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Journal of Chinese Integrative Medicine ›› 2011, Vol. 9 ›› Issue (9): 1014-1021.doi: 10.3736/jcim20110913

• Original Experimental Research • Previous Articles     Next Articles

Scutellarin benzyl ester partially secured the ischemic injury by its anti-apoptosis mechanism in cardiomyocytes of neonatal rats

Hao Dai1, Jun Gu1,2, Ling-zhi Li1,2, Li-mei Yang1, Hui Liu3, Jian-yu Li1,2()   

  1. 1. Division of Pharmaceutical Chemistry,Medical College of Chinese People’s Armed Police Forces,Tianjin 300162, China;
    2. Tianjin Key Laboratory of Occupational and Environmental Hazards Biomarkers, Tianjin 300162, China
    3. Institute of Hepatobiliary Diseases, Third Central Hospital of Tianjin Medical University, Tianjin 300170, China
  • Received:2011-04-14 Accepted:2011-05-27 Online:2011-09-20 Published:2011-09-15
  • Contact: Li Jian-yu E-mail:lijianyu118@163.com

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Objective: To investigate the protective effects of scutellarin benzyl ester on neonatal rats’ cardiomyocytes injured by ischemia and its anti-apoptosis mechanism.

Methods: The cardiomyocytes in primary culture were prepared from ventricular tissue of 1- to 3-day-old Sprague-Dawley rats and the cells in good condition were assigned to five groups: control group, ischemic model group and three scutellarin benzyl ester groups (doses of 100, 50 and 25 μmol/L, respectively). The model of ischemic injury was established in the primary culture of cardiomyocytes under glucose-free anoxic condition. After ischemia for 6 h, the metabolic ability of the cells was detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay and the activity of lactate dehydrogenase (LDH) in the media was measured by biochemistry approaches. The nuclear damage was revealed by Hoechst-propidium iodide staining. The percentage of apoptotic cells was monitored by flow cytometry. The expression levels of cytochrome c and caspase-3 mRNAs and proteins were determined by reverse transcription-polymerase chain reaction and Western blotting, respectively.

Results: After exposure to ischemic condition, the cell viability of the model group was degraded compared with that of the control group (P<0.01) and scutellarin benzyl ester (high and medium doses) could attenuate the loss of cell viability induced by ischemia (P<0.01 and P<0.05). In addition, each dose of scutellarin benzyl ester could significantly reduce the release of LDH from cardiomyocytes injured by ischemia (P<0.01). In morphology, the injured nuclei presented significant changes such as condensation of chromatin, and shrinkage and fragmentation of nuclei, which could be attenuated remarkably by pretreatment with scutellarin benzyl ester. Furthermore, scutellarin benzyl ester could significantly decrease the percentage of apoptosis induced by ischemia (P<0.01) and inhibit the increased expression levels of cytochrome c and caspase-3 mRNAs and proteins (P<0.01).

Conclusion: Scutellarin benzyl ester has a remarkable protective effect against myocardial ischemic injury and the protective mechanism may associate with its anti-apoptosis effect by inhibiting cytochrome c release and caspase-3 activation.

Key words: Erigeron breviscapus, scutellarin benzyl ester, prodrugs, myocytes, cardiac, ischemia, apoptosis, rats

"

Group n OD value
Control 6 0.517±0.054
Model 6 0.215±0.025**
High-dose scutellarin benzyl ester
(100 μmol/L)		 6 0.287±0.023**△△
Medium-dose scutellarin benzyl ester
(50 μmol/L)		 6 0.253±0.023**△
Low-dose scutellarin benzyl ester
(25 μmol/L)		 6 0.210±0.021**

"

Group n LDH
Control 6 78.8±12.9
Model 6 288.1±30.7**
High-dose scutellarin benzyl ester
(100 μmol/L)		 6 64.2±22.9△△
Medium-dose scutellarin benzyl ester
(50 μmol/L)		 6 101.4±22.7△△
Low-dose scutellarin benzyl ester
(25 μmol/L)		 6 141.3±23.1**△△

Figure 1

Changes of nuclei tested by Hoechst-prodium iodide staining (Microscopy, ×400)A: Control; B: Model; C: Scutellarin benzyl ester (100 μmol/L)."

Figure 2

Changes of apoptosis percentage tested by flow cytometryA: Control group; B: Model group; C: Scutellarin benzyl ester (100 μmol/L) group."

"

Group n Apoptosis percentage
Control 6 4.52±0.29
Model 6 30.14±1.89**
High-dose scutellarin benzyl ester
(100 μmol/L)		 6 18.93±1.45**△△
Medium-dose scutellarin benzyl ester
(50 μmol/L)		 6 22.10±1.27**△△
Low-dose scutellarin benzyl ester
(25 μmol/L)		 6 29.89±2.12**△△

Figure 3

Changes in the expression of cytochrome c mRNA tested by RT-PCRA: Control group; B: Model group; C: Scutellarin benzyl ester (100 μmol/L) group; D: Scutellarin benzyl ester (50 μmol/L) group; E: Scutellarin benzyl ester (25 μmol/L) group. **P<0.01, vs control group; △△P<0.01, vs model group. RT-PCR: reverse transcription-polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase."

Figure 4

Changes in the expression of caspase-3 mRNA tested by RT-PCRA: Control group; B: Model group; C: Scutellarin benzyl ester (100 μmol/L) group; D: Scutellarin benzyl ester (50 μmol/L) group; E: Scutellarin benzyl ester (25 μmol/L) group. **P<0.01, vs control group; △△P<0.01, vs model group. RT-PCR: reverse transcription-polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase."

Figure 5

Changes in the expression of cytochrome c protein tested by Western blotting A: Control group; B: Model group; C: Scutellarin benzyl ester (100 μmol/L) group; D: Scutellarin benzyl ester (50 μmol/L) group; E: Scutellarin benzyl ester (25 μmol/L) group. **P<0.01, vs control group; △△P<0.01, vs model group."

Figure 6

Changes in the expression of caspase-3 protein tested by Western blotting A: Control group; B: Model group; C: Scutellarin benzyl ester (100 μmol/L) group; D: Scutellarin benzyl ester (50 μmol/L) group; E: Scutellarin benzyl ester (25 μmol/L) group. **P<0.01, vs control group; △△P<0.01, vs model group."

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