Patient demographics and clinical characteristics

A total of 644 participants were included in this study and divided into four groups. The healthy control group comprised 57 healthy individuals, among which 33 (7.35%) were male, and 24 (12.31%) were female, with an average age of 42.6 ± 13.51 years. The Chronic Hepatitis B group comprised 254 patients, of which 148 (32.96%) were male and 106 (54.36%) were female, with an average age of 41.11 ± 10.71 years. The Cirrhosis group comprised 206 patients with cirrhosis following chronic HBV infection, of which 158 (35.19%) were male and 48 (24.62%) were female, with an average age of 49.24 ± 10.58 years. The Liver cancer group comprised 127 patients with hepatitis B-related hepatocellular carcinoma, of which 110 (24.50%) were male and 17 (8.72%) were female, with an average age of 53.17 ± 11.87 years. There were significant differences among the four study groups in terms of AST, ALT, TBIL, HBV DNA, HBsAg, and AFP levels (P < 0.001). The serum HBsAg level in the Chronic Hepatitis B group was higher than that in the Cirrhosis group and the Liver Cancer group. The serum TBIL level in the Cirrhosis group was significantly higher than that in the Healthy Control group, Chronic Hepatitis B group, and Liver Cancer group. The Liver Cancer group exhibited higher levels of serum AST, ALT, AFP, and HBV DNA load (Table 1).

Expression levels of CTH, IL-6, CRP, and IL-10 in the healthy control group and chronic HBV infection group

We used ELISA kits to measure serum levels of CTH, IL-6, CRP, and IL-10 in the control and the chronic HBV infection group (Fig. 1). Compared to the control group (7.69 ± 1.83 ng/ml), the serum CTH levels were significantly increased in the Chronic Hepatitis B group (9.98 ± 1.9 ng/ml), the Cirrhosis group (11.54 ± 1.79 ng/ml), and the Liver Cancer group (13.52 ± 2.21 ng/ml) (P < 0.0001) (Fig. 1a). In the Liver Cancer group (153.43 ± 36.71 pg/ml), the expression level of IL-6 was significantly higher than that in the LC group (130.87 ± 30.62 pg/ml), the Chronic Hepatitis B group (110.62 ± 31.43 pg/ml), and the Healthy Control group (103.24 ± 36.68 pg/ml) (P < 0.0001). There was no significant difference between the CHB group and the Healthy Control group (P > 0.05) (Fig. 1b). In the liver cancer group, the expression level of CRP (16.6 ± 4.54 ng/ml) was significantly higher than that in the LC (13.33 ± 3.74 ng/ml), CHB (11.60 ± 4.02 ng/ml), and healthy control (9.98 ± 2.41 ng/ml) groups (P < 0.0001) (Fig. 1c). For the cytokine IL-10, there was a significant difference between the healthy control group (309.56 ± 106.22 pg/ml) and the chronic hepatitis B (196.22 ± 103.51 pg/ml) (P < 0.05), cirrhosis (178.8 ± 85.61 pg/ml) (P < 0.001), and liver cancer (109.07 ± 74.68 pg/ml) groups (P < 0.0001). There was no significant difference between the CHB group and the LC group (P > 0.05). As the disease progressed, the expression level of IL-10 gradually decreased (Fig. 1d).

Expression of CTH, IL-6, CRP, and IL-10 in HI, CHB, LC, and HCC groups (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). CHB, chronic hepatitis B; CRP C-reactive protein; CTH Cystathionine γ-lyase; HCC hepatocellular carcinoma; HI healthy control group; IL interleukin.

Association between serum levels of CTH, IL-6, CRP, and IL-10 and liver inflammation grading

As shown in Fig. 2, the levels of CTH (13.12 ± 2.4 ng/ml), IL-6 (155.52 ± 40.5 pg/ml), and CRP (16.28 ± 4.97 ng/ml) in patients with significant inflammation (G2-4) were significantly higher than those in patients with no or mild inflammation (G0-1) (10.79 ± 2.17 ng/ml, 127.18 ± 32.2 pg/ml, and 12.26 ± 5.68 ng/ml, respectively; P < 0.0001). The IL-10 level (99.50 ± 85.02 pg/ml) in patients with significant inflammation (G2-4) was significantly lower than that in patients with no or mild inflammation (G0-1) (149.62 ± 89.37 pg/ml), P < 0.0001.

The correlation of serum CTH, IL-6, CRP, IL-10 levels with liver inflammation grades. CRP C-reactive protein; CTH Cystathionine γ-lyase; IL interleukin.

The efficacy of serum CTH, IL-6, CRP, and IL-10 in diagnosing significant inflammation

The diagnostic accuracy of serum CTH, IL-6, CRP, and IL-10 for significant inflammation was assessed using the Receiver Operating Characteristic (ROC) curve (Fig. 3 and Table 2). The area under the curve (AUC) for predicting significant hepatitis using CTH, IL-6, CRP, and IL-10 levels were 0.77, 0.70, 0.73, and 0.69, respectively (95% Confidence Intervals:[0.69–0.85)], [0.62–0.79], [0.64–0.82], and [0.60–0.78), respectively, with sensitivities of 81.2%, 45.9%, 77.6%, and 67.1%, respectively, and specificity is 62.3%, 86.9%, 65.6%, and 68.9% respectively.

Receiver operating characteristic curve of serum CTH, IL-6, CRP, and IL-10 levels in predicting significant liver inflammation. CRP C-reactive protein; CTH Cystathionine γ-lyase; IL interleukin.

Correlation between CTH, IL-6, CRP, and IL-10 and pathological parameter G

In the HBV infection group, CTH (r = 0.50, P < 0.0001), IL-6 (r = 0.33, P < 0.0001), and CRP (r = 0.37, P < 0.0001) showed a significant positive correlation with G, while IL-10 (r =  − 0.34, P < 0.0001) showed a significant negative correlation with G (Fig. 4a). In the Chronic Hepatitis B group, CTH (r = 0.41, P = 0.01) showed a significant positive correlation with G, while IL-6 (r = 0.17, P = 0.33), CRP (r = 0.23, P = 0.17), and IL-10 (r =  − 0.11, P = 0.53) did not show significant correlations with G (Fig. 4b). In the Cirrhosis group, CTH (r = 0.42, P = 0.04) exhibited a significant positive correlation with G, whereas IL-6 (r = 0.32, P = 0.12), CRP (r = 0.29, P = 16), and IL-10 (r =  − 0.32, P = 0.12) did not show significant correlations with pathological G (Fig. 4c). In the Liver cancer group, CTH (r = 0.47, P < 0.0001), IL-6 (r = 0.38, P < 0.0001), and CRP (r = 0.36, P = 0.001) are significantly positively correlated with G, while IL-10 (− 0.35, P = 0.001) shows a significant negative correlation with G (Fig. 4d).

Immunohistochemistry images and correlation analysis of CTH, IL-6, CRP, and IL-10 with pathological parameter G in HBV infection group, CHB, LC, and HCC groups. (a) represents CHB immunohistochemistry; (b) represents LC immunohistochemistry; Figure c represents HCC immunohistochemistry. The bar chart indicates HBsAg( +). AFP alpha-fetoprotein; CHB chronic hepatitis B; CRP C-reactive protein; CTH Cystathionine γ-lyase; HBV Hepatitis B virus; HCC hepatocellular carcinoma; IL interleukin; LC cirrhosis.

Serum CTH, IL-6, CRP, IL-10, AFP, and the combined efficacy of CTH + AFP in HCC diagnosis

The ROC curve shows that the area under the curve (AUC) for diagnosing HCC using CTH, IL-6, CRP, IL-10, and AFP is 0.83, 0.77, 0.78, 0.76, and 0.82, respectively. The combined diagnosis of HCC with AFP + CTH had an AUC of 0.88, which is superior to any single test value mentioned above (95% confidence intervals: [0.79–0.87], [0.72–0.81], [0.73–0.82], [0.72–0.81], [0.77–0.86], and [0.84–0.91]), with sensitivities of 64.6%, 77.2%, 68.5%, 60.6%, 70.9%, and 73.2%, respectively, and specificities of 83.3%, 65.0%, 79.8%, 81.7%, 82.2%, and 85.2%, respectively (Fig. 5 and Table 3).

Serum CTH, IL-6, CRP, and IL-10 levels in predicting HCC receiver operating characteristic curve. CHB chronic hepatitis B; CRP C-reactive protein; CTH Cystathionine γ-lyase; HBV hepatitis B virus.

Association of serum CTH, IL-6, CRP, and IL-10 levels with HCC BCLC staging

In patients with early (BCLC A stage, n = 82 cases), intermediate (BCLC B stage, n = 17 cases), and advanced (BCLC C stage, n = 28 cases) HCC, the expression of CTH in advanced stage (15.33 ± 1.98 ng/ml) HCC patients was higher than in early (12.8 ± 2.02 ng/ml) and intermediate (13.99 ± 1.64 ng/ml) patients (P < 0.0001) (Fig. 6a,b). The expression level of IL-6 in patients with late-stage (164.68 ± 25.47 pg/ml) HCC was significantly higher than that in patients with early-stage (148.53 ± 39.96 pg/ml) HCC (P < 0.05). The expression of CRP in patients with late-stage (18.92 ± 4.81 ng/ml) HCC was higher than that in patients with early-stage (15.92 ± 4.48 ng/ml) and mid-stage (16.06 ± 3.12 ng/ml) HCC (P < 0.05). There was no significant difference in IL-10 expression among patients with early-stage, mid-stage, and late-stage HCC patients.

According to the classifications of BCLC system, the correlations (a) serum CTH concentrations (b) serum IL-6 concentrations (c) serum CRP concentrations (d) serum IL-10 concentrations in different-stage hepatocellular carcinoma patients. *P < 0.05 was defined as statistically significant. CRP C-reactive protein; CTH Cystathionine γ-lyase; IL interleukin.

Expression levels of serum CTH, IL-6, CRP, and IL-10 in the CHB, LC, preoperative HCC, and postoperative 6-month HCC groups

Almost all HBV-related HCC in China develops from LC, therefore, further analysis was conducted on the differences in the expression of CTH, IL-6, CRP, and IL-10 between the CHB group, LC group, preoperative group, and postoperative 6-month group. In the postoperative 6-month group, the CTH expression level (10.57 ± 2.02 ng/ml) was significantly lower than that of the cirrhosis group (11.54 ± 1.79 ng/ml) (P < 0.05), with no significant difference compared to the chronic Hepatitis B group (Fig. 7a). The pro-inflammatory cytokine IL-6 expression level in the postoperative 6-month group (142.64 ± 33.34 pg/ml) was significantly higher than that in the chronic Hepatitis B group (110.62 ± 31.43 pg/ml) and the cirrhosis group (130.87 ± 30.62 pg/ml) (Fig. 7b). The expression level CRP in the postoperative 6-month group (12.77 ± 5.48 ng/ml) showed no significant difference compared to the chronic Hepatitis B group (11.6 ± 4.02 ng/ml) and the cirrhosis group (13.33 ± 3.74 ng/ml) (Fig. 7c). In the postoperative 6-month non-recurrence group (131.47 ± 46.84 pg/ml), the expression level of the anti-inflammatory cytokine IL-10 was significantly lower than that in the chronic Hepatitis B group (196.22 ± 103.51 pg/ml) and the cirrhosis group (178.8 ± 85.61 pg/ml) (Fig. 7d, Table 4).

Expression of CTH, IL-6, CRP, and IL-10 in the CHB group, LC group, pre-operative HCC group, and post-operative 6-month group (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). CHB chronic hepatitis B; CRP C-reactive protein; CTH Cystathionine γ-lyase; HCC hepatocellular carcinoma; IL interleukin; LC cirrhosis.

Expression levels of CTH, IL-6, CRP, and IL-10 in pre-operative, post-operative, and recurrent HCC

There is no significant difference in serum expression levels of CTH, IL-6, CRP, and IL-10 between the pre-operative and recurrent groups. However, compared to the pre-surgery group, the expression level of CTH in the post-surgery group significantly decreased, and as HCC recurred, the CTH level increased again. The level of IL-10 showed the opposite trend, with the post-surgery group having significantly higher IL-10 levels than the pre-surgery group, and the IL-10 level decreased with the recurrence of HCC. There was no significant difference in the levels of IL-6 and CRP between the post-surgery group and the recurrence group (Tables 3, 5, Fig. 8).

Expression Levels of CTH, IL-6, CRP, and IL-10 in pre-surgery, post-surgery, and recurrence groups of HCC. CRP C-reactive protein; CTH Cystathionine γ-lyase; HCC hepatocellular carcinoma; IL interleukin.