Our current study reveals that c-secretase inhibitor DAPT treatment reduced the numbers of myofibroblast-like cells and simultaneously inhibited expression of snail, vimentin, and TGF-b1 in parallel withFigure 5. DAPT ameliorated liver fibrosis induced by CCl4 in rats. A. Hematoxylin and eosin, Masson’s trichrome, and Sirius red staining were used to examine pathological alterations and collagen deposition. Data shown are the representative of 8 animals. B. Semiquantitative analysis of the Masson’s trichrome staining result. C. Assay of hydroxyproline content. *P,0.05 versus rats in fibrosis group; #P.0.05 versus rats in fibrosis group. enhanced expression of E-cadherin in fibrotic liver. These finding suggests that the reversion of EMT contributes to the resolution of hepatic fibrosis. It has also been suggested that the activation of HSCs might be considered an EMT phenomenon . The elements of the Notch signaling pathway, including Jagged1, Notch1, Notch2, and Hes1, have been identified as TGF-b1-responsive genes in kidney epithelia . Our previously study showed that activated Notch signaling was found in HSC-T6 cells, and that transient knockdown of Notch3 antagonized TGF-b1-induced expression of a-SMA and collagen I in HSC-T6 . In this study, the results show that the up-regulation of Notch signaling is implicated in the activation of HSCs in vivo. Moreover, treatment with DAPT effectively reduces the expression of a-SMA, snail, and vimentin in HSC-T6 cells. These data suggest that DAPT attenuated hepatic fibrosis at least partially through the inhibition of the EMT process of HSCs activation. The knockdown of Notch3 using siRNA was found to have the same impact on EMT in HSC-T6 cells as that of DAPT. This confirms that the Notch signaling pathway is a key regulator of EMT in HSC-T6 cells. The conventional strategy for the treatment of hepatic fibrosis involves reducing the activation and proliferation of HSCs andinducing apoptosis. However, it has been reported that activated HSCs promote liver development and regeneration . This suggests that it may not be appropriate to simply target HSCs to treat liver fibrosis. In the process of liver fibrosis, stimulation of hepatocyte regeneration and inhibition of apoptosis is essential to treating hepatic fibrosis . In the present study, DAPT treatment was found not to inhibit hepatocyte proliferation. In contrast, DAPT was found likely to inhibit hepatocyte apoptosis to some degree in vivo. We also found that the expression of TGF-b1 was upregulated in the fibrotic livers, and some hepatocytes close to the fibrotic area expressed high levels of TGF-b1, which can induce apoptosis in hepatocytes and stimulate ECM deposition in hepatic fibrosis . We infer that one potential mechanism underlying the protecting effect of DAPT may involve the suppression of TGFb1 expression, which contributes to hepatocyte proliferation and protects hepatocytes from apoptosis. Inhibiting c-secretase can prevent the cleavage of the Notch receptor, blocking Notch signal transduction [30,31]. The clinical trials with c-secretase inhibitors have revealed several adverse events, such as gastrointestinal toxicity [45,46]. Other approaches that target the Notch signaling were recently evaluated andFigure 6. DAPT treatment was found to inhibit EMT in fibrotic livers in rats. Immunohistochemical staining was performed to detect the expression of vimentin, snail, E-cadherin, and TGF-b1 in livers from normal, fibrosis, and DAPT-treated rats.
showed promising effects accompanied by a lack of intestinal toxicity in preclinical models [47,48]. These studies shed light on the clinical implications of c-secretase inhibitor. In summary, the present investigation indicates that the Notch signaling pathways become activated in a rat model of liver fibrosis induced by CCl4 and that inhibition of Notch signaling exerts potent anti-fibrotic effects in preclinical models. Our study provides the first evidence for the striking suppressive effects of DAPT on hepatic fibrosis. These findings suggest that inhibition of Notch signaling might be a novel option for hepatic fibrosis therapy.bers: 2011-237. This study was approved by ethics committee of Huazhong University of Science and Technology.
Reagents and Antibodies
N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S- phenylglycine t-butyl ester (DAPT), a highly active c-secretase inhibitor, and carbon tetrachloride (CCl4) were purchased from Sigma-Aldrich Corporation (U.S.). LipofectamineTM 2000 transfection reagent was obtained from Invitrogen (Carlsbad, CA, U.S.). The antibodies used for Western blot analysis and immunohistological staining are listed in Table S1.
Materials and Methods
See Materials and Methods S1 for detailed experimental materials and methods.
Quantitative Real-time PCR Analysis
Total RNA from each sample was extracted using Trizol Reagent (Invitrogen, U.S.) and was reverse-transcribed using PrimeScript RT Master Mix Kit (Takara, Japan) according to the manufacturer’s instructions. Primers for these transcripts are listed in Table S2.
All the animal related procedures were performed according to the ethical guidelines of the Animal Care and Use Committee of Huazhong University of Science and Technology. Permit num-
Figure 7. Effects of DAPT treatment on cell proliferation and apoptosis. A. Immunohistochemical and immunofluorescence staining were used to determine the expression of PCNA and TUNEL in fibrosis induced by CCl4 in liver tissues from normal, fibrosis, and DAPT-treated rats. B, C. Regions with positive PCNA or TUNEL staining were quantified using ImageJ as described in Materials and Methods. The white bars represent 50 mm. # P.0.05 versus DAPT-treated rats; *P,0.01 versus normal rats; ##P.0.05 versus rats in fibrosis group; **P,0.05 versus rats in fibrosis group.Figure 8. DAPT treatment inhibited EMT in HSC-T6 cells. Expression of snail, vimentin, Hes1, and a-SMA in HSC-T6 cells cultured with DAPT (0.1, 0.2, 0.4, and 0.8 mmol/l) or DMSO (control) for 48 h were analyzed by Western blot analysis. The expression was normalized against b-actin. *P,0.05 versus control group. Western Blotting Analysis
Western blotting analysis was performed according to the manufacturer’s recommended method (Bio-Rad Laboratories, Hercules, CA, U.S.).
Treatment of Hepatic Fibrosis in Rats
Male Spragueawley rats (180?20 g) were purchased from the Experimental animal center of Tongji Medical College of Huazhong University of Science and Technology (Wuhan, China). Forty male SD rats were randomly allocated into five groups of 8 rats each: Group A served as normal group. These rats receivedolive oil (the vehicle for CCl4, 3 mL/kg) twice a week for 8 weeks. Rats in group B (model group) received subcutaneous injections of CCl4 (3 mL/kg) dissolved in olive oil (2:3 ratio) twice a week for 8 weeks to induce liver fibrosis as previously described [49,50]. Rats in group C received CCl4 twice a week for 12 weeks and received antifibrotic treatment with DAPT (10 mg/kg) at 8 weeks by intraperitoneal injection daily for another 4 weeks. Rats in group D were treated with CCl4 as in group C and received DAPT (50 mg/kg) at 8 weeks daily for another 4 weeks. Rats in group E served as fibrosis group were given the equivalent volumes of CCl4 and DMSO (the vehicle for DAPT). Rats in groups A and B were sacrificed at 4 weeks, 8 weeks, and 12 weeks after the firsttreatment, and the rest of the rats were killed 12 weeks after the initial treatment.