Compound Astragalus And Salvia Miltiorrhiza Extract Retards Hepatocellular Carcinoma Progression By Inhibiting Fibrosis And PAI-1Transcription Activity

Background and AimsOur previous studies have showed Compound Astragalus and Salvia miltiorrhizaExtract(CASE) significantly inhibited cell proliferation and invasion in hepatoma HepG2cell lines in vitro However, the exact pharmacological effects of CASE in hepatocytecarcinoma are still far from clear.So we utilized a rat model of DEN-inducedhepatocarcinogenesis, to investigate the effects of CASE on the development of hepaticfibrosis-carcinoma in vivo experiments.Methods1. DEN-Induced Hepatic Fibrosis-Carcinoma in RatFifty male Sprague-Dawley rats were randomly divided into the DEN group and thenormal control group. The rats of DEN group were administrated0.2%DEN solution bygavage five times a week for14weeks, and the control group received vehicle. Theanimals were sacrificed at the end of4,8,12,16week, respectively. Liver tissues werestained with hematoxylin-eosin (HE) and Van Gibson’s collagen fiber stain (V.G) forhistopathologic examination. The protein expression of pSmad3L and PAI-1was measuredby immunohistochemical method and Western blot method.2. Compound Astragalus and Salvia Miltiorrhiza Extract Retards HepatocellularCarcinoma Progression by Inhibiting Fibrosis and PAI-1transcription Activity2.1Animals model of HCC and CASE treatmentThe rats were randomly divided into five groups: control group, DEN alone group, CASE groups (60,120, and240mg/kg group). The rats in control group were administered with0.5%carboxymethylcellulose (CMC-Na) for16weeks, while the rats in other groups weretreated with0.2%DEN in CMC-Na by gavage5times a week for14weeks to inducehepatocarcinogenesis. During the experimental period, the rats in CASE groups wereconcomitantly administered CASE in0.5%CMC-Na by oral gavage at the doses of60,120, or240mg/kg per day for16weeks, while the rats in the DEN group wereconcomitantly administered equal amounts of0.5%CMC-Na for16weeks2.2Serum biochemistry and hepatic Hyp contentSerum enzymes levels were measured with an automatic biochemical analyzer. Hyaluronicacid (HA) in serum was measured by gamma radioimmunoassay counter. Hepatic Hypcontent was determined using commercial kits.2.3The protein expression of pSmad3L and PAI-1was measured byImmunohistochemical analysesParaffin sections were deparaffinized in xylene and rehydrated. Nonenzymatic antigenretrieval was done by heating the sections to121℃in sodium citrate buffer for10min.Primary Abs used in this study included mouse monoclonal anti-α-SMA Ab (α,α-SMA,Abcam) and anti-GST P1(Sigma, USA). Finally, the sections were developed with3,3’-diaminobenzidine counterstained with hematoxylin, and mounted under cover slips.2.4Cell culture and treatmentHepG2cells were grown as sub-confluent monolayer cultures in Dulbecco’s modifiedEagle’s medium (DMEM) supplemented with10%fetal bovine serum (FBS). These cellswere kept in95%air and5%CO2at37°C. The cells were incubated in the absence orpresence of CASE (20,40or80μg/ml) for14h.2.5Real-time quantitative RT-PCR was used to detect the PAI-1mRNA level inHepG2cellsTotal RNA was extracted from HepG2cells after incubation and was reverse transcribed tocDNA.Using each primer for human PAI-1gene and human β-actin gene.Calculate the relative quantization of PAI-1Mrna which normalized to the housekeeping geneβ-actin.Melting curves were generated to confirm amplification of specific transcripts.Results1. DEN administration successfully induced hepatocarcinogenesis in ratsIn the DEN group, at12weeks, hepatic lobules were encysted and separated byhyperplastic collagen bundles. The normal structure of lobules was destroyed, and pseudolobules formed. At the end of16weeks, the remaining surviving rats all developedhepatocarcinoma. The hepatoma cells were arranged in cords or mass with obvious atypia.The structure of liver tissue was normal in control group.There were almost no positiveexpression of pSmad3L and PAI-1in normal liver tissue. In the DEN group, the amountand intensity of positive expression gradually increased from4to12weeks and reachedmaximum to16weeks2. Compound Astragalus and Salvia Miltiorrhiza Extract Retards HepatocellularCarcinoma Progression by Inhibiting Fibrosis and PAI-1transcription Activity2.1Inhibition of DEN-induced hepatoma incidence and multiplicity by CASEThe incidence (100%) and multiplicity (3.622.13) of development of HCC in DENtreated rats at16th week were significantly decreased by the treatment of CASE in adose-dependent manner.2.2Effect of CASE on serum biochemistry and hepatic Hyp contentAt12th week the elevated serum ALT, AST, ALP, GGT, HA and Hyp content levels weresignificantly decreased by the treatment of CASE in a dose-dependent manner. At16thweek, three dosages of CASE significantly decreased these elevated levels of ALP, HA,DBIL, IBIL and increased the content of TRP and ALB.2.3Effect of CASE on α-SMA protein expressionAt12th week, the elevated amounts of-SMA positive cells were markedly decreased byCASE treatment in a dose-dependent manner. At16th week, the elevated amounts of α-SMA positive cells were markedly decreased by CASE treatment at the dose of120and240mg/kg2.4Effect of CASE on GST protein (GST-P) expressionAt16th week, widespread malignant cells were observed in DEN treated rat livers.Numbers and areas of GST-P-positive foci in DEN treated alone group at16th week weresignificantly decreased compared with that at12th week, although they are higher thanthose in the normal control group. The higher expression of GST-P was significantlyreduced in the CASE-treated group compared with the model group (DEN group) both at12th week and16th week.2.5Effect of CASE on PAI-1transcriptional activity in HepG2cells induced byTGF-β1The transcriptional activities were markedly decreased in a dose-dependent manner inHepG2cells by CASE treatment.Conclusions1. DEN administration successfully induced fibrosis at12weeks andhepatocarcinogenesis at16weeks in rats.During the process of DEN induced hepaticfibrosis-carcinoma in rat, the expression of pSmad3L and PAI-1gradually increased.2. CASE treatments were found to have a marked effect in retarding DEN-inducedhepatocarcinogenesis by inhibiting hepatoma incidence and multiplicity3. CASE might exhibit their protective effects on DEN-induced HCC by retardinghepatocarcinogenesis4. CASE might be effective on suppression of DEN induced liver fibrosis in rats andretard tumor formation by ameliorating the extent of fibrosis.5. CASE could delay the process of DEN induced hepatocellular carcinoma in rats.6. CASE markedly decreased the transcriptional activity of PAI-1in HepG2cells inducedby TGF-β1in a dose-dependent manner, providing further evidence on CASE’s mediation and interference with the TGF-β1/Smad signaling pathway.