Analysis of the nitric oxide-cyclic guanosine monophosphate pathway in experimental liver cirrhosis suggests phosphodiesterase-5 as potential target to treat portal hypertension

Table 1 Nucleotide sequences of forward and reverse primers

Gene	Forward primer (5´-3´)	Reverse primer (5´-3´)	Product length (bp)
eNOS	5´-AAGTGGGCAGCATCACCTAC-3´	5´-GCCTGGGAACCACTCCTTTT-3´	211
iNOS	5´-CTCACTGGGACTGCACAGAA-3´	5´-TGTTGAAGGGTGTCGTGAAA-3´	128
PDE-5	5´-GCGGAGGAAGAAACAAGGGA-3´	5´-ATCGGCAAAGAACCTCGTGT-3´	196
sGCa1	5´-GCCCCACGACATACAGGTTA-3´	5´-GCGGCTCACTAATCTACCCC-3´	229
sGCb1	5´-AATTACGGTCCCGAGGTGTG-3´	5´-ACCAGCATTGAGGTTGAGGAC-3´	147
18sRNA (reference)	5´-GTAACCCGTTGAACCCCATT-3´	5´-CCATCCAATCGGTAGTAGCG-3´	151
srsf4 (reference)	5´-GGTTCTGGACGCAGTGGATA-3´	5´-CTCCTTCGTTTTTGCGTCCC-3´	193

Table 2 Mean ± standard deviation of enzyme gene expression

	CON (n = 11) Mean ± SD	FIB (n = 6) Mean ± SD	CIR (n = 8) Mean ± SD
eNOS (fold exp.)	1.0 ± 0.4	1.5 ± 0.3'	2.2 ± 1.0
iNOS (fold exp.)	n.d.1	4.6 ± 3.0	5.3 ± 2.3
PDE5 (fold exp.)	1.0 ± 1.0	7.7 ± 0.9	11.0 ± 3.1
sGCa1 (fold exp.)	1.0 ± 0.3	1.4 ± 0.3	1.7 ± 0.4
sGCb1 (fold exp.)	1.0 ± 0.5	2.1 ± 0.3	3.0 ± 1.3

¹Since iNOS expression in CON was below detection limit, it was arbitrarily set to “1.0”. iNOS: Inducible NO synthase.

Fold expression is referring to CON. qRT-PCRs were performed in duplicates, thus values are based on mean values of duplicates.

Table 3 Median ± interquartile range of serum cyclic guanosine monophosphate concentrations

	CON (n = 11)Median ± IQR	FIB (n = 6)Median ± IQR	CIR (n = 8)Median ± IQR	CON + Sil (n = 12)Median ± IQR	CIR + Sil (n = 10)Median ± IQR
cGMP (pmol/mL)	152 ± 86	100 ± 68	91 ± 22	249 ± 153	168 ± 52

ELISAs were performed in duplicates, thus values are based on mean values of duplicates. IQR: Interquartile range; cGMP: Cyclic guanosine monophosphate.

Table 4 Pairwise comparisons between groups are determined according to Dunn[35]

		eNOS	iNOS	PDE5	sGCa1	sGCb1	cGMP
CON vs FIB	sig. (pairwise)	0.0241	-	0.0021	0.065	0.0061	0.151
	sig. (adjusted)	0.072	-	0.0061	0.195	0.0181	0.453
CON vs CIR	sig. (pairwise)	0.0011	-	0.0011	0.0011	0.0011	0.0181
	sig. (adjusted)	0.0031	-	0.0031	0.0031	0.0031	0.054
FIB vs CIR	sig. (pairwise)	0.280	0.732	0.201	0.194	0.267	0.495
	sig. (adjusted)	0.840	1.000	0.603	0.582	0.801	1.000
CON vs CON + Sil	sig. (pairwise)	n.m.2	n.m.2	n.m.2	n.m.2	n.m.2	0.0121
	sig. (adjusted)	n.m.2	n.m.2	n.m.2	n.m.2	n.m.2	0.0241
CIR vs CIR + Sil	sig. (pairwise)	n.m.2	n.m.2	n.m.2	n.m.2	n.m.2	0.0091
	sig. (adjusted)	n.m.2	n.m.2	n.m.2	n.m.2	n.m.2	0.0181

¹Significant differences (P < 0.05) are marked by an.

²Means: Not measured.

Adjusted comparisons are corrected according to Bonferroni.

Table 5 Median ± interquartile range of the absolute values of body weight, portal venous pressure, mean arterial pressure, and heart rate at different time points

	CON			FIB			CIR
	NaCl (n = 19)	Sil 0.1 mg/kg (n = 18)	Sil 1.0 mg/kg (n = 18)	NaCl (n = 7)	Sil 0.1 mg/kg (n = 15)	Sil 1.0 mg/kg (n = 7)	NaCl (n = 7)	Sil 0.1 mg/kg (n = 7)	Sil 1.0 mg/kg (n = 10)
	Median ± IQR	Median ± IQR	Median ± IQR	Median ± IQR	Median ± IQR	Median ± IQR	Median ± IQR	Median ± IQR	Median ± IQR
Body weight (g)	375 ± 15	370 ± 28	380 ± 27	359 ± 35	363 ± 28	361 ± 24	336 ± 40	350 ± 46	337 ± 15
PVP_0 (mmHG)	6.4 ± 0.7	6.6 ± 0.4	6.3 ± 0.7	6.0 ± 0.9	6.2 ± 1.3	6.2 ± 0.9	6.8 ± 1.6	7.5 ± 1.7	7.7 ± 1.1
PVP_10 (mmHG)	6.0 ± 0.9	6.6 ± 0.6	6.6 ± 0.9	5.7 ± 1.2	5.5 ± 1.3	5.1 ± 1.4	6.8 ± 1.4	7.1 ± 1.6	7.1 ± 2.2
PVP_30 (mmHG)	5.9 ± 0.7	6.3 ± 0.7	6.4 ± 0.8	5.3 ± 1.4	5.4 ± 1.3	4.8 ± 1.4	6.5 ± 1.2	6.6 ± 1.0	6.3 ± 1.3
PVP_60 (mmHG)	5.9 ± 0.7	6.3 ± 0.8	6.4 ± 1.1	5.1 ± 1.2	5.2 ± 1.2	4.8 ± 1.5	6.6 ± 2.0	6.3 ± 1.0	5.8 ± 1.2
MAP_0 (mmHG)	89 ± 12	106 ± 14	101 ± 28	65 ± 30	59 ± 21	58 ± 28	49 ± 19	47 ± 15	60 ± 19
MAP_10 (mmHG)	83 ± 20	94 ± 14	85 ± 27	55 ± 25	45 ± 10	42 ± 8	42 ± 11	41 ± 5	38 ± 16
MAP_30 (mmHG)	79 ± 21	88 ± 17	77 ± 21	50 ± 14	43 ± 12	37 ± 6	41 ± 12	35 ± 8	36 ± 8
MAP_60 (mmHG)	76 ± 21	82 ± 23	74 ± 28	44 ± 11	40 ± 8	37 ± 7	38 ± 3	34 ± 5	34 ± 7
HR_0 (bpm)	373 ± 40	377 ± 47	368 ± 31	320 ± 30	335 ± 30	314 ± 22	307 ± 38	308 ± 76	365 ± 28
HR_10 (bpm)	362 ± 55	383 ± 44	380 ± 50	313 ± 23	339 ± 41	351 ± 29	286 ± 33	316 ± 71	374 ± 22
HR_30 (bpm)	356 ± 57	366 ± 52	373 ± 41	291 ± 20	310 ± 43	310 ± 21	268 ± 40	313 ± 57	350 ± 36
HR_60 (bpm)	347 ± 39	359 ± 43	362 ± 49	284 ± 26	304 ± 41	295 ± 33	254 ± 36	302 ± 47	324 ± 30

Table 6 Relative median of differences (%) ± interquartile range of the parameters portal venous pressure, mean arterial pressure, and heart rate

	CON			FIB			CIR
	NaCl (n = 19)	Sil 0.1 mg/kg (n = 18)	Sil 1.0 mg/kg (n = 18)	NaCl (n = 7)	Sil 0.1 mg/kg (n = 15)	Sil 1.0 mg/kg (n = 7)	NaCl (n = 7)	Sil 0.1 mg/kg (n = 7)	Sil 1.0 mg/kg (n = 10)
	RMD (%) ± IQR	RMD (%) ± IQR	RMD (%) ± IQR	RMD (%) ± IQR	RMD (%) ± IQR	RMD (%) ± IQR	RMD (%) ± IQR	RMD (%) ± IQR	RMD (%) ± IQR
PVPnorm	- 3 ± 7	- 6 ± 10	- 3 ± 6	- 9 ± 11	- 8 ± 8	- 7 ± 6	- 3 ± 7	- 13 ± 7	- 19 ± 26
MAPnorm	- 10 ± 17	- 8 ± 16	- 7 ± 19	- 21 ± 24	- 14 ± 21	- 10 ± 10	- 17 ± 16	- 14 ± 11	- 17 ± 23
HRnorm	- 4 ± 6	- 4 ± 5	- 4 ± 5	- 8 ± 5	- 8 ± 14	- 12 ± 13	- 6 ± 6	- 8 ± 11	- 14 ± 101

¹Significant differences (P < 0.05) between Sil 0.1mg/kg and CON or Sil 1.0 mg/kg and CON are marked by an “¹”. RMD: Relative median of differences; IQR: Interquartile range; PVP: Portal venous pressure; MAP: Mean arterial pressure; HR: Heart rate.