The Dynamic Change Of PTEN Expression During Fibrogenesis And Reversal Of Rat Induced CCl₄ And Its Relation With The Activation And Proliferation Of Hepatic Stellate Cells In Vivo

Hepatic fibrosis, results from excessive deposition of extracellular matrix (ECM) in the liver. More fibrous tissues will be produced if the chronic liver injury continues, and finally turn to be liver cirrhosis. Liver fibrosis is reversible, while the liver cirrhosis is not. Since hepatic fibrosis is the former process of liver cirrhosis, it is necessary to explore its pathogenesis and some potential approaches for its reversal.Hepatic stellate cells (HSC) are known to be the key factors in liver fibrogenesis, especially when they are activated by some stimulation like injury, et al, although HSC are in quiescent conditions normally. Activated HSC produce mass of cytokines and ECM components mainly composed of collogen. Apoptosis will be the only way for activated HSC, as they cannot return quiescent, which promotes inducing apoptosis a target as well as inhibiting activation and proliferation of HSC for treating liver fibrosis.Phosphatase and tensin homolog deleted on chromosome ten (PTEN), the first tumor-suppressing gene found to exhibit double phosphatase activity. The dysfunction or absence of PTEN is highly related to the formation and progression of various tumors. Recently, studies on PTEN have gradually extended beyond tumors. For instance, PTEN was found to decrease in BDL (bile duct ligation) rat liver fibrosis, and also was found negative correlation with rat activated HSC in vivo. However, its relationship with activated HSC in vivo in reversal of liver fibrosis is still unclear. This study was designed to provide experiment basis for preventing and treating hepatic fibrosis through investigating the connection between PTEN expression and apoptosis and proliferation of HSC. Objective: This study was designed to investigate the dynamic expression of PTEN in fibrogenesis and reversal of rat fibrogenic liver tissues, and its relation with the activation and proliferation of hepatic stellate cells in vivo.Methods: A rat model of hepatic fibrosis was established by hypodermic injection of CCl₄ mixed with olive oil at the concentration of 40% for 5 weeks (2 ml·kg^-1, twice a week); the reversal model was made through 4 weeks'normal feeding based on hypodermic injection of 40%CCl₄ for 5 weeks at first. 108 male Wistar rats were randomly divided into following groups:, model group(containing 1 wk, 2 wk, 3 wk, 4 wk and 5 wk), model control group, reversal group(containing Re 1 wk, Re 2 wk, Re 3 wk and Re 4 wk), reversal control group. Haematoxylin and eosin staining (H&E staining) and Masson's trichrome staining (MT staining) were used to determine histopathology changes. The expression of PTEN in liver tissues was measured by immunofluorescence staining, real-time fluorescence quantitative PCR (real-time PCR) and western blot. And alpha-smooth muscle actin (α-SMA), a marker of the activated HSC, was detected by immunohistochemical staining and immunofluorescence. The co-expression between PTEN andα-SMA, PTEN and terminal deoxynucleotidy transferrase UTP-nick end labeling (TUNEL),α-SMA and TUNEL were observed by confocal laser scanning microscopy.Results:â‘ H&E and MT staining confirmed the successful models. Hepatic cells swelled, and appeared fatty degeneration, necrosis and regeneration, the fibrosis were spread from the vascular smooth muscle cells to portal area and damaged hepatic cells, the latter appeared fatty degeneration, necrosis and regeneration, the continuous CCl₄ led to the enlargened fibrosis area in model group; the fibrosis tissues turned to decrease from Re 1 wk to Re 4 wk, while the other histopathological changes gradually turned to be normal, especially for the hepatic cells;â‘¡In normal rat liver,α-SMA was occasionally detected in vascular smooth muscle cells, and the expression level was low. After CCl₄ administration, a significant increased expression of α-SMA from 1 wk to 5 wk in model group (0.51±0.02, 0.74±0.02, 1.29±0.02, 1.40±0.01, 1.65±0.02, P<0.01), compared with control group (0.07±0.01), P<0.01. Immunofluorescence staininig was used to find higher level ofα-SMA expression than control group (0.12±0.04, P<0.01) gradually decreased after the end of 5 weeks'CCl₄ administration, it revealed significant differences between Re 1 wk, Re 2 wk, Re 3 wk and Re 4 wk (1.31±0.11, 1.14±0.03, 0.97±0.01, 0.55±0.11), P<0.05;â‘¢PTEN protein, existing in wildspread areas in normal rat liver tissues, decreased significantly from 1 wk to 5 wk (2.23±0.02, 1.96±0.03, 1.61±0.02, 1.37±0.02, 0.98±0.01, P<0.01), compared with control group(2.67±0.02), P<0.01. After the end of CCl₄, the PTEN protein expression increased significantly from Re 1 wk to Re 4 wk (1.01±0.11, 1.46±0.09, 1.62±0.22, 1.99±0.30), P<0.01; however it was still under the normal lever of control group (2.31±0.12);â‘£Western blot showed that PTEN protein expression in rat hepatic tissues were significant lower at 1 wk, 2 wk, 3 wk, 4 wk and 5 wk (1.33±0.10, 1.12±0.13, 0.83±0.10, 0.55±0.08, 0.41±0.09) in model group than that of control group (1.75±0.32), P<0.01; and marked differences were found between the 5 time points. At Re 1 wk, Re 2 wk, Re 3 wk and Re 4 wk, PTEN protein expression were still lower than the control group(0.56±0.07), but it increased gradually, and significant differences were found between them (0.10±0.03, 0.21±0.05, 0.36±0.08, 0.44±0.06). PTEN protein declined during the progressive liver fibrosis and enhanced during its reversal;⑤Real-time fluorescent quantitation PCR assay was used to detected the expression change of PTEN mRNA, and the 2-△△Ct accounted for the results. PTEN mRNA in rat liver tissues were significant lower at 1 wk, 2 wk, 3 wk, 4 wk and 5 wk in model group (0.67±0.01, 0.58±0.04, 0.48±0.02, 0.38±0.04, 0.28±0.08) than that of control group (1.00), P<0.01, and marked differences were found between the 5 time points, P<0.01. At Re 1 wk, Re 2 wk, Re 3 wk and Re 4 wk, the relative expression of PTEN mRNA was still lower than the control group(1.00), but it increased gradually, and significant differences were found between them (0.53±0.01, 0.74±0.01, 0.80±0.03, 0.93±0.06), P<0.01. PTEN mRNA declined during the progressive liver fibrosis and enhanced during its reversal;â‘¥The activated HSC expressing PTEN accounted for an decreasing percentage of total activated HSC from 1 wk to 5 wk (0.90±0.05, 0.75±0.03, 0.41±0.03, 0.29±0.02, 0.23±0.03), compared with control group (1.00±0.00), P<0.01, there were also significant differences between 1 wk, 2 wk, 3 wk, 4 wk and 5 wk, P<0.01. The activated HSC expressing PTEN accounted for an increasing percentage of total activated HSC from Re 1 wk to Re 4 wk (0.35±0.02, 0.57±0.02, 0.66±0.02, 0.71±0.02), compared with control group (1.00±0.00), P<0.01, there were also significant differences between Re 1 wk, Re 2 wk, Re 3 wk and Re 4 wk, P<0.01;⑦The percentage of apoptotic activated HSC to total activated HSC decreased from 1 wk to 5 wk (7.12±0.03, 6.35±0.02, 4.11±0.02, 3.68±0.02, 1.50±0.03, P<0.01), compared with control group (0.00±0.00), P<0.01; the percentage of apoptotic activated HSC to total activated HSC from Re 1 wk to Re 4 wk was enhanced (1.20±0.23, 1.35±0.12, 4.20±0.17, 6.51±0.45, P<0.01), showing significant differences comparing with control group (0.00±0.00), P<0.01;⑧In normal rat and 1 week's rat liver tissues, no apoptosis cells were observed. There were some yellow cells-the apoptosis cells expressing PTEN, the percentage of which decreased from 2 wk to 5 wk (0.70±0.01, 0.64±0.01, 0.50±0.02, 0.33±0.01, P<0.01), compared with control group (0.00±0.00), P<0.01. From Re 1 wk to Re 4 wk, PTEN expression were rised, the number of apoptosis cells were also higer than before, the percentage of apoptosis cells expressing PTEN was enhanced much higher (0.78±0.02, 0.80±0.01, 0.92±0.02, 0.95±0.02), showing significant differences comparing with control group (0.00±0.00), P<0.01, however, there were no marked differences between Re 1 wk and Re 2 wk, Re 3 wk and Re 4 wk, P>0.05;⑨Pearson correlation analysis was used to find significant negative correlation between PTEN andα-SMA expression. There were also significant positive correlations between PTEN expression and the percentage of PTEN-positive activated HSC, PTEN expression and the apoptotic index of activated HSC, PTEN expression and the percentage of PTEN-positive apoptotic cells. r values were -0.979, 0.962, 0.991, 0.979, respectively (P<0.05).Conclusion: These data supported that PTEN decreased in both liver tissues and HSC in vivo of rats with hepatic fibrosis, and increased in reversal of liver fibrosis. The dynamic expression of PTEN in rat liver tissues had a significant negative correlation with the activation and proliferation of HSC and had a significant positive correlation with the apoptosis of activated HSC in vivo.