| Liver fibrosis is the mechanism of compensation and reparation after chronic hepatic injury, which is a necessary pathologic stage from chronic hepatitis to cirrhosis. The main pathological characteristic of liver fibrosis is the increased concentration and deposition of extracellular matrix (ECM). Hepatic stellate cells (HSC) are increasingly being recognized as the key players in liver fibrosis which is the main source of ECM. The activation and proliferation of HSC play a important role in fibrosis process. So inhibiting the proliferation or inducing apoptosis of activated HSC play a key role in reversing the liver fibrosis.Heme Oxygenase-1(HO-1) is a microsomal enzyme and rate-limiting enzyme for heme degradation in a wide range of human and mammalian tissues. HO-1can catalyze the heme degrading into biliverdin, iron atoms and carbon monoxide (CO) which play vital physiological roles in anti-inflammation, anti-oxidation and regulation of apoptosis. Many reseachers recently confirmed that HO-1had protective effects on liver cells, such as liver transplantation, acute liver injury, ischemia/reperfusion injury and so forth. In chronic liver disease, induction of HO-1is important to prevent the development of liver fibrosis effectively. Peroxisome Proliferator-Activated Receptor (PPAR) is a ligand activated transcription factors. PPARy is one of the subtypes which is mainly expressed in HSC. Up-regulation of PPARy resulted in a significant reduction of HSC activation, and reversed the development of liver fibrosis. Nuclear Factor-kappa B (NF-κB) is an important nuclear transcription factor. Up-regulating the activation of NF-κB would promote the proliferation of HSC, the releasing of cytokines and decrease HSC apoptosis which play important role in liver fibrosis. Studies have found that PPARy can inhibit the transcription and DNA synthesis of NF-κB by binding p50/p65subunits to form transcriptional repressor complexes directly or by binding p300and CBP co-activating factors to inhibit the transcription and expression of NF-κB competitively. Studies in other areas have shown that co-regulation existed between HO-1and PPARy, and there was an NF-κB binding site in the HO-1promoter region. Up-regulation of HO-1could decrease NF-κB activity drastically and inhibiting the serum levels of TNF-α and IL-6significantly.Reseach about the defense mechanism of HO-1againsting many kinds of liver injuries concentrate on the role of anti-inflammation, anti-oxidation and apoptosis regulation induced by its degrading production such as biliverdin, iron atoms and CO. But no reports have been found about the regulation of liver signals and molecules by HO-1. So, in this study we design animal and cells research to explored the effects of HO-1on rat liver fibrosis and HSC proliferation or apoptosis, and the expression of PPARγ,NF-κB and the downstream signals or inflammation cytokines which are aim to explore the signals and molecules mechanism of HO-1on the proliferation and apoptosis of activated HSC and the prevention of liver fibrosis.Reseach about the defense mechanism of HO-1againsting many kinds of liver injuries concentrate on the role of anti-inflammation, anti-oxidation and regulation of apoptosis induced by degrading production such as biliverdin, iron atoms and CO. But no reports have been found about the regulation of liver signals and molecules by HO-1. So, in this study we designed animal and cells research to explore the effects of HO-1on rat liver fibrosis and HSC proliferation or apoptosis, and the expression of PPARγ,NF-κB and the downstream signals or inflammation cytokines which were aim to explore the signals and molecules mechanism of HO-1on the proliferation and apoptosis of activated HSC and the prevention of liver fibrosis.The project contain three parts as below:Part1:Effects of HO-1induction or inhibition on liver fibrosis and the potential mechanismsPart2:Effects of HO-1induction or inhibition on the proliferation and apoptosis of activated HSCPart3:The signaling and molecular mechanisms of HO-1on HSC proliferation and apoptosisPart1:Effects of HO-1on liver fibrosis and the potential mechanismObjective:To explore the impacts of HO-1induction or inhibition on liver fibrosis and the potential mechanism by using liver fibrosis modelsMethods:Liver fibrosis models in rats was constructed by using composite factors which were randomly divided into five groups:①Normal group,②4weeks model,③6weeks model,④Znpp-IX treated model,⑤Hemin treated model. Two opposite reagents were used to induce or inhibite the expression of HO-1such as hemin (20μmol/kg) and ZnPP-IX (30μimol/kg) which were peritoneal injected to rats every other day from week4to week6. Histopatho logical changes were evaluated by hematoxylin and eosin (HE) and Masson staining. The expressions of HO-1and a-SMA in hepatic tissues were measured by immunohistochemical staining. Liver function including ALT, AST, ALB and TBIL were detected by using an automated biochemistry clinical analyzer. Levels of serum hyaluronic acid (HA) and Type Ⅲ procollagen (PⅢP) were determined by Enzyme-linked immunoadsorbent assay (ELISA). The content of hydroxyproline (Hyp) in the liver specimens were determined by using spectrophotometric method. The mRNA expression of a-SMA,HO-1,PPARy and NF-κB were determined by Real-time PCR. The protein expression of HO-1,PPARy and NF-κB were determined by Western blot.Results:(1) HE and Masson's staining showed rat modes of hepatic fibrosis were established successfully by using composite factors mainly with CC14. The degree of collagen proliferation, hepatic lobule destruction and the pseudolobuli formation in livers of Hemin treated model was significantly lower than the6week model control and Znpp-IX treated model (P<0.05).(2) The expression of HO-1mRNA and protein increased gradually during the process of liver fibrosis by using immunohistochemistry, Western blot and Real-time PCR (P<0.01). Hemin promoted the expression of HO-1compared with model control(P<0.01), but Znpp-IX diminished it (P<0.01).(3) The expression of a-SMA with immunohistochemistry showed that in hepatic tissues of normal rats, there was weakly expression of a-SMA in the smooth muscle cells of the vessel wall. With the development of hepatic fibrosis the a-SMA positive cells in hepatic tissues of rats increased significantly. The expression of a-SMA could be decreased by Hemin (P<0.01), but promoted by Znpp-IX (P<0.01). The relative expression of HO-1mRNA was consistent with the results above.(4) The degree of hepatic injury was increased gradually during the process of liver fibrosis as evidenced by significantly higher serum concentrations of ALT, AST, TBIL and lower concentration of ALB. Hemin treatment could change the injury of liver function by ameliorating the increased of ALT, AST and TBIL, and the decrease of ALB compared with model control (P<0.05). But the results in Znpp-IX treated group was opposite (P<0.05).(5) Results about the changes of collagen metabolism in livers of each group showed that the levels of HA,PⅢP in serum and Hyp in liver tissues increased in the process on liver fibrosis (P<0.05). Hemin treatment decreased the levels of HA,PⅢP and Hyp compared with6week model control and Znpp-IX treated group (P <0.05).(6) The expression of PPARy mRNA and protein decreased gradually during the process of liver fibrosis by using Real-time PCR and Western blot (P<0.01). Especially in Znpp-Ⅸ treated group PPARy decreased more evidently compared with6week model group, although no statistically significant (P=0.195).But the expression of PPARy was promoted significantly in Hemin treated group (P<0.05).(7) The expression of NF-κB mRNA and protein increased gradually during the process of liver fibrosis by using Real-time PCR and Western blot (P<0.01). NF-κB increased more significantly compared with6week model group especially in Znpp-IX treated group (P<0.05),but decreased apparently in Hemin treated group (P<0.05)Conclusions:The induction of HO-1in liver of fibrosis rats model could decrease the a-SMA expression, collagen synthesis, liver injury and the degree of fibrosis, thus prevent liver fibrosis progression. Then, our data demonstrated a potential mechanism that the liver protective effect of HO-1could be achieved by enhancing the expression of PPARγ and decreasing the expression of NF-κB in liver tissues.Part2:Effects of HO-1on the proliferation and apoptosis of activated HSCObjective:To explore the effects of HO-1induction or inhibition on activated HSC proliferation and apoptosisMethods:Activated HSC-T6were used as object of study which were incubated with Hemin (10μmol/l,20μmol/l,40μmol/l) or Znpp-Ⅸ (5μmol/l,10μmol/l,20μmol/l) for12h,24h and48h. MTT assay was used to determine cell proliferation, trypan blue staining was used to observe the survival rate of HSC-T6and lactate dehydrogenase (LDH) release assay was used to observe the toxicity of Hemin and Znpp-Ⅸ to HSC-T6. Through above the best concentration and action time about Hemin and Znpp-Ⅸ were chosen for the following experiments. Cells were grouped as follows:①Normal control group,②Hemin treated group,③Znpp-Ⅸ treated group,④Hemin and Znpp-Ⅸ co-treated group. MTT assay was used to determine cell proliferation. ELISA was used to detect the levels of HA and PⅢP in cell cultured supernatant. The expressions of HO-1and α-SMA in HSC-T6were measured by immunocytochemical staining, Real-time PCR and Western blot. TUNEL assay and AnnexinV-FITC/propidium iodide (PI) co-labed flow cytometry (FCM) were used to detect cell apoptosis. Changes of Bcl-2and Caspase-3mRNA in HSC-T6were monitored by Real-time PCR.Results:(1) The best concentration of Hemin inducting HO-1expression was20μmol/l (13μg/ml), and ZnPP-IX inhibiting HO-1expression was10μmol/l (3μg/ml) which had been proved to be effective and no toxicity to cultured HSC-T6. The best action time was24h.(2) The expression of HO-1in Hemin treated group was more extensively and strong than normal control and other treated group by using immunocytochemical staining. Results from Western blot and Real-time PCR also showed that HO-1expression was significantly higher in Hemin treated group compared with normal control (P<0.01), but lower in Znpp-IX treated group although no significant difference with normal control. HO-1expression in Hemin and Znpp-Ⅸ co-treated group was lower than merely Hemin treated group (P<0.05).(3) The expression of α-SMA about extent and intensity was lower in Hemin treated group than normal control and other treated group by using immunocytochemical staining. The mRNA and protein expression about α-SMA were lower in Hemin treated group (P<0.01), but higher in Znpp-Ⅸ treated group than normal control (P<0.01) by using Real-time PCR and Western blot. In Hemin and Znpp-Ⅸ co-treated group, a-SMA expression was higher than merely Hemin treated group (P<0.05).(4)The proliferation of HSC-T6decreased16.15%in Hemin treated group (MTT value was0.867±0.023), but increased12.28%in Znpp-Ⅸ treated group (MTT value was 1.161±0.015) compared with normal control (P<0.01). The proliferation of HSC-T6in Hemin and Znpp-Ⅸ co-treated group was higher than merely Hemin treated group (P<0.05).(5) Results about the changes of collagen metabolism in cultured HSC-T6supernatant of different groups showed that the levels of HA,PⅢP decreased significantly in Hemin treated group (P <0.01), but increased in Znpp-Ⅸ treated group compared with normal control (P<0.01) Levels of HA and PⅢP in Hemin and Znpp-Ⅸ co-treated group were higher than merely Hemin treated group (P<0.01).(6) TUNEL assay showed that the apoptotic rates of HSC-T6in Hemin treated group (23.5%±2.02%) increased6.23folds compared with normal control (3.25%±0.63%)(P<0.01). There was no significant difference between Znpp-Ⅸ treated group (4.00%±0.82%) and normal control (P=0.574). But the apoptotic rate in Hemin and Znpp-Ⅸ co-treated group (16.25%±1.38%) was lower than merely Hemin treated group (P <0.01). Results about the apoptotic rates of HSC analyzed by AnnexinV-FITC/PI co-labed FCM were coincidence with TUNEL.(7) Real-time PCR analysis showed that the expression of Bcl-2mRNA was decreased significantly in Hemin treated group compared with normal control (P<0.05), but increased in Znpp-Ⅸ treated group (P<0.05). Bcl-2mRNA in Hemin and Znpp-Ⅸ co-treated group was higher than merely Hemin treated group (P<0.05). In contrast, the expressions of Caspase-3mRNA was increased in Hemin treated group (P<0.01) but decreased in Znpp-Ⅸ treated group compared with normal control. Caspase-3mRNA in Hemin and Znpp-Ⅸ co-treated group was lower than merely Hemin treated group (P<0.05)Conclusions:The induction of HO-1could inhibit HSC-T6proliferation and collagen metabolism, and induce HSC-T6apoptosis by regulating the expression of apoptosis correlated protein such as Bcl-2and Caspase-3.Part3:The signaling and molecular mechanisms of HO-1on HSC proliferation and apoptosisObjective:To investigate the signaling and molecular mechanisms of HO-1on the proliferation and apoptosis of activated HSC-T6.Methods:Cells were grouped as follows:①Normal control group,②Hemin treated group,③Hemin+GW9662treated group,④Znpp-Ⅸ treated group,⑤Znpp-Ⅸ+Rosiglitazone treated group. The expression of HO-1in HSC-T6was detected by Real-time PCR and Western blot. The expression of PPARγ and NF-κB in HSC-T6were measured by immunocytochemical staining or immunofluorescence, Real-time PCR and Western blot. The contents of TGF-β1and IL-6in the supernatants of HSC-T6were detected by ELISA.Results:(1) Western blot and Real-time PCR demonstrated that the expression of HO-1was higher in Hemin treated group than this in normal control (P<0.01). But in Hemin+GW9662group the expression of HO-1mRNA decreased19.96%and HO-1protein decreased17.28%compared with Hemin treated group (P<0.05). The expression of HO-1was lower in Znpp-IX treated group than this in normal control (P<0.01). But in Znpp-IX+Rosiglitazone group the expression of HO-1mRNA increased18.32%and HO-1protein increased13.7%compared with Znpp-IX treated group (P<0.05).(2) The protein expression of PPARy detected by Western blot was higher in Hemin treated group than normal control (P<0.01), but the protein expression of NF-κB p65was lower than normal control (P <0.01). In contrast, in Znpp-IX treated group the expression of PPARy was lower but NF-κB p65was higher than normal group (P<0.01). In Hemin+GW9662treated group, PPARy decreased17.78%but NF-κB p65increased28.94%compared with merely Hemin treated group (P<0.05). In Znpp-IX+Rosiglitazone treated group, PPARγ increased18.6%but NF-κB p65decreased23.16%compared with merely Hemin treated group (P<0.01). The mRNA expression of PPARγ and NF-κB detected by Real-time PCR were coincidenced with the protein expression above.(3) Results about the expression of PPARγ from immunocytochemical staining showed that they all located in nucleus of HSC-T6. PPARγ expressed weakly in normal control, but strengthen in Hemin treated group. When GW9662pretreated with Hemin, the expression of PPARy was lower than Hemin merely treated group. PPARy expressed more weaker in Znpp-IX treated group than normal group. But in Rosiglitazone pretreated group, the expression of PPARy was higher than Znpp-IX merely treated group. Results about the expression of NF-κB p65from immunofluorescence were opposite to PPARy.(4) The changes of TGF-β1and IL-6in HSC-T6supernatant were same. Levels of TGF-β1and IL-6were lower in Hemin treated group (P<0.01), but significantly higher in Znpp-IX treated group (P<0.01) than normal control. In Hemin+GW9662treated group, levels of TGF-β1and IL-6all increased compared with Hemin treated group (P<0.05). In Znpp-Ⅸ+Rosiglitazone treated group, levels of TGF-β1and IL-6all decreased compared with Znpp-IX treated group.Conclusions:The induction of HO-1could up-regulate the expression of PPARy and negatively regulate the expression of NF-κB at same time in activated HSC-T6, and then impact the release of inflammatory cytokines such as TGF-β1and IL-6, and thus inhibit the proliferation and apoptosis of activated HSC-T6.
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