Characteristics Of The Gene Expression Profiles Of Hepatic Stelate Cells In Mice Infected With Schistosoma Japonicum And Functions Of Vascular Endothelial Growth Factor In Liver Fibrosis Of Schistosomiasis

Schistosomiasis is the second zoonotic parasitic disease which is seriously harmful to human health and currently the population of about 200 million people are infected in the world. Schistosomiasis japonica in China is popular. Even though after many years of control efforts for greatly reducing prevalence of the disease, but it is still one of the major public health problems.The major pathological damage of schistosomiasis japonica is the egg granuloma response caused by egg deposition in liver and intestine in the acute phase of infection, which gradually develops to liver fibrosis in the chronic phase. Hepatic stellate cells (HSCs) as the principal cells producing extra-cellular matrix (ECM) may be activated by some factors, which is a key for fibrogenesis of liver. In recent years, there were quite a number of studies on the pathogenesis of schistosomiasis, but very few systematic and dynamic observations on the developmental process of schistosomiasis in the gene expression profiles of HSCs. Praziquantel (PZQ), since its good efficacy, low toxicity, rapid metabolism and other advantages such as safe, is the first choice of the drug for treating schistosomiasis, and by killing adult worms it can partially reverse liver fibrosis. However, liver fibrogenesis is that a multi-factors and multi-step processes are involved, which makes us poorly understand many participating genes in the occurrence and development of liver fibrosis. Therefore, at present there are no good prevention and treatment measures and means for liver fibrosis.In this study, the mice infected with Schistosuma juponicum were used as animal model, the primary HSCs were isolated as the key material, and the gene expression microarray technology and bioinformatics analysis were applied in order to investigate the dynamic characteristics of the gene expression profiles in HSCs and the related molecular mechanisms during the occurrence and development of schistosomiasis, and then observe the effect of PZQ on the gene expression profiles of HSCs in the chronic phase of infection. Through these studies we hope to filter out which hepatic factors play a key role in the outcome of liver fibrosis, and they may be served as the potential targets for treatment of liver fibrosis. To this end, we conduct the following three main parts of the study:1. Dynamic observation and analysis of the gene expression patterns of HSCs isolated from the mice infected with Schistosoma japonicumIn order to investigate the pathogenic mechanisms of schistosomiasis at gene level, we established the mouse model of schistosomiasis. Five time points were chosen during the process of mice infected with Schistosoma japonicum, i.e. uninfected (normal control),3 weeks post-infection (ahead of eggs laid by worms),6 weeks post-infection (acute infection),12 weeks post-infection (chronic infection) and 18 weeks post-infection (late chronic infection). HSCs were isolated by enzyme digestion and density gradient centrifugation, and gene microarrays were used to detect the expression patterns of HSCs from different stages of infection. Pathways and Go analysis of significantly changed genes in development of schistosomiasis in microarrays were also analyzed by bioinformatics. Our results showed that the purity of primary HSCs was up to 90% and the viability was more than 95%. Results of microarrays showed that there were 6762 genes of differential expression in development of schistosome infection. According to the tendency of expressed genes, 22 kinds of expression tendency were summarized. The top 4 tendencies of significances (P＜0.001) among them were described as follows. The first one was the genes which were up-regulated at 3 weeks post-infection (p.i) and continued at the high level of expression until the end of experiment. The second one was the genes that were up-regulated at 3 weeks p. i and continued to express at higher level through the acute and chronic phases of infection. The third one was the genes which started to be up-regulated at acute phase and continued at a high level till chronic stage. The fourth one was the genes that were down-regulated at 3 weeks p.i and continued at low level of expression until the end of the experiment. GO and pathway analysis showed that the genes in these four kinds of expression tendency may take part in apoptosis, immune response, collagen catabolic process, MAPK signaling pathway, VEGF signaling pathway and so on. Therefore, these four kinds of genes might play a key role in regulating the processes of egg production, acute infection and chronic infection.2. Effect of PZQ-anti-parasite treatment on the gene expression patterns of HSCs from chronic schistosomiasisAlthough lots of researches were performed to reveal the molecular mechanisms of liver fibrosis, the accurate mechanisms in liver fibrosis caused by different diseases were not clear and there were still several questions needed to be resolved. Herein, we established the mouse model of Schistosoma japonicum infection and administration of PZQ (250mg/kg/day for 3 days) was used as anti-parasite treatment. Our aim was to show the effect of PZQ-anti-parasite treatment on expression pattern of HSCs and further investigate the reversible mechanisms of liver fibrosis in schistosomiasis japonica. First of all, we established the mouse models of schistosomiasis including chronic stage (12 weeks p.i) group and PZQ-treatment (6 weeks p.i with PZQ treatment) group. Normal un-infected mice were used in our experiments as control. HSCs were isolated by enzyme digestion and density gradient centrifugation, and experiments of microarray were performed to detect the expression of HSCs. The genes, significantly changed after PZQ treatment were analyzed by bioinformatics to show their bio-functions. The cell process regulation net and gene interaction net were constructed by Pathway Studio to find out the key genes in the reversal of liver fibrosis in schistosomiasis japonica after PZQ treatment. We also validated some changed genes by Real Time PCR. The results showed that there were 1075 changed genes which participated 14 biological processes (P＜0.05). Among these processes, the processes of immune and defense contained most of the genes (132 genes). Besides, the genes expressing deferent functions such as processes of cell adhesion, cytokine-/chemokine-mediated immune, cell cross-talk, cell mobility, extracellular matrix protein-mediated signal pathway, apoptosis and carbohydrate metabolism,etc. were also significantly regulated.Thus, the gene expression profiles of HSCs were significantly changed after schistosome infection, while the expressions of some changed genes which participated to regulate the activation, migration, cell morphology and apoptosis were reversed after PZQ treatment. Interestingly, the relationship between VEGF or IL-6 and other changed genes was very complicated and the two genes located in the center of the gene-regulation net. We supposed that VEGF and IL-6 might play a key role in liver fibrosis of schistosomiasis.3. Function of VEGF in liver fibrosis of schistosomiasis japonicaIt was found out that VEGF was up-regulated significantly at chronic stage, but reversed after PZQ treatment, based on the results obtained from microarray detection. Analysis of bioinformatics further suggested that VEGF was one of the most important genes among the changed genes after PZQ treatment, and was involved in the regulations of cell migration, chemiotaxis, proliferation of fibroblasts and cell morphology. VEGF is likely to participate in the formation of hepatic egg granuloma and fibrosis. Thus, it is worth being further investigated. In order to search for the functional mechanisms of VEGF in schistosomiasis japonica, we established the model of mice infected with schistosomes and administration of PZQ was used as pathogenic cure, and then the expression of VEGF in liver of infected mice and PZQ-treated mice was detected. Meanwhile, we observed the effect of SWAP or SEA on cell proliferation and gene expression of VEGF in the cell lines of human hepatic stellate cells, LX-2 and human vascular endothelial cells, HUVEC. We also observed the effects of VEGF on HSCs, such as activation, proliferation and collagen production in vitro to investigate the effect and its mechanisms of VEGF from paracrine secretion on HSCs. This research will provide the basement of further study on VEGF in liver fibrosis of schistosomiasis and find the target of bio-therapy with VEGF. Our results showed that VEGF-positive cells were more frequent in infected group, compared with control, while in PZQ-treated group we also saw few positive cells, compared with control mice. This suggested that PZQ treatment significantly inhibited the expression of VEGF in liver during chronic schistosomiasis. We also observed that the number of a-SMA and VEGF double positive cells in infected group were more than that in control group, while the number was also low in PZQ-treated group, suggesting that PZQ treatment suppressed the expression of VEGF in both liver and HSCs. In vitro, SWAP and SEA inhibited the activation and expression of collagen on HSCs, which was not consistent with our results that HSCs were activated and produce collagen in mice with schistosomiasis, and the level of VEGF on HSCs after SWAP and SEA stimulation was not as high as that in vivo of schistosomiasis mice. These results suggested that the antigens from worms might not activate HSCs directly to produce collagen and VEGF, and the increased VEGF might be not due to the the effect of SWAP/SEA on HSCs directly. Because endothelial cell is the main source of VEGF, we thus cultured human vascular endothelial cell (HUVEC) and detected the level of VEGF by Real Time PCR and ELISA after stimulation with different concentrations of SWAP/SEA. SWAP of 20μg/mL significantly increased the VEGF in culture supernatant (P＜0.001), and showed the dose-dependent correlation, While SEA displayed this effect at 10μg/mL. It is indicated that SEA had the more ability to induce the expression of VEGF. The gene expression was consistent with the protein level. After the stimulation of LX-2 using VEGF, mRNA expressions of a-SMA and Collal were increased, suggesting that VEGF could activate HSCs and increased the expression of collagen. In addition, LX-2 was proliferated after the stimulation of 10ng/mL VEGF.The following results were obtained from this study:1. We isolated primary HSCs from mouse liver, and their purity was more than 90% and their viability was more than 95%, which satisfied the need of related experiments2. Six thousand seven hundred and sixty two differential expressed genes were found during schistosome infection and twenty two kinds of expression tendency were summarized. The top 4 tendencies of significances (P<0.001) among them were as follows. The first one was the genes which were up-regulated at 3 weeks post-infection (p.i) and continued at the high level of expression until the end of experiment. The second one was the genes which were up-regulated at 3 weeks p.i and continued to express at higher level through the acute and chronic phases of infection. The third one was the genes that started to be up-regulated at acute phase and continued at a high level till chronic stage. The fourth one was the genes that were down-regulated at 3 weeks p. i and continued at low level of expression until the end of the experiment.3. There were 1075 changed genes which participated 14 biological processes (P＜0.05). Among these processes, the process of immune and defense contained most of the genes (132 genes). Besides, the genes expressing deferent functions such as processes of cell adhesion, cytokine-/chemokine-mediated immune, cell cross-talk, cell mobility, extracellular matrix protein-mediated signal pathway, apoptosis and carbohydrate metabolism,etc. were also significantly regulated. Thus, the gene expression profiles of HSCs were significantly changed after schistosome infection, while the expressions of some changed genes which participated to regulate the activation, migration, cell morphology and apoptosis were reversed after PZQ treatment. Interestingly, the relationship between VEGF or IL-6 and other changed genes was very complicated and the two genes located in the center of the gene-regulation net.4. VEGF-positive cells were more frequent in infected group, compared with normal control, while in PZQ-treated group there were few positive cells, which were much less than those in infected mice group. This suggested that PZQ treatment significantly inhibited the expression of VEGF in liver during chronic schistosomiasis. The number of a-SMA and VEGF double positive cells in infected group were more than that in normal control group, while the number was also low in PZQ-treated group, when compared with infected group, suggesting that PZQ treatment suppressed the expression of VEGF in both liver and HSCs.5. In vitro, SWAP and SEA inhibited the activation and expression of collagen on HSCs. This was not consistent with our results that HSCs were activated and produce collagen in mice with schistosomiasis, and the level of VEGF on HSCs after SWAP and SEA stimulation was not as high as that in mice with schistosome infection. These results suggested that the antigens from parasites might not activate HSCs directly to produce collagen and VEGF, and the increased VEGF might be not due to the effect of SWAP/SEA on HSCs directly.6. After stimulation with different concentrations of SWAP/SEA, SWAP of 20μg/mL significantly increased VEGF in culture supernatant (P＜0.001) of HUVEC, and showed the dose-dependent correlation, while SEA displayed this effect at 10μg/mL. Thus, SEA had the more ability to induce the expression of VEGF of HUVEC. The gene expression was consistent with the protein level.7. After stimulation of LX-2 using VEGF, mRNA expressions of a-SMA and Collal were increased, suggesting that VEGF could activate HSCs and increased the expression of collagen. In addition, 10ng/mL of VEGF could stimulate proliferation of LX-2.