\*

\*\*

##

\*

vs. normal group, ##: *P*<0.05 vs. control group, \*\*: *P*<0.05

\*\* \*\*

\*

\*

\*\*

vs. normal group, ##: *P*<0.05 vs. control group, \*\*: *P*<0.05

Fig. 6. Fuzheng Huayu Recipe and SA-B could decrease the avtivity of serum ALT and AST, and serum level of TBiL, but increase serum concentration of Alb in DMN model rats normal group, n=14; control group, n=9; FZHY group, n=13; SA-B group, n=14.

normal controls FZHY SA-B

**3.1.3.2.4 The concentration of Hyp in the liver tissue of cirrhotic rats was decreased by** 

Compared with normal group, concentration of Hyp in the liver tissue was significantly increased at the end of experiment in controls (*P*<0.01). The concentration of Hyp, however, was significantly lower both in FZHY group and SA-B group than in controls (*P*<0.05). (Fig. 7)

Fig. 5. Fuzheng Huayu Recipe and SA-B could relieve collagen fiber deposition in the liver tissue of DMN model rats A: normal group, B: control group, C: FZHY group, and D: SA-B group. The staining was by Sirius red (200)

Fig. 5. Fuzheng Huayu Recipe and SA-B could relieve collagen fiber deposition in the liver tissue of DMN model rats A: normal group, B: control group, C: FZHY group, and D: SA-B

normal controls FZHY SA-B

\*

\*\*\*\*

\*

## vs. control group, \*\*: *P*<0.01

\*

\*\*

vs. normal group, ##: *P*<0.01

\*

\*\*

#

##

#

ALT(U/L) AST(U/L)

group. The staining was by Sirius red (200)

serum activity of transaminase(U/L)

**A B**

**C D** 

Fig. 6. Fuzheng Huayu Recipe and SA-B could decrease the avtivity of serum ALT and AST, and serum level of TBiL, but increase serum concentration of Alb in DMN model rats normal group, n=14; control group, n=9; FZHY group, n=13; SA-B group, n=14.

#### **3.1.3.2.4 The concentration of Hyp in the liver tissue of cirrhotic rats was decreased by Chinese herb medicine**

Compared with normal group, concentration of Hyp in the liver tissue was significantly increased at the end of experiment in controls (*P*<0.01). The concentration of Hyp, however, was significantly lower both in FZHY group and SA-B group than in controls (*P*<0.05). (Fig. 7)

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Portal vein pressure (mmHg)

n=14.

**3.1.4 Summary** 

ET-1(pg/mg)

Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 103

##

vs. normal group, ##: *P*<0.01 vs. control group, \*: *P*<0.05, \*\*: *P*<0.01

\* \*\*

\* \*

vs. normal group, ##: *P*<0.01 vs. control group, \*: *P*<0.05

Fig. 8. Fuzheng Huayu Recipe and SA-B could decrease portal vein pressure in DMN model

normal controls FZHY SA-B

Fig. 9. Fuzheng Huayu Recipe and SA-B could decrease content of ET-1 in the liver tissue of DMN model rats normal group, n=14; control group, n=9; FZHY group, n=13; SA-B group,

normal controls FZHY SA-B

The elevation of portal vein pressure is positively correlated with the increase of ET-1 concentration in the liver tissue during the process of liver cirrhosis. FZHY Recipe and SA-B

rats normal group, n=12; control group, n=9; FZHY group, n=13; SA-B group, n=14.

##

#### **3.1.3.2.5 Portal vein pressure of the cirrhotic rats was declined and the tissue concentration of ET-1 was dropped by Chinese herb medicine**

The portal vein pressure of control rats was significantly increased in the end of experiment compared with normal rats (*P*<0.01). The increased range was up to 8mmHg. The portal pressure,however, was significantly lower in the rats treated with FZHY Recipe or SA-B than controls (*P*<0.05). (Fig. 8) The hepatic tissue concentration of ET-1 was obviously higher in controls than in normal group (*P*<0.01). But concentration of ET-1 was remarkably declined in FZHY group and SA-B group compared with controls (*P*<0.05). (Fig. 9.)

Fig. 8. Fuzheng Huayu Recipe and SA-B could decrease portal vein pressure in DMN model rats normal group, n=12; control group, n=9; FZHY group, n=13; SA-B group, n=14.

Fig. 9. Fuzheng Huayu Recipe and SA-B could decrease content of ET-1 in the liver tissue of DMN model rats normal group, n=14; control group, n=9; FZHY group, n=13; SA-B group, n=14.

#### **3.1.4 Summary**

102 Portal Hypertension – Causes and Complications

##

\*

\*

vs. normal group, ##: *P*<0.01 vs. control group, \*: *P*<0.05

vs. normal group, ##: *P*<0.01 vs. control group, \*: *P*<0.05

normal controls FZHY

##

Fig. 7. Fuzheng Huayu Recipe and SA-B could decrease concentration of Hyp in the liver tissue of DMN model rats A. normal group, n=12; control group, n=9; FZHY group, n=12. B.

normal controls SA-B

The portal vein pressure of control rats was significantly increased in the end of experiment compared with normal rats (*P*<0.01). The increased range was up to 8mmHg. The portal pressure,however, was significantly lower in the rats treated with FZHY Recipe or SA-B than controls (*P*<0.05). (Fig. 8) The hepatic tissue concentration of ET-1 was obviously higher in controls than in normal group (*P*<0.01). But concentration of ET-1 was remarkably declined in FZHY group and SA-B group compared with controls

**3.1.3.2.5 Portal vein pressure of the cirrhotic rats was declined and the tissue** 

normal group, n=13; control group, n=9; SA-B group, n=12.

**B**

Hyp(ug/g)

**A**

Hyp(ug/g)

(*P*<0.05). (Fig. 9.)

**concentration of ET-1 was dropped by Chinese herb medicine** 

The elevation of portal vein pressure is positively correlated with the increase of ET-1 concentration in the liver tissue during the process of liver cirrhosis. FZHY Recipe and SA-B

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Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 105

Fig. 10. Fuzheng Huayu Recipe inhibited hepatic fibrosis The liver sections were stained with Masson and showed hepatic fibrosis. A: Fibrous septa (arrows indicated) and pseudolobule (arrow's head indicated) have formed in controls. B: Fibrous septa (arrow

> **Groups n Hyp(μg/g liver wet weight)**  normal 7 112.37±11.93 controls 11 292.83±36.78\* FZHY 14 215.03±75.46#

**3.2.3.2 Fuzheng Huayu Recipe could effectively inhibit sinusoid capillarization in fibrotic** 

It was observed that the structure of sinusoids is clearly and the phenotype of SEC is thin by microscopy in normal group. There were many fenestrae in SEC cytoplasm and no basement membrane covered on SEC in perisinusoid side. In controls, the sinusoids were twisted and narrow, the fenestrae in SEC were reduced or disappeared, and basement membrane was observed. (Fig.11)Factor Ⅷ related antigen (Ⅷ R·Ag), -smooth muscle actin (-SMA) and laminin (LM), the important indexes for hepatic sinusoid capillarization, were positive staining in liver tissure. But collagen type Ⅳ (Col Ⅳ), which is a composition of functional membrane but not a composition of basement membrane, expressed majorly in fibrous septa and HSC but less and discontinuously in perisinusoids in controls. Compared with controls, however, the narrow sinusoids were reduced and some sinusoids returned to normal phenotype, basement membrane looked discontinuous thiner, and the positive degree of Ⅷ R·Ag, -SMA and LM were lower and their positive area was significantly smaller by image analysis in the liver of FZHY group than in that of controls (*P*<0.05). Col Ⅳ expressed still continuously in perisinusoids in FZHY group. It indicated that the normal structure of sinusoids was most kept and it meansed that the alteration of hepatic sinusoid

indicated) is less and pseudolobule is absent in FZHY group (100×).

Note: compared with normal, \*: *P*<0.05; compared with controls, #: *P*<0.05

Table 4. Liver Hydroxyproline level in liver tissues ( *x* ±**s**)

capillarization was reversed. (Fig.12)

**rats** 

can dramatically decrease the cirrhosis-induced elevation of portal vein pressure and concentration of ET-1 in the liver tissue through their effect on anti-fibrosis in the liver.

#### **3.2 Experiment 2: Chinese herb medicine can relieve capillarization of sinusoid in cirrhotic rats**

#### **3.2.1 Aims**

To investigate the effects of FZHY Recipe on capillarization of hepatic sinusoids.

#### **3.2.2 Methods**

32 Sprague-Dawley male rats, weighing about 150g, were randomly divided into normal group (n=7) and model group (n=25). The modeling mathod was the same as Experiment 1 for making cirrhosis. The cirrhotic rats were more randomly divided into control group (n=11), and FZHY group (n=14). The drug, dose and method of administration for each group were the same as Experiment 1, too. But the duration of treatment was for 4 weeks.

Rat's blood, under etherization condition, was collected from inferior vena cava, and was separated to serum. The sample of liver (1.0cm×0.8cm×0.3cm) was immediately taken and fixed in neutral formalin, embedded in paraffin, sectioned at 5 m thick. The sections of liver were stained with HE, Masson and immunohistochemistry. Three rats were randomly selected from each group respectively for observation of hepatic microstructure. The each liver sample was cut to a 1 mm3 cube, fixed with 2.5% glutaraldehyde and 1% osmium tetroxide for 2h, embedded with araldite 618, and cut to ultrathin sections. The sections were observed by the electron microscope (H-600). 100mg liver tissue was used to detect Hyp.

#### **3.2.3 Results**

#### **3.2.3.1 Fuzheng Huayu Recipe had the potent effect against hepatic fibrosis of the model rat**

It was observed that hemorrhage, necrosis and extensive connective tissue hyperplasia in the liver tissues of model rats. The fiber septa reached out its branches towards the liver lobule. (Fig. 10 and Tab. 3) The level of hydroxyproline was significantly increased. Compared with controls, liver inflammation, necrosis of hepatocytes and hepatic fibrosis was reduced in FZHY group. The concentration of liver hydroxyproline was significantly lower in FZHY group than in controls (*P*<0.05). (Tab. 4)


Note: compared with controls, \*: U=4.06, *P*<0.01

Table 3. Hyperplasia degree of collagenous fibers in each group

can dramatically decrease the cirrhosis-induced elevation of portal vein pressure and concentration of ET-1 in the liver tissue through their effect on anti-fibrosis in the liver.

32 Sprague-Dawley male rats, weighing about 150g, were randomly divided into normal group (n=7) and model group (n=25). The modeling mathod was the same as Experiment 1 for making cirrhosis. The cirrhotic rats were more randomly divided into control group (n=11), and FZHY group (n=14). The drug, dose and method of administration for each group were the same as Experiment 1, too. But the duration of treatment was for 4 weeks.

Rat's blood, under etherization condition, was collected from inferior vena cava, and was separated to serum. The sample of liver (1.0cm×0.8cm×0.3cm) was immediately taken and fixed in neutral formalin, embedded in paraffin, sectioned at 5 m thick. The sections of liver were stained with HE, Masson and immunohistochemistry. Three rats were randomly selected from each group respectively for observation of hepatic microstructure. The each liver sample was cut to a 1 mm3 cube, fixed with 2.5% glutaraldehyde and 1% osmium tetroxide for 2h, embedded with araldite 618, and cut to ultrathin sections. The sections were observed by the electron microscope (H-600). 100mg liver tissue was used to detect Hyp.

**3.2.3.1 Fuzheng Huayu Recipe had the potent effect against hepatic fibrosis of the model** 

It was observed that hemorrhage, necrosis and extensive connective tissue hyperplasia in the liver tissues of model rats. The fiber septa reached out its branches towards the liver lobule. (Fig. 10 and Tab. 3) The level of hydroxyproline was significantly increased. Compared with controls, liver inflammation, necrosis of hepatocytes and hepatic fibrosis was reduced in FZHY group. The concentration of liver hydroxyproline was significantly

**Groups n Hyperplasia degree of collagenous fibers** 

normal 7 7 0 0 0 0 controls 11 0 1 1 4 5 FZHY \* 14 0 0 8 4 2

0 + ++ +++ ++++

lower in FZHY group than in controls (*P*<0.05). (Tab. 4)

Table 3. Hyperplasia degree of collagenous fibers in each group

Note: compared with controls, \*: U=4.06, *P*<0.01

**3.2 Experiment 2: Chinese herb medicine can relieve capillarization of sinusoid in** 

To investigate the effects of FZHY Recipe on capillarization of hepatic sinusoids.

**cirrhotic rats 3.2.1 Aims** 

**3.2.2 Methods** 

**3.2.3 Results** 

**rat** 

Fig. 10. Fuzheng Huayu Recipe inhibited hepatic fibrosis The liver sections were stained with Masson and showed hepatic fibrosis. A: Fibrous septa (arrows indicated) and pseudolobule (arrow's head indicated) have formed in controls. B: Fibrous septa (arrow indicated) is less and pseudolobule is absent in FZHY group (100×).


Note: compared with normal, \*: *P*<0.05; compared with controls, #: *P*<0.05

Table 4. Liver Hydroxyproline level in liver tissues ( *x* ±**s**)

#### **3.2.3.2 Fuzheng Huayu Recipe could effectively inhibit sinusoid capillarization in fibrotic rats**

It was observed that the structure of sinusoids is clearly and the phenotype of SEC is thin by microscopy in normal group. There were many fenestrae in SEC cytoplasm and no basement membrane covered on SEC in perisinusoid side. In controls, the sinusoids were twisted and narrow, the fenestrae in SEC were reduced or disappeared, and basement membrane was observed. (Fig.11)Factor Ⅷ related antigen (Ⅷ R·Ag), -smooth muscle actin (-SMA) and laminin (LM), the important indexes for hepatic sinusoid capillarization, were positive staining in liver tissure. But collagen type Ⅳ (Col Ⅳ), which is a composition of functional membrane but not a composition of basement membrane, expressed majorly in fibrous septa and HSC but less and discontinuously in perisinusoids in controls. Compared with controls, however, the narrow sinusoids were reduced and some sinusoids returned to normal phenotype, basement membrane looked discontinuous thiner, and the positive degree of Ⅷ R·Ag, -SMA and LM were lower and their positive area was significantly smaller by image analysis in the liver of FZHY group than in that of controls (*P*<0.05). Col Ⅳ expressed still continuously in perisinusoids in FZHY group. It indicated that the normal structure of sinusoids was most kept and it meansed that the alteration of hepatic sinusoid capillarization was reversed. (Fig.12)

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Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 107

Fig. 11. Fuzheng Huayu Recipe could inhibit sinusoid capillarization by observation of electron micrographs S: sinusoid; C: collagen. A: Hepatic sinusoid appeare as a typical capillary surrounded by continuous basement membrane. (arrow's heads indicated) in the liver of fibrotic rat. The perisinusoid is stenosis (arrows indicated) and microvilli of hepatocyte are almost absent in the space (15000×). B: Sinusoid capillarization is not formed in the liver of rat treated with Fuzheng Huayu Recipe. The fenestrae are observed (arrow's heads indicated) in cytoplasm of SEC. A mass of microvilli of hepatocyte stretchs into perisinusoid is exist (arrow indicated) but basement membrane is absent (10000×).

Fig. 11. Fuzheng Huayu Recipe could inhibit sinusoid capillarization by observation of electron micrographs S: sinusoid; C: collagen. A: Hepatic sinusoid appeare as a typical capillary surrounded by continuous basement membrane. (arrow's heads indicated) in the liver of fibrotic rat. The perisinusoid is stenosis (arrows indicated) and microvilli of

hepatocyte are almost absent in the space (15000×). B: Sinusoid capillarization is not formed in the liver of rat treated with Fuzheng Huayu Recipe. The fenestrae are observed (arrow's heads indicated) in cytoplasm of SEC. A mass of microvilli of hepatocyte stretchs into perisinusoid is exist (arrow indicated) but basement membrane is absent (10000×).

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30min after injection.

injection of ET-1.

**3.3.3 Results** 

**injection of ET-1** 

Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 109

(1.0g/kg body weight) and the ET-1 high-dose group (3.0g/kg body weight). Rats were injected with 200l NS solution into rat via femoral vein by a pump with a rate of 80μl/min in NS solution group. As the same method, rats were injected with 200ml ET-1 solution with the dose of 0.3g/kg body weight, 1.0g/kg body weight or 3.0g/kg body weight respectively in 3 ET-1 groups. Then, monitored the changes of carotid pressure and portal vein pressure in each group at 2min, 4min, 6min, 8min, 10min, 15min, 20min, 25min and

20 Sprague-Dawley male rats with about 330g body weight were randomly divided into four groups: normal group, SA-B group, BQ-123 (ETAR antagonist) group and BQ-788 (ETBR antagonist) group (n=5 in each group). In normal group, BQ-123 group and BQ-788 group, rats were gavaged with water at a dose of 1ml/100g body weight and rats in SA-B group were gavaged with SA-B solution (containing 125mg/100ml) at the same dose. The gavage was performed twice per day for 5 days. The experiment started at 1 hour after the final gavage. First, via rat's femoral vein, rats was injected with BQ-123 (12.5µg/kg body weight) in BQ-123 group and with BQ-788 (15µg/kg body weight) in BQ-788 group. 30min later, rats were injected with a dose (3µg/kg body weight) of ET-1 solution by a pump with rate of 80μl/min in each group. Then, the portal vein pressure was measured before and after

A piece of PE-50 tube, which full with heparin solution, was inserted into the left carotid artery of the rat. Following that, connect the tube to a pressure transducer in order to measure carotid pressure and heart rate. Then a piece of PE-10 tube, which full with heparin solution, was inserted from superior mesenteric vein to the middle of portal vein. Connect

**3.3.3.1 A kind of model of rat with portal hypertension was successfully established by** 

There was no obvious effect of solution volume on carotid pressure and portal vein pressure after injection of NS solution. ET-1 solution with equal volume of NS solution was injected into the rats in low- and medium-dose groups. The carotid pressure was not noticeable rise or fall, only minor fluctuations within 20 min after injection. The carotid pressure, however, was increased slightly after injection of ET-1 in high-dose group but the alteration was not significant (Fig.13). Portal vein pressure was increased in short time after injection of ET-1 although the alteration was not same in three different dose groups. The alteration of pressure was most remarkable in high-dose group than in others. After reaching to peak level, portal vein pressure was stable for 4min, 4min and 10min in low-, medium- and highdose groups, respectively (Fig.14 and Tab. 5). The results indicated that the increase of portal vein pressure was solely caused by the injection of ET-1 and without related with the volume of solution in this kind of animal model. This inducing method would not cause a significant increase of systemic blood pressure. Compared with the existing portal vein perfusion model, this kind of portal hypertension model is more similar to the "living" state. It was also avoided that increase of portal vein pressure caused by sudden increase of partial

**3.3.2.2 To treat rats with drugs in portal hypertension models** 

**3.3.2.3 To measure carotid pressure, heart rate and portal vein pressure** 

the tube to a pressure transducer for measurement of portal vein pressure.

Fig. 12. Fuzheng Huayu Recipe could alter the express of indexes for hepatic sinusoid capillarization by immunohistochemistry staining in fibrotic liver of rats Arrows show the positive area. A: Ⅷ R·Ag is around the vessels with strong staining in controls. B: The positive area of Ⅷ R·Ag is decreased in FZHY group. C: Col Ⅳ expresses majorly in fibrous septa and HSC but less and discontinuously in perisinusoids in controls. D: Col Ⅳ expresses almost continuously in perisinusoids in FZHY group. E: The express of LM is observed on the wall of vessels and sinusoids in the liver of controls. F: The express of LM is only observed on the wall of vessels in the liver of FZHY group. G: -SMA expresses strongly in perisinusoids, HSC and fibous septa in controls. H: -SMA expresses dramaticlly less in FZHY group (100×).

#### **3.2.4 Summary**

Fuzheng Huayu Recipe had the potent effect on against liver fibrosis. The one of mechanisms of the recipe perhaps is associated with inhibition of hepatic sinusoid capillarization.

#### **3.3 Experiment 3: SA-B could decrease portal vein pressure in rats induced by ET-1**

#### **3.3.1 Aims**

To establish a rat model of portal hypertension induced by ET-1 and investigate the effect of SA-B on decreasing portal vein pressure in the model.

#### **3.3.2 Methods**

#### **3.3.2.1 To establish portal hypertension model of rats induced by ET-1**

48 Sprague-Dawley male rats were randomly divided into four groups: the NS solution group, the ET-1 low-dose group (0.3g/kg body weight), the ET-1 medium-dose group (1.0g/kg body weight) and the ET-1 high-dose group (3.0g/kg body weight). Rats were injected with 200l NS solution into rat via femoral vein by a pump with a rate of 80μl/min in NS solution group. As the same method, rats were injected with 200ml ET-1 solution with the dose of 0.3g/kg body weight, 1.0g/kg body weight or 3.0g/kg body weight respectively in 3 ET-1 groups. Then, monitored the changes of carotid pressure and portal vein pressure in each group at 2min, 4min, 6min, 8min, 10min, 15min, 20min, 25min and 30min after injection.

#### **3.3.2.2 To treat rats with drugs in portal hypertension models**

20 Sprague-Dawley male rats with about 330g body weight were randomly divided into four groups: normal group, SA-B group, BQ-123 (ETAR antagonist) group and BQ-788 (ETBR antagonist) group (n=5 in each group). In normal group, BQ-123 group and BQ-788 group, rats were gavaged with water at a dose of 1ml/100g body weight and rats in SA-B group were gavaged with SA-B solution (containing 125mg/100ml) at the same dose. The gavage was performed twice per day for 5 days. The experiment started at 1 hour after the final gavage. First, via rat's femoral vein, rats was injected with BQ-123 (12.5µg/kg body weight) in BQ-123 group and with BQ-788 (15µg/kg body weight) in BQ-788 group. 30min later, rats were injected with a dose (3µg/kg body weight) of ET-1 solution by a pump with rate of 80μl/min in each group. Then, the portal vein pressure was measured before and after injection of ET-1.

#### **3.3.2.3 To measure carotid pressure, heart rate and portal vein pressure**

A piece of PE-50 tube, which full with heparin solution, was inserted into the left carotid artery of the rat. Following that, connect the tube to a pressure transducer in order to measure carotid pressure and heart rate. Then a piece of PE-10 tube, which full with heparin solution, was inserted from superior mesenteric vein to the middle of portal vein. Connect the tube to a pressure transducer for measurement of portal vein pressure.

#### **3.3.3 Results**

108 Portal Hypertension – Causes and Complications

Fig. 12. Fuzheng Huayu Recipe could alter the express of indexes for hepatic sinusoid capillarization by immunohistochemistry staining in fibrotic liver of rats Arrows show the positive area. A: Ⅷ R·Ag is around the vessels with strong staining in controls. B: The positive area of Ⅷ R·Ag is decreased in FZHY group. C: Col Ⅳ expresses majorly in fibrous septa and HSC but less and discontinuously in perisinusoids in controls. D: Col Ⅳ expresses almost continuously in perisinusoids in FZHY group. E: The express of LM is observed on the wall of vessels and sinusoids in the liver of controls. F: The express of LM is only observed on the wall of vessels in the liver of FZHY group. G: -SMA expresses strongly in perisinusoids, HSC and

fibous septa in controls. H: -SMA expresses dramaticlly less in FZHY group (100×).

Fuzheng Huayu Recipe had the potent effect on against liver fibrosis. The one of mechanisms of the recipe perhaps is associated with inhibition of hepatic sinusoid

**3.3 Experiment 3: SA-B could decrease portal vein pressure in rats induced by ET-1** 

To establish a rat model of portal hypertension induced by ET-1 and investigate the effect of

48 Sprague-Dawley male rats were randomly divided into four groups: the NS solution group, the ET-1 low-dose group (0.3g/kg body weight), the ET-1 medium-dose group

SA-B on decreasing portal vein pressure in the model.

**3.3.2.1 To establish portal hypertension model of rats induced by ET-1** 

**3.2.4 Summary** 

capillarization.

**3.3.1 Aims** 

**3.3.2 Methods** 

#### **3.3.3.1 A kind of model of rat with portal hypertension was successfully established by injection of ET-1**

There was no obvious effect of solution volume on carotid pressure and portal vein pressure after injection of NS solution. ET-1 solution with equal volume of NS solution was injected into the rats in low- and medium-dose groups. The carotid pressure was not noticeable rise or fall, only minor fluctuations within 20 min after injection. The carotid pressure, however, was increased slightly after injection of ET-1 in high-dose group but the alteration was not significant (Fig.13). Portal vein pressure was increased in short time after injection of ET-1 although the alteration was not same in three different dose groups. The alteration of pressure was most remarkable in high-dose group than in others. After reaching to peak level, portal vein pressure was stable for 4min, 4min and 10min in low-, medium- and highdose groups, respectively (Fig.14 and Tab. 5). The results indicated that the increase of portal vein pressure was solely caused by the injection of ET-1 and without related with the volume of solution in this kind of animal model. This inducing method would not cause a significant increase of systemic blood pressure. Compared with the existing portal vein perfusion model, this kind of portal hypertension model is more similar to the "living" state. It was also avoided that increase of portal vein pressure caused by sudden increase of partial

Traditional Chinese Medicine

Portal vein pressure

(mmHg)

*P*<0.01

controls, ##: *P*<0.01, ###: *P*<0.001

by ET-1 (X±S)

Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 111

medium-dose group high-dose group

**Groups n Initial pressure Highest** 

0 5 7 9 11 13 15 20 25 30 35

Time(min)

Fig. 14. Effect of ET-1 with different doses on rat portal vein pressure After detecting portal vein pressure for 5min, NS solution and ET-1 with same volume but different doses were constantly injected and portal vein pressure was detected for 30min. There was no obvious effect of NS solution on portal vein pressure. The pressure was increased at 5min when starting injection of ET-1 and kept high level for 4min, 4min and 10min in low-, mediumand high-dose groups after the pressure reaching to a peak level at 9 min, respectively.

Portal vein pressure

(mmHg)

 (mmHg) (mmHg) (mmHg) NS 8 6.6±0.3 6.5±0.4 -0.1±0.2 low-dose 10 6.1±0.8 7.5±0.8\*\* 1.3±0.3 medium-dose 10 6.3±0.5 8.1±0.6\*\* 1.8±0.5## high-dose 9 6.0±0.6 8.5±0.9\*\* 2.5±0.5##☆☆

Note: compared with the initial pressure in the interior-group, \*\*: *P*<0.01; compared with NS group, *: P*<0.001; compared with low-dose group, ##*: P*<0.01; compared with medium-dose group, ☆☆:

**Groups n Initial pressure Highest pressure Increased range**  (mmHg) (mmHg) (mmHg) control 5 6.1±0.7 8.6±1.1\*\*\* 2.5±0.4 SA-B 5 6.1±0.7 7.8±1.6\*\* 1.6±0.5## BQ-123 5 5.9±1.1 7.1±1.0 1.2±0.2### BQ-788 5 5.9±0.9 6.9±1.1 0.98±0.5###

Table 5. Effect of ET-1 with different doses on portal vein pressure of rats (X±S)

Note: compared with initial pressure interior-group, \*\*: *P*<0.01, \*\*\*: *P*<0.001; compared with the

**3.3.3.2 SA-B could inhibit the rise of portal vein pressure in rats induced by ET-1** 

Table 6. Effect of SA-B, BQ-123 or BQ-788 on reducining portal vein pressure of rats induced

Portal vein pressur significantly increased after ET-1 solution (3μg/kg body weight) was injected to rats pretreated with water by gavage. Although portal vein pressure of the rats,

**pressure Increased range** 

0 5 7 9 11 13 15 20 25 30 35

Time (min)

blood volume by quickly direct injection of ET-1 in portal vein. This kind of model would better reflect the true effect of ET-1 compared with other kind of model. Therefore, this kind of model could use not only for investigation of efficacy and mechanism of drug, but also provided an ideal experimental subject for screening drugs to treat portal hypertension.

medium-dose group high-dose group

Fig. 13. Effect of ET-1 with different doses on rat carotid pressures After detecting carotic pressure for 5min, NS solution or ET-1 with same volume but different doses were constantly injected and carotic pressure was detected for 30min. The pressure was not increased during detected time in NS group, and low- and medium-dose groups. But the carotid pressure was increased slightly in high-dose group.

medium-dose group high-dose group

blood volume by quickly direct injection of ET-1 in portal vein. This kind of model would better reflect the true effect of ET-1 compared with other kind of model. Therefore, this kind of model could use not only for investigation of efficacy and mechanism of drug, but also provided an ideal experimental subject for screening drugs to treat portal hypertension.

Portal vein pressure

Carotid pressure

(mmHg)

(mmHg)

Portal vein pressure

(mmHg)

0 5 7 9 11 13 15 20 25 30 35

Time(min)

0 5 7 9 11 13 15 20 25 30 35

Time(min)

0 5 7 9 11 13 15 20 25 30 35

Time(min)

NS group low-dose group

medium-dose group high-dose group

NS group low-dose group

carotid pressure was increased slightly in high-dose group.

0 5 7 9 11 13 15 20 25 30 35

Time (min)

0 5 7 9 11 13 15 20 25 30 35

Time (min)

0 5 7 9 11 13 15 20 25 30 35

Time (min)

Portal vein pressure

(mmHg)

Carotid pressure

Carotid pressure

(mmHg)

(mmHg)

Fig. 13. Effect of ET-1 with different doses on rat carotid pressures After detecting carotic pressure for 5min, NS solution or ET-1 with same volume but different doses were constantly injected and carotic pressure was detected for 30min. The pressure was not increased during detected time in NS group, and low- and medium-dose groups. But the

Fig. 14. Effect of ET-1 with different doses on rat portal vein pressure After detecting portal vein pressure for 5min, NS solution and ET-1 with same volume but different doses were constantly injected and portal vein pressure was detected for 30min. There was no obvious effect of NS solution on portal vein pressure. The pressure was increased at 5min when starting injection of ET-1 and kept high level for 4min, 4min and 10min in low-, mediumand high-dose groups after the pressure reaching to a peak level at 9 min, respectively.


Note: compared with the initial pressure in the interior-group, \*\*: *P*<0.01; compared with NS group, *: P*<0.001; compared with low-dose group, ##*: P*<0.01; compared with medium-dose group, ☆☆: *P*<0.01



Note: compared with initial pressure interior-group, \*\*: *P*<0.01, \*\*\*: *P*<0.001; compared with the controls, ##: *P*<0.01, ###: *P*<0.001

Table 6. Effect of SA-B, BQ-123 or BQ-788 on reducining portal vein pressure of rats induced by ET-1 (X±S)

#### **3.3.3.2 SA-B could inhibit the rise of portal vein pressure in rats induced by ET-1**

Portal vein pressur significantly increased after ET-1 solution (3μg/kg body weight) was injected to rats pretreated with water by gavage. Although portal vein pressure of the rats,

Traditional Chinese Medicine

**3.4.1 Aims** 

**3.4.2 Methods** 

blocker group.

after injection of ET-1.

**microcirculation** 

3 groups.

Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 113

To establish portal hypertension model of mice induced by ET-1 and investigate the effect of

36 male Kunming mices, 30±5g body weight, were randomly divided into three groups: control group (n=12), SA-B group (n=12) and blocker group (n=12). Mice were gavaged with SA-B (1mg/ml) in SA-B group and gavaged with water in control group and blocker group. The volume of gvage was 0.5 ml per mouse once a day for 3days. Mice were injected with BQ-123(2g/kg body weight)by tail vein at 0.5h before start of below experiment in

Mice were injected with ET-1(1.6g/kg body weight)at constant velocity by tail vein. 6 mice were randomly taken out from each group to measure blood flow volume in liver microcirculation using a laser-Doppler flow instrument before and after injection of ET-1. Other 6 mice in each group were measured the blood flow rate in liver microcirculation by an inverted fluorescence microscope with microscopic live video technology before and

Each mouse was injected with 2% pentobarbital sodium solution at a dose of 2ml/kg body weight for anesthesia. After opening abdomen the liver was put on a detector connected with a laser-Doppler flow instrument to record the blood flow volum in the perfusion of liver microcirculation for 5min. After a pause, ET-1 was injected by a syringe pump at a constant rate of 80l/ min through tail vein. Then, the blood flow volum was recorded again for 5min. The average blood flow volume was calculated from the stable datas during the

Each mouse was injected with 2% pentobarbital sodium solution at a dose of 2ml/kg body weight for anesthesia. After opening abdomen the liver was put on a piece of glass plate which was on an inverted fluorescence microscope. After 0.05ml pre-labeled erythrocytes with FITC was injected into the inferior vena cava, a suitable site was focused to observe the liver microcirculation. Some FITC-RBCs were observed to be moving quickly in microvasculature or sinusoids and recorded with digital video. ET-1(1.6g /kg body weight) was injected by a syringe pump at a constant rate of 80l/ min through tail vein of mouse then moving FITC-RBCs were immediately recorded again with digital video. The moving range of FITC-RBCs in record was calculated by the analytic software to compare the differences of blood flow rate in liver microcirculation in

**3.4 Experiment 4: SA-B can decrease portal vein pressure in mice induced by ET-1** 

**3.4.2.1 To establish a kind of portal hypertension model mice induced by ET-1** 

**3.4.2.2 To measure the average blood flow volume in the perfusion of liver** 

former 5min record and late 5min record, respectively.

**3.4.2.3 To measure the blood flow rate in liver microcirculation** 

SA-B on decreasing portal vein pressure in this kind of model.

which were pretreated with SA-B solution by gavage or with BQ-123 or BQ-788 by injection, was also increased after ET-1 injection, however, the ranget-increased was obviously less than that in controls (*P*<0.01, *P*<0.001) (Tab.6). Furthermore, the stable time, when portal vein pressure kept high level after reaching peak level, was short in SA-B group. It was just 6 min in SA-B group but was 10min in controls. (Fig. 15)

Fig. 15. SA-B could inhibit portal vein pressure of rats induced by ET-1 Portal vein pressure began increase at 5min when starting injection of ET-1. The peak level of pressure was at 9min in SA-B group. Then the pressure kept high level until to at 15min. In controls, however, the pressure continuously increased until to highest peak level at 15min and began to decrease. The pressure level was still hinger (a little bit higher than a peak level in SA-B group) until to at 25min.


Table 7. Compare of the carotid artery pressure and heart rate in SA-B group and controls

#### **3.3.3.3 SA-B did not affect on carotid pressure and heart rate in normal rat**

Rats were pretreated with SA-B solution by gavage for 5d, but the carotid pressure and heart rate were no significant different between in SA-B group and controls. (Tab.7)

#### **3.3.4 Summary**

A kind of rat model of portal hypertension is successfully established by injection of ET-1. SA-B can down regulate the raised portal vein pressure by antagonizing the effect of extraneous ET-1.

#### **3.4 Experiment 4: SA-B can decrease portal vein pressure in mice induced by ET-1**

#### **3.4.1 Aims**

112 Portal Hypertension – Causes and Complications

which were pretreated with SA-B solution by gavage or with BQ-123 or BQ-788 by injection, was also increased after ET-1 injection, however, the ranget-increased was obviously less than that in controls (*P*<0.01, *P*<0.001) (Tab.6). Furthermore, the stable time, when portal vein pressure kept high level after reaching peak level, was short in SA-B group. It was just

Fig. 15. SA-B could inhibit portal vein pressure of rats induced by ET-1 Portal vein pressure began increase at 5min when starting injection of ET-1. The peak level of pressure was at 9min in SA-B group. Then the pressure kept high level until to at 15min. In controls, however, the pressure continuously increased until to highest peak level at 15min and began to decrease. The pressure level was still hinger (a little bit higher than a peak level in

Time

13min

15min

20min

25min

30min

35min

**pressure**

 (mmHg) (Beats/min) control 3 130.9±13.2 439±21 SA-B 5 145.6±10.1 439±21 Table 7. Compare of the carotid artery pressure and heart rate in SA-B group and controls

Rats were pretreated with SA-B solution by gavage for 5d, but the carotid pressure and

A kind of rat model of portal hypertension is successfully established by injection of ET-1. SA-B can down regulate the raised portal vein pressure by antagonizing the effect of

**Heart rate** 

Control SA-B

**Groups n Carotid artery** 

**3.3.3.3 SA-B did not affect on carotid pressure and heart rate in normal rat** 

heart rate were no significant different between in SA-B group and controls. (Tab.7)

6 min in SA-B group but was 10min in controls. (Fig. 15)

SA-B group) until to at 25min.

0.0 2.0 4.0 6.0 8.0 10.0

Portal vein pressure(mmHg)

0min

5min

7min

9min

11min

**3.3.4 Summary** 

extraneous ET-1.

To establish portal hypertension model of mice induced by ET-1 and investigate the effect of SA-B on decreasing portal vein pressure in this kind of model.

#### **3.4.2 Methods**

#### **3.4.2.1 To establish a kind of portal hypertension model mice induced by ET-1**

36 male Kunming mices, 30±5g body weight, were randomly divided into three groups: control group (n=12), SA-B group (n=12) and blocker group (n=12). Mice were gavaged with SA-B (1mg/ml) in SA-B group and gavaged with water in control group and blocker group. The volume of gvage was 0.5 ml per mouse once a day for 3days. Mice were injected with BQ-123(2g/kg body weight)by tail vein at 0.5h before start of below experiment in blocker group.

Mice were injected with ET-1(1.6g/kg body weight)at constant velocity by tail vein. 6 mice were randomly taken out from each group to measure blood flow volume in liver microcirculation using a laser-Doppler flow instrument before and after injection of ET-1. Other 6 mice in each group were measured the blood flow rate in liver microcirculation by an inverted fluorescence microscope with microscopic live video technology before and after injection of ET-1.

#### **3.4.2.2 To measure the average blood flow volume in the perfusion of liver microcirculation**

Each mouse was injected with 2% pentobarbital sodium solution at a dose of 2ml/kg body weight for anesthesia. After opening abdomen the liver was put on a detector connected with a laser-Doppler flow instrument to record the blood flow volum in the perfusion of liver microcirculation for 5min. After a pause, ET-1 was injected by a syringe pump at a constant rate of 80l/ min through tail vein. Then, the blood flow volum was recorded again for 5min. The average blood flow volume was calculated from the stable datas during the former 5min record and late 5min record, respectively.

#### **3.4.2.3 To measure the blood flow rate in liver microcirculation**

Each mouse was injected with 2% pentobarbital sodium solution at a dose of 2ml/kg body weight for anesthesia. After opening abdomen the liver was put on a piece of glass plate which was on an inverted fluorescence microscope. After 0.05ml pre-labeled erythrocytes with FITC was injected into the inferior vena cava, a suitable site was focused to observe the liver microcirculation. Some FITC-RBCs were observed to be moving quickly in microvasculature or sinusoids and recorded with digital video. ET-1(1.6g /kg body weight) was injected by a syringe pump at a constant rate of 80l/ min through tail vein of mouse then moving FITC-RBCs were immediately recorded again with digital video. The moving range of FITC-RBCs in record was calculated by the analytic software to compare the differences of blood flow rate in liver microcirculation in 3 groups.

Traditional Chinese Medicine

**4.2.1 To observe contraction of HSC** 

experiment was repeated for 3 times.

**4.2.2 To detect the concentration of free calcium ([Ca2+]i) in HSC** 

**4.3.1 SA-B inhibited contraction of HSC induced by ET-1** 

group was between the low- and high-dose groups. (Fig. 17)

**4.1 Aims** 

**4.2 Methods** 

repeated 2 times.

**4.3 Results** 

Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 115

To investigate the effects of SA-B on inhibiting contraction of human HSC induced by ET-1.

Human HSC was isolated from the normal section of transplant patients'liver by enzymatic digestion and density-gradient centrifugation with Nycodenz solution.14. Passaged HSC was planted on the collagen gel which was pre-poured into 12-wells plates. After cells had adhered, HSC was divided into 5 groups: 1) serum-free group in which HSC was cultured with serum-free M199; 2) ET group in which HSC was treated with ET-1(10 -8 mol/L); 3) low-dose group in which HSC was treated with ET-1 and 10-7mol/L SA-B; 4) medium-dose group in which HSC was treated with ET-1 and 10-6mol/L SA-B; 5) high-dose group in which HSC was treated with ET-1 and 10-5mol/L SA-B. The edge of the gel was scraped out wall of the well by a syringe needle after adding drugs. The diameter of gel was observed at 2h, 4h, 6h, and 12 h during HSC culture and a gel image analysis system was used to calculate the area of gel. The area of gel can indicate the contractibility of HSC. The

HSC was planted in 6-wells plates. Flu-3/AM was added into media with final concentration of 5μmol/L. Then HSC was in a incubator with 37℃, 5%CO2-95% humid air and dark for 30min. HSC was washed twice with serum-free M199 for moving the dye and divided into 5 groups as above. HSC in each group was observed under a laser confocal microscope before and after adding relevant drugs. The observation conditions were Kr/Ar laser, excitation wavelength 488nm, emission wavelength 505-530nm. The experiment was

Area of gel was significantly bigger in low-dose group (2209.02±177.96μm2), medium-dose group (2164.95±111.84μm2) and high-dose group (2374.73±218.38μm2) than in ET group (156.23±102.16m2). The statistical difference was dramatically (*P*<0.01). But there was no obviously difference among in 3 SA-B treatment groups. (Fig. 16) The morphological changes of HSC showed dose-dependent after incubation with SA-B. The phenotype of HSC in low-dose group was similar to that in ET group. The phenotype of HSC in high-dose group was similar to that in serum-free group. The phenotype of HSC in medium-dose

**4.3.2 SA-B suppressed the intracellular concentration of [Ca2+]i in HSC induced by ET-1**  Compared with ET group in which fluorescence of HSC was very strong, [Ca2+]i fluorescence image appeared the opposite phenomenon in 3 SA-B treatment groups. Only a few cells appeared slightly enhanced fluorescent and the intracellular concentration of

#### **3.4.3 Results**

#### **3.4.3.1 SA-B reduced average blood flow volume in liver microcirculation**

The blood flow volume in liver microcirculation was decreased in 3 groups after injection of ET-1. The reduced range was smaller in SA-B group and in block group than in controls (*P*<0.01). (Tab. 8)

#### **3.4.3.2 SA-B reduced average blood flow rate in liver microcirculation**

The blood flow rate in liver microcirculation was significantly decreased after injection of ET-1 (*P*<0.01). But the reduced range was smaller in SA-B group and in blocker group than in controls (*P*<0.05 and *P*<0.01). (Tab. 9)


Note: compared with the controls, \*\*: *P*<0.01

Table 8. SA-B and BQ-123 could inhibit reduction of average blood flow volume in mice induced by ET-1 (X ± S)


Note: compared with initial blood flow rate,☆☆: *P*<0.01; compared with controls, \*: *P*<0.05,\*\*: *P*<0.01

Table 9. SA-B and BQ-123 could inhibit reduction of the liver blood flow rate in mice induced by ET-1 (X ± S).

#### **3.4.4 Summary**

The average blood flow volume and blood flow rate in mice liver microcirculation were reduced by extraneous ET-1. SA-B could inhibit the effect of ET-1 to improve liver microcirculation.

#### **4. The experiment** *in vitro*

It is believed that recipe of herb medicine is not suitable for research of mechanism *in vitro*. For more studying the mechanism of Fuzheng Huayu Recipe on reducing portal hypertension, we can only select SA-B as investigated subject *in vitro*.

#### **4.1 Aims**

114 Portal Hypertension – Causes and Complications

The blood flow volume in liver microcirculation was decreased in 3 groups after injection of ET-1. The reduced range was smaller in SA-B group and in block group than in controls

The blood flow rate in liver microcirculation was significantly decreased after injection of ET-1 (*P*<0.01). But the reduced range was smaller in SA-B group and in blocker group than

**Average blood flow volume in liver microcirculation**(**BPU**)

**Before ET-1 injection** 

Table 8. SA-B and BQ-123 could inhibit reduction of average blood flow volume in mice

**Blood flow rate in liver microcirculation**(**mm/s**)

**injection** 

Table 9. SA-B and BQ-123 could inhibit reduction of the liver blood flow rate in mice

Control 6 0.60±0.01 0.36±0.02☆☆ 0.23±0.02 SA-B 6 0.62±0.04 0.39±0.01☆☆ 0.18±0.04 \* blocker 6 0.60±0.05 0.43±0.01☆☆ 0.17±0.05 \*\* Note: compared with initial blood flow rate,☆☆: *P*<0.01; compared with controls, \*: *P*<0.05,\*\*: *P*<0.01

The average blood flow volume and blood flow rate in mice liver microcirculation were reduced by extraneous ET-1. SA-B could inhibit the effect of ET-1 to improve liver

It is believed that recipe of herb medicine is not suitable for research of mechanism *in vitro*. For more studying the mechanism of Fuzheng Huayu Recipe on reducing portal

hypertension, we can only select SA-B as investigated subject *in vitro*.

control 6 1186.83±41.14 1060.50±18.33 126.33±27.51 SA-B 6 1269.50±59.90 1189.33±40.33 80.17±20.30 \*\* blocker 6 202.00±36.54 1131.00±23.67 71.00±12.82 \*\*

**After ET-1** 

**After ET-1** 

**injection Reduced range** 

**injection Reduced range** 

**3.4.3.1 SA-B reduced average blood flow volume in liver microcirculation** 

**3.4.3.2 SA-B reduced average blood flow rate in liver microcirculation** 

**3.4.3 Results** 

(*P*<0.01). (Tab. 8)

in controls (*P*<0.05 and *P*<0.01). (Tab. 9)

Note: compared with the controls, \*\*: *P*<0.01

**Groups n Before ET-1** 

**Groups n**

induced by ET-1 (X ± S)

induced by ET-1 (X ± S).

**4. The experiment** *in vitro* 

**3.4.4 Summary** 

microcirculation.

To investigate the effects of SA-B on inhibiting contraction of human HSC induced by ET-1.

#### **4.2 Methods**

#### **4.2.1 To observe contraction of HSC**

Human HSC was isolated from the normal section of transplant patients'liver by enzymatic digestion and density-gradient centrifugation with Nycodenz solution.14. Passaged HSC was planted on the collagen gel which was pre-poured into 12-wells plates. After cells had adhered, HSC was divided into 5 groups: 1) serum-free group in which HSC was cultured with serum-free M199; 2) ET group in which HSC was treated with ET-1(10 -8 mol/L); 3) low-dose group in which HSC was treated with ET-1 and 10-7mol/L SA-B; 4) medium-dose group in which HSC was treated with ET-1 and 10-6mol/L SA-B; 5) high-dose group in which HSC was treated with ET-1 and 10-5mol/L SA-B. The edge of the gel was scraped out wall of the well by a syringe needle after adding drugs. The diameter of gel was observed at 2h, 4h, 6h, and 12 h during HSC culture and a gel image analysis system was used to calculate the area of gel. The area of gel can indicate the contractibility of HSC. The experiment was repeated for 3 times.

#### **4.2.2 To detect the concentration of free calcium ([Ca2+]i) in HSC**

HSC was planted in 6-wells plates. Flu-3/AM was added into media with final concentration of 5μmol/L. Then HSC was in a incubator with 37℃, 5%CO2-95% humid air and dark for 30min. HSC was washed twice with serum-free M199 for moving the dye and divided into 5 groups as above. HSC in each group was observed under a laser confocal microscope before and after adding relevant drugs. The observation conditions were Kr/Ar laser, excitation wavelength 488nm, emission wavelength 505-530nm. The experiment was repeated 2 times.

#### **4.3 Results**

#### **4.3.1 SA-B inhibited contraction of HSC induced by ET-1**

Area of gel was significantly bigger in low-dose group (2209.02±177.96μm2), medium-dose group (2164.95±111.84μm2) and high-dose group (2374.73±218.38μm2) than in ET group (156.23±102.16m2). The statistical difference was dramatically (*P*<0.01). But there was no obviously difference among in 3 SA-B treatment groups. (Fig. 16) The morphological changes of HSC showed dose-dependent after incubation with SA-B. The phenotype of HSC in low-dose group was similar to that in ET group. The phenotype of HSC in high-dose group was similar to that in serum-free group. The phenotype of HSC in medium-dose group was between the low- and high-dose groups. (Fig. 17)

#### **4.3.2 SA-B suppressed the intracellular concentration of [Ca2+]i in HSC induced by ET-1**

Compared with ET group in which fluorescence of HSC was very strong, [Ca2+]i fluorescence image appeared the opposite phenomenon in 3 SA-B treatment groups. Only a few cells appeared slightly enhanced fluorescent and the intracellular concentration of

Traditional Chinese Medicine

**A B** 

**C D** 

**E** 

system microscope, Japan, 200×).

Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 117

Fig. 17. The morphological changes of HSC in each group a: serum-free group; b: ET group; c: low-dose group; d: medium-dose group; e: high-dose group. HSC grew exuberantly on gel and the pseudopodium of HSC was a lot, thin and long in serum-free group. After induced by ET-1 for 12h, however, HSC became obviously small with contraction. The quantity and the pseudopodium of HSC was decreased in ET group. The phenotype of HSC in low-dose group was similar to that in ET group. The phenotype of HSC in high-dose group was similar to that in serum-free group. The phenotype of HSC in medium-dose group was between the low- and high-dose groups (By OLYMPUS I × 50 / I × 70 inverted

[Ca2+]i was obviously lower in 3 SA-B treatment groups than in ET group. The intracellular concentration of [Ca2+]i was weak and only two relative low-lying peaks were in low-dose group within 120sec. The alteration of intracellular concentration of [Ca2+]i was smaller in medium-dose group and high-dose group during observation. (Fig.18). The results indicated that one of mechanisms of SA-B on inhibiting contraction of HSC may be related with that SA-B suppressed the intracellular concentration of [Ca2+]i in HSC.

★★ VS model group *P*<0.01 ☆☆ VS serum-free group *P*<0.01

Fig. 16. SA-B could inhibit contraction of human HSC a: serum-free group; b: ET group; c: low-dose group; d: medium-dose group; e: high-dose group. Human HSC was cultured on collangen gel. Actived HSC are able to contract to lead to area of gel becoming small. In serum-free group, contraction capacity of HSC was weak so that the area of gel was nearly invariant. Active HSC induced by ET-1 contracted strongly. The area of gel was significantly smaller in ET group than in serum-free groups. SA-B could inhibit contraction of HSC induced by ET-1. The area of gel was bigger in 3 SA-B treatment groups than in ET group.

[Ca2+]i was obviously lower in 3 SA-B treatment groups than in ET group. The intracellular concentration of [Ca2+]i was weak and only two relative low-lying peaks were in low-dose group within 120sec. The alteration of intracellular concentration of [Ca2+]i was smaller in medium-dose group and high-dose group during observation. (Fig.18). The results indicated that one of mechanisms of SA-B on inhibiting contraction of HSC may be related

d

e

Fig. 16. SA-B could inhibit contraction of human HSC a: serum-free group; b: ET group; c: low-dose group; d: medium-dose group; e: high-dose group. Human HSC was cultured on collangen gel. Actived HSC are able to contract to lead to area of gel becoming small. In serum-free group, contraction capacity of HSC was weak so that the area of gel was nearly invariant. Active HSC induced by ET-1 contracted strongly. The area of gel was significantly smaller in ET group than in serum-free groups. SA-B could inhibit contraction of HSC induced by ET-1. The area of gel was bigger in 3 SA-B treatment groups than in ET group.

☆☆

Groups

★★ ★★ ★★

medium-dose

★★ VS model group *P*<0.01 ☆☆ VS serum-free group *P*<0.01

with that SA-B suppressed the intracellular concentration of [Ca2+]i in HSC.

a

b

c

Fig. 17. The morphological changes of HSC in each group a: serum-free group; b: ET group; c: low-dose group; d: medium-dose group; e: high-dose group. HSC grew exuberantly on gel and the pseudopodium of HSC was a lot, thin and long in serum-free group. After induced by ET-1 for 12h, however, HSC became obviously small with contraction. The quantity and the pseudopodium of HSC was decreased in ET group. The phenotype of HSC in low-dose group was similar to that in ET group. The phenotype of HSC in high-dose group was similar to that in serum-free group. The phenotype of HSC in medium-dose group was between the low- and high-dose groups (By OLYMPUS I × 50 / I × 70 inverted system microscope, Japan, 200×).

Traditional Chinese Medicine

**5. Conclusion** 

liver diseases.

**7. Reference** 

**6. Acknowledgements** 

Yuanwei is gratefully acknowledged.

1996,110:534-548.

Anat Rec (Hoboken). 2008,291(6):661-71.

hepatitis. J Hepatol. 2009,50(3):631-3.

Hepatol 1995, 22: 700-706.

Hepatopancreatobiliary Surgery. 2000,12(1):53-54.

Can Improve Liver Microcirculation and Reduce Portal Hypertension in Liver Cirrhosis 119

The Chinese medicine, Fuzheng Huayu Recipe, is able to reduce portal hypertension in liver cirrhosis both in clinic and *in vivo*. The effect of Fuzheng Huayu Recipe is related with reversing hepatic fibrosis and capillarization, and decreasing concentration of ET-1 in the liver tissue. SA-B is an extracted component of Fuzheng Huayu Recipe. Several studies showed that the effect in SA-B was similar to Fuzheng Huayu Recipe on reversing hepatic fibrosis and decreasing concentration of ET-1 in the liver tissue. SA-B inhibited contraction of HSC through suppressing the intracellular concentration of [Ca2+]i. Based on above effects, intrahepatic vessel resistance was reduced which leaded to improve liver microcirculation. It has distinctively character that Chinese herb medicine is used to treat hepatic portal hypertension which is caused by failure of liver microcirculation in chronic

This work was supported by grants from National Natural Science Foundation of China 30672489; Leading Academic Discipline Project of Shanghai Municipal Education Commission J50307; E-Institute of TCM Internal Medicine, Shanghai Municipal Education Commission E03008; Innovation Research Team in Universities, Shanghai Municipal Education Commission; and Leading Academic Discipline Project of Hepatology, SATCM (2011sh). Translation assistance from Lu Chao, Wang Meifeng, Zhang Jing, Wu Mei and Pan

McCuskey RS. The hepatic microvascular system in health and its response to toxicants.

Wu Zhiyong. Regulation of liver microcirculation and portal hypertension. J.

Pinzani M, Milani S, Defranco R, et al. Endothelin 1 is overexpressed in human cirrhotic

Vollmar B, Menger. The hepatic microcirculation: mechanistic contributions and therapeutic

Spengler U. Hepatic microcirculation: a critical but neglected factor for the outcome of viral

Nishida J, McCuskey RS, McDonnell D. Protective Role of NO in Hepatic Microcirculatory Dysfunction during Endotoxemia. Am J Physiol. 1994,267(6 Pt 1):G1135-41. Ramadori G. The stellate cell of the liver. Virchows Arch B Cell Pathol 1991, 61:147-158. Pinzani M. Hepatic stellate (ITO) cells: expanding roles for a liver-specific pericyte. J

ZHANG Jie, XU Lieming, ZHANG Wenwei. Study of Effect and Mechanism of Salvianolic-

by Endothelin-1. Chinese J Integrative Medicine. 2009,29(1):60-64.

acid B Salt on Inhibiting the Contraction of Human Hepatic Stellate Cells Induced

targets in liver injury and repair. Physiol Rev. 2009,89(4):1269-339.

liver and exerts multiple effects on activated hepatic stellate cells. Gastroenterology

Fig. 18. SA-B suppressed the intracellular concentration of [Ca2+]i in HSC Fluorescent of Flu-3/AM which represents the intracellular concentration of [Ca2+]i was observed. The expression of fluorescent in HSC was very stronger in ET group than in serum-free group. But the expression of fluoresent in HSC was dose-dependent in 3 SA-B treatment groups. The higher the dose of SA-B was, the weaker expression of fluorescent was (By Zeiss510 laser confocal microscope, German, 200×).

#### **5. Conclusion**

118 Portal Hypertension – Causes and Complications

**Groups:** 

serum-free ET

high-dose

low-dose medium-dose

Fig. 18. SA-B suppressed the intracellular concentration of [Ca2+]i in HSC Fluorescent of Flu-

3/AM which represents the intracellular concentration of [Ca2+]i was observed. The expression of fluorescent in HSC was very stronger in ET group than in serum-free group. But the expression of fluoresent in HSC was dose-dependent in 3 SA-B treatment groups. The higher the dose of SA-B was, the weaker expression of fluorescent was (By Zeiss510

laser confocal microscope, German, 200×).

The Chinese medicine, Fuzheng Huayu Recipe, is able to reduce portal hypertension in liver cirrhosis both in clinic and *in vivo*. The effect of Fuzheng Huayu Recipe is related with reversing hepatic fibrosis and capillarization, and decreasing concentration of ET-1 in the liver tissue. SA-B is an extracted component of Fuzheng Huayu Recipe. Several studies showed that the effect in SA-B was similar to Fuzheng Huayu Recipe on reversing hepatic fibrosis and decreasing concentration of ET-1 in the liver tissue. SA-B inhibited contraction of HSC through suppressing the intracellular concentration of [Ca2+]i. Based on above effects, intrahepatic vessel resistance was reduced which leaded to improve liver microcirculation. It has distinctively character that Chinese herb medicine is used to treat hepatic portal hypertension which is caused by failure of liver microcirculation in chronic liver diseases.

#### **6. Acknowledgements**

This work was supported by grants from National Natural Science Foundation of China 30672489; Leading Academic Discipline Project of Shanghai Municipal Education Commission J50307; E-Institute of TCM Internal Medicine, Shanghai Municipal Education Commission E03008; Innovation Research Team in Universities, Shanghai Municipal Education Commission; and Leading Academic Discipline Project of Hepatology, SATCM (2011sh). Translation assistance from Lu Chao, Wang Meifeng, Zhang Jing, Wu Mei and Pan Yuanwei is gratefully acknowledged.

#### **7. Reference**


**7** 

*Argentina* 

**Role of Manganese** 

Amalia Delfante2, Pablo Souto2,

 *and Department of Pathophysiology, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, 2Laboratory of Portal Hypertension,* 

*University of Buenos Aires, Buenos Aires,* 

Juan Pablo Prestifilippo1,2, Silvina Tallis2,

*1Institute of Pharmacological Research (ININFA), National Research Council of Argentina (CONICET)* 

Juan Carlos Perazzo2 and Gabriela Beatriz Acosta1,2

*School of Pharmacy and Biochemistry & Hepatic Encephalopathy,* 

**as Mediator of Central Nervous System:** 

**Alteration in Experimental Portal Hypertension** 

Portal hypertension (PH) is a major syndrome that frequently accompany chronic liver diseases such as cirrhosis. Prehepatic PH develops a splanchnic hyperdynamic circulation and hyperemia with increased splanchnic resistance and production of collateral vessels that drive splanchnic blood flow to systemic circulation (Chojkier & Groszmann, 1981). Several substances have been proposed as mediators of this hypodynamic circulatory state including prostacyclins, nitric oxide and endotoxins (Bosch et al., 1992; Reiner & Groszmann, 1999; Palma et al., 2005). PH is found in patients with cirrhosis, and in portal vein thrombosis. It is characterized by an increase in splanchnic blood flow and pressure, among others caused by abdominal blood flow resistance, secondary to important liver

Recent studies have demonstrated that experimental PH in rats is also a sub-clinic model of Minimal Hepatic Encephalopathy (MHE) (Butterworth et al., 2009), since rats with PH develop hyperammonemia, electrophysiology alterations, blood-brain barrier (BBB) breakdown, hippocampal mitochondrial dysfunction and changes in frontal cortex and hippocamus on glutamate uptake (Scorticati et al., 2004; Lores-Arnaiz et al., 2005; Eizayaga

Chronic hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome associated with liver dysfunction, such as cirrhosis. The pathophysiology of HE is poorly understood and there are few high-quality diagnostic tests and markers. As a result, its treatment has

**1. Introduction** 

parenchyma alterations (fibrosis or cirrhosis).

et al., 2006; Acosta et al., 2009; Bustamante et al., 2011).

