Multi-parameter magnetic resonance imaging of zebularine in liver fibrosis treatment and calcineurin/NFAT3 mechanism

Table 1 Primer sequences for real-time polymerase chain reaction

Gene (human)	Forward	Reverse
CoL1a1	5’-AGACGAAGACATCCCACCA-3’	5’-GTCGCAGACGCAGATCC-3’
ACTA2 (α-SMA)	5’-GTTACGAGTTGCCTGATGG-3’	5’-AGGTGGTTTCATGGATGC-3’
SEPTIN9	5’-GTGAAGAACTCAGAACCCTCG-3	5’-TCAATGGACAGCTCAGTGC-3’
PPAR-γ	5’-AGATCATTTACACAATGCTGGC-3’	5’-TAAAGTCACCAAAAGGCTTTCG-3’
PTEN	5’-GACCAGAGACAAAAAGGGAGTA-3’	5’-ACAAACTGAGGATTGCAAGTTC-3’
RCAN1.4	5’-TTTAGCTCCCTGATTGCCTGT-3’	5’-AAAGGTGATGTCCTTGTCATACG-3’
PTCH1	5’-TTTTCTGCTGTTTTACAAGCCC-3’	5’-CATGGTAATCTGCGTTTCATGG-3’
β-actin (loading control)	5’-TCCCTGGAGAAGAGCTACGA-3’	5’-TGAAGGTAGTTTCGTGGATGC-3’

RCAN: Regulator of calcineurin; CoL1a1: Type I collagen; α-SMA: α-smooth muscle actin.

Table 2 Antibody characteristics

Protein	Application	Origin	Dilution
α-SMA	WB and IHC-P	ab5694, Abcam, United Kingdom	1:200
RCAN1.4	WB	ab140131, Abcam, United Kingdom	1:10000
CaN	WB	55147-1-AP, Proteintech, China	1:1000
NFAT3	WB and IHC and IF	bs-6461R, Bioss, China	1:1000
β-tubulin	WB and IHC and IF	10094-1-AP, Proteintech, China	1:5000
GAPDH	WB and IHC and IF	ab9485, Abcam, United Kingdom	1:2500
Lamin B1	WB and IHC and IF	12987-1-AP, Proteintech, China	1:5000
HRP-conjugated affinipure goat anti-rabbit IgG (High + Low)	ELISA, WB	SA00001-2, Proteintech, China	1:5000

WB: Western blot; IF: Immunofluorescence; IHC: Immunohistochemistry; ELISA: Enzyme-linked immunosorbent assay; IgG: Immunoglobulin; HRP: Horseradish peroxidase; RCAN: Regulator of calcineurin; α-SMA: α-smooth muscle actin; CaN: Calcineurin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Table 3 Sequence-specific parameters

Parameter/sequence	T1WI	T2WI	EPI-DWI	T1 MAP	T2 MAP
TR	585	3243	2000	15	3000
TE	11.32	87.38	80	3.87	17.4, 34.8, 52.2, 69.6, 87.0, 104.4
Flip angle (°)	150°	140°	90°	5°, 25°	180°
Voxel size (mm³) (X × Y × Z)	0.16 × 0.16 × 1	0.30 × 0.30 × 1	0.59 × 0.59 × 2	0.13 × 0.13 × 1	0.50 × 0.50 × 2
FOV (mm²)	40 × 40	40 × 40	50 × 80	75 × 83	90 × 100
Slices	20	20	15	20	15
Acquisition time	4:47	5:24	3:22	3:8	5:39

T1WI: T1-weighted imaging; T2WI: T2-weighted imaging; EPI: Echo planar imaging; DWI: Diffusion-weighted imaging; TR: Repetition time; TE: Echo time; FOV: Field of view; T1 MAP: T1 mapping; T2 MAP: T2 mapping.

Table 4 Diagnostic performance of magnetic resonance parameters in the assessment of liver fibrosis

Parameters	T1pre (ms)	△T1 (ms)	ADC (× 10-3, mm2/second)	T2	Combined diagnosis
AUC	0.926	0.961	0.809	0.926	0.989
95%CI	0.902-0.951	0.945-0.976	0.768-0.851	0.899-0.953	0.982-0.996
P value	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
Sensitivity	0.889	0.898	0.810	0.829	0.940
Specificity	0.852	0.907	0.708	0.940	0.977

ADC: Apparent diffusion coefficient; AUC: Area under the curve; CI: Confidence interval.

Table 5 Correlation matrix between magnetic resonance parameters and histological features of liver fibrosis

Parameters	Fibrosis		Inflammation
	rs	P value	rs	P value
T2	0.861	< 0.001	0.795	< 0.001
△T1	0.853	< 0.001	0.763	< 0.001
T1pre T1	0.779	< 0.001	0.673	< 0.001
ADC	-0.692	< 0.001	-0.577	< 0.001

ADC: Apparent diffusion coefficient.

Table 6 Comparison of magnetic resonance parameters between the control, carbon tetrachloride, and carbon tetrachloride + zebularine groups

Parameters	2 weeks				4 weeks				6 weeks
	Control	CCl4	CCl4 + zebularine	P value	Control	CCl4	CCl4 + zebularine	P value	Control	CCl4	CCl4 + zebularine	P value
T1pre (millisecond)	632.554 ± 91.474	783.493 ± 47.454	790.09 ± 57.283	1.000	650.917 ± 14.543	845.448 ± 98.6	694.370 ± 52.447	< 0.001	632.712 ± 27.834	934.073 ± 38.4291	760.6564 ± 61.1215	< 0.001
△T1	445.771 ± 91.877	657.431 ± 51.533	633.599 ± 72.49	1.000	445.996 ± 69.364	724.751 ± 102.433	584.67 ± 55.173	< 0.001	439.015 ± 53.865	816.836 ± 41.547	666.994 ± 64.864	< 0.001
T2 (millisecond)	24.54 ± 1.253	30.124 ± 2.269	29.9 ± 1.592	1.000	25.278 ± 1.473	33.544 ± 2.575	30.698 ± 1.582	0.12	25.777 ± 1.304	37.305 ± 1.547	34.641 ± 1.489	< 0.001
ADC (× 10-3, mm2/second)	1.397 ± 0.190	1.076 ± 0.129	1.271 ± 0.135	< 0.001	1.178 ± 0.099	1.028 ± 0.106	1.454 ± 0.147	0.012	1.388 ± 0.229	0.946 ± 0.043	1.164 ± 0.155	0.005

ADC: Apparent diffusion coefficient; CCl₄: Carbon tetrachloride. The P values for the carbon tetrachloride group, compared to the control group, were < 0.001 at all three time periods, therefore, the P values presented in Table 6 correspond specifically to comparisons between the liver fibrosis and treatment groups.