Gastroenterol Res

Gastroenterology Research, ISSN 1918-2805 print, 1918-2813 online, Open Access

Article copyright, the authors; Journal compilation copyright, Gastroenterol Res and Elmer Press Inc

Journal website http://www.gastrores.org

Original Article

Volume 1, Number 1, December 2008, pages 20-28

Ginkgo Biloba Extract EGb 761 Alleviates Hepatic Fibrosis and Sinusoidal Microcirculation Disturbance in Patients with Chronic Hepatitis B

Figure 2. Light microscopic observation after EGb 761 treatment. Light microscopy observations of liver biopsy samples. (a, b), H&E staining, before treatment, swollen and vacuolated hepatocytes, spotty and patchy acidophilic bodies were seen, inflammatory cells infiltration; aggregates of red blood cells in sinusoids (a); after EGb 761 treatment, acidophilic bodies were rare, no obstructed sinusoids (b). (c, d) Masson 3 staining, collagen fibrin hyperplasia, thickened fibrous septae (arrows, c); after EGb 761 treatment, collagen fibrin decreased, fibrous septa became thinner (arrow, d). (e, f) Meticular fibrin staining, reticular fibrin hyperplasia, fibrous septae thickened (arrow, e); after EGb 761 treatment, reticular fibrin significantly decreased, fibrous septae became thinner (f). Original magnification, a-f, x100.

**Table 1.** Baseline characteristics of the subjects at entry
Characteristics	Treated	Controls	p
No. patients	32	32
Age (y)	44.7 ± 10.8	42.9 ± 11.3	NS
Weight (kg)	63.7 ± 11.7	62.2 ± 9.8	NS
Male/female	27/13	22/12	NS
Hepatitis history (y)	3.4 ± 1.8	3.3 ± 1.7	NS

Table 1. Baseline characteristics of the subjects at entry

Characteristics

Treated

Controls

No. patients

Age (y)

44.7 ± 10.8

42.9 ± 11.3

Weight (kg)

63.7 ± 11.7

62.2 ± 9.8

Male/female

27/13

22/12

Hepatitis history (y)

3.4 ± 1.8

3.3 ± 1.7

**Table 2.** Biochemistry and virology evaluation
Group	n	ALT (IU/L)	ALB (g/L)	TBIL (µmol/L)	PT (s)	HBV DNA (x10⁷copies/ml)
Data are expressed as mean ± SD. Fisher’s exact test, EGb 761 versus Control. NS, not significant (P>0.05). Comparisons of blood levels of ALT, TBil, PT, ALB, and HBV DNA titres between baseline and after treatment in the same group; and comparisons inter-groups. ALT, alanine aminotransferase; TBIL, total bilirubin; ALB, albumin; PT, prothrombin. P<0.05 VS baseline, *P<0.01 VS baseline
Treated	Baseline	32	75.7±18.5	34.9±4.4	39.3±21.2	15.2±3.4	4.5±2.7
	Week 4	32	37.3±16.3^*	38.2±5.9^*	17.1±9.5^**	13.2±2.1^*	4.4±2.4
Controls	Baseline	28	83.3± 11.4	35.1±4.8	38.2±25.4	15.0±3.5	4.3±2.5
	Week 4	28	35.2±18.6^*	38.3±6.1^*	17.6±8.1^*	13.1±1.9^*	4.4±2.3

Table 2. Biochemistry and virology evaluation

Group

ALT (IU/L)

ALB (g/L)

TBIL (µmol/L)

PT (s)

HBV DNA (x10⁷copies/ml)

Data are expressed as mean ± SD. Fisher’s exact test, EGb 761 versus Control. NS, not significant (P>0.05). Comparisons of blood levels of ALT, TBil, PT, ALB, and HBV DNA titres between baseline and after treatment in the same group; and comparisons inter-groups. ALT, alanine aminotransferase; TBIL, total bilirubin; ALB, albumin; PT, prothrombin. *P<0.05 VS baseline, **P<0.01 VS baseline

Treated

Baseline

75.7±18.5

34.9±4.4

39.3±21.2

15.2±3.4

4.5±2.7

Week 4

37.3±16.3^*

38.2±5.9^*

17.1±9.5^**

13.2±2.1^*

4.4±2.4

Controls

Baseline

83.3± 11.4

35.1±4.8

38.2±25.4

15.0±3.5

4.3±2.5

Week 4

35.2±18.6^*

38.3±6.1^*

17.6±8.1^*

13.1±1.9^*

4.4±2.3

**Table 3.** Serum TGF- β1, PAF, ET-1
Group	n	TGF-β₁ (µg/L)	PAF (µg/L)	ET-1 (µg/L)
Comparisons of TGF-β, PAF and ET-1 in two groups before and after treatment. TGF-β₁: transforming growth factor β₁; PAF platelet activate factor; ET-1 endothelin-1. P<0.05 VS baseline; * P<0.01 VS baseline, ^△P<0.05 VS week 4 in treated group; ^△△P<0.01 VS week 4 in treated group.
Treated	Baseline	32	58.43±11.04	13.23±9.79	68.13±21.71
	Week 4	32	17.61±5.06^*	7.62±6.54^*	47.61±15.34^**
Controls	Baseline	28	57.69±10.23	12.44±9.63	65.46±20.67
	Week 4	28	61.17±11.45^△	11.65±8.96^△	61.17±16.45^△△

Table 3. Serum TGF- β1, PAF, ET-1

Group

TGF-β₁ (µg/L)

PAF (µg/L)

ET-1 (µg/L)

Comparisons of TGF-β, PAF and ET-1 in two groups before and after treatment. TGF-β₁: transforming growth factor β₁; PAF platelet activate factor; ET-1 endothelin-1. *P<0.05 VS baseline; ** P<0.01 VS baseline, ^△P<0.05 VS week 4 in treated group; ^△△P<0.01 VS week 4 in treated group.

Treated

Baseline

58.43±11.04

13.23±9.79

68.13±21.71

Week 4

17.61±5.06^*

7.62±6.54^*

47.61±15.34^**

Controls

Baseline

57.69±10.23

12.44±9.63

65.46±20.67

Week 4

61.17±11.45^△

11.65±8.96^△

61.17±16.45^△△

**Table 4.** HAI score and collagen levels
Group	n	Inflammation score	Fibrosis score	Collagen (%)
Hepatic inflammation, fibrosis and collagen deposit before and after treatment in two groups. *P<0.05 VS baseline; ^△P<0.05 VS 4 weeks in treated group. Collagen density was represented by the collagen percentage in each field, data was mean ± SD.
Baseline	26	15.7±6.3	3.2±0.6	21.6±9.3
Week 4	26	10.7±4.8^*	2.2±0.3^*	14.7±6.4^*
Baseline	21	15.6±5.9	3.3±0.4	22.7±9.8
Week 4	21	14.2±6.6^△	3.1±0.5^△	21.2±9.3^△

Table 4. HAI score and collagen levels

Group

Inflammation score

Fibrosis score

Collagen (%)

Hepatic inflammation, fibrosis and collagen deposit before and after treatment in two groups. *P<0.05 VS baseline; ^△P<0.05 VS 4 weeks in treated group. Collagen density was represented by the collagen percentage in each field, data was mean ± SD.

Baseline

15.7±6.3

3.2±0.6

21.6±9.3

Week 4

10.7±4.8^*

2.2±0.3^*

14.7±6.4^*

Baseline

15.6±5.9

3.3±0.4

22.7±9.8

Week 4

14.2±6.6^△

3.1±0.5^△

21.2±9.3^△

**Table 5.** Sinusoidal microcirculation observation (per 100 sinusoids). (a-d) Before EGb 761 treatment, (a) Hepatocytes lysis, necrosis (arrow), collagen fibrin deposit; (b)mircrothrombosis in the sinusoid (arrow), collagen fibrin deposit in the Disse space; (c) sinusoidal endothelial cells fenestrae disappered, collagen fibrin deposits in the Disse space (arrow); (d) collagen fibrin deposit in sinusoids and Disse space (arrow) with clogged sinusoidal lumen. (e-f) After EGb 761 treatment, (e) Hepatocellular organelle degeneration, however cellular morphology, structure and intercellular connection were normal; (f) there was no obstructed sinusoids and Disse space, without microthrombosis and ECM deposits. Original magnification, a-f, X 28000.
	n	Microthrombosis	Collagen	Endothelial Damage	Capillarization
Ultrastructural observation of sinusoidal microcirculation in the two groups before and after treatment. Microthrombosis, collagen deposit, endothelial damage and capilliarization were examined for each sinusoid, 100 sinusoids were observed for each group before or after treatment. P<0.05 VS baseline;* P<0.01 VS baseline; ^△P<0.05 VS week 4 in control group.
Treated	Baseline	100	56	38	59	32
	Week 4	100	32^**△	23^*△	41^*△	17^*△
Controls	Baseline	100	58	37	61	30
	Week 4	100	53	34	57	29

Table 5. Sinusoidal microcirculation observation (per 100 sinusoids). (a-d) Before EGb 761 treatment, (a) Hepatocytes lysis, necrosis (arrow), collagen fibrin deposit; (b)mircrothrombosis in the sinusoid (arrow), collagen fibrin deposit in the Disse space; (c) sinusoidal endothelial cells fenestrae disappered, collagen fibrin deposits in the Disse space (arrow); (d) collagen fibrin deposit in sinusoids and Disse space (arrow) with clogged sinusoidal lumen. (e-f) After EGb 761 treatment, (e) Hepatocellular organelle degeneration, however cellular morphology, structure and intercellular connection were normal; (f) there was no obstructed sinusoids and Disse space, without microthrombosis and ECM deposits. Original magnification, a-f, X 28000.

Microthrombosis

Collagen

Endothelial Damage

Capillarization

Ultrastructural observation of sinusoidal microcirculation in the two groups before and after treatment. Microthrombosis, collagen deposit, endothelial damage and capilliarization were examined for each sinusoid, 100 sinusoids were observed for each group before or after treatment. *P<0.05 VS baseline;** P<0.01 VS baseline; ^△P<0.05 VS week 4 in control group.

Treated

Baseline

100

Week 4

100

32^**△

23^*△

41^*△

17^*△

Controls

Baseline

100

Week 4

100