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Journal of Chinese Integrative Medicine ›› 2008, Vol. 6 ›› Issue (10): 1017-1023.doi: 10.3736/jcim20081006

• Original Experimental Research • Previous Articles     Next Articles

Regulation of aromatase P450 expression by puerarin in endometrial cell line RL95-2

Yan-wei Li1, Zai-long Cai2, Wei Shen1, Sheng-sheng Yang3, Chao-qin Yu1()   

  1. 1. Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
    2. Clinical Research Center, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
    3. Department of Biochemistry and Molecular Biology, Second Military Medical University, Shanghai 200433, China
  • Received:2008-07-24 Online:2008-10-20 Published:2008-10-15

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Objective: 

To study the effects of puerarin on the aromatase P450 (P450arom) mRNA expression and the effects of low-dose puerarin on transcription factors of the P450arom gene (PⅡ) 5'-flanking region.


Methods: 

The effects of puerarin on the P450arom mRNA expression were determined by real-time polymerase chain reaction (RT-PCR). The 5'-flanking region was amplified by PCR using human genomic cDNA as a template. By means of the restriction sites and sequence confirmation, the PCR product was cloned into reporter vector. Series of sequential deletion reporter constructs were transiently transfected into RL95-2 cells which were treated with or without puerarin. Luciferase activity was measured by Dual-Luciferase Reporter Assay System and Luminoskan Ascent luminometer. Furthermore, by using web-based search program, the most possible cis-acting elements and transcription factors were evaluated.


Results: 

The data demonstrated that low-dose puerarin treatment could decrease P450arom expression at mRNA level compared to dimethyl sulphoxide (DMSO) treatment (P<0.01), and puerarin (10 –7 mol/L) had a time-course effect on P450arom mRNA expression, which reached the bottom at 12 h (P<0.01). Cells transfected with the –763/+8 bp constructs showed decrease in relative luciferase activity after puerarin (10 –7 mol/L) treatment compared to DMSO treatment (P<0.05), indicating an essential regulatory site between –763 bp and –543 bp responsible for the transcription suppression by puerarin. Furthermore, the most possible transcription factors, which turned out to be AP-1(c-jun/c-fos) at –410/–401 bp were also evaluated. The activity of exogenous AP-1 was reduced after 12 hours of puerarin treatment (P<0.05). The inhibition of c-jun mRNA also showed a time-course effect, which bottomed out at 12 h in parallel with that of P450arom (P<0.01). The protein level of c-jun was also down-regulated by puerarin (10 -7mol/L) treatment at 12 h.


Conclusion: 

The suppression of P450arom expression and activity may be associated with the down-regulation of transcription factor AP-1/c-jun. This partially explains the mechanisms whereby puerarin treats endometriosis.

Key words: puerarin, aromatase P450, endometriosis

CLC Number: 

  • R711.71

Figure 1

Effects of puerarin on P450arom expression and activity A: Structure of puerarin; B: mRNA expression of P450arom in RL95-2 cells treated with different concentration of puerarin, by RT-PCR, normalized by β-actin mRNA; C: Time-course experiment on P450arom expression at 10–7 mol/L puerarin. Values were expressed as mean±SE of three separate experiments. *P<0.05, **P<0.01, vs DMSO line."

Figure 2

P450arom PⅡ responsible for the transcription suppression by puerarin A: RL95-2 cells were transfected with –1 017/+8 bp, –763/+8 bp, –543/+8 bp and –234/+8 bp constructs and were then treated with puerarin or DMSO for 12 hours. Luciferase activity was normalized by cotransfected pRL-CMV and these results represented the mean±SE of three different experiments. *P<0.05 with respect to the DMSO-treated samples; B: Sequence of the cloned 5'-flanking region of P450arom PⅡ from –417 bp to –336 bp. Putative transcription factor binding sites were identified using the TESS job and cis-acting elements were indicated in boxes. Two potential AP-1/c-jun binding sites with the highest scores were observed."

Figure 3

Suppression of 4×AP-1-luciferase activity and c-jun mRNA expression by puerarin A: To find out whether AP-1 varies along with P450arom by puerarin, the activity and mRNA levels of AP-1 were assayed. RL95-2 cells were transfected with 4×AP-1-luciferase plasmid and treated with DMSO (control) or puerarin (10–7 mol/L) for 12 hours; B: Cells were collected at indicated time point after treated with puerarin (10–7 mol/L) or DMSO and then used for RNA extraction and quantitative RT-PCR; C: Western blot analysis showed a significant decrease of c-jun protein expression by puerarin (10–7 mol/L) treatment for 12 hours, compared with the amount of β-actin. The target gene mRNA levels were normalized by the amount of β-actin mRNA levels. These results represented the mean±SE of three separate experiments. *P<0.05, **P<0.01, vs DMSO line."

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