypertension as one of the MetS parameters, which are in line with previous studies [37]. Our results showed circulating BMP9 levels reduced with the number of MetS components. Xu et al. [23] also found that plasma BMP9 concentrations were significantly associated with MetS and circulating BMP9 levels reduced progressively with an increasing number of MetS components.

Although our study observed close association of serum BMP9 with the severity of MetS and NAFLD, there are some limitations including the small sample size, and the cross-sectional study design did not allow us to establish causality between serum BMP9 and NAFLD development. Future longitudinal studies with larger and more diverse participants are needed. Additionally, the absence of a liver biopsy-proven NAFLD cohort limits our comprehension for the connection of NASH and BMP9. Nonetheless, our findings suggest that BMP9 is highly has the potential to be used as a noninvasive biomarker for monitoring the severity and progression of NAFLD and MetS.