| Background The illustration of the mechanism of liver fibrosis helps to prevent or delay the development of fibrosis. As the largest immune organ, the spleen features a bidirectional and phasic immune function that facilitates anti-infection but the other way round, greatly compromises the body via activation of immune cells and release of inflammatory factors. Studies show that splenectomy during liver fibrosis progression is both conducive to repair of liver injuries and suppression of liver fibrosis as well as enhancement of liver function. Nevertheless, the role of the spleen in liver fibrosis remains unclear. In light of its reduction of blood supply to liver tissues, splenectomy is generally believed capable of aggravating damages of liver functions. However, opposite results has been obtained in the related research. Therefore, a hypothesis can be safely drawn that the spleen may influence liver tissue repair and liver function through some cytokine or signaling pathway. Therefore, the spleen can be through some cytokines or signaling pathways that affect liver tissue repair and improve liver function.PTEN gene, phosphatase and tensin homolog deleted on chromosome ten, is a tumor suppressor gene discovered in1997and possesses both protein phosphatase activity and lipid phosphatase activity. A number of tumor diseases were found with an abnormal expression of PTEN gene, such as pulmonary fibrosis, bronchial asthma, rheumatoid arthritis, liver fibrosis, etc. Previous studies showed that PTEN expression declined with liver fibrosis progression, and its expression in vitro was negatively correlated with HSC (Hepatic Stellate Cells) activity and proliferation capacity. On the other hand, the activation of HSC acts as a central event in liver fibrosis. And under such stimulus as cytokines, inflammatory mediators, acetaldehyde and oxygen free radicals, HSC can transform into myofibroblast-like cells which are capable of expression of alpha-SMA and can migrate to the injury sites and proliferate to produce large amounts of collagen. Furthermore,alpha-SMA signals the activation of HSC, whose expression in liver tissues can reflect the degree of HSC activation and proliferation.In conclusion, spleen through influence the expression of PTEN and play to the role of the delay of liver fibrosis, remains to be further discussed in the process of liver fibrosis. Therefore, this study aimed to ascertain the effect of the spleen on PTEN expression during the progression of liver fibrosis by splenectomy so as to unveil the possible mechanism of spleen in suppression of liver fibrosis.Aim This study proceeded to investigate their correlation and explore the relevant mechanisms of splenectomy in delaying the progression of liver fibrosis.Methods135healthy Sprague-Dawley male adult rats weighing230-270g were obtained from the Experimental Animal Center of Southern Medical University (Certificate No.4402101312). All rats were housed in plastic cages with free access to clean food and water. Experiments were performed in compliance with the national ethical guidelines for the care and use of laboratory animals. The rats were randomly divided into three groups (45rats in each group):group A with bile duct ligation and splenectomy (BDL+SP, group B with bile duct ligation and spleen sham operation (BDL+SSP), group C with sham bile duct ligation and spleen sham operation (SBDL+SSP).Groups A and B in the process of experimental rats were killed26, another19broken neck method was used to put to death.All groups underwent laparotomy with4ml/kg of10%chloral hydrate injected intraperitoneally to induce deep anesthesia. Each was managed as follows. Group A: peripheral ligaments of the spleen were dissociated and blood vessels ligated (double-ligated) with1-0silk. Splenectomy was performed with accessory spleen (if existed) also removed. Common biliary duct was ligated at both proximal and distal ends and was cut in-between. Then sterile stabs were applied to examine bile and blood leakage before closure. Group B:the spleen was retained with other operations same as group A. Group C:a sham operation was administered that consisted of exposure but no ligation of the common bile duct. All the above operations were performed under aseptic conditions. Postoperatively,10rats in each group were terminated of life under anesthesia respectively at1,3and5weeks and their livers were harvested. Liver tissue specimens were fixed in4%phosphate-buffered paraformaldehyde and stained with hematoxylin and eosin (H&E), Sirius red staining or immunohistochemical staining.All data were expressed as mean±standard deviation (x±s) and analyzed with SPSS13.0software. The comparison of mean variability among all groups was conducted with one-way ANOVA analysis. The correlation between the western blots result of PTEN and a-SMA expression in stained liver tissue was analyzed with Pearson’s correlation analysis. p<0.05was considered statistically significant.Results With the extension of the modeling time, H&E showed a histologically normal phenotype in group C and visualized varying degrees of degeneration and necrosis of liver cells in Group A and group B,with liver fibrosis gradually formed. At the fifth week, extensive proliferation of liver connective tissues were portrayed, which interconnected, encased and divided the adjacent proliferated septa to form in some areas pseudo-lobules. As for Sirius red staining, group C was disclosed with a few collagen fibers at mere portal area and central vein wall, and group A and group B with an increasing number of collagen fibers as modeling prolonged and with the former chalking up a higher value at the same time point.PTEN immunohistochemical staining showed expression of PTEN in broad liver tissues of normal rats but only in a remarkably decreased number of cells as liver fibrosis exacerbated, with group B suggesting a sharper decline than group A at the same time point. α-SMA immunohistochemistry indicated expression present only in a limited liver tissues in group C but on the rise in group A and group B as modeling proceeded with group A fewer than group B.In the real-time fluorescence quantitative PCR, a mRNA amplification curve was rendered automatically by mapping the fluorescence value with cycle number. The samples were manifested with good reproducibility and consistent amplification efficiency. PTEN gene expressions in rats with hepatic fibrosis at1,3weeks and5weeks were significantly higher in group A (1.48±0.21,0.72±0.12,0.57±0.14) than that in group B(0.79±0.21,0.56±0.14,0.33±0.11) with P<0.01. Moreover, a significant difference was found between Group A and group B at any time point (P <0.05). PTEN mRNA expression corresponded to PTEN protein with significant differences concluded between the two groups in expression of PTEN protein (P <0.01).α-SMA was ascertained with a weak positive expression in smooth muscle cells of the vascular wall in normal rat liver tissues but gradually increased with the development of hepatic fibrosis, and the expression of α-SMA in rat liver tissue was significantly distinctly between the two groups at different time points(p<0.01).Pearson’s correlation analysis of PTEN protein expression and alpha SMA expression showed that the expression of PTEN and alpha SMA expression in liver fibrosis tissues of rats were significantly negatively correlated (r=-0.86, P<0.05). Conclusion In the model of rats with liver fibrosis, splenectomy can up-regulate the expression of PTEN gene and reduce the secretion of a-SMA, thereby deterring the progression of liver fibrosis. Besides, Bile duct ligation combined with splenectomy is an effective way to build models of liver fibrosis loss of function of the spleen. |
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