Experimental Study Of Nandrolone Phenypropionate On Regulation Of Androgen Receptor-mediated Target Gene Transcriptional Activity In Burned Rats And Cultured Rat's Hepatocyte

Objective: to investigate the mechanism of Nandrolone Phenylpropionate (NP) on regulation of androgen receptor- mediated target gene transcriptional activity in burned rats and cultured rat's hepatocyte, so as to better application of anabolic steroids in clinic practice. Method: The study included two parts.Part 1. Animal tissues experiment in vivo. 36 Wistar rats were divided into 3 groups randomly: (i)experimental group(NP group) that rats were burned with 20% total body surface areas (TBSA) of deep II ° scalding and treated by 5mg/kg NP injection q.o.d after injured 2 days; (ii)control group that rats were the same scalding models treated by saline as placebo at the same days; (iii)blank group that rats were normal without any disturb. At the burned 21th day, all rats were killed. 90mg± 10mg liver, testis and ovary tissues were cut. The total RNA of tissues was extracted by Trizol? method for reverse transcription. The expression levels of gene SRC-1, IGF-1 and c-myc of ratliver, testis and ovary were measured by real-time fluorescent quantitative PCR with TaqMan probe.Part 2. Hepatocyte experiment in vitro. 4 pairs of small interfering RNA (siRNA) of rat SRC-1 were designed basing on genebank data online and double-stranded RNA (dsRNA) were annealed in vitro for RNA interference (RNAi). By DNA recombine technic, siRNA expression vectors were formed with plasmid pSUPER EGFP1 and conversed into E.coli DH5a for amplification steadily. The positive siRNA expression vectors were collected purely after colony PCR screening, bi-enzyme-cut verification and DNA sequence check. The best siRNA design was picked out from the 4 pairs of siRNA by fluorescent quantitative PCR with TaqMan probe after transfecting cultured hepatocyte by Iiposomes2000-introduced method. The other same generation cultured hepatocyte were divided into 4 groups: (i)+NP+RNAi group that hepatocyte were transfected by the best siRNA expression vector and treated by 10|ig/ml NP at transfected 4 hours; (ii)-NP+RNAi group that hepatocyte were only transfected by the best siRNA expression vector without NP treatment; (iii)+NP-RNAi group that hepatocyte were only treated by NP without siRNA vector transfection; (iiii)-NP-RNAi group that hepatocyte were normally cultured without any disturb. After transfected 24 hours , all 4 groups of hepatocyte were collected and splitted for the templat of reverse transcription. The expression levels of gene SRC-1, IGF-1 and c-myc of hepatocyte were measured by fluorescent quantitative PCR with TaqMan probe.Result: Part 1. The experimental results showed that the expression levels of SRC-1 in sexual glands (testis and ovary) and IGF-1 in liver, testis andovary are different significantly between NP group and control group (PO.05). NP can increase the expression of SRC-1 in sexual glands and IGF-1 in the three kinds of tissues. The expression of SRC-1 in liver and c-myc in liver, testis and ovary are similar between NP group and control group (P>0.05). NP can not induce the expression of SRC-1 in liver and c-myc in the three kinds of tissues. The expression of SRC-1 in NP groups are related with the physiological abundant of different tissues SRC-1 in blank groups (r=0.953, t Test P<0.05).Part 2. The experimental data exhibit the expression level of SRC-1, IGF-1 and c-myc in hepatocyte are different significantly between +NP+RNAi group and +NP-RNAi, as well as between -NP+RNAi group and -NP-RNAi group (P<0.05), which means RNAi effects is very well and the expression of IGF-1 and c-myc are decreased obviously along with the SRC-1 silence. As while +NP+RNAi group compared with -NP+RNAi group, +NP-RNAi group vs -NP-RNAi group, the expression of SRC-1, IGF-1 and c-myc in hepatocyte are similar (P>0.05), which means NP can not abolish the RNAi inhibition effects on the SRC-1, IGF-1, c-myc expression and can not induce the expression of SRC-1, IGF-1 and c-myc under this dosage in vitro. Conclusion: 1. NP play the important role on the anabolic effects by regulation of androgen receptor-mediated target gene transcriptional activity. The expression and recruitment of SRC-1 is necessary and sufficient to the expression of IGF-1 and c-myc.2. NP is helpful to the expression of SRC-1 in sexual glands, but SRC-1 maybe is not the key tissue-specific coactivator effected by anabolic steroids NP. The more detail research on the androgen receptor transcriptionalregulation mechanism is needed in the future.3. Cross-talk to the IGF cellular information system pathway is one of the mechanism of NP effects. NP can increase the expression level of IGF-1 gene.4. NP can not induce the expression of c-myc gene coordinately and less result in hormone-dependent cancer within therapy dosage so that be used safely.