The Mechanism Of The Herbal Compound “Diwu Yanggan” Reducing The Liver Precancerous Degree In 2-Acetylaminofluorene/Partial Hepatectomy Rats

Objectives:To investigate the effects of the herbal compound DWYG on oval cell proliferation in 2-acetylaminofluorene(2-AAF)/partial hepatectomy(PH) rats and determine its mechanism.Methods:Male Wistar rats(180 ± 10 g) were purchased from Hu Bei Province Experimental Animal Research Center. The rats were fed pellet food and water ad libitum in plastic cages at 21 ± 2oC and kept on a 12-hour light-dark cycle. Animal welfare and experimental procedures were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals(The Ministry of Science and Technology of China, 2006) and the related ethical regulations of our hospital. All efforts were made to minimize animals’ suffering and to reduce the number of animals used.The rats were distributed into four groups: normal, sham, vehicle and DWYG. For the sham group, rats’ abdomens were opened, and 1 ml whole blood was taken from the portal vein. No partial hepatectomy(PH) was performed. For vehicle and DWYG groups, the rats received PH. The three groups were then orally administered20 mg/kg 2-AAF once a day for one week. Meanwhile, the the sham and vehicle groups were orally administered distilled water(10 ml/kg) until sacrificed, while the rats of the DWYG group were orally administrated 10 ml/kg DWYG until sacrificed. Sixteen rats in each group were sacrificed on day 8, 10, 14, 17, 19 and 22 post-PH. Liver sections and bone marrow were collected. The survival rate was recorded and analyzed.For hematoxylin-eosin(H&E) staining, liver paraffin sections were dewaxed and rehydrated, and the sections were stained with H&E. Histological observations were made by light microscopy. For immunohistochemical staining, paraffin sections were dewaxed and hydrated and stained with the indicated primary antibodies.The oval cell markers CD34, alpha-fetoprotein(AFP), cytokeratin-19(CK-19) and hematopoietic cell markers CD45, thymus cell antigen 1, theta(Thy1.1) and hepatocyte marker albumin(ALB) were examined with immunohistochemistry. The percentage of CD34/CD45 double-positive cells in bone marrow was detected by flow cytometry. Cytokine levels were measured with the Bio-plex suspension array system. Wnt1、Wnt3、β-catenin、FZD2、GSK3β、C-myc、cyclin D1、Ep CAM was detected by western blot.Results:DWYG significantly increased the survival rates of 2-AAF/PH rats and promoted liver regeneration and reduce the degree of liver Precancerous. Compared to the vehicle group, DWYG-treated livers exhibited significantly healthier-looking livers. H&E staining revealed that hepatic cords of 2-AAF/PH rats were destroyed and small neoformatively basophilia liver cell nodes had formed. Moreover, there were increased bile canaliculi and central veins,and oval cells formed abundant long-cable-like tubular structures. On day 19, vehicle group livers exhibited increased proliferative mitotic oval cells. Strikingly, DWYG ameliorated the above pathological changes and structural disorganization.In the following experiments, we investigated changes in immunohistochemical indices in 2-AAF/PH livers treated with vehicle or DWYG. We examined protein levels of the oval cell markers AFP, CD34, and CK19, the hepatocyte marker ALB, and the hematopoietic cell markers CD45 and Thy 1.1 by immunohistochemistry, and analyzed their mean integral optical density(OD) DWYG significantly and transiently increased the OD of the oval cell marker AFP on day 14(0.00704±0.00621 vs. 0.00030± 0.00032, p<0.01), which then decreased on day 17(0.00001 ± 0.00004 vs. 0.00143±0.00279, p<0.01) and day 19(0.00144±0.00273 vs. 0.00504±0.00494, p<0.05), compared to the vehicle group. Similarly, DWYG significantly increased CK-19 OD on day 8(0.00714±0.00457 vs. 0.00138±0.00215, p<0.01), which then decreased on day 17(0.00169±0.00229 vs. 0.00836±0.00770, p<0.01), compared to the vehicle group. However, DWYG significantly decreased a third oval cell marker CD34 on day 17(0.00000±0.00000 vs. 0.00004±0.00006, p<0.01), day 19(0.00001± 0.00002 vs. 0.00106±0.00149, p<0.01) and day 22(0.00000±0.00000 vs. 0.00004±0.00004, p<0.01). DWYG significantly decreased the hematopoietic cell marker CD45 OD on day 8(0.00182±0.00336 vs. 0.02223±0.01637, p<0.01), day 10(0.00311 ± 0.00680 vs. 0.01182 ± 0.00432, p<0.01) and day 22(0.00000 ± 0.00000 vs. 0.00062 ± 0.00056, p<0.01). It also significantly decreased Thy 1.1 OD on day 19(0.00116±0.00154 vs. 0.00488 ± 0.00628, P<0.01). DWYG significantly decreased thehepatocyte marker ALB OD on day 14(0.00030±0.00039 vs. 0.00193±0.00228, p<0.01), day 17(0.00092±0.00106 vs. 0.01740±0.01170, P<0.01), day 19(0.00602±0.00253 vs. 0.00989±0.00447, P<0.01) and day 22(0.00009±0.00017 vs. 0.00044±0.00019, P<0.01). These results suggest that DWYG has distinct influences on liver immunohistochemical indices at different time points after 2-AAF/PH induction.Because we saw a decrease in differentiated cell marker expression and a transient expression in progenitor oval cell markers, we aimed to determine changes in each population. We next examined the ratio of CD34 and CD45 double-positive cells in the bone marrow of 2-AAF/PH rat. As shown in Figure 4, the percentage of CD34/CD45 double-positive cells significantly increased(p<0.05) in the vehicle group(1.36 ± 0.10) compared to normal(0.80 ± 0.10) and sham groups(0.54 ± 0.03). DWYG significantly(p<0.05) increased the percentage of CD34/CD45 double-positive cells on day 10 and day 14. These results suggest that DWYG promotes the differentiation of bone marrow cells in 2-AAF/PH rats.DWYG groups can be earlier(in the first eight days and before) to activate Wnt / β-catenin pathway, and late days(14-22 days after hepatectomy), DWYG can inhibit sustained activation of Wnt / β-catenin pathway.In the following experiments, we detected changes in cytokine levels at different time points after 2-AAF/PH induction. levels of IL-1, GRO/KC and VEGF were significantly(p<0.05) higher in the vehicle group compared to the normal group, while the levels of IL-6, IFN-γ, TNF-αand TGF-β1 were significantly lower in the vehicle group compared to the normal group(p<0.05). Compared to the vehiclegroup, DWYG rescued cytokine levels more closely to those in the normal group, and the levels of IL-1, GRO/KC and VEGF gradually returned to normal levels. These results imply that DWYG treatment allows the recovery of cytokine levels to normal levels, allowing for liver regeneration after 2-AAF/PH treatment.Conclusions:In this study, we found that 2-AAF suppressed liver regeneration because it was toxic to the liver, resulting in a high death rate of 2-AAF/PH rats. The death rate of rats in the vehicle group was significantly higher compared to that of normal and sham groups. DWYG significantly rescued the death rate of 2-AAF/PH rats and improved pathological liver damage in 2-AAF/PH rats, with a more complete recovery.Hepatic oval cells are the seed of liver regeneration, but can also be hepatic carcinoma(HCC) progenitors cells, suggesting that oval cells are characteristic hepatic stem cells during liver regeneration and cancer initiation. Disruption of the microenvironment is an important factor for oval cell transition into HCC stem cells. 2-AAF/PH is a hepatic precancerous model, and we found that the precancerous lesions in the vehicle group were more severe than those in the normal and sham groups. DWYG bidirectionally regulated oval cell proliferation and differentiation, as it promoted bone-marrow stem cell and oval cell proliferation in early stages(8-14 days post-PH) to support liver regeneration; it also suppressed excessive oval cell proliferation in later stages(17-22 days post-PH) and abnormal differentiation to prevent cancerous hepatic cell lesions.DWYG increased the percentage of CD34/CD45 double-positivecells in 2-AAF/PH rats, which may be associated with DWYG’s promotion of bone marrow hematopoietic cell proliferation, accelerating oval cell generation to achieve liver regeneration. We did not observe excessive bone marrow hematopoietic cell proliferation upon the completion of liver regeneration. Combined with the increased CD34 protein levels, we hypothesize that the promotion of bone marrow cells proliferation would increase the percentage of CD34-positive oval cells to accelerate liver regeneration. Thy1.1 and CD45 are hematopoietic stem cell markers. Compared to vehicle, DWYG significantly decreased Thy1.1 and CD45 protein levels and accelerated peak ALB expression. These results suggest that DWYG efficiently promotes oval cell differentiation to facilitate liver regeneration.In conclusion, our results suggest that DWYG increases 2-AAF/PH rat survival, suppresses hepatic pre-carcinoma changes, and restores hepatic tissue structure and function. We suggest that DWYG acts by promoting bone marrow stem cell and oval cell proliferation during early stages(8-14 days post-PH) to facilitate liver regeneration, while suppressing bone marrow stem cell differentiation to hepatic oval cells during later stages(17-22 days post-PH) to prevent hepatic carcinogenesis. DWYG may modulate liver regeneration by modulating the hepatic stem cell microenvironment, controlling oval cell proliferation and bone marrow stem cell differentiation. Regulation of Wnt / β-catenin pathway is one mechanism of DWYG.