Dual therapy with vildagliptin and sacubitril/valsartan alleviates portal hypertension and inhibits soluble epoxide hydrolase in cirrhotic rats

Figure 1 Portal and systemic hemodynamics in choline-deficient, L-amino acid-defined, high-fat diet-fed rats. A: Experimental design of choline-deficient, L-amino acid-defined, high-fat diet-fed rats; B and C: Portal vein pressure (B) and portal blood flow (C) at the end of experiment; D: Pearson’s correlation between portal vein pressure and portal blood flow in all of experimental rats; E: Intrahepatic vascular resistance (calculated as portal vein pressure/portal blood flow); F-H: Heart rates (F), mean arterial pressure (G), and cardiac index (H) (calculated as cardiac output/100 g body weight) at the end of experiment; I: Systemic vascular resistance (calculated as mean arterial pressure/cardiac index); J: Superior mesenteric artery (SMA) resistance (calculated as mean arterial pressure/SMA blood flow). Data are the mean ± SD (n = 6). ^aP < 0.05. ^bP < 0.01. Significant difference between groups determined by Student’s t-test. CSANFD: Choline-supplemented L-amino acid-defined, normal-fat diet; CDAHFD: Choline-deficient, L-amino acid-defined, high-fat diet; DPP4-I: Dipeptidyl peptidase-4 inhibitor; ARNI: Angiotensin receptor-neprilysin inhibitor; Veh: Vehicle (saline)-treated groups; DP4i: Dipeptidyl peptidase-4 inhibitor (vildagliptin)-treated group; Car: Carvedilol-treated group; ARNI group: Angiotensin receptor-neprilysin inhibitor (sacubitril/valsartan)-treated group; CS: Choline-supplemented L-amino acid-defined, normal-fat diet-fed group; CD: Choline-deficient, L-amino acid-defined, high-fat die-fed groups. “Both group” defined dipeptidyl peptidase-4 inhibitor and angiotensin receptor-neprilysin inhibitor-treated group.

Figure 2 Intrahepatic vasoconstriction and vascular resistance in choline-deficient, L-amino acid-defined, high-fat diet-fed rats. A: Hepatic level of endothelin-1; B: Hepatic message RNA (mRNA) levels of cystathionine γ-lyase and dimethylarginine dimethyl amino-hydrolase 1; C: Western blotting for the phosphorylation of endothelial nitric oxide (NO) synthase (eNOS), inducible NO synthase (iNOS) and moesin in liver tissues; D-F: Quantitative phosphorylation rate of phospho-eNOS/total eNOS (D), phospho-iNOS/total iNOS (E) and phospho-moesin/total moesin (F). Actin was used as the loading control; G: Hepatic mRNA levels of guanosine triphosphate cyclohydrolase 1; H: Hepatic level of NO (quantified as the total amount of nitrate and nitrite); I: Hepatic level of cyclic guanosine monophosphate. The mRNA expression levels were measured by quantitative real-time polymerase chain reaction, and glyceraldehyde-3-phosphate dehydrogenase was used as internal control. Quantitative values are indicated as fold changes to the values of choline-supplemented L-amino acid-defined, normal-fat diet-fed group + vehicle (saline)-treated group (B, D-G). Data are the mean ± SD (A, B, G-I: n = 6; D-F: n = 3). ^aP < 0.05. ^bP < 0.01. Significant difference between groups determined by Student’s t-test (A, B, G-I) or Mann-Whitney U test (D-F). mRNA: Message RNA; eNOS: Endothelial nitric oxide synthase; iNOS: Inducible nitric oxide synthase; p-eNOS: Phospho-endothelial nitric oxide synthase; p-iNOS: Phospho-inducible nitric oxide synthase; Veh: Vehicle (saline)-treated groups; DP4i: Dipeptidyl peptidase-4 inhibitor (vildagliptin)-treated group; ARNI group: Angiotensin receptor-neprilysin inhibitor (sacubitril/valsartan)-treated group; CS: Choline-supplemented L-amino acid-defined, normal-fat diet-fed group; CD: Choline-deficient, L-amino acid-defined, high-fat die-fed groups. “Both group” defined dipeptidyl peptidase-4 inhibitor and angiotensin receptor-neprilysin inhibitor-treated group.

Figure 3 Hepatic soluble epoxide hydrolase status and epoxyeicosatrienoic acids level in choline-deficient, L-amino acid-defined, high-fat diet-fed rats. A: Immunofluorescence with soluble epoxide hydrolase (sEH) in liver tissues. Scale bar: 50 μm. Nuclei were stained by 4’,6-diamidino-2-phenylindole; B: Quantification of sEH positive area in liver tissues; C: SEH activity in liver tissues; D: Hepatic levels of 11,12-epoxyeicosatrienoic acids (EET) and 14,15-EET; E and F: Protein (E) and message RNA (mRNA) (F) levels of angiotensin II receptor type 1 (AT1R) in liver tissues; G: Pearson’s correlation between sEH expression and AT1R protein level in all of experimental rats; H and I: Protein (H) and mRNA (I) levels of nicotinamide adenine dinucleotide phosphate hydrogen oxidase 4 (NOX4) in liver tissues; J: Pearson’s correlation between sEH expression and NOX4 protein level in all of experimental rats. The mRNA expression levels were measured by quantitative real-time polymerase chain reaction, and glyceraldehyde-3-phosphate dehydrogenase was used as internal control. Quantitative values are indicated as fold changes to the values of choline-supplemented L-amino acid-defined, normal-fat diet-fed group + vehicle (saline)-treated group (B-D, F, I). Data are the mean ± SD (n = 6, B-F, H, I). ^aP < 0.05. ^bP < 0.01. Significant difference between groups determined by Student’s t-test. mRNA: Message RNA; DAPI: 4’,6-diamidino-2-phenylindole; CSANFD: Choline-supplemented L-amino acid-defined, normal-fat diet; CDAHFD: Choline-deficient, L-amino acid-defined, high-fat diet; sEH: Soluble epoxide hydrolase; Veh: Vehicle (saline)-treated groups; DP4i: Dipeptidyl peptidase-4 inhibitor (vildagliptin)-treated group; ARNI group: Angiotensin receptor-neprilysin inhibitor (sacubitril/valsartan)-treated group; CS: Choline-supplemented L-amino acid-defined, normal-fat diet-fed group; CD: Choline-deficient, L-amino acid-defined, high-fat die-fed groups. “Both group” defined dipeptidyl peptidase-4 inhibitor and angiotensin receptor-neprilysin inhibitor-treated group.

Figure 4 Sinusoidal capillarization and intrahepatic angiogenesis in choline-deficient, L-amino acid-defined, high-fat diet-fed rats. A: Immunofluorescence with cluster of differentiation 34 in liver tissues. Scale bar: 50 μm. Nuclei were stained by 4’,6-diamidino-2-phenylindole; B: Quantification of cluster of differentiation 34-positive sinusoidal capillarization; C: Hepatic message RNA (mRNA) levels of capillary markers (platelet endothelial cell adhesion molecule-1, endothelin-1 and laminin subunit beta-1); D: Hepatic mRNA levels of sinusoidal endothelial markers (lymphatic vessel endothelial hyaluronan receptor 1, plasmalemma vesicle-associated protein and stabilin-2; E: Hepatic mRNA levels of Hedgehog signaling-related genes (Shh, Gli2, Gli3, Sfrp1 and Opn); F: Hepatic mRNA levels of genes related to vascular injury (vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and neurogenic locus notch homolog protein 1); G: Hepatic mRNA levels of proangiogenic factors (Vegfa, Ptgs2, Pigf and Vwf). The mRNA expression levels were measured by quantitative real-time polymerase chain reaction, and glyceraldehyde-3-phosphate dehydrogenase was used as internal control. Quantitative values are indicated as fold changes to the values of choline-supplemented L-amino acid-defined, normal-fat diet-fed group + vehicle (saline)-treated group (B-G). Data are the mean ± SD (n = 6, B-G). ^aP < 0.05; ^bP < 0.01. Significant difference between groups determined by Student’s t-test. CD34: Cluster of differentiation 34; mRNA: Message RNA; DAPI: 4’,6-diamidino-2-phenylindole; CSANFD: Choline-supplemented L-amino acid-defined, normal-fat diet; CDAHFD: Choline-deficient, L-amino acid-defined, high-fat diet; Pecam1: Platelet endothelial cell adhesion molecule-1; ET-1: Endothelin-1; Lamb1: Laminin subunit beta-1; Lyve1: Lymphatic vessel endothelial hyaluronan receptor 1; Plvap: Plasmalemma vesicle-associated protein; Stab2: Stabilin-2; Vcam1: Vascular cell adhesion molecule 1; Icam1: Intercellular adhesion molecule 1; Notch1: Neurogenic locus notch homolog protein 1; Veh: Vehicle (saline)-treated groups; DP4i: Dipeptidyl peptidase-4 inhibitor (vildagliptin)-treated group; ARNI group: Angiotensin receptor-neprilysin inhibitor (sacubitril/valsartan)-treated group; CS: Choline-supplemented L-amino acid-defined, normal-fat diet-fed group; CD: Choline-deficient, L-amino acid-defined, high-fat die-fed groups. “Both group” defined dipeptidyl peptidase-4 inhibitor and angiotensin receptor-neprilysin inhibitor-treated group.

Figure 5 Glycemic status and steatohepatitis in choline-deficient, L-amino acid-defined, high-fat diet-fed rats. A: Changes in the body weights during the experimental period; B: Liver/body weight at the end of experiment; C: Serum glucose levels after 6 and 12 weeks of the experiment; D: Hematoxylin and eosin (HE) staining in liver tissues. Scale bar: 50 μm; E: Non-alcoholic fatty liver disease activity score based on HE staining; F and G: Serum alanine aminotransferase and albumin levels after 6 and 12 weeks of the experiment. Data are the mean ± SD (n = 6, A-C, E-G). ^aP < 0.05; ^bP < 0.01. ¹P vs choline-supplemented L-amino acid-defined, normal-fat diet-fed group + vehicle (saline)-treated group at 12 weeks (C, F and G) determined by Student’s t-test. Significant difference between groups (A and B). CSANFD: Choline-supplemented L-amino acid-defined, normal-fat diet; CDAHFD: Choline-deficient, L-amino acid-defined, high-fat diet; HE: Hematoxylin and eosin; Veh: Vehicle (saline)-treated groups; DP4i: Dipeptidyl peptidase-4 inhibitor (vildagliptin)-treated group; Car: Carvedilol-treated group; ARNI group: Angiotensin receptor-neprilysin inhibitor (sacubitril/valsartan)-treated group; CS: Choline-supplemented L-amino acid-defined, normal-fat diet-fed group; CD: Choline-deficient, L-amino acid-defined, high-fat die-fed groups. “Both group” defined dipeptidyl peptidase-4 inhibitor and angiotensin receptor-neprilysin inhibitor-treated group.

Figure 6 Hepatic fibrosis development in choline-deficient, L-amino acid-defined, high-fat diet-fed rats. A: Sirius-red staining and immunofluorescence with alpha-smooth muscle actin (α-SMA) in liver tissues. Scale bar: 50 μm. Nuclei were stained by 4’,6-diamidino-2-phenylindole; B: Quantification of Sirius-red-stained fibrotic area; C: Hepatic content of hydroxyproline; D: Quantification of α-SMA-positive hepatic stellate cells; E: Hepatic message RNA (mRNA) levels of profibrogenic markers (Acta2, Col1a1, Tgfb1 and Ctgf). The mRNA expression levels were measured by quantitative real-time polymerase chain reaction, and glyceraldehyde-3-phosphate dehydrogenase was used as internal control. Quantitative values are indicated as fold changes to the values of choline-deficient, L-amino acid-defined, high-fat die-fed groups + vehicle (saline)-treated group (B, D and E). Data are the mean ± SD (n = 6, B-E). ^aP < 0.05. ^bP < 0.01. Significant difference between groups determined by Student’s t-test. CSANFD: Choline-supplemented L-amino acid-defined, normal-fat diet; CDAHFD: Choline-deficient, L-amino acid-defined, high-fat diet; mRNA: Message RNA; DAPI: 4’,6-diamidino-2-phenylindole; α-SMA: Alpha-smooth muscle actin; Veh: Vehicle (saline)-treated groups; DP4i: Dipeptidyl peptidase-4 inhibitor (vildagliptin)-treated group; ARNI group: Angiotensin receptor-neprilysin inhibitor (sacubitril/valsartan)-treated group; CS: Choline-supplemented L-amino acid-defined, normal-fat diet-fed group; CD: Choline-deficient, L-amino acid-defined, high-fat die-fed groups. “Both group” defined dipeptidyl peptidase-4 inhibitor and angiotensin receptor-neprilysin inhibitor-treated group.

Figure 7 Portal pressure, sinusoidal capillarization and hepatic fibrosis in bile duct-ligated rats. A: Experimental design of bile duct-ligated rats; B and C: Portal vein pressure (B) and portal blood flow (C) at the end of experiment; D: Immunofluorescence with cluster of differentiation 34, hematoxylin and eosin and Sirius-red staining in liver tissues; E: Quantification of cluster of differentiation 34-positive sinusoidal capillarization; F: Quantification of Sirius-red-stained fibrotic area; G: Hepatic message RNA (mRNA) levels of Acta2 and Col1a1. The mRNA expression levels were measured by quantitative real-time polymerase chain reaction, and glyceraldehyde-3-phosphate dehydrogenase was used as internal control. Quantitative values are indicated as fold changes to the values of sham-operated group + vehicle (saline)-treated group (E) or bile duct-ligated groups + vehicle (saline)-treated group (F and G). Data are the mean ± SD (n = 4, B, C and E-G). ^aP < 0.05; ^bP < 0.01. Significant difference between groups determined by Mann-Whitney U test. BDL: Bile duct-ligated; mRNA: Message RNA; DAPI: 4’,6-diamidino-2-phenylindole; CD34: Cluster of differentiation 34; HE: Hematoxylin and eosin; Sham: Sham-operated group; Veh: Vehicle (saline)-treated groups; DP4i: Dipeptidyl peptidase-4 inhibitor (vildagliptin)-treated group; ARNI group: Angiotensin receptor-neprilysin inhibitor (sacubitril/valsartan)-treated group; “Both group” defined dipeptidyl peptidase-4 inhibitor and angiotensin receptor-neprilysin inhibitor-treated group.