| Schistosomiasis is a tropical parasitic disease caused by infection with the helminthschistosoma, widely prevalent in Asia, Africa and Latin America. Six major schistosomespecies are known to infect human, and only one of the six species, Schistosomajaponicum(S. japonicum), endemic in our country. S. japonicum remains a major publichealth problem in China today.454districts still has schistosome infection, and the numberof people infected was estimated360000. The pathological basis of schistosomiasis is thehost’s immune response to the parasite and the soluble egg antigens secreted by the matureparasite eggs. In the early stage of schistosome infection, during which the host is exposedto migrating immature parasites, the dominant immune response is T helper1(Th1)-like,which is characterized by the elevation of cytokines such as TNF-α、IL-1、IL-6and IFN-γetc. As the parasites mature, and begin to produce eggs deposited in the host tissue, theimmune response switches quickly to a Th2response which is characterized by theelevation of cytokines such as IL-4、IL-13、IL-10etc. The primary cause of death inschistosomiasis is the formation of liver egg granulomas and secondary hepatic fibrosis.Liver fibrosis is a injury repair response after liver egg granulomas which will obstructblood flow through the liver, resulting in the symptoms of portal hypertension such asascites, splenomegaly and the esophageal and gastric varices etc. The activated hepaticstellate cells (HSC) are considered the key effector cells driving liver fibrosis and the mainsource of collagen deposited in liver in various animal models of liver fibrosis and humandiseases leading to liver fibrosis. Resting HSCs usually locate in the perisinusoidal spacewith vitamin A containing lipid droplets. Resident hepatic stellate cells (HSCs)transdifferentiate into proliferative, contractile, and fibrogenic myofibrosblast undervarious pathological circumstances, and secrete excessive extracellular matrix (ECM)depositing in the liver tissue resulting in fibrosis. Many studies have shown that HSCs playa vital role in the progression of schistosomiasis hepatic fibrosis. Therefore, inhibiting theactivation or activity of HSCs may be able to prevent the pathogenesis and progression ofschistosomiasis liver fibrosis.MicroRNAs (MiRNAs) are a class of highly conserved, small non-coding RNAsmolecules that posttranscriptionally regulate gene expression. miRNAs in animal cellsprevent the translation process of target gene by specifically binding the3’-untranslatedregions (3’-UTRs) of target mRNAs with their seed sequence to regulate a series ofphysiological and pathological processes, such as development, cell proliferation and differentiation, apoptosis, signal transduction, and tumorigenesis etc. Current studies haveshown that miRNAs play an essential role in the pathogenesis and progression of hepaticdiseases caused by a variety of pathological factors such as infection, ethanol or chemicalinduction, metabolic diseases etc. The role of miRNA in the development of liver fibrosisalso has been reported. Hepatic stellate cells are considered the key effector cells drivingliver fibrosis, and studies have shown that miRNAs directly participate in the process ofactivation, apoptosis, and regulation of collagen synthesis of HSC. These studies not onlyhelp us better understand the mechanism of liver fibrosis, but also help us find new targetsto alleviate or even reverse the hepatic fibrosis. However, little is known about the role ofmiRNAs in the schistosomiasis liver fibrosis.Our lab had identified the dys-regulated miRNAs in the progression of hepaticschistosomiasis of a mouse model by performing microarray analysis, and the expressionlevel of miR-203in the infected liver samples decreased on day42,49,56,70post-infection and the value was0.49、0.33、0.41、0.48respectively. By real-time PCR, wefound the expression level of miR-203in the infected liver samples decreased on day42,49,56,70and84, and the value was0.47、0.23、0.31、0.18and0.40respectively. Based onthe results above and bioinformatics analysis, we selected miR-203and its effect inschistosomiasis liver fibrosis as the object.In this study, we investigated the functions of miR-203in a murine schistosomiasismodel, using a recombinant adeno-associated virus (rAAV) vector which expressedpri-miR-203. After treated with rAAV8-pri-miR-203or PBS by tail injection on day10post-infection, mice were sacrificed to harvest the liver and serum samples on day42,56,70after infection. Indicators of liver egg granulomas and fibrosis were determined. Weexpected that up-regulation of miR-203could prevent schistosomiasis hepatic fibrosis, andthen revealed the molecular mechanism. Methods and results are as follows:1、 Confirm the microarray results: total RNA was extracted from liver samplers ofmice infected with cercaria of S. Japonicum for10,26,35,42,48,56,70and84days andliver samples of uninfected mice in the microarray analysis. Reverse transcription reactionsare performed to synthesize cDNAs and real-time PCR were performed to detect theexpression level of miR-203to verificate microarray results. The expression level ofmiR-203detected by RT-PCR was in well agreement with the results of microarrayhybridization: the expression level of miR-203in the infected liver samples significantlydecreased on day42,49,56,70,84post-infection and the value was0.47,0.23,0.31,0.18, 0.40respectively, compared with the uninfected liver samples.2、 Effect of up-regulation of miR-203on the survival days of mice infected with S.Japonicum: Mice were exposed to a lethal dose of schistosome infection (30cercariae),and then were treated with6×109rAAV8-pri-miR-203or PBS by tail injection on day10post-infection. Survival days of each mouse were recorded in the monitored80days. Theresult shown that rAAV8-pri-miR-203protected the mice from the lethal infection ofschistosome (P<0.05): five mice in PBS treated group gradually died after54to65daysinfection, however, one mouse survived beyond the monitored80days; two mice in therAAV8-pri-miR-203treated group died on day59and74post-infection, and the other fourmice survived beyond the monitored80days.3、 Up-regulation of miR-203and the effect on the schistosomiasis liver fibrosis:Mice were exposed to a mild dose of schistosome parasites (16cercariae), and then treatedwith5×109rAAV8-pri-miR-203or PBS for control by tail injection on day10post-infection. Liver and serum samples were obtained on day42,56and70post-infectionto detect the indictors of liver fibrosis、liver injury and immune response. The result shownthat rAAV8-pri-miR-203significantly increased the expression level of miR-203in liveron day42、day56post-infection (P<0.01)，and the value was7.69、2.18respectively,compared with the normal group. However, there were no significant difference betweenrAAV8-pri-miR-203and PBS group on day70(0.67±0.10vs0.53±0.13).rAAV8-mediated up-regulation of miR-203and the effect on the schistosomiasis liverfibrosis as follow:（1）up-regulation of miR-203significantly decreased hydroxyprolinecontent in liver by49.17%(P<0.01)、35.36%(P<0.01)、31.02%(P<0.05) on day42,56,70after infection respectively, compared with mice treated with PBS.（2）The expressionlevel of ColⅠof rAAV8-pri-miR-203group was significantly reduced on day42(37.71±21.55vs173.62±33.67, P<0.01), compared with and PBS group, but there were nosignificant difference on day56、70after infection.（3） The expression level of ColⅢ ofrAAV8-pri-miR-203group was significantly reduced on day42(12.35±2.97vs63.23±5.8,P<0.01),56(5.95±2.16vs20.05±8.91, P<0.05) after infection respectively, compared withand PBS group, and there were no significant difference on day70.（4）ALT ofrAAV8-pri-miR-203group was significantly reduced on day70(74.91±5.38vs100.75±16.70, P<0.05) after infection, compared with and PBS group, but there were nosignificant difference on day42、56after infection. However, other indicators had nosignificant difference between rAAV8-pri-miR-203and PBS group at three time points. 4、 The expression level of miR-203in HSC: Mice were exposed to a mild dose ofschistosome parasites (16cercariae), and then isolated and purify the HSCs on day10,26,35,42,56post-infection by in situ perfusion and density gradient centrifugation. PrimaryHSCs were isolated from3to5infected or normal mice, and total RNA was extracted andsubjected to RT reaction to synthesize cDNAs. Real-time PCR were performed to detectedmiR-203expression level at different time points. The purity of HSCs isolated was above95%, the yield of each mice was about3.6~4.8×106cell. RT-PCR results shown thatmiR-203expression level was significantly down-regulated in HSCs from infected liverscompared with uninfected livers on day42and56post-infection, and the expression levelis-6.25and-3.45respectively.5、 Statistical analysisStatitical analysis of values was performed with SPSS software (16.0version), resultsare reported as mean±s.d. and compared between groups using two-tailed Student’s t-testor one-way ANOVA, Survival analysis using Log-rank test. Data were consideredstatistically significant for P values less than0.05.Conclusion:In this study, we investigated the functions of miR-203in a murine schistosomiasismodel, using a recombinant adeno-associated virus (rAAV) vector which expressedpri-miR-203. By increasing the expression of miR-203in infecte mice liver we observe theexpression of miR-203and investigate its effect in schistosomiasis liver fibrosis. We use amurine schistosomiasis model by infected with cercaria of S. Japonicum.Our results showed that:（1）mice administered with5×109rAAV8vector on day10after infection significantly increased the expression of miR-203in liver and up-regulationof miR-203in liver prevented the production of collagen, but the expression of miR-203was decreased as time progresses;（2）Up-regulation of miR-203can decreased theexpression level of Col and hydroxyproline content, it indicated that up-regulation ofmiR-203in liver may attenuate the liver fibrosis of mice infected with S. Japonicum;（3）rAAV-mediated up-regulation of miR-203protected infected mice from the lethal effectsof Schistosomiasis;（4）Isolated the HSCs from infected and uninfected livers at differenttime points, and we found that miR-203was significantly down-regulated in HSCs ofinfected mice, our results suggested that miR-203could play an important role in theprocess of activation of HSCs during schistosomiasis liver fibrosis. This study show thatmiR-203play a important role in schistosomiasis hepatic fibrosis and laid a foundation for further study of miRNA-mediated treatment of schistosomiasis hepatic fibrosis. |
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