| BACKGROUND&OBJECTIVEThe liver, one of the organs with abundant blood supply in the human body, acts as the carrier of detoxification, digestion, and secretion, which make it vulnerable to a variety of factors, such as hepatotropic virus, bile accumulation, alcohol, drugs, bacteria, parasitic infections, metabolic diseases, and autoimmune disorders. Hepatic fibrosis is an intermediate link of the slow protraction to the outcome of a variety of acute and chronic liver diseases, a healing response to chronic liver injury of the body, and also a pathological change of chronic liver disease. Its pathophysiological basis is the activation of hepatic stellate cell, which synthesizes and secretes copious amounts of extracellular matrix (ECM), and undermines the normal lobular structure. Schistosomiasis is one of the primary parasitic diseases in tropics and subtropics of developing countries. In China, zoonotic schistosomiasis japonica, also called "snail fever," is associated with chronic liver and intestinal fibrosis. Major foci of endemicity occur in the marsh and lake regions along the Yangtze River basin, where the elimination of transmission has proven difficult, with approximately1million people and several hundred thousand livestock currently infected. Although the current prevalence is controlled by effective drugs (praziquantel), schistosome infections remain a threat to seven provinces in the south of the Yangtze River. Schistosomiasis-induced hepatic fibrosis is the most prevalent among the impairments to the body. Schistosome eggs are deposited in the hepatic portal system, secreting soluble egg antigen, which activates hepatic stellate cells (HSCs) with large amounts of deposited ECM, thereby ultimately developing schistosomal hepatic fibrosis. With the development of the course of hepatic fibrosis, the disease is often manifested by the hardening of the liver texture at its advanced stage, accompanied by enlarged spleen, portal hypertension, ascites formation, and others during decompensation.Necrosis of the liver cells and accumulation of inflammatory cells are the initial factors for hepatic fibrosis, and the cells involved mainly include sinusoidal endothelial cells, Kupffer cell, hepatocyte, and HSCs. It is a key issue that HSCs are activated by various mediating factors, such as the transforming growth factor (TGF-β1), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), which will result in the imbalance between the synthesis and degradation of ECM. The deposition of excessive ECM forms hepatic fibrosis. Studies on the mechanism of hepatic fibrosis have placed emphasis on the activation of initial factors and mainly probed the aspects of HSC proliferation, migration, cytokine secretion, and ECM synthesis and degradation.Epoxyeicosatrienoic acids (EETs) are bioactivators produced by arachidonic acid (AA) via the metabolic pathway of cytochrome P450(CYP450) epoxygenases. AA is the precursor for a variety of important bioactive substances for cardiovascular function in the human body; it mainly exists on the inner surface of cytomembrane in its esterified form and may be released into the cytoplasm via the hydrolysis of phosphatidase A2under the action of the lipolytic hormone. AA has been studied for several years, and the best-known findings are the metabolic pathways of cyclooxygenase and lipoxygenase. Four kinds of EETs (5,6-,8,9-,11,12-, and14,15-EET) are produced through the pathways ofcytochrome P450epoxygenase. Hydrolysis of EETs mainly by the soluble form oxide hydrolase (soluble epoxide hydrolase, sEH) metabolites generated the corresponding Dihydroxyeicosatrienoic acids (DHET) which have been considered to have minor biological effects.Regarding EETs, epoxygenase metabolites have been extensively studied in the cardiovascular and renal systems at the initial stage. EETs perform various functions, such as vasodilation, inhibition of platelet aggregation, inhibition of vascular smooth muscle cell inflammation and smooth muscle cell migration, enhancement of fibrinolysis, and promotion of endothelial cell proliferation and neovascularization. The biological effects of EETs include a wide, diversified range of subjects and vary for different tissue-derived cells. Previous studies have found that EETs can alleviate the apoptosis of endothelial cells and cardiomyocytes induced by tumor necrosis factor (TNF-α) by inhibiting the dephosphorylation of ERKI/2and activating the PI3K/AKT signaling pathway. EETs can inhibit the adherence of inflammatory cells to endothelial cells and reduce inflammatory cell infiltration in the tissue by inhibiting the nuclear translocation of the nuclear factor NF-kB, or through inhibiting the phosphorylation of the natural inhibitor IkB of NF-kB. Additionally, they can inhibit vascular smooth muscle cell migration by activating the cAMP-PKA pathway. The findings suggest that EETs exert a variety of biological functions, and they may also play a critical role in organ fibrosis. Recently, researchers conducted a series of extensive research in KKAy diabetes mice and bleomycin-induced pulmonary fibrosis mice. Results have shown that by inhabiting the migration of macrophage to cardiac muscle, reducing the secretion of TGF-β1, and upregulating MMP-2expression, CYP2J2epoxygenase gene and its metabolites (EETs) effectively inhibit myocardial fibrosis in KKAy diabetes mice and retard myocardial remodeling, and that by oral gavaging of sEH inhibitor (TPPU), increasing the concentration of EETs in lung alleviated the bleomycin-induced inflammatory and collagen deposition and inhibited the pulmonary fibrosis. The present study provides a novel thought and direction for further studies on the pathogenesis of organ fibrosis.The pathogenesis of hepatic fibrosis (an organ fibrosis) is complex, and its corresponding effective prevention and treatment strategies have yet to be investigated further. In liver fibrosis, the major source of hepatic myofibroblasts and excessive ECM come from HSCs that have been activated by inflammatory cytokine and stimulated to migrate into injury area. EETs are known to have significant effect of anti-inflammatory and potent suppression of vascular smooth muscle cell migration, therefore, they are easily associated with the pathogenesis of liver fibrosis. However, the biological effects of EETs in the course of hepatic fibrosis have been rarely reported. Recent findings in our laboratory showed that higher expression of some cytokines inducing HSCs migration (PDGF, TGF-β1, and MCP-1) in schistosomiasis mice than in control group, along with the changes of cytoskeletal. Thus, we assume that EETs have the role of slowing down the progressive course of hepatic fibrosis infected by schistosoma japonicum through alleviating inflammatory injury and inhibiting activated HSCs migration.We proposed the establishment of an animal model (mice) with schistosomal hepatic fibrosis to detect the content of EETs in the liver during the course of disease, as well as the downstream sEH. Then, we treated mice with TPPU, a sEH enzyme specific inhibitor, increased the endogenous EET content, observed the progressive course of hepatic fibrosis, and explored the related mechanisms. We observed the effects of EETs on PDGF-induced LX-2cell activation and migration abilities in vivo, and further demonstrated the related in vivo mechanisms, with the aim of providing a new theoretical base and tactics for the pathogenesis and treatment of hepatic fibrosis. The experimental purposes are as follows:1. In vivo study:Inhibitory effect of EETs on the hepatic fibrosis in schistosomiasis mice.:1). Study the variation of EETs content in the hepatic tissue during the course of schistosomal hepatic fibrosis and the dynamic changes of the downstream metabolic enzyme (sEH) during the course, as well as initially identify the biological effects of EETs in hepatic fibrosis.2). Adopt TPPU (a sEH specific inhibitor) to increase the endogenous EET content in the mice models with schistosomal hepatic fibrosis and further explore the relationship between EETs and the course of schistosomal hepatic fibrosis, as well as the related molecular mechanisms.2. In vitro study:Inhibitory effect of EETs against LX-2cell migration induced by PDGF.1). Based on the experimental data of the animal model, discuss in detail the difference between the effects of four types of EET isomers and specific links of their roles in pathogenesis of hepatic fibrosis2). Based on clarification of the specific links, further explore the possible molecular signaling pathwaysMETHODS1. Animal model1). A total of16SPF balb/c mice,6weeks of age and weighing20g, were randomly divided into two groups after adaptive feeding a week:, control group (n=8) and infected group (n=8). The mice from the infected group, which suffered from percutaneous infection by schistosoma japonicum cercariae (20+2per mouse), were observed for12weeks. Specific experimental methods were as follows:the oncomelania was placed in fresh water and soaked for1h until the cercaria overflowed. Mice anesthetized with1%pentobarbital intraperitoneally were fixed to the sterile operation table with the abdomen upward and a skin preparation (1cm×1cm) at the lower abdomen. Two drops of infected water were placed on a coverslip and observed under a microscope. After counting the cercariae, the prepared skin was covered with the contaminated side of the coverslip for15min. After removing the coverslip, the mice were returned back to their cages after they regained consciousness. The entire operation was completed in a greenhouse, and all laboratory workers required appropriate protective measures. The control group was treated using the same procedures, except for the fact that the infected water was substituted with physiological saline. At12weeks after infection, the mice underwent treatment. The corresponding blood and tissue samples were retained and photographed for future use.2). A total of48balb/c mice,6weeks of age and weighing20g, were randomly divided into two groups after adaptive feeding a week:8wk control (n=8),8wk infected (n=8),8wk sEHi-treated (TUUP, n=8),12wk control (n=8),12wk infected (n=8), and12wk sEHi-treated (TPPU, n=8). The infected and sEHi-treated groups represented the schistosome infection models established as described above. At6wk after infection by Schistosoma japonicum cercaria, the mice in the sEHi-treated group underwent TPPU (sEH-specific inhibitor) intervention. The specific methods were as follows:the mice were intragastrically administered with TPPU at2.5mg/kg/day and at a daily dose of0.2ml. TPPU was dissolved in physiological saline, and a sufficient amount of polyethylene glycol400(PEG400), which acted as the latent solvent and stabilizer, was added. Meanwhile, the infected group was intragastrically administered with the same volume of a mixture of physiological saline and PEG400at10:00a.m. everyday. At8and12weeks after infection, samples were obtained from the mice and photos were taken for recordkeeping.2. Prior to treating each group, the weights of the mice were obtained and recorded. After treatment, liver and spleen tissues were obtained, with the longest diameter measured and the weights obtained. The blood samples were centrifuged, and the supernatant was obtained and analyzed for biochemical indicators (alanine aminotransferase and aspartate aminotransferase).3. Frozen hepatic tissue (100mg) was separately weighed and obtained from the mice of each group. After pyrolysis with tissue lysate, the supernatant was extracted and measured for11,12/14,15-DHET content through ELISA method before and after acidification (the difference is the EET content in the tissue). The variation in sEH expression was analyzed.4. The structural changes in the liver tissues were observed, including collagen deposition and distribution, through HE/Masson staining of the paraffin sections prepared from the stained liver tissues of the mice.5. Through a-SMA/fascin movement protein fluorescent staining, frozen sections of certain parts of the mice liver tissues were visualized through confocal microscopy.6. The HSC activation-related indicators in the liver tissues of mice were detected, and the effects of the increase of endogenous EETs on HSC activation were observed:HSC activation markers (a-SMA, desmin, and vimentin), HSC migration-related proteins (fascin, dynein, and dynactin), and collagen synthesis and degradation balance indicators (collagen Ⅰ, collagen Ⅲ, MMP-9, and TIMP-1) through real-time PCR detection; a-SMA and fascin protein expression levels through Western blot; and effects of HSC migration on MCP-1and ICAM-1expression levels through ELISA method.7. PDGF-induced LX-2cell migration system in vitro:cells were cultured in DMEM medium containing10%fetal bovine serum at37℃C incubator with5%CO2. When the cell growed to about90%confluence,0.25%trypsin was used to digest and passage, when cells growed to70%-80%confluence under microscope, discarded the medium, cells were cultured in DMEM medium containing0.1%fetal bovine serum for starvation of12h to pursue subsequent treatments.4x103cells/well were cultivated in a Transwell cell culture chamber. PDGF cytokine with a final concentration of10ng/ml was added to the fluid under the chamber.8. While observing the effects of EETs on PDGF-induced LX-2cell migration, the cells were pre-treated for30min with EETs (four types of isomers) or LY294002after the cells were passaged to the Transwell chamber. PDGF cytokine was added as above, and the cells were counted at12and24h.9. The expressions of a-SMA, desmin, vimentin, collagen I, collagen III, and fascin in LX-2cells were detected through real-time PCR before and after treatment. a-SMA, fascin, and PI3k/Akt phosphorylation were detected through Western blot. Finally, the expressions of cytoskeletal filament, fascin, and the a-SMA protein were detected via co-immunoprecipitation and confocal assays.RESULTS1. Mice models with schistosomal hepatic fibrosis were successfully established. Loose cytoplasm and feathery degeneration of hepatocytes around the portal regions of liver tissues of the mice of the infected group, accompanied by spotty necrosis or acidophilic necrosis, were detected through HE staining after12weeks. Inflammatory cell infiltration and egg granuloma visibly formed in the portal regions, and collagenous fibers were distributed around the egg granuloma and portal regions. Masson staining revealed that the liver tissues in the infected group of mice exhibited deposition of a considerable amount of collagen fibers.2. ELISA results showed that the11,12-EET content in the infected group (43.69±9.07pg/ug protein) were significantly reduced compared with the control group (79.67±7.05pg/ug protein)(P<0.05). Although the14,15-EET content of the infected group (9.54±1.70pg/ug protein) was lower than that of the control group (14.34±1.97pg/ug protein), no statistical difference (P>0.05) was found. The ratio of EET to DHET indirectly reflects the sEH enzyme activity and is inversely proportional to it. Moreover, the sEH enzyme activity in the infected group significantly increased compared with the control group (P<0.05).3. Mice with schistosomal hepatic fibrosis after sEH inhibitor (TPPU) intervention revealed the general and morphological tissue changes after examination. Overall sampling of the liver and spleen showed that the control group had rosy and smooth livers, as well as red spleens with sharp edges; the infected group had dark green livers with marked cholestasis, visibly milky granulomatous spots on the surfaces, as well as a hard texture, splenomegaly, and darker spleen with blunt edge; the sEHi-treated group livers were dark red, had harder texture than the control group, and spleens that had sizes ranging between those of the control group and the infected group. Comparison of the liver (spleen) indices (liver (spleen) to body weight ratio) showed that after12weeks, the liver (spleen) index of the sEHi-treated group (0.068±0.008/0.009±0.001) was significantly reduced compared with the infected group (0.096±0.007/0.015±0.002)(P<0.05). HE staining of hepatic tissue revealed that the measured egg granuloma diameter of the sEHi-treated group was significantly reduced at8weeks (0.44±0.03cm) and12weeks(0.61±0.04cm) compared with the same time in infected group (0.71±0.04cm)(0.73±0.04cm)(P<0.05). However, the percentage of collagen area of the sEHi-treated group detected by Masson staining at8weeks (21.77±7.87%) and12weeks (30.25±8.06%) was reduced compared with the same time in infected group (24.83±6.35%)(45.20±10.49%), there were not statistical difference (P>0.05).4. After the intragastric administration of sEHi, the supernatant was taken from the liver homogenate of mice after lysis, and the11,12-/14,15-DHET content in the hepatic tissues before and after acidification were respectively detected through ELISA. The calculated11,12-EET content in the hepatic tissues of the sEHi-treated group at8weeks (83.30±12.66pg/ug protein) and12weeks (79.02±20.55pg/ug protein) significantly increased compared with the infected group (29.51±5.36pg/ug protein)(P<0.05). The14,15-EET content of the sEHi-treated group at8weeks (32.63±6.38pg/ug protein) and12weeks (32.83±7.22pg/ug protein) also sharply increased compared with the infected group (14.27±1.969pg/ug protein)(P<0.05). 5. Real-time PCR results showed that at12weeks after infection, the expressions of the HSC activation-related genes of the sEHi-treated group, such as a-SMA, desmin, and vimentin, significantly declined compared with the infected group (P<0.05). The expression of collagen I mRNA of the sEHi-treated group also reduced compared with the infected group (P<0.05). In addition, the expression of TIMP-1, an enzyme closely related to the collagen degradation balance, was significantly reduced in liver tissues of the sEHi-treated group (P<0.05), which is conducive to the degradation of collagen. No significant difference was found between the two groups in the expression of MMP-9(P>0.05), indicating that the increase of endogenous EETs has numerous effects on the progressive course of hepatic fibrosis.6. Further analysis of HSC activation under the actions of increasing EETs.Real-time PCR results show that the expressions of fascin and dynactin, two HSC migration-related proteins, of the sEHi-treated group were significantly downregulated compared with those of the infected group (P<0.05). The expression of dynein of the sEHi-treated group just at12weeks after infection showed a downregulating trend, but no statistical difference was found (P>0.05). The expression levels of extracellular factors influencing cell migration, such as MCP-1and ICAM, were analyzed via ELISA. At12weeks after infection, the MCP-1content in the liver tissues of the sEHi-treated group (138.1±15.81pg/ug protein) was significantly lower than that in the infected group (215.1±21.78pg/ug protein)(P<0.05), but was significantly higher compared with the control group (61.32±6.18pg/ug protein)(P<0.05). The expression of ICAM-1in the liver tissues of the mice was similar to that of MCP-1, and the expression of ICAM-1in the sEHi-treated group (183.4±16.43pg/ug protein) obviously declined compared with that in the infected group (260.1±26.46pg/ug protein)(P<0.05). Co-immunoprecipitation and confocal assays revealed that in terms of the expressions of a-SMA (the HSC activation marker, red) and fascin (a movement protein, green), almost no a-SMA expression was detected in the control group but a slight fascin expression was noted. In the infected group, HSC activation and a-SMA expression were significantly upregulated. The expression of fascin also increased, and the overlapping part of the red and green lights was significantly enlarged, indicating that HSC activation with hepatic fibrosis accompanied increasing cell migration. However, the overlapping part of the sEHi-treated group was reduced compared with that of the infected group. Western blot confirmed that at12weeks after infection, the expressions of a-SMA and fascin in the sEHi-treated group were downregulated to different extents compared with the infected group. In the course of schistosomiasis-induced hepatic fibrosis, the increase of endogenous EETs was proven not only to inhibit HSC activation, but also inhibit HSC migration-related protein.7. In vitro, PDGF-induced LX-2cell migration Transwell model was established, and the cells were treated for20min with four types of EET isomers (5,6-;8,9-;11,12-and14,15-EET) at the concentration of1μM. Observations of the effects of four types of isomers on the PDGF-induced LX-2cell migration showed that at12h after the addition of PDGF (10ng/ml), the cell migration of the11,12-EET group was significantly reduced compared with that of the PDGF group, with statistical difference (P<0.05), and was significantly higher than that in the control group and the DMSO group (P<0.05). Cell count at24h revealed that the cell migrations of the11,12-EET and14,15-EET groups were significantly reduced compared with the PDGF group,(P<0.05), and were higher than those in the control and DMSO groups (P<0.05). The cell migrations of the5,6-and8,9-EET groups, whether at12or24h, were significantly higher than those in the control and DMSO groups (P<0.05), but was not significantly different from the PDGF group (P>0.05). The effects of11,12-EET and14,15-EET on the PDGF-induced LX-2cell migration were respectively observed at different time points (2,6,12,24, and48h). Staining with crystal violet showed that the cell migrations of the EET group at2and6h declined, but did not exhibit statistical difference (P>0.05). The cell migrations of the11,12-EET and14,15-EET groups at12and24h showed a significant decrease compared with the PDGF group. The same observation was noted at12and24h as above, and after48h, no statistical difference was found between the EETs group and the PDGF group (P>0.05). Transwell results showed that11,12-and14,15-EET have inhibitory effects on PDGF-induced LX-2cell migration, during which11,12-EET was manifested most notably. 8. The stimulating effects of PDGF on the expression levels of Akt and p-Akt in LX-2cells at5,10,30, and60min were detected through Western blot. Results indicate that after the stimulation of PDGF, the expression of p-Akt was significantly upregulated, whereas the expressions of p-Akt in the cells pre-treated with11,12-EET at the same points were downregulated compared with the PDGF group, indicating that11,12-EET inhibits PDGF-induced Akt phosphorylation. Further observation of the effects of EET and PI3K-Akt signaling pathways in cell migration demonstrated that11,12-EET and LY249002(specific PI-3K inhibitor,10uM) had similar effects, and can significantly inhibit cell migration at-12and24h after PDGF stimulated the LX-2cells (P<0.05). No statistical difference was observed between the11,12-DHET group and the PDGF group in the cell migration (P>0.05).9. The gene expression levels of movement-related proteins in the cell model were detected through real-time PCR, and results showed that the a-SMA, fascin, dynein, and dynactin expression levels of the11,12EET group and the LY249002group were significantly downregulated compared with the PDGF group. This result indicates that11,12-EET can inhibit LX-2cell migration induced by PDGF, probably through decreasing PI3K-Akt phosphorylation. Meanwhile, The results of detecting expressions of F-actin (green, a filament protein) and fascin (red, a movement protein) in LX-2cells by co-immunoprecipitation and confocal assays showed that F-actin is mostly concentrated at the synapses of LX-2cells of the PDGF and the11,12-DHET groups, whereas fascin exists in the perinuclear region of the cytoplasm and the distal synapse. However, the F-actin expressions of the11,12-EET group and the LY249002group were relatively weak, with slight expression in the cytoplasm and no significant aggregation at the synapse. Fascin expression was downregulated and mostly concentrated at the perinuclear region of the cytoplasm, with little expression at the distal synapse. Detection results of the expressions of a-SMA and fascin via Western blot showed that the expression of a-SMA and fascin of the11,12-EET and LY249002groups were significantly downregulated compared with the PDGF and11,12-DHET groups. STATISTICAL ANALYSISData are presented as mean±SEM. Comparisons were carried out with One-way ANOVA and Newman-Keuls tests for post hoc analyses. Significance was accepted at a value of P <0.05.CONCLUSIONS1. In the course of schistosomal hepatic fibrosis, the contents of11,12-EET and14,15-EET in the hepatic tissues dynamically change, indicating that EETs are involved in the progression of hepatic fibrosis. Additionally, during the development of fibrosis, the EET content in hepatic tissues declines, which is closely related to the increased activity of the downstream metabolic enzyme (sEH).2. Treating the mice with schistosomal hepatic fibrosis through oral administration of TPPU (sEH enzyme inhibitor) everyday increases the endogenous11,12-and14,15-EET contents in the liver.3. After infection with S. japonicum cercaria and at6wk after intragastric administration with TPPU (equivalent to12wk after infection), the increase in EETs significantly inhibits the size of schistosome eggs and alleviates the inflammatory reaction around the egg. In addition, the liver and spleen indexes decline, and the course of schistosomiasis hepatic fibrosis is retarded.4. The molecular mechanism under which EETs slow down the course of schistosomiasis can be obtained by inhibiting the activation and migration of HSCs, collagen synthesis and secretion, increase of collagen degradation, HSC activation marker expression, HSC movement-related protein expression, downregulated HSC-induced cell migration factor expression, and collagen I and downregulated TIMP-1expressions, among others.5. Four types of exogenous EETs isomers were added, and their effects on PDGF-induced LX-2cell migration were observed. Among them,11,12-EET and14,15-EET exert significant inhibitory effects on cell migration, which is mainly manifested in the downregulated LX-2movement protein expression achieved via inhibiting the PDGF-induced PI3K-Akt signaling pathway phosphorylation.SUMMARY:EETs alleviate the course of schistosomal hepatic fibrosis by inhibiting HSC activation and migration, reducing collagen synthesis, and increasing collagen degradation, among others.11,12-EET partially lowers the ability of PDGF to induce LX-2cell migration by inhibiting PI3K-Akt phosphorylation. |
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