Assessment of Pyroptosis-Related Indicators as Potential Biomarkers and Their Association with Severity in Patients with Liver Cirrhosis

Figures & data

Table 1 The Clinical Characteristics of Enrolled Individuals

	Serum Samples		Tissue Specimens
	Liver Cirrhosis (n=151)	HCs (n=62)	Liver Cirrhosis (n=12)	HCs (n=5)
Age year (IQR)	53 (18)	54 (16)	50 (16)	53 (14)
Gender
Male n (%)	97 (64.23)	36 (58.06)	7 (58.33)	3 (60)
Fale n (%)	54 (35.76)	26 (41.94)	5 (41.67)	2 (40)
Aetiology
Viral hepatitis		NA	7 (58.33)	NA
HBV n (%)	87 (73.73)
HCV n (%)	31 (26.27)
Alcohol n (%)	15 (9.93)	NA	2 (16.67)	NA
Cholestatic/Autoimmune n (%)	18 (11.92)	NA	3 (25)	NA
Child-Pugh Classification.
A n (%)	60 (39.73)	NA	2 (16.66)	NA
B n (%)	46 (30.46)	NA	5 (41.67)	NA
C n (%)	45 (29.81)	NA	5 (41.67)	NA
Phase
Compensated stage n (%)	48 (31.79)	NA	4 (33.33)	NA
Decompensated stage n (%)	103 (68.21)	NA	8 (66.67)	NA
APRI score	2.11 (2.52)	0.29 (0.11)	2.1 (2.84)	0.26 (0.1)
AST (U/L)	63 (89)	24 (6)	52 (68.55)	23 (4.5)
PLT	92 (78)	204 (76)	67 (45)	207 (72)

Abbreviations: IQR, interquartile range; HCs, healthy controls; APRI, AST to Platelet Ratio Index; NA, not applicable.

Figure 1 Expression of GSDMD, IL-1ß and IL-18 in patients with liver cirrhosis. (A-C) Representative IHC staining for GSDMD, IL-1ß and IL-18 in liver cirrhosis patients. Black scale bars, 100µm. (D) ELISA analysis of serum GSDMD levels in HC and liver cirrhosis patients. (E) ELISA analysis of serum IL-1ß levels in HC and liver cirrhosis patients. (F) ELISA analysis of serum IL-18 levels in HC and liver cirrhosis patients. (G) Correlation between serum IL-1ß and GSDMD in liver cirrhosis patients. (H) Correlation between serum IL-18 and GSDMD in liver cirrhosis patients. ***p<0.001.

Abbreviations: HC, health control; IQR, data represents the median.

Figure 2 Serum levels of GSDMD, IL-1ß and IL-18 in patients with various etiologies. (A) ELISA analysis of serum GSDMD levels in liver cirrhosis patients with three main etiologies, including viral infection, alcohol consumption and autoimmunity. (B) ELISA analysis of serum IL-1ß levels in liver cirrhosis patients with three main etiologies, including viral infection, alcohol consumption and autoimmunity. (C) ELISA analysis of serum IL-18 levels in liver cirrhosis patients with three main etiologies, including viral infection, alcohol consumption and autoimmunity. Data represents the median (IQR). *p<0.05; ***p<0.001.

Table 2 Significant Factors Associated with GSDMD, IL-1ß and IL-18 by Step Forward Multiple Linear Regression Analysis in Liver Cirrhosis Patients

Variables	GSDMD				IL-1ß				IL-18
	Univariate*		Multivariate^+		Univariate*		Multivariate^+		Univariate*		Multivariate^+
	ß	p value	ß	p value	ß	p value	ß	p value	ß	p value	ß	p value
Age (Years)	−0.492	0.059			−1.049	0.283			−1.852	0.32
Gender
Male^a
Female	−1.649	0.768			−22.072	0.517			−40.429	0.375
Aetiology
Viral hepatitis^a
Alcohol	15.151	0.084			−95.263	0.069			−189.248	0.006	65.041	0.001
Autoimmune	−20.27	0.013	0.069	0.191	−157.97	< 0.001	−49.842	0.008	−292.305	< 0.001	−240.457	0.002
AST (U/L)	0.042	0.003	0.008	0.883	0.231	0.006	0.146	0.066	0.288	0.008	0.096	0.205
PLT	−0.019	0.672			0.158	0.561			0.155	0.67
APRI score	3.048	< 0.001	−0.016	0.774	14.225	0.003	0.117	0.16	16.228	0.012	0.020	0.800
Child-Pugh classification	31.33	< 0.001	31.343	<0.001	78.197	< 0.001	59.919	0.003	118.197	< 0.001	101.793	< 0.001
Compensated status	7.707	0.178			132.87	< 0.001	0.116	0.197	191.081	< 0.001	0.133	0.13

Notes: a, reference group; ß, regression coefficient; *, univariate coefficients; ⁺, a stepwise multivariate regression analysis was performed; APRI: AST to Platelet Ratio Index. p value < 0.05 was considered statistically significant.

Figure 3 Correlation of serum GSDMD, IL-1ß and IL-18 levels with disease severity in cirrhosis patients. (A-C) ELISA analysis of serum levels of GSDMD (A), IL-1ß (B) and IL-18 (C) in liver cirrhosis patients with Child-Pugh classification A, B and C. (D-F) Correlation between serum GSDMD (D), IL-1ß (E) and IL-18 (F) with Child-Pugh score in liver cirrhosis patients. (G-I) Correlation between serum GSDMD (G), IL-1ß (H) and IL-18 (I) with APRI score in liver cirrhosis. (J-L) Distribution of GSDMD (J), IL-1ß (K) and IL-18 (L) in compensated and decompensated cirrhosis. *p<0.05; **p<0.01; ***p<0.001.

Abbreviation: Ns, no statistical significance.

Table 3 Serum Levels of GSDMD, IL-1ß and IL-18 in Patients with Liver Cirrhosis with Complications

	GSDMD	p value	IL-1ß	p value	IL-18	p value
LC (Ascites)
- (n=48)	48.5 (48.47)	p=0.32	59.98 (163.58)	p =0.0111	99.63 (227.55)	p=0.0104
+ (n=16)	44.29 (31.53)		159 (268.26)		294.7 (429.3)
LC (BEV)
- (n=48)	48.5 (48.47)	p=0.1299	59.98 (163.58)	p =0.0250	99.63 (227.55)	p=0.0008
+ (n=29)	38.88 (49.59)		143.4 (218.81)		327.5 (425.57)
LC (SBP)
- (n=48)	48.5 (48.47)	p=0.0006	59.98 (163.58)	p<0.0001	99.63 (227.55)	p<0.0001
+ (n=42)	74.13 (47.45)		282.1 (308.7)		516.9 (526.6)
LC (HE)
- (n=48)	48.5 (48.47)	p=0.0658	59.98 (163.58)	p=0.2129	99.63 (227.55)	p=0.4357
+(n=16)	78.88 (58.78)		80.36 (238.68)		175.2 (460.28)
LC (Ascites vs SBP)
Ascites (n=16)	44.29 (31.53)	p=0.0001	159 (268.26)	p=0.09	294.7 (429.3)	p=0.18
SBP (n=42)	74.13 (47.45)		282.1 (308.7)		516.9 (526.6)

Notes: Data are presented as median (interquartile range).

Abbreviations: BEV, bleeding esophageal varices; SBP, spontaneous bacterial peritonitis; HE, hepatic encephalopathy.

Figure 4 Differentiating power of serum GSDMD, IL-1ß and IL-18 for liver cirrhosis with various complications. (A) ROC curves of serum GSDMD, IL-1ß and IL-18 for predicting liver cirrhosis with ascites. (B) ROC curves of serum GSDMD, IL-1ß and IL-18 for predicting liver cirrhosis with EV. (C) ROC curves of serum GSDMD, IL-1ß and IL-18 for predicting liver cirrhosis with HE. (D) ROC curves of serum GSDMD, IL-1ß and IL-18 for predicting liver cirrhosis with SBP. (E) ROC curves of serum GSDMD, IL-1ß and IL-18 for predicting SBP occurrence in the setting of ascites for liver cirrhosis patient.