Herbal Compound "Songyou Yin" Inhibited Malignant Potential Of Hepatocellular Carcinoma With Liver Fibrosis Through Regulating Hepatic Stellate Cell

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors of digestive system, and incidence of HCC is rising worldwide. Meanwhile, the rapid proliferation and invasive ability of HCC have led to the diagnoses of most of the patients late and lost the opportunities of resection. Therefore, more than 70% of HCC patients need to accept noncurative treatments, such as hepatic arterial chemoembolization, sorafenib therapy or systemic chemotherapy. Unfortunately, adjuvant therapies are not very effective. Especially, in our country, hepatocellular carcinoma showed a higher morbidity and mortality, and most patients have a background of hepatic f ibrosis. More and more studies have found that hepatocellular carcinoma and liver fibrosis or cirrhosis have a close relationship, and fibrosis, cirrhosis, and liver cancer are results of interaction between liver parenchymal cells and mesenchymal cells. Further study found that hepatic stellate cell(HSC), as the liver mesenchymal cells of the main components are activated in response to liver damage and associated with inflammation, which can transdifferentiate into myofibroblasts (MFB) expressing alpha smooth muscle actin(α-SMA) and synthesizing a large number of extracellular matrix(ECM), becoming the main source of cells for liver fibrosis of chronic liver extracellular matrix and collagen production. Therefore, activated hepatic stellate cell (aHSC) are recognized as central in the development and progress of liver fibrosis. And all of these components in liver microenvironment, may have contributed to transform hepatic stem cells or liver cells into the liver cancer cells, and further increase cancer cells proliferation, invasion and migration. Thus, hepatocellular carcinoma cells and hepatic stellate cells must have a close relationship, this relationship maybe become a new target in the treatment of HCC, and through regulation of HSC in the tumor microenvironment would be a potential strategy to prevent and treat HCC.Traditional Chinese medicines have been used to treat malignancies since ancient times. As an important supplement to the treatment of liver fibrosis and HCC, which offer a feasible way for us and increasingly highlight its role. It is based on this understanding, in addition to the previous work, we applied our development of traditional Chinese medicine prescription "Songyou Yin" by combining five flavor components including astragalus, salvia, hawthorn, turtle shell and wolf berry to intervene the orthotopic xenografts in nude mice models with liver fibrosis and focused on liver fibrosis through the study of its role on hepatic stellate cells in the microenvironment, finally found the mechanism of "Songyou Yin" inhibiting malignant potential of hepatocellular carcinoma with liver fibrosis.Objective:Applying traditional Chinese medicine compound "Songyou Yin" (SYY) on the orthotopic xenografts in nude mice models with liver fibrosis, to prove that "Songyou Yin" influence on activated hepatic stellate cell (aHSC) of hepatic fibrosis microenvironment, and then inhibit malignant potential of hepatocellular carcinoma, and further to explore the possible mechanism.Methods:1.5-6 weeks old nude mice (n=30) received subcutaneous injection of 5ul/g carbon tetrachloride (CC14/olive oil:50:50) weekly. Since the beginning of the fourth week of injection,6 nude mice were sacrificed by spine dislocation for every two weeks until the tenth week, changes of liver specimen were dynamically observed via tissue sections and immunohistochemistry techniques to identify the degree of fibrosis in nude mice.2.20 nude mice were divided randomly into 4 groups, and the tumor tissues of HCCLM3 were transplanted into the livers of all mice, three days later, two groups were subcutaneously injected with carbon tetrachloride (5ul/g/week), another two groups were treated in equal doses of saline, at the same time, both each group has a group began drinking SYY (2g/Kg/day), all nude mice were killed after 6 weeks. Hepatic changes and tumor of liver changes were observed, liver tissue was extracted, formalin-fixed, paraffin and stained. Nude mice (n= 45) of 5-6 week old were divided randomly into 9 groups. And 4 groups were used as the experimental groups,4 groups were used as controls and 1 group as the observation group. The experimental groups of nude mice were treated with different magnitude of highly metastatic human hepatocellular carcinoma cell line HCCLM3 (0.1ml) and human hepatic stellate cells (0.1ml LX2,1×105) mixed cells by subcutaneous injection. The control groups were injected the magnitude of HCCLM3 cells (0.2ml) and without LX2 mixed, and the last group was subcutaneously injected with LX2 cells alone (2 ×105,0.2ml). After 12 weeks, all the mice were sacrificed by spine dislocation, and the subcutaneous tumor formation were observed. Nude mice(n=15) of 5-6 week old were randomly divided into 3 groups, two groups were treated with 1×106 LX2 (0.1ml) and 1×105 HCCLM3 (0. lml) mixed cells via subcutaneous injection, and one group in the above two groups as the experimental group began drinking SYY (2g/Kg/day), another group served as the control group with regular diet. The third group in nude mice was treated by subcutaneous injection of 1 ×106 (0.2ml) LX2 cells with regular diet. After 6 weeks, all the mice were sacrificed, and the subcutaneous tumor and lung metastasis were observed. The liver and lung tissues were extracted, formalin fixed, paraffin, and stained. CCK8 and lactate dehydrogenase release experiment (LDH) testing were performed to detect inhibition of proliferation and toxic effect "Songyou Yin" on LX2. Normal LX2 cells were stimulated at dose of 8mg/ml for 48 hours, and then took the supernatant as HCCLM3 and Hep3B cells conditioned medium respectively as the experimental group, the supernatant of LX2 cells normal medium was used as conditioned culture medium for the control groups. And then the same concentration of SYY was added to above two cells, set up a normal medium as control group, each group for above cells were determined by colony formation assay, CCK8 assay and invasion assay separately. The total RNA were extracted from SYY treatment LX2 cells for 48 hours as the experimental group, and the total RNA of control group was from normal cultured LX2 cells, then whole-genome microarray analyses were performed to get the key regulatory genes and related signal pathways. Western blotting was used to verify key protein involved the core genes encode and related signaling pathways, and enzyme linked immunosorbent assay (Elisa) was performed to clarify related secretory proteins in the supernatant of two groups.4. All statistical calculations were performed by the SPSS 13.0. P-values less than 0.05 was considered a statistically significant difference.Results:1. Hepatic fibrosis models in nude mice were successfully established with intermittent subcutaneous carbon tetrachloride injection. Immunohistochemistry results revealed that stellate cells as the center of hepatic fibrosis formation were positively correlation between activation stage "α-SMA" protein expression and the degree of liver fibrosis.2. Carbon tetrachloride-induced liver fibrosis background hepatocellular carcinoma weight was greater than orthotopic xenografts to normal liver background. The growth of hepatocellular carcinoma in nude mice with background of liver fibrosis was significantly inhibited by treatment with the daily drinking 2g/Kg of SYY, same dose of SYY for the growth of hepatocellular carcinoma with background of normal liver had no significant inhibitory effect. Immunohistochemistry staining (α-SMA staining) showed that a large of activated hepatic stellate cells existed in tumor and peritumoral tissue. Cell numbers less than 1×105 of HCCLM3 tumor cells could not form subcutaneous tumor, LX2 cells also could not form subcutaneous tumor independently. In contrast, cell numbers less than 1×10 HCCLM3 tumor cells mixed with the same numbers of LX2 cells could form the subcutaneous tumor. Tumor growth in nude micesubcutaneousinoculatedHCCLM3 and LX2 mixed cells was significantly inhibited by drinking SYY. And mixture of nude mice subcutaneous tumor tissue slices with HE and Sirius staining revealed that there were a large number of fibroblasts and collagen in the two groups of subcutaneous tumors. IOD values of α-SMA and PCNA-positive staining in drinking SYY group were significantly lower compared with control group. PCNA and a-SMA immunofluorescence double staining in two groups of subcutaneous tumor tissue also showed the same results, SYY also inhibited mixed subcutaneous tumor pulmonary metastasis in nude mice. CCK8 and lactate dehydrogenase release experiments showed that "Songyou Yin" had no acute toxicity and inhibition of proliferation to hepatic stellate cell line(LX2).SYY treated LX2 conditioned medium cultured HCCLM3 and Hep3B cells indicated that the cloning capacity was weaken, and the two cells proliferation and invasion ability were significantly attenuated. However, two kinds of tumor cells treated by SYY directly in the above three experiments did not changes significantly compared with the control group. Based on the whole-genome chip, there were 1205 up-regulated and 1323 down-regulated genes, some genes were closely related to proliferation and growth of HSC (PIK3CD, AKT, IGF1R, TGF-β). PI3K/AKT signaling pathway was selected as target for further study. Western-blot demonstrated that the expression of key proteins that regulate PI3K/AKT signaling pathway, which was significantly reduced in SYY treated LX2 cells. Elisa test clarified that the expression of cytokines secreted by aHSC in the supernatant, which promoted tumor growth factor, was significantly decreased through down-regulation of PI3K/AKT signaling pathway.Conclusion:1. Applying intermittent subcutaneous injection of carbon tetrachloride, we successfully established a model of experimental liver fibrosis in nude mice; 2. The degree of activated hepatic stellate cell was related positively with the progress of liver fibrosis; 3. "Songyou Yin" (SYY) significantly inhibited the proliferation of hepatocellular carcinoma with liver fibrosis; 4. Mixed inoculation of activated hepatic stellate cell (aHSC) and HCC notably promoted the formation of HCC; 5. SYY treatment inhibited the growth of subcutaneous mixed tumor of aHSC and HCC; 6. SYY treatment inhibited pulmonary metastasis of subcutaneous mixed tumor of aHSC and HCC; 7. The inhibitory effect of SYY on subcutaneous mixed tumor of aHSC and HCC were closely related with aHSC; 8. SYY inhibited malignant potential of HCC through affecting the paracrine of aHSC; 9. Down-regulation of PI3K/AKT signaling pathway was one of the main mechanisms of SYY inhibiting malignant potential of HCC with liver fibrosis through regulating activated hepatic stellate cell.