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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 E-mail:huangqfl937@yahoo.com.cn

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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."

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