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Journal of Chinese Integrative Medicine ›› 2008, Vol. 6 ›› Issue (1): 45-50.doi: 10.3736/jcim20080110

• Original Experimental Research • Previous Articles     Next Articles

Effects of Tribulus terrestris L. saponion on apoptosis of cortical neurons induced by hypoxia-reoxygenation in rats

Xue-mei Liu1, Qi-fu Huang2(), Yun-ling Zhang1, Jin-li Lou1, Hong-sheng Liu1, Hong Zheng1   

  1. 1. Center of Science Experiment, Dongfang Hospital, Beijing University of Chinese Medicine, Beijing 100078, China
    2. Department of Pathology, Beijing University of Chinese Medicine, Beijing 100029, China
  • Online:2008-01-20 Published:2008-01-15
  • Contact: HUANG Qi-fu

Objective:To observe the effects of Tribulus terrestris L. saponion (TTLS) on apoptosis in cortical neurons induced by hypoxia-reoxygenation in rats.

Methods:Primary culture of rat cortical neurons was performed in vitro. A model of apoptosis of cortical neurons was established by hypoxia and reoxygenation. Hypoxia for 3 h in neural cells was induced with mixture of 95% N2 and 5% CO2, and then reoxygenation in neural cells was induced with mixture of 95% O2 and 5% CO2 for 12 h. Different concentrations of TTLS were administered to traditional Chinese herbal medicine-treated group separately during hypoxia and reoxygenation. The apoptosis rate was analyzed quantitatively by flow cytometry with Annexin V-FITC and propidium iodide staining. Mitochondria membrane potential was observed by a confocal laser-scanning microscope with JC-1 fluorescence. Caspase-3/7 activity in cytoplasm was measured by fluorescent plate reader. Bax protein expression was observed by immunohistochemical technique.

Results:The percentage of apoptosis was significantly increased, mitochondria membrane potential was obviously decreased, fluorescence of caspase-3/7 activity was increased, and Bax protein was abundantly expressed followed by 3 h of hypoxia and 12 h of reoxygenation (P<0.01). TTLS could inhibit the depression of membrane potential induced by hypoxia and reoxygenation, decrease the activity of caspase-3/7, reduce the expression of Bax protein, and inhibit the apoptosis of the cortical neurons.

Conclusion:Hypoxia and reoxygenation can induce apoptosis of rat cortical neurons. TTLS can decrease the apoptosis induced by hypoxia and reoxygenation. The mechanism might be related to stabilization of mitochondria membrane potential, inhibition of caspase activity and reduction of Bax protein expression.

Key words: Tribulus terrestris, neurons, apoptosis, mitochondria, anoxia

CLC Number: 

  • R338.1

Table 1

Effects of different doses of TTLS on viability of rat cortical neurons injured by hypoxia-reoxygenation ($\bar{x}$±s)"

Group n OD
Normal control 8 0.336±0.006
Untreated 8 0.287±0.004*
0.003 g/L TTLS-treated 8 0.290±0.005
0.006 g/L TTLS-treated 8 0.295±0.006
0.012 g/L TTLS-treated 8 0.291±0.002
0.025 g/L TTLS-treated 8 0.383±0.006△△
0.050 g/L TTLS-treated 8 0.387±0.003△△

Table 2

Effects of TTLS on apoptosis rate of rat cortical neurons injured by hypoxia-reoxygenation ($\bar{x}$±s, %)"

Group n Apoptosis rate
Normal control 6 3.56±0.20
Untreated 6 24.45±3.87**
0.025 g/L TTLS-treated 6 12.02±0.78△△

Figure 1

Apoptosis rate of cortical neurons in rat tested by flow cytometry with Annexin V-FITC/PI staining A: normal control group; B: untreated group; C: 0.025 g/L TTLS-treated group."

Figure 2

Changes of mitochondrial membrane potential in cortical neurons tested by laser scanning confocal microscopy with JC-1 staining A: normal control group; B: untreated group; C: 0.025 g/L TTLS-treated group."

Figure 3

Effects of TTLS on membrane potential of rat cortical neurons injured by hypoxia-reoxygenation. **P<0.01 vs normal control group; △△P<0.01 vs untreated group; n=10."

Table 3

Effects of TTLS on caspase-3/7 activity in rat cortical neurons injured by hypoxia-reoxygenation ($\bar{x}$±s)"

Group n Caspase-3/7 activity
Normal control 10 22 086.20±1 446.93
Untreated 10 59 512.90±3 302.67**
0.025 g/L TTLS-treated 10 34 437.50±3 016.62△△

Table 4

Effects of TTLS on Bax protein expression of rat cortical neurons injured by hypoxia-reoxygenation ($\bar{x}$±s)"

Group n Bax expression
Normal control 3 442.11±63.02
Untreated 3 999.49±158.93**
0.025 g/L TTLS-treated 3 584.96±136.75△△

Figure 4

Effects of TTLS on Bax protein expression of rat cortical neurons injured by hypoxia-reoxygenation A: normal control group; B: untreated group; C: TTLS-treated group."

[1] Chu SD, Qu WJ, Li M , et al. Research advance on chemical component and pharmacological action of Tribulus terrestris. Zhongguo Ye Sheng Zhi Wu Zi Yuan. 2003; 22(4):4-7. Chinese with abstract in English.
doi: 10.3969/j.issn.1006-9690.2003.04.002
褚书地, 瞿伟菁, 李穆 , 等. 蒺藜化学成分及其药理作用研究进展.中国野生植物资源. 2003; 22(4):4-7.
doi: 10.3969/j.issn.1006-9690.2003.04.002
[2] Jiang ZW, Lu WW, Zhang ZQ , et al. Protective effect of total saponins from fruit of Tribulus terrestris on experimental cerebral ischemia of rat. Zhong Cao Yao. 2002; 33(11):1020-1022. Chinese with abstract in English.
doi: 10.3321/j.issn:0253-2670.2002.11.026
姜宗文, 吕文伟, 张志强 , 等. 蒺藜果总皂苷对大鼠实验性脑缺血的保护作用.中草药. 2002; 33(11):1020-1022.
doi: 10.3321/j.issn:0253-2670.2002.11.026
[3] Wang XH, Li H, Wen K , et al. Effect of anti-cerebral ischemia of gross saponin from Tribulus terrestris. Jilin Da Xue Xue Bao (Yi Xue Ban). 2004; 30(1):60-62. Chinese with abstract in English.
doi: 10.3969/j.issn.1671-587X.2004.01.019
王秀华, 李红, 温克 , 等. 注射用蒺藜皂苷的抗脑缺血作用. 吉林大学学报(医学版). 2004; 30(1):60-62.
doi: 10.3969/j.issn.1671-587X.2004.01.019
[4] Zhang J, Zhang YL , Lou et al. Mechanism of toxin damaging brain collaterals in rats with hippocampus ischemia of acute multi-infarction. Tianjin Zhong Yi Yao. 2006; 23(4): 316-319. Chinese with abstract in English.
张锦, 张允岭, 娄金丽 , 等. 从急性多发性脑梗死大鼠海马缺血损伤探讨毒损脑络机制.天津中医药. 2006; 23(4):316-319.
[5] Choi DW . Ionic dependence of glutamate neurotoxicity.[J] Neurosci. 1987; 7(2):369-379.
doi: 10.1523/JNEUROSCI.07-02-00369.1987
[6] Brewer GJ, Torricelli JR, Evege EK , et al. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal,a new serum-free medium combination.[J] Neurosci Res. 1993; 35(5) : 567-576.
doi: 10.1002/(ISSN)1097-4547
[7] Saikumar P, Dong Z, Weinberg JM , et al. Mechanisms of cell death in hypoxia/reoxygenation injury. Oncogene. 1998; 17(25):3341-3349.
doi: 10.1038/sj.onc.1202579 pmid: 9916996
[8] Susin SA, Zamzami N, Kroemer G . Mitochondria as regulators of apoptosis: doubt no more. Biochim Bio- phys Acta. 1998; 1366(1-2):151-165.
[9] Ly JD, Grubb DR, Lawen A . The mitochondrial membrane potential (A^m) in apoptosis; an update. Apoptosis. 2003; 8(2):115-128.
doi: 10.1023/A:1022945107762 pmid: 12766472
[10] Harris MH, Thompson CB . The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability. Cell Death Differ. 2000; 7(12):1182-1191.
doi: 10.1038/sj.cdd.4400781 pmid: 11175255
[11] Ashe PC, Berry MD . Apoptotic signaling cascades. Prog Neuropsychopharmacol Biol Psychiatry. 2003 ; 27(2):199-214.
[12] Hardwick JM, Polster BM . Bax, along with lipid conspirators, allows cytochrome c to escape mitochondria. Mol Cell. 2002; 10(5):963-965.
doi: 10.1016/S1097-2765(02)00751-7 pmid: 12453403
[13] Jiangsu New Medical College Chinese Medicine Dictionary (Vol 1). Shanghai: Shanghai Scientific and Technical Publishers. 2005: 1274-1276. Chinese.
江苏新医学院. 中药大辞典(上册).上海: 上海科学技术出版社. 2005: 1274-1276.
[14] Su YG, Yang XY, Chen HZ. Clinical and research status quo of Xinnao Shutong. Shengzhen Zhong Xi Yi Jie He Za Zhi. 1999; 9(1):39-40. Chinese.
宿燕岗, 杨学义, 陈灏珠 . 心脑舒通的临床应用及研究现状.深圳中西医结合杂志. 1999; 9(1):39-40.
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