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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (1): 76-84.doi: 10.3736/jcim20120112

• Original Experimental Research • Previous Articles     Next Articles

Effects of extracts from Panax ginseng, Panax notoginseng and Ligusticum chuanxiong on expression of β-galactosidase and signal pathway p16-cyclin D/CDK-Rb in vascular smooth muscle cells

Tao Li-li1,Lei Yan2()   

  1. 1. Cardiovascular Center, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091, China
    2. Medical Experiment Center, China Academy of Chinese Medical Sciences, Beijing 100700, China
  • Received:2011-08-04 Accepted:2011-09-12 Online:2012-01-20 Published:2018-09-21
  • Contact: Lei Yan E-mail:leiy999@163.com

OBJECTIVE: To observe the pathological changes of vascular aging in spontaneously hypertensive rats (SHRs), and the effects of extracts from Panax ginseng, Panax notoginseng (Burk.) and Ligusticum chuanxiong on vascular aging.METHODS: Vascular smooth muscle cells (VSMCs) isolated from thoracic aorta of Wistar-Kyoto (WKY) rats and SHRs were primarily cultured and divided into WKY group, SHR group, valsartan group, low-dose extract group and high-dose extract group. Aging situation of VSMCs was detected by senescence-associated β-galactosidase (SA-β-gal) staining; the cell cycle was analyzed by flow cytometry; mRNA and protein expressions of p16, cyclin D1, cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (Rb) were detected by real-time fluorescence quantitative polymerase chain reaction (PCR) and Western blotting.RESULTS: SA-β-gal stain showed that compared with the WKY group, number of SA-β-gal-positive cells in the SHR group was increased significantly (P<0.01); extracts from Panax ginseng, Panax notoginseng (Burk.) and Ligusticum chuanxiong reduced the number of SA-β-gal-positive cells in the SHR group (P<0.01). Flow cytometry analysis showed that the number of VSMCs in G1 phase was reduced and in S phase was increased significantly (P<0.05) in the SHR group compared with the WKY group, and the number of VSMCs in G1 phase was increased and in S phase was reduced significantly in the extracts groups (P<0.05). PCR result showed that compared with the WKY group, the mRNA expressions of p16 and Rb in the SHR group were reduced and those of cyclin D1 and CDK4 were increased significantly, the mRNA expressions of p16 and Rb in the extracts group were increased and mRNA expressions of cyclin D1 and CDK4 were reduced significantly. Western blotting result showed that compared with the WKY group, the protein expression of p16 in the SHR group was reduced and the protein expressions of cyclin D1, CDK4 and phospho-Rb were increased significantly (P<0.05), while the protein expression of p16 in the extracts group was increased and the protein expressions of cyclin D1, CDK4 and phospho-Rb were reduced significantly (P<0.05).CONCLUSION: The extracts from Panax ginseng, Panax notoginseng (Burk.) and Ligusticum chuanxiong can delay vascular aging of SHRs, which works by p16-cyclin D/CDK-RB pathways to inhibit VSMC proliferation.

Key words: hypertension, vascular aging, plant extracts, senescence-associated β-galactosidase;, gene, p16, cyclin D1, cyclin-dependent kinase 4, retinoblastoma protein

Figure 1

Growth morphology of VSMCs and identificationA: Cells obtained from aorta of rats had a spindle shape (Inverted microscopy, ×200); B: Cells displayed a hill-and-valley pattern typical for VSMCs (Inverted microscopy, ×100); C: Percentage of α-actin-positive smooth muscle cells was more than 95% (Inverted microscopy, ×400). VSMCs: vascular smooth muscle cells."

Table 1

SA-β-gal-positive VSMCs in different groups ($\bar{x}$±s, %)"

Group n SA-β-gal-positive VSMCs
Wistar-Kyoto 3 11.14±2.21
SHR 3 32.89±3.14**
Valsartan 3 15.96±1.59△△
Low-dose extract 3 24.36±2.00△△
High-dose extract 3 20.78±1.86△△

Figure 2

SA-β-gal-positive VSMCs in different groups (×400) A: Wistar-Kyoto group; B: Spontaneously hypertensive rat group; C: Valsartan group; D: Low-dose extract (20 mg/L) group; E: High-dose extract (40 mg/L) group. SA-β-gal: senescence-associated β-galactosidase; VSMC: vascular smooth muscle cell."

Table 2

Cell cycle analysis of VSMCs ($\bar{x}$±s, %)"

Group n G1 phase G2 phase S phase
Wistar-Kyoto 3 91.23±1.10 6.75±0.83 2.02±1.21
SHR 3 67.40±0.85* 17.43±0.34* 15.17±0.99*
Valsartan 3 87.90±8.49 10.53±0.96 2.71±1.57
Low-dose extract 3 81.67±0.81 15.12±0.17 3.22±0.91
High-dose extract 3 85.43±0.96 7.53±0.56 7.03±1.52

Figure 3

Cell cycle analysis of VSMCsA: Wistar-Kyoto group; B: Spontaneously hypertensive rat group; C: Valsartan group; D: Low-dose extract (20 mg/L) group; E: High-dose extract (40 mg/L) group. VSMC: voscular smooth muscle cell."

Table 3

Expressions of p16, cyclin D1, CDK4 and Rb mRNAs in VSMCs ($\bar{x}$±s)"

Group n p16 Cyclin D1 CDK4 Rb
Wistar-Kyoto 3 3.31±0.06 0.27±0.02 1.01±0.02 2.87±0.04
SHR 3 0.79±0.38* 1.25±0.24* 3.14±0.19* 0.57±0.49*
Valsartan 3 2.52±0.12 0.45±0.03 1.24±0.01 2.30±0.17
Low-dose extract 3 1.39±0.11 0.77±0.10 3.01±0.30 1.17±0.32
High-dose extract 3 1.76±0.21 0.63±0.15 2.70±0.09 1.74±0.05

Table 4

Expressions of p16, cyclinD1 and CDK4 proteins and pRb in VSMCs ($\bar{x}$±s)"

Group n p16 Cyclin D1 CDK4 pRb
Wistar-Kyoto 3 1.19±0.31 0.82±0.06 1.37±0.13 0.50±0.14
SHR 3 0.51±0.06* 2.17±0.35* 2.17±0.27* 1.34±0.08*
Valsartan 3 0.89±0.23 0.93±0.07 0.95±0.20 0.56±0.01
Low-dose extract 3 1.04±0.14 1.52±0.32 1.81±0.13 1.19±0.21
High-dose extract 3 1.03±0.09 1.47±0.15 2.00±0.35 0.86±0.07

Figure 4

Expressions of p16, cyclinD1, CDK4 proteins and pRb in VSMCsA: Wistar-Kyoto group; B: SHR group; C: Valsartan group; D: Low-dose extract (20 mg/L) group; E: High-dose extract (40 mg/L) group. SHR: spontaneously hypertensive rat; VSMC: vascular smooth muscle cell; CDK4: cyclin-dependent kinase 4; pRb: phospho-retinoblastoma protein."

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