Compound Astragalus And Salvia Miltiorrhiza Extract Exerts Anti-hepatic Fibrosis And Anti-hepatocellular Carcinoma Effects By Regulating TGF-β/Smad Signal And Expression Of MiR-145 And MiR-21

Background Hepatocellular carcinoma(HCC) with high mortality has been a major public health problem threatening human’s lives. A natural history of chronic inflammation and severe fibrosis has been found in most HCC cases. To develop new drugs to inhibit hepatic fibro-carcinogenesis is important to HCC prevention and treatment, which is dependent with elucidating the underlying mechanism of HCC and searching for new specific targets for drugs. Transforming growth factor-beta(TGF-b)/Smad signaling has been intensively studied and is closely related with hepatic fibro-carcinogenesis, which attracts special attention in pharmacological research. Activated TGF- b type I receptor(TbRI) upon TGF-b binding can directly phosphorylate Smad2 and Smad3 in their C-termini. TGF-b can alternatively active mitogen-activated protein kinase(MAPK) pathways including extracellular signal-regulated kinase(ERK), c-Jun N-terminal kinase(JNK), p38 MAPK, then cause the phosphorylation of Smad2/3 at specific sites in their linker regions. TbRI and MAPK differentially phosphorylate Smad2 and Smad3 to create multiple phosphorylated(phospho-) isoforms, including C-tail phosphorylated Smad2/3(p Smad2 C and p Smad3C), linker phosphorylated Smad2/3(p Smad2 L and p Smad3L), and dually phosphorylated Smad2/3(p Smad2L/C and p Smad3L/C). Chronic inflammation can shift hepatocytic Smad signaling from tumor suppressive p Smad3 C signaling to carcinogenic p Smad3 L and fibrogenic p Smad2 L /C signaling, increasing the risk of hepatic fibro-carcinogenesis. For modulating transcription of target genessuch as plasminogen activator inhibitor(PAI-1), Smad phosphor-isoforms need to form a complex with Smad4, then translocate into nucleus with the help of Imp7 and Imp 8. Smad7 can antagonize TGF-β signaling through multiple mechanisms both in the cytoplasm and nucleus. Tumor suppressive mi R-145 and carcinogenic mi R-21 are closely associated with HCC development, which interact with TGF-b/Smad signal in multiaspects. Compound Astragalus and Salvia miltiorrhiza Extract(CASE) consists of astragalosides, astragalus polysaccharide and salvianolic acids extracted from Astragalus membranaceus Bunge(Leguminosae) and Salvia miltiorhiza Bunge(Lamiaceae). Our previous data showed that CASE can exert anti-fibrosis and anti-HCC effects by modulating TGF-b/Smad signal in vitro. Then we have proved that CASE inhibits DEN-induced hepatic fibro-carcinogenesis and shift carcinogenic p Smad3 L and fibrogenic p Smad2 L /C signaling to tumor suppressive p Smad3 C signaling. But the effect of CASE on Smad4, Smad7, PAI-1, Imp7 and Imp8 in vivo remain unclear. And whether mi R-145 and mi R-21 are targets of CASE or not keeps in the dark. Therefore, we propose to make it clear and illustrate the mechanism of CASE’s anti-fibro-carcinogenesis effect by in vivo or in vitro experiments.Objectives 1. To observe the effects of CASE on the expression of Smad4, Smad7, PAI-1, Imp7 and Imp8 during the process of DEN-induced hepatic fibrosis-carcinoma in rats. 2. To investigate the effects of CASE on the expression of mi R-145 and mi R-21 during the process of DEN-induced hepatic fibrosis-carcinoma in rats and in Hep G2 cells.Methods 1. Animals model of hepatic fibrosis-carcinoma and CASE treatment 180 male Sprague-Dawley rats were randomly divided into six groups, including thenormal control group, DEN alone group, three CASE(60, 120 and 240mg/kg) treatment groups and positive control drug(liver aid tablets) treatment group. The rats in the normal control group were administered with 0.5% CMC-Na for 16 weeks, while the rats in the other five groups were treated with 0.2% DEN in 0.5% CMC-Na by gavage for 14 weeks to induce hepatocarcinogenesis. The rats in the three CASE groups were concomitantly administered CASE at the doses of 60, 120, or 240 mg/kg per day, respectively, for 16 weeks. And the rats in the positive control drug treatment group were administered liver aid tablets at the dose of 921mg/kg. The rats were sacrificed at 12 th week or 16 th week after the start of DEN administration. 2. Cell culture and treatment Hep G2 cells were starved for 24 h in serum-free medium, in the absence or presence of TGF-b1 for different periods of 0, 3, 6, 12, and 24 hours. The total RNA was isolated for subsequent q RT-PCR experiment to detect mi R-145 and mi R-21. Hep G2 cells were incubated in serum-free medium for 24 h without or with CASE(20, 40, 80μg/ml). TGF-b1 was added in the medium 3h before RNA extraction except the blank control. The total RNA was isolated for subsequent q RT-PCR experiment to detect mi R-145. After starving overnight, Hep G2 cells were incubated in serum-free medium for 24 h without or with CASE(20, 40, 80μg/ml). Simultaneously,TGF-b1 was added in the medium except the blank control. The total RNA was isolated for subsequent q RT-PCR experiment to detect mi R-21. 3. The expression of Smad4 and PAI-1 proteins was measured by immunohistochemical analysis Tissue microarray was prepared. The sections were incubated with primary antibodies(Abs). Primary Abs used in this study included mouse polyclonal to Smad4 Abs and rabbit polyclonal to PAI-1 Abs. The sections were then incubated with peroxidase-labeled polymer conjugated to goat antimouse/rabbit immunoglobulin.Finally, the sections were developed with 3, 3′-diaminobenzidine counterstained with hematoxylin and mounted under cover slips. Mean density were evaluated by Image J software, assigning score of 0–1 based on the percentage of positive-stained cells. 4. The expression levels of Smad4, Smad7, PAI-1, Imp7 and Imp8 in liver tissues were measured by western blot method The proteins were extracted from frozen liver tissues. The expression of Smad4, Smad7, PAI-1, Imp7 and Imp8 were monitored by western blot using corresponding antibody. The GAPDH was measured as the reference. 5. The expression of mi R-145 and mi R-21 in liver tissues and Hep G2 cells was detected by real-time PCR The total RNA was isolated from frozen liver tissues and Hep G2 cells. c DNA was prepared by reverse transcriptase. Expression of mi R-21 and mi R-145 was determined by SYBR Green Ⅰ real-time PCR with small nuclear RNA U6 as an endogenous control for normalization. The melt curves of PCR products after amplification were analyzed to confirm amplification of specific transcripts.Results 1. Effects of CASE on the expression of Smad4 during DEN-induced hepatic fibrosis-carcinoma On 12 th week, immunohistochemical analysis showed that the Smad4 was highly expressed in DEN group. CASE treatment markedly decreased the elevated Smad4 expression induced by DEN at different doses. Western blot measurement showed that only CASE(120 and 240 mg/kg) could decrease the elevated Smad4 expression induced by DEN. On 16 th week, immunohistochemical analysis showed that the Smad4 was highly expressed in adjacent tissues and was negative in hepatoma nodules in DEN group. Comparing with the expression in adjacent tissues of DEN group, CASE treatmentmarkedly decreased the Smad4 expression in dose-dependent manner. Western blot measurement showed the same effect of CASE. 2. Effects of CASE on the expression of PAI-1 during DEN-induced hepatic fibrosis-carcinoma On 12 th and 16 th week, CASE treatment markedly decreased the elevated PAI-1 expression in rats induced by DEN at three different doses, which showed by immunohistochemical analysis and western blot measurement. 3. Effects of CASE on the expression of Smad7 during DEN-induced hepatic fibrosis-carcinoma The data of western blot measurement showed that CASE treatment markedly increased the depressed Smad7 expression both in 12 th week and 16 th week rats induced by DEN. 4. Effects of CASE on the expression of Imp7 and Imp8 during DEN-induced hepatic fibrosis-carcinoma On 12 th week, western blot measurement showed that CASE treatment markedly decreased the elevated Imp7 and Imp8 expression in rats induced by DEN at three different doses. On 16 th week, western blot measurement showed that CASE treatment markedly decreased the elevated Imp7 and Imp8 expression in rats induced by DEN at high dose. CASE treatment had no effect on the Imp7 and Imp8 expression at low or medium doses. 5. Effects of CASE on the expression of mi R-145 during DEN-induced hepatic fibrosis-carcinoma On 12 th week, DEN markedly induced mi R-145 expression. CASE treatment upregulated mi R-145 expression further at different doses. On 16 th week, the expression of mi R-145 in DEN model group was lower than that in normal control group. CASE upregulated the expression of mi R-145 at medium and high doses, but did not restore to normal level. CASE at low dose had no effect on theexpression of mi R-145. 6. Effects of CASE on the expression of mi R-21 during DEN-induced hepatic fibrosis-carcinoma On 12 th week, CASE treatment upregulated mi R-21 expression at different doses. The expression levels in CASE treatment groups were higher than that in DEN model group. On 16 th week, the expression of mi R-21 in DEN model group was lower than that in normal control group. CASE further downregulated the expression of mi R-21 at different doses. 7. Effects of CASE on the expression of mi R-145 in Hep G2 cells In Hep G2 cells, the expression of mi R-145 was markedly downregulated after TGF-b1 treatment for 3h, 6h, 12 h compared with control. CASE treatment for 24 h restored the expression of mi R-145 at different doses, especially at high dose. 8. Effects of CASE on the expression of mi R-21 in Hep G2 cells In Hep G2 cells, the expression of mi R-21 was markedly upregulated after TGF-b1 treatment in time-dependent manner. CASE treatment for 24 h significantly decreased the expression of mi R-21 induced by TGF-b1 at different doses.Conclusions 1. Inhibiting the expression of PAI-1 may be an important mechanism in CASE’s anti-fibrosis and anti-HCC effects. The expression of PAI-1 may be an applicable marker for evaluating the treatment effect. 2. Inhibiting the expression of Smad4 may be associated with CASE’s anti-fibrosis and anti-HCC effects. 3. CASE treatment may exert anti-fibrosis and anti-HCC effects by upregulating the expression of Smad7 via negatively regulation on TGF-b/Smad signaling. 4. CASE may decrease the translocation into nucleus of fibrogenic signal andcarcinogenic signal by inhibiting the expression of Imp7 and Imp8. 5. CASE treatment can upregulate the expression of mi R-145 and downregulate the expression of mi R-21, which may be helpful to its anti-HCC effect. But the effect on mi R-145 and mi R-21 may not associate with CASE’s anti-fibrosis effect.