Effect Of Reversine On Experimental Hepatic Fibrosis Model In Rats

Hepatic fibrosis (HF) occurs as a result of initial liver injury and chronicinflammation, among others. In HF, the extra-cellular matrix (ECM) is synthesizedrather than degraded, hence resulting in the excessive deposition of ECM. Therefore,liver fibrosis is a crucial stage in cirrhosis. In China, cirrhosis is a common diseasethat causes a high mortality rate. It has become a big problem to human health.Approximately30million people have died of this disease. Thus, anti-fibrosistreatments have great medical significance because it can prevent the development ofcirrhosis, reduce the number of liver cancer and cirrhosis cases, and improve the livesand survival rates of patients. In our recent study, we found that reversine, a smallcell-permeable synthetic chemical, has the ability to inhibit hepatic stellate cell (HSC)activation and proliferation. Therefore, an experiment was designed to improve theeffects of reversine in preventing HF through the inhibition of HSC activation andproliferation. The experiment was divided into two parts:(1) in vitro, in which weobserved the functions of the HSCs affected by reversine, and (2) in vivo, in whichreversine was used to treat liver fibrosis in rat models.PurposeTo explore the effect of reversine on experimental hepatic fibrosis model in rats.Methods1. Liver fibrosis rat models were prepared by subcutaneously injecting healthy maleSprague Dawley (SD) rats with thioacetamide. The models were monitored via B ultrasound imaging. VTQ was used to detect the Vs values of the liver fibrosismodels. Furthermore, the composite scores of the models were taken afterspecimen collection and preservation, H&E staining, α-SMAimmunohistochemistry, and digital medical image analysis.2. A different group of liver fibrosis rat models was prepared by subcutaneouslyinjecting rats with thioacetamide. The primary HSCs were separated by using thedensity gradient solution of Nycodenz. The morphologies of the HSCs wereobserved under fluorescence microscopy. Then, the cells were inoculated, and thenext generation was bred and identified via α-SMA immunofluorescence.3. Through in vitro experimentation, we observed the functions of the HSCs affectedby reversine under a microscope. We also detected the proliferation of these HSCsby using CCK-8. The OD value was detected using a microplate reader at450nmwavelength. Then, the rate of cell proliferation was calculated.4. Another group of liver fibrosis rat models was prepared by subcutaneouslyinjecting healthy male SD rats with thioacetamide. Then, subcutaneous injectionsof reversine (250μg/kg) were administered to the liver fibrosis models withinthree weeks. Thereafter, VTQ was used to detect the Vs values of the liver fibrosismodels. Then, after specimen collection and preservation, H&E staining, α-SMAimmunohistochemistry, and digital medical image analysis, the effects of reversinewere evaluated by using the composite score.Results1. The Vs value of the normal group was1.18±0.181, whereas that of theexperimental group was2.47±0.511. The degree of HF and the Vs values werepositively correlated (P<0.01). The H&E stained liver tissues of normal ratsshowed portal areas, normal hepatocytes, and no obvious inflammatory reaction,degeneration, and necrosis. On the other hand, the liver tissues of the experimentalgroup showed that the normal lobular structure was destroyed, had swollenhepatocytes, and was significantly inflamed. The α-SMA immunohistochemistryresults of the normal rats showed a clear lobule structure without collagen fiberhyperplasia. Meanwhile, the normal lobular structure of the experimental groupwas destroyed and the portal area was expanded. Plenty of mesenchymal cells were positively expressed. Using ImageJ in the digital medical image analysis,the α-SMA immunohistochemistry area was7.052±2.730in the normal group and193.037±16.318in the experimental group. In the pathological stages, the normalgroup was at the S0stage, whereas the experimental group was between the S2and S3stages. The experimental rats had varying degrees of liver fibrosis.2. About1.2*10^7to2.0*10^7HSCs were obtained from each liver fibrosis ratmodel. Freshly isolated HSCs were round, had strong refractions, and contained asignificant number of lipid droplets in the cytoplasm. Moreover, by usingfluorescence microscopy, we observed that the HSCs had a blue-greenfluorescence. The immunofluorescence staining results also revealed that theα-SMAwas91.9%positive.3. The HSC morphologies changed after being exposed to reversine for10hour. Thecells appeared shrunken and became round, and nuclear chromatin condensationoccurred. Three reversine concentrations and10μL of CCK-8were used tointervene with the HSC proliferation. The proliferation rates during incubation at37°C for4hour were8.56%,10.77%, and18.31%in groups1(with reversineconcentration of25ng/mL),2(with reversine concentration of50ng/mL), and3(with reversine concentration of75ng/mL), respectively. After10hour, HSCproliferation was clearly inhibited by reversine (25ng/mL), and the degree ofinhibition increased with increasing reversine concentrations (P<0.05).4. The Vs value of the normal group was1.23±0.180, whereas that of theexperimental group was2.27±0.231. The Vs value of the experimental group afterreversine intervention was1.18±0.139. The degree of HF was positivelycorrelated with the Vs value. Thus, after the reversine intervention, the Vs value ofthe experimental group was reduced. This finding shows that liver fibrosis hasimproved after reversine intervention (P<0.01). The H&E stained liver tissues ofthe treated rats showed that the portal area of fibrous tissue proliferation decreasedsignificantly. The collagen fibers around the liver pseudolobule have also beenreduced. Confusion arrangement in the hepatic cord was improved. Hepatocyteswelling was reduced, and no obvious focal necrosis was observed. The α-SMAimmunohistochemistry results of the treated rats showed that the lobule structureshave been repaired. Incomplete yellowish brown mesenchymal cells can also be observed. Using ImageJ in the digital medical image analysis, the α-SMAimmunohistochemistry area was found to be20.196±5.835in the normal group,189.636±28.322in the experimental group, and72.195±25.141in the treatedgroup. HSC expression was reduced significantly from the experimental group tothe treatment group (P<0.01). In terms of pathological stages, the normal groupwas at the S0stage, whereas the experimental group was between the S2and S3stages. All experimental rats were at the S1stage after the reversine intervention.Conclusion1. The liver fibrosis models of SD rats were successfully prepared by usingthioacetamide and were monitored via B ultrasound imaging. These liver modelshad more HSCs than the normal liver.2. Another group of liver fibrosis models was also successfully prepared bysubcutaneously injecting rats with thioacetamide. The primary HSCs weresuccessfully separated using the density gradient solution of Nycodenz.3. Through in vitro experimentation, we found that reversine inhibits HSCproliferation and induces apoptosis. The best inhibitory effect was achieved with areversine concentration of25ng/mL after10hour. The HSCs showed apoptoticmorphology characteristics, that is, the appeared shrunken, became round, andunderwent shedding.4. Reversine can reduce α-SMA expression in liver fibrosis rat models. It can alsoinhibit HSC proliferation and treat liver fibrosis. The inhibitory effects ofreversine on HSC proliferation were consistent in vivo and in vitro.