| Hepatic fibrosis, the liver's wound healing response to virtually all forms of chronic liver injury, can result from several chronic liver diseases caused by many pathogenic factors. Given enough time, fibrosis will progress to cirrhosis. The main pathological characteristic of hepatic fibrosis is the increased irregular deposition of extracellular matrix (ECM). Currently, it is believed that the activation of hepatic stellate cells (HSC), which play a pivotal role in fibrosis process, into cells with spontaneous proliferation and strong fibrogenic activity appears to be the dominant driving force in fibrosis. While the apoptosis of HSC increases significantly during the reparative process following liver injury. So the inhibiting proliferation or inducing apoptosis of activated HSC play a key role in the process of reversion of hepatic fibrosis.Phosphatase and tensin homolog deleted on chromosome ten (PTEN), the first tumor-suppressing gene found to exhibit phosphatase activity, negatively regulates cell cycle, inhibits the proliferation and promotes the apoptosis of tumor cells. As such, it is reasonable to conclude that dysfunction or absence of PTEN is intimately related to the formation and progression of human tumors.Nevertheless, in recent years, PTEN research has gradually extended beyond cancer, focusing on its role in other disease states. Studies demonstrated that lowered expression and phosphatase activity of PTEN is found in lung fibroblasts of patients with idiopathic pulmonary fibrosis. Moreover, studies also documented that PTEN inhibits the proliferation and induces the apoptosis of lung fibroblasts cultured in vitro. A study on PTEN and myocardial fibrosis showed that knocking out PTEN in mice leads to an increased ratio of heart to body weight, decreased cardiac contractility and, eventually, interstitial fibrosis. This suggests that the low expression or deactivation of PTEN involves itself in the pathogenesis of lung fibrosis and myocardial fibrosis. In the liver, the absence of PTEN in specific hepatic cells may result not only in hepatocellular carcinoma, but also in non-alcoholic steatohepatitis, a condition closely related to hepatic fibrosis. Currently, though, the expression and function of PTEN in hepatic fibrosis, especially its effects on the proliferation and apoptosis of HSC, remain unclear. So, using the rat bile duct ligation (BDL) model, this study explored the dynamic expression of PTEN in the process of hepatic fibrosis in rats and its relation with the proliferation and apoptosis of HSC in vivo. And we set out to determine the effects of PTEN over-expression, via adenoviral transduction of wild type PTEN and its mutant G129E gene, on the proliferation and apoptosis as well as cell cycle of activated HSC cultured in vitro. Meanwhile, the signaling transduction pathways of PTEN on the proliferation, apoptosis and of cell cycle of activated HSC were studied. It was designed to provide new viewpoint and defined strategy for preventing and treating hepatic fibrosis.The project contain four parts as below:Part 1:The dynamic expression of PTEN in fibrogenic liver tissues in rats and its relation with the proliferation and apoptosis of hepatic stellate cells in vivoObjective: To investigate the dynamic expression of PTEN in liver tissues in the process of hepatic fibrosis in rats and its relation with the proliferation and apoptosis of HSC in vivo.Methods: The rat model of hepatic fibrosis used in this study was established by means of common bile duct ligation (BDL). HE and Masson's trichrome staining were used to determine histopathology changes of liver tissues. At 4 time points, the expressions of PTEN in hepatic tissues and activated HSC of rats were measured by immunohistochemical staining, Western blot, Real-time Q-PCR and immunofluorescence confocal laser scanning microscopy, respectively. And alpha-smooth muscle actin (α-SMA), an activated HSC marker in rat liver tissues, was detected by immunohistochemical staining. Furthermore, apoptotic HSC in rat liver tissues were determined by dual staining both of the terminal deoxynucleotidy transferrase UTP-nick end labeling (TUNEL) and ofα-SMA immunohistochemistry.Results: (1) HE and Masson's trichrome staining showed rat modes of hepatic fibrosis with BDL were established successfully. With each consecutive week after BDL, increased fibrosis, degeneration and necrosis were observed in rat liver cells. Not surprisingly, a disruption of normal architecture and a decrease in normal hepatic cells were concomitantly observed. (2)α-SMA immunohistochemistry showed that in hepatic tissues of normal rats, there was only weakly positive expression ofα-SMA in the smooth muscle cells of the vessel wall. With the development of hepatic fibrosis, though, theα-SMA positive cells in the hepatic tissues of rats increased significantly. At weekly time points after BDL, the optical density values ofα-SMA in rat liver tissues, 0.16±0.01, 0.17±0.01, 0.21±0.01, 0.26±0.02 (1, 2, 3 and 4 weeks, respectively) increased significantly with each passing week, P<0.01. Furthermore, optical density values from BDL rats were all significantly higher than those from the sham operation group, 0.07±0.01 (P<0.01). (3) Dual staining both of TUNEL and ofα-SMA immunohistochemistry showed that few apoptotic HSC in normal livers appeared, with the developing of liver fibrosis,increased activated HSC, the number of apoptotic HSC increased too. But at weekly time points after BDL, the apoptotic index of activated HSC in rat liver tissues, 4.57%±0.41%,4.02%±0.48%,3.45%±0.37%,2.88%±0.50% (1, 2, 3 and 4 weeks, respectively) decreased gradually with aggravation of liver fibrosis (P<0.01). (4) PTEN immunohistochemical examination of hepatic tissues from wild-type rats showed widespread staining of PTEN protein in the cytoplasm, and, to a lesser extent, in the nuclei. With the progression of hepatic fibrosis, PTEN signal in the rat liver correspondingly decreased in the portal area, the fibrous septa and the proliferated peripheral cells of the bile duct. Nevertheless, there were no significant changes in the intracellular localization of PTEN protein. At weekly time points after BDL, optical density values of PTEN in rat liver tissues, 0.15±0.01,0.12±0.02,0.09±0.01,0.07±0.01 (1, 2, 3, 4 weeks, respectively), decreased significantly with each subsequent week, P<0.01. Furthermore, optical density values from BDL rats were significantly lower than those from the the sham operation group, 0.21±0.02 (P<0.01). (5) PTEN andα-SMA immunofluorescence double labeled hepatic tissue slices were examined under a confocal laser scanning microscope. Single channel scanning displayed PTEN andα-SMA positive signal as green and red fluorescence foci, respectively. Alongside the green and red foci, after merging the images of single channel scanning, yellow foci corresponding to colocalized PTEN andα-SMA were found in the hepatic tissue slices. Since, in rat liver tissue, only activated HSC and a few vascular smooth muscle cells expressα-SMA during hepatic fibrosis, the yellow spots marked PTEN-positive, activated HSC. The yellow foci were observed primarily in the cytoplasm. Analysis of the images indicated that, at weekly time points after BDL, PTEN-positive, activated HSC, accounted for 79.97%±5.49%, 73.83%±5.04%, 66.68%±4.58%, 60.20%±4.65% (1, 2, 3 and 4 weeks, respectively) of theα-SMA positive expression cells (total activated HSC). Thus, with the development of hepatic fibrosis, the ratio of activated HSC of PTEN positive expression to total activated HSC significantly decreased (P<0.01). (6) Western blot at weekly time points after BDL showed that the expression levels of PTEN protein in fibrotic rat liver tissues, 1.20±0.13, 1.07±0.16, 0.88±0.08, 0.73±0.07 (1, 2, 3, and 4 weeks, respectively) decreased significantly with increasing severity of hepatic fibrosis, P<0.01. Furthermore, all values from BDL rats were significantly lower than those from the sham operation group, 1.37±0.14 (P<0.01). (7) Expression levels of PTEN mRNA in rat liver tissues were measured with Real-time Q-PCR. PTEN mRNA expressions in rat liver tissues were compared by using the method of fold increase (2-△△C t method). The expression level of PTEN gene in the sham operation group was assigned a reference value of 1. In the fibrotic liver tissues of BDL rats, the mRNA expression levels of PTEN were 0.66-, 0.53-, 0.44- and 0.37-fold (1, 2, 3 and 4 weeks, respectively), all were significantly lower than that in the sham operation group, and down-regulated gradually with the development of hepatic fibrosis (P<0.01). (8) Pearson's correlation analysis showed the expression of PTEN had a significant negative correlation with the expression ofα-SMA, a significant positive correlation with the percentage of PTEN-positive activated HSC and the apoptotic index of activated HSC in fibrosis liver tissues in rats. r values were -0.92, 0.78, 0.76, respectively (P<0.01).Conclusions: Rat models of hepatic fibrosis with bile duct ligation (BDL) are established succesfully. During liver fibrosis in rats, the activation and proliferation of HSC accelerate gradually, whereas the apoptotic index of activated HSC decreases gradually. The expressions of PTEN mRNA and protein are down-regulated gradually in fibrogenic rat liver tissues, PTEN expression in HSC in vivo also decreases with progression of fibrosis. Thus, the dynamic expression of PTEN in rat liver tissues has a significant negative correlation with the activation and proliferation of HSC in vivo, and a significant positive correlation with the apoptotic index of activated HSC in vivo.Part 2:Effects of PTEN on the proliferation and apoptosis of activated hepatic stellate cell in vitroObjective: To investigate effects of over-expression of wild type PTEN and its mutant G129E (only exhibit protein phosphatase and lose lipids phosphatase activity) on the proliferation and apoptosis of activated HSC culcured in vitro.Methods: Amplifications of adenoviral vectors (Ad-PTEN, Ad-G129E and Ad-GFP) were performed in AD293 cells and viral titer estimates were conducted. The wild type PTEN and its mutant G129E gene were transduced into activated HSC in vitro via adenoviral vector, respectively. PTEN expression in HSC was then measured by Western blot and Real-time Q-PCR. Changes in Bcl-2 and Bax in HSC were monitored by Western blot. And MTT assay was used to determine cell proliferation and a TUNEL assay and propidium iodide (PI) labed flow cytometry (FCM) were used to detect cell apoptosis. Cells were grouped as follows: (1) Control group, cells were cultured under the same conditions, except DMEM (without FBS and antibiotics) was used in place of the adenovirus; (2) Ad-GFP group, HSC were infected with adenovirus expressing green fluorescent protein (GFP) alone; (3) Ad-PTEN group, HSC were infected with adenovirus harboring genes for both wild type PTEN and GFP; (4) Ad-G129E group, HSC were infected with adenovirus harboring genes for both PTEN mutant G129E and GFP.Results: (1) Adenoviral vectors (viral titers of Ad-PTEN, Ad-G129E and Ad-GFP: 1.2 x 109, 1.5 x 109, 1.8 x 109 pfu/ml, respectively) for experiment were obtained via performing repeated amplifications of virus in AD293 cells. (2) PTEN expression in HSC was detected at 72 hours after adenoviral infection. Real time Q-PCR was used to extrapolate relative mRNA expression levels of PTEN in HSC. PTEN mRNA expression in HSC was compared by using the method of fold increase (2-△△C t method). PTEN mRNA expression levels in Ad-GFP group, Ad-PTEN group and Ad-G129E group were 0.993-, 1.569- and 1.561-fold, respectively (the expression value of the untreated control group was arbitrarily assigned an expression value of 1). Obviously, the expression of PTEN mRNA was significantly higher in the Ad-PTEN group and Ad-G129E group than those in both the control group and the Ad-GFP group, P<0.01. Western blot analysis recapitulated the Real time Q-PCR data by showing that expression levels of PTEN protein in Ad-PTEN group (1.66±0.09), Ad-G129E group (1.65±0.09) were significantly higher than those in control group (1.10±0.07) and Ad-GFP group (1.09±0.07), P<0.01. Furthermore, no significant differences were observed in the expressions of PTEN mRNA and protein between Ad-PTEN group and Ad-G129E group or between control group and Ad-GFP group (P>0.05). Overall, wild type PTEN gene and G129E gene were successfully transduced and expressed in HSC. (3)There was no significant difference in the proliferation of HSC at 24 hours after transduction of Ad-PTEN or Ad-G129E (P>0.05). At 48 and 72 hours after transduction, though, a precipitous time-dependent drop in proliferation was observed in the Ad-PTEN group or Ad-G129E group. Inhibition rates were 14.03% and 23.12% in Ad-PTEN group, 9.52% and 12.63% in Ad-G129E group, respectively, when compared with the control group (P<0.01). Obviously, the inhibitory effect of wild type PTEN on HSC proliferation was more powerful than that of G129E. Moreover, the A value between control and Ad-GFP groups showed no notable difference at various time points (P>0.05). (4) TUNEL assay showed that at 72 hours after adenoviral transduction, the apoptotic rates of HSC in Ad-PTEN group (29.81%±2.52%), Ad-G129E group (26.37%±1.97 %) were greatly higher than those in control group (1.98%±0.25%) and Ad-GFP group (2.16%±0.28%), P<0.01, and Ad-PTEN group increased notably in the apoptotic rates of HSC compared with Ad-G129E group too (P<0.01). In addition, no significant difference was found in the apoptotic rates of HSC between control group and Ad-GFP group (P>0.05). (5) At 72 hours after adenovirus transduction, the apoptotic rates of HSC analyzed by PI labeled FCM in Ad-PTEN group (20.84%±1.44%), Ad-G129E group (17.54%±1.76%) increased markedly compared with control group (1.12%±0.57%) and Ad-GFP group (1.21%±0.22%), P<0.01. And the action of wild type PTEN on HSC apoptosis was more powerful than that of Ad-G129E (P<0.01). Furthermore, no significant difference was observed in apoptotic rate of HSC between control group and Ad-GFP group (P>0.05). (6) Western blot analysis showed that the expression of Bcl-2 decreased significantly at 72 hours after Ad-PTEN or Ad-G129E infection compared to control group and Ad-GFP group (1.16±0.03 or 1.24±0.05vs 1.37±0.06 and 1.34±0.08, respectively, P<0.01), and Bcl-2 expression in Ad-PTEN group was significantly lower than that in Ad-G129E group too (P<0.01). In contrast, the expressions of Bax in HSC at 72 hours after infection of Ad-PTEN (1.50±0.05) or Ad-G129E (1.41±0.05) heightened markedly compared with those in control group (1.28±0.06) and Ad-GFP group (1.26±0.08), P<0.01. And Bax expression in Ad-PTEN group inceased notably compared with that in Ad-G129E group as well (P<0.01). Moreover, there were no significant differences in the expressions of Bcl-2 and Bax between control group and Ad-GFP group (P>0.05).Conclusions: Exogenous wild type PTEN gene and G129E gene are successfully transduced and expressed in activated HSC in vitro respectively, inhibit the proliferation and induce apoptosis of them. At the same time, the expression of apoptosis regulating gene Bax is increased and Bcl-2 is decreased in activated HSC. Moreover, the action of wild type PTEN is more powerful than that of Ad-G129E.Part 3:Regulatory effects of PTEN on cell cycle of activated hepatic stellate cells in vitroObjective: To investigate regulatory effects of over-expression of wild type PTEN and its mutant G129E on cell cycle of activated HSC culcured in vitro. Methods: The wild type PTEN gene and its mutant G129E gene were transduced into activated HSC cultured in vitro mediated by adenoviral vector, respectively. PI labed FCM was then used to detect cell cycle phase of activated HSC. And the expressions of PTEN, cyclinD1, cyclin dependent kinase 4 (CDK4) and P27kip1 in HSC were measured by Western blot and Real-time Q-PCR, respectively. Cells were grouped in accordance with part 2.Results: (1) Western blot and Real-time Q-PCR demonstrated that exogenous wild type PTEN gene and G129E gene were successfully transduced and expressed in activated HSC cultured in vitro (the result was clearly in accordance with the part 2). (2) At 72 hours after adenovirus infection, Cell cycle phase of HSC in each group was detected by PI labed FCM. At G0/G1 phase, the number of HSC in Ad-PTEN group (67.68%±2.75%), Ad-G129E group (61.17%±3.41%) increased greatly compared with those in control group (53.01%±2.37%) and Ad-GFP group (53.85%±3.08%), P<0.01, and the number of HSC in Ad-PTEN group was significantly higher than that in Ad-G129E group (P<0.01). At S phase, the number of HSC decreased notably in Ad-PTEN group (14.42%±1.81%), Ad-G129E group (18.17%±2.43%) compared with those in control group (22.17%±1.99%) and Ad-GFP group (21.54%±1.74%), P<0.01, and the number of HSC in Ad-PTEN group was lower than that in Ad-G129E group too (P<0.01). At G2/M phase, the number of HSC in Ad-PTEN group (17.90%±2.70%), Ad-G129E group (20.66%±2.37%) decreased significantly compared to those in control group (24.82%±3.81%) and Ad-GFP group (24.62%±3.15%), P<0.01, P<0.05, while there was no significant difference in the number of HSC between Ad-PTEN group and Ad-G129E group (P>0.50). Furthermore, at any phase, no significant difference was found in the number of HSC between control group and Ad-GFP group (P>0.50). (3) At 72 hours after adenoviral infection, the expression of cyclinD1 protein in HSC was analyzed by Western blot, the expression of cyclinD1 protein in Ad-PTEN group (1.12±0.07), Ad-G129E group (1.23±0.05) decreased greatly compared to those in control group (1.45±0.05) and Ad-GFP group (1.47±0.08), P<0.01, and cyclinD1 protein expressio in Ad-PTEN group was significantly lower than that in Ad-G129E group too (P<0.01). The Real time Q-PCR was further used to detect the expression level of cyclinD1 mRNA in HSC, the method of fold increase (2-△△Ct method) was used to calculate relative mRNA expression level of cyclinD1 in HSC in each group. If the mRNA expression level of cyclinD1 in control group was assigned a value of 1, then cyclinD1 mRNA expressions in Ad-GFP group, Ad-PTEN group and Ad-G129E group were 1.011-, 0.773- and 0.838-fold compared with control group, respectively. Obviously, the expressions of cyclinD1 mRNA decreased significantly in Ad-PTEN group and Ad-G129E group compared with those in both control group and Ad-GFP group (P<0.01), and the expression of cyclinD1 mRNA in Ad-PTEN group was markedly lower than that in Ad-G129E group (P<0.01). Moreover, no significant differences were observed in the protein and mRNA expressions of cyclinD1 between control group and Ad-GFP group (P>0.05). (4) At 72 hours after adenoviral infection, Western blot analysis showed the expressions of CDK4 protein in Ad-PTEN group (1.05±0.07), Ad-G129E group (1.18±0.06) were markedly lower than those in control group (1.41±0.03) and Ad-GFP group (1.43±0.06), P<0.01, and CDK4 protein expression in Ad-PTEN group reduced notably compared with that in Ad-G129E group as well (P<0.01). Real time Q-PCR was further used to detect the expression level of CDK4 mRNA in HSC, the method of fold increase (2-△△C t method) was used to calculate relative mRNA expression level of CDK4 in HSC in each group. If the mRNA expression level of CDK4 in control group was assigned a value of 1, then CDK4 mRNA expression levels in Ad-GFP group, Ad-PTEN group and Ad-G129E group were 1.007-, 0.738- and 0.822-fold, respectively. The expression of CDK4 mRNA down-regulated notably in Ad-PTEN group and Ad-G129E group compared with those in control group and Ad-GFP group (P<0.01), and the expression of CDK4 mRNA in Ad-PTEN group was significantly lower than that in Ad-G129E group too (P<0.01). In addition, no significant differences were observed in the protein and mRNA expressions of CDK4 between control group and Ad-GFP group (P>0.05). (5) At 72 hours after adenoviral infection, Western blot analysis showed that the protein expressions of P27kip1 in HSC in Ad-PTEN group (1.40±0.03), Ad-G129E group (1.28±0.08) increased significantly compared to those in control group (1.11±0.04) and Ad-GFP group (1.12±0.04), P<0.01, and the protein expression of P27kip1 in Ad-PTEN group was notably higher than that in Ad-G129E group as well (P<0.01). Real time Q-PCR was used to extrapolate relative mRNA expression level of P27kip1 in HSC. P27kip1 mRNA expression in HSC in each group was compared by using the method of fold increase (2-△△C t method). If the mRNA expression level of P27kip1 in control group was assigned a value of 1, then the mRNA expressions of P27kip1 in Ad-GFP group, Ad-PTEN group and Ad-G129E group were 1.008-, 1.264- and 1.157-fold compared with control group, respectively. Obviously, the expressions of P27kip1 mRNA increased greatly in Ad-PTEN group and Ad-G129E group compared with those in control group and Ad-GFP group (P<0.01), and the expression of P27kip1 mRNA in Ad-PTEN group was markedly higher than that in Ad-G129E group too (P<0.01). Moreover, no significant difference was found in expressions of P27kip1 protein and mRNA between control group and Ad-GFP group (P>0.05).Conclusions: The over-expression of not only wild type PTEN but also G129E can inhibit transition of activated HSC in vitro from G1 to S phase, arrest cell cycle of them at G0/G1 phase, and thus negatively regulate cell cycle progression of them. Meanwhile, at both transcriptional and translational levels, the expressions of cyclinD1 and CDK4 are down-regulated and P27kip1 expression is up-regulated in activated HSC in vitro. In addition, wild type PTEN is more powerful than Ad-G129E for above-mentioned effects. Part 4: Signaling transduction mechanisms of PTEN on the activated hepatic stellate cell behaviorsObjective: To investigate the signaling transduction mechanisms of PTEN on the proliferation and apoptosis as well as cell cycle of activated HSC.Methods: Using cell culture techniques in vitro, the wild type PTEN gene and its mutant G129E gene were transduced into activated HSC in vitro mediated by adenoviral vector, respectively. And the protein expressions of serine-threonine protein kinase B (Akt), p-Akt (Thr308), extracellular signal regulated kinase1 (ERK1) and p-ERK1 in activated HSC were measured by Western blot. And the mRNA expressions of Akt and ERK1 were detected by Real-time Q-PCR. Cells were grouped in accordance with part 2.Results: ((1) Western blot and Real-time Q-PCR demonstrated that exogenous wild type PTEN gene and G129E gene were successfully transduced and expressed in activated HSC cultured in vitro (the result was clearly in accordance with the part 2). (2) Western blot and Real-time Q-PCR showed that there were no significant differences in the mRNA and protein expression of Akt in HSC at 72 hours after transduction of Ad-PTEN or Ad-G129E (P>0.05). While Western blot analysis documented the expression of p-Akt (Thr308) in HSC at 72 hours after adenoviral infection decreased greatly in Ad-PTEN group (0.63±0.04) compared to those in Ad-G129E group (0.93±0.03), control group (0.95±0.04) and Ad-GFP group (0.94±0.03), P<0.01. Moreover, no significant differences were observed in the expressions of p-Akt (Thr308) among Ad-G129E group, control group and Ad-GFP group (P>0.05). (3) At 72 hours after transduction of Ad-PTEN or Ad-G129E, there were no significant differences in the protein and mRNA expression of ERK1 in HSC (P>0.05). While Western blot analysis documented the protein expression of p-ERK1 in HSC at 72 hours after adenoviral infection reduced greatly in Ad-PTEN group (0.65±0.04), Ad-G129E group (0.68±0.07) compared to those in control group (0.84±0.07) and Ad-GFP control (0.85±0.06), P<0.01. In addition, no significant differences were found in the protein expressions of p-ERK1 between Ad-PTEN and Ad-G129E group or between control group and Ad-GFP group (P >0.05).Conclusions: The PTEN phosphatase negatively regulates the phosphoinositol-3-kinase (PI3K)/Akt and ERK1/2 signal transduction pathways via inhibiting phosphorylation of Akt and ERK1 in activated HSC, and thus curbs the proliferation, induces apoptosis and negatively regulates cell cycle of activated HSC in vitro.
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