1. BBD improved the general condition of mice of the treatment group compared with that of mice of the model group.
As for the control group, the mice were in good mental condition with normal activity ,stable body weight, bright fur, light red limbs and mouth corners, slightly yellow urine , and moderate stool humidity. Compared with control group, the general condition of mice in the model and treatment group was worse. The mental state was worse with declined activity level, slow movement and weight loss. The Fur on head and back was lost and dull. The color of limbs, mouth corners and tail circumference became yellow, the color of urine became yellow and the volume decreased, stool became dry. The mice became emotional, defensive, and had aggressivity and aggressive behavior. After administration, compared with the model group, the general condition of mice in the treatment group improved. As the time of administration increase, the mental state ,activity level, fur and urine improved. During the administration period, there were no obvious adverse reactions occurred in each model group.
2. BBD made the liver function indexes decreased and improved the liver function.
Compared with the control group, liver functional indexes (TBA, TBil, DBil, ALT, AST, ALP, GGT) in the model group were significantly increased (P<0.01). Compared with the model group, liver functional indexes(TBA, TBil, DBil, ALT, AST, ALP, and GGT) in the treatment group were significantly decreased (P<0.01),the differences among all groups were statistically significant.(See Table 2)
Compared with the control group, the liver fibrosis indexes (LN, HA, PC-Ⅲ and C-Ⅳ) in the model group and the treatment significantly increased(P<0.01). Compared with the model group, the liver fibrosis indexes (LN, HA, PC-Ⅲ and C-Ⅳ) in the treatment group significantly decreased(P<0.01), and the differences between the groups were statistically significant. (See Table 3)
3. BBD improved liver tissue structure and inhibited collagen synthesis in the liver.
3.1 HE staining
In the control group, liver cells were neatly arranged and cord-like radiated without necrosis or degeneration. The structure of liver lobules was clear and complete, the structure of portal area was normal, and the epithelial cells of bile duct were neatly arranged without blockage. No abnormal hyperplasia of connective tissue was observed in the central vein and portal area, and no inflammatory cell infiltration was observed. In the model group, mice liver cells cord arrangement was disorder and irregular, liver cell became swelling, and had fat necrosis, balloon-like degeneration, large area of necrosis, connective tissue in the necrosis area significantly increased, forming fibrous septum, which destroyed liver lobular structure and then gradually became false lobules, finally developed to liver fibrosis. Meanwhile, it could be found that the significant hyperplasia of bile duct epithelial cells, the obstruction of bile duct and the infiltration of a large number of inflammatory cells in the portal area. Compared with model group, the arrangement and hierarchy of liver cells in the treatment group were more regular, the cell size was more uniform, and the morphology and structure were clearer. The edema of liver cells, fatty necrosis of hepatocytes, necrotic area of liver, obstruction of bile duct and inflammatory cell infiltration in portal area were significantly reduced. The liver injury was improved obviously, and the tissue structure and cell morphology were close to normal condition compared with model group. (see Figure1)
3.2 Masson staining
A small amount of collagen fibers were seen in the wall of the control group, which is in the normal range. Compared with the control group, a large number of bright blue collagen fibers deposited in the liver tissue of the model group, and the staining was deeper. More collagen fibers were found in the liver tissue, fiber spacing varied in width, and pseudo-lobules were formed. The change of collagen fibers in the treatment group were between the control group and the model group. (see Figure 2)
3.3 Picric acid-Sirius red staining(Ⅰ and III collagen fibers observed by polarized light microscopy)
In the control group, a small amount of type Ⅰ and Ⅲ collagen in mouse liver tissue can be observed, which were distributing in the portal area, blood vessel wall and bile duct wall. By contrast, in the model group, the type Ⅰ collagen in liver tissue increased significantly, which was distributing in the space around the hepatic sinus and the lobular vein and partially extending into the lobules, then forming fibrous septa between the leaves. There were a large number of orange-red crude fibers in the portal area with strong refraction. Type Ⅲ collagen is increased and scattered around type Ⅰ collagen. Compared with the model group, in the treatment group, collagen fibers in liver tissue are mainly composed of green type Ⅲ collagen which were radiated in filaments and showed weak birefringence. The green type Ⅲ collagen mainly distributed in the portal area and small blood vessel wall, meanwhile, it also can be observed around the hepatic sinus. Type Ⅲ collagen was accompanied by type Ⅰ collagen which showed red or yellow coarse fibrousa and strong birefringence. (See Figure 3)
3.4 Collagen fiber area analysis
Collagen fiber area analysis showed that the collagen area of the model group increased significantly compared with the control group, and the differences was statistically significant (P<0.01).Compared with the model group, the collagen area of the treatment group significantly decreased, and the differences was statistically significant (P<0.01).There was no significant differences of the area of fibrosis between Masson staining and picric acid-Sirius red staining between the same group (P>0.01). (See Table 4)
4. BBD inhibited expression levels of TGF-β1 and Smad3 proteins in mouse liver tissues
4.1 TGF-β1 expression
A few brownish yellow positive granules in liver tissue of control group were observed. No positive expression was found in hepatocytes and interstitial cells, and a small amount of positive expression was found in hepatic sinus space. A large number of colored particles can be seen in the model group. In the fibrosis portal areas and increased fibrous tissue areas, it showed mainly that a large number of positive staining and cytoplasmic expression were observed in interstitial cells, cytoplasm of inflammatory cells, spaces between hepatic sinuses and wall of central venous vessels. It can be seen that TGF-β1 is mostly derived from mesenchymal cells. The expression of TGF-β1 protein in the treatment group was lower than that of the model group, the number of positive cells was significantly reduced, and the degree of staining was significantly reduced, the TGF-β1 protein mainly expressed on the interstitial cell membrane and in the cytoplasm. Compared with the normal group, the expression of TGF-β1 protein in the model group and the treatment group increased significantly, and the differences were statistically significant (P<0.01). Compared with the model group, the expression of TGF-β1 protein in the treatment group was decreased obviously, and the differences were statistically significant (P<0.01). (See Figure 4, Table 5)
4.2 Smad3 expression
There was no positive expression of hepatocytes in the control group, and a small amount of chromogenic particles were observed in the portal areas. Compared with control group, a large number of positive chromogenic particles were observed in the model group, mainly locating in the portal areas, hepatic sinus spaces, fibrous areas, and around the blood vessel wall, and the expression of Smad3 protein was mainly cytoplasmic expression. The positive expression of the treatment group was less than that of the model group, and the positive particles were significantly reduced. Compared with the control group, the expression of Smad3 protein positive particles in the model group and treatment group increased significantly, and the differences were statistically significant (P<0.01).Compared with the model group, the expression of Smad3 protein positive particles in the treatment group was decreased obviously, and the differences was statistically significant (P<0.01). (See Figure 5, Table 5)