Gut Dysbiosis and Increased Intestinal Permeability Drive microRNAs, NLRP-3 Inflammasome and Liver Fibrosis in a Nutritional Model of Non-Alcoholic Steatohepatitis in Adult Male Sprague Dawley Rats

Figures & data

Figure 1 (A) Determination of body weight parameters (p <0.001): a significant difference between groups is observed starting in the sixth week. * significant effect of the high-fat and choline-deficient diet according generalized estimating equations (GEE) followed by Bonferroni’s test. (B) Delta Lee index, *significant effect of the high-fat and choline-deficient diet (p = 0.017). Data expressed as mean ± standard deviation, Student’s t-test. Each experimental group with 10 male Sprague Dawley rats.

Figure 2 (A) Abdominal circumference, (B) abdominal adipose tissue, (C) macroscopic appearance of liver in the control group, (D) macroscopic appearance of liver in the intervention group, (E) liver weight and (F) liver weight/body weight ratio of the animal. *Significant effect of the high-fat and choline-deficient diet (p < 0.001). Data expressed as mean ± standard deviation, Student’s t-test. Each experimental group with 10 male Sprague Dawley rats.

Table 1 Biochemical Parameters of Animals Fed with HFCD Diet versus Standard Diet

Variable ^#	Control (n=10)	Intervention (n=10)	p *
ALT (U/L)	27.5 (± 9.0)	48.0 (± 20.7)	0.010
AST (U/L)	76. 1 (± 18.9)	73.4 (± 17.8)	0.745
Albumin (g/dL)	2.2 (± 0.2)	2.1 (± 0.3)	0.532
Glucose (mg/dL)	278.2 (± 56.8)	353.4 (± 64.3)	0.013
Total cholesterol (mg/dL)	78.7 (± 11.7)	101.8 (± 27.5)	0.033
LDL cholesterol (mg/dL)	16.7 (± 2.7)	23.4 (± 6.6)	0.011
HDL cholesterol (mg/dL)	62.7 (± 31.3)	31.3 (± 6.6)	0.006
Triglycerides (mg/dL)	79.3 (± 14.7)	105.6 (± 23.7)	0.011

Notes: ^#Variables described as mean ± standard deviation; *Student’s t-test, p < 0.05 considered significant.

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; HDL, high-density lipoprotein; HFCD, high-fat and choline-deficient; LDL, low-density lipoprotein.

Figure 3 Quantitative analysis of fat deposition in liver tissue *Significant effect of the high-fat and choline-deficient diet (p < 0.001). Data expressed as mean ± standard deviation, Student’s t-test. Each experimental group with 10 male Sprague Dawley rats.

Table 2 Changes in Liver Concentration of Pro- and Anti-Inflammatory Cytokines Related to Pathophysiology of NASH

Variable ^#	Control (n=10)	Intervention (n=10)	p *
TNF-α (pg/mg)	9720.3 (± 3048.0)	13,904.3 (± 3231.5)	0.008
IL-1β (pg/mg)	2186.0 (± 302.9)	2934.3 (± 456.0)	0.001
IL-6 (pg/mg)	50,378.5 (± 8347.8)	49,175.3 (± 7329.2)	0.736
IL-10 (pg/mg)	7116.8 (± 1158.4)	5125.9 (± 781.0)	< 0.001
TNF-α/IL-10 ratio	1.4 (± 0.6)	2.8 (± 1.0)	0.001
IL-1β/IL-10 ratio	0.3 (± 0.1)	0.6 (± 0.1)	< 0.001
IL-6/IL-10 ratio	7.2 (± 1.3)	9.7 (± 1.7)	0.002

Notes: ^#Variables described as mean ± standard deviation; *Student’s t-test, p < 0.05 considered significant.

Abbreviations: IL, interleukin; NASH, non-alcoholic steatohepatitis; TNF, tumor necrosis factor.

Table 3 Gene Expression of Gut Permeability Markers and Hepatic Inflammation Involved in NASH Pathogenesis

Variables ^#	Control (n=10)	Intervention (n=10)	p *
Evaluation performed on intestinal tissue
Ocln	0.87 (0.6–1.8)	0.29 (0.1–0.5)	0.003
F11r (JAM-1)	1.5 (0.5–2.7)	0.3 (0.2–0.6)	0.041
Cldn1	1.1 (0.3–1.8)	2.7 (1.2–3.3)	0.495
Cldn4	1.4 (0.6–1.6)	0.8 (0.5–2.2)	0.657
Evaluation performed on liver tissue
Tlr4	17.3 (0.2–9.0)	50.5 (2.2–40.7)	0.041
Tlr9	13.3 (11.6–15.1)	14.8 (14.5–18.3)	0.033
Tlr2	1.3 (0.5–2.0)	1.0 (0.1–1.8)	0.364
Myd88	1.6 (0.4–2.4)	5.1 (2.9–7.7)	< 0.001
Nlrp3	3.9 (1.4–4.2)	13.8 (3.8–20.5)	0.019
Casp1	2.1 (0.2–3.1)	82.3 (10.1–148.0)	< 0.001
Tnfa	17.3 (0.3–1.6)	111.4 (1.0–193.2)	0.028
Il6	1.2 (0.6–1.7)	9.8 (1.3–6.2)	0.013
Il18	2.2 (0.4–3.2)	14.9 (2.0–25.0)	0.007
Il1b	1.2 (0.7–1.7)	2.5 (1.1–3.9)	0.082
Il10	1.3 (0.6–1.7)	1.4 (0.6–2.1)	0.940
Ppara	1.4 (0.5–2.2)	0.4 (0.1–0.4)	0.008

Notes: ^#Variables described as median (25^th - 75^th percentiles) *Mann–Whitney U-test, p < 0.05 considered significant. β-actin gene was used as a housekeeping gene.

Abbreviations: Casp-1, caspase-1; Cldn, claudin; IL, interleukin; Jam, junctional adhesion molecule; MyD-88, myeloid differentiation primary response-88; NASH, non-alcoholic steatohepatitis; NLRP-3, Nod-like receptor protein-3; Ocln, occludin; PPAR, peroxisome proliferator-activated receptor; TLR, toll-like receptor; TNF, tumor necrosis factor.

Figure 4 Gene expression of the circulating microRNAs. (A) mir-122 (p = 0.041), (B) miR-145 (p = 0.010) and (C) miR-143 (p = 0.199). *Significant effect of the high-fat and choline-deficient diet. Data expressed as median (25th-75th percentile), Mann–Whitney U-test. Each experimental group with 10 male Sprague Dawley rats.

Figure 5 Gut microbiota features in control and intervention. (A) Hierarchical clustering dendrogram based on the Bray-Curtis dissimilarity index, (B) Number of OTUs, (C) Relative abundance, (D) distribution of the eight observed phyla and (E) Histogram of the LDA scores (log10) computed for the four differentially abundant phyla. Each experimental group with 10 male Sprague Dawley rats.

Table 4 Distribution of Liver Histopathological Findings

Variable ^#	Control (n=10)	Intervention (n=10)	p *
	n (%)	n (%)
General NAFLD scoring system for rodent models			< 0.001
No NAFLD	10 (100) *	0 (0.0)
NAFL	0 (0.0)	3 (30) *
NASH	0 (0.0)	7 (70) *

Notes: ^#Variables described as frequency (%). *Chi-square tests complemented by adjusted residual analysis, p < 0.05 considered significant.

Abbreviations: NAFLD, non-alcoholic fatty liver disease; NAFL, non-alcoholic fatty liver; NASH, non-alcoholic steatohepatitis.

Figure 6 Liver histological evaluation. Images referring to the control (A) and intervention (C) groups, H&E staining and both at 40X magnification. Picrosirius staining in the control (B) and intervention (D) groups, both at 10X magnification. (E) Quantification of collagen fibers. *Significant effect of the high-fat and choline-deficient (p < 0.001). Data expressed as median (25th-75th percentile), Mann–Whitney U-test. Each experimental group with 10 male Sprague Dawley rats.

Figure 7 Small intestine histological evaluation. Images referring to the control group without injury (A); moderate injuries (B) and severe lesions (C) in the intervention group. H&E staining and both at 40X magnification. Each experimental group with 10 male Sprague Dawley rats.

Table 5 Correlations Between NASH Markers and Microbiota Composition

Variables ^#	Inflammatory Response		Intestinal Permeability		Microbiota Composition
	Index TNF-α/IL-10 (pg/mg)	Index IL-1β/IL-10 (pg/mg)	Ocln	F11r (JAM-1)
NAFLD scoring	0.751**	0.832**	−.719**	−.525*	0.694**
Index TNF-α/IL-10 (pg/mg)		0.729**	−.463*	−.208	0.234*
Index IL-1β/IL-10 (pg/mg)			−.457*	−.361	0.522*
Ocln				0.722**	0.090
F11r (JAM-1)					0.146*

Notes: ^#Variables was represented for Spearman correlation coefficient. Moderate (0.3 < r < 0.6 - blue) and strong (0.6 < r < 0.9 - green) correlation of Spearman’s. ** Correlation is significant at the 0.01 level. * Correlation is significant at the 0.05 level.

Abbreviations: IL, interleukin; JAM, junctional adhesion molecule; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; Ocln, occludin; TNF, tumor necrosis factor.