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Journal of Chinese Integrative Medicine ›› 2007, Vol. 5 ›› Issue (2): 183-188.doi: 10.3736/jcim20070217

• Original Experimental Research • Previous Articles     Next Articles

Conditioned mediums of different rat cerebral microvascular endothelial cells against damage of ischemia and ischemia/reperfusion neurons

Xue-mei Qing, Peng-tao Li, Jing-hong Hu, Wei-hong Li, Jin-cai Hou, Huan Du, Bing Wang, Lin Suo   

  1. School of Preclinical Medicine, Beijing University of Chinese Medicine, Beijing 100029, China
  • Online:2007-03-31 Published:2007-03-15

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Objective: Using the method of lactate dehydrogenase (LDH) assay, to observe the activities of rat cerebral microvascular endothelial cells (CMECs) intervened by Tongluo Jiunao Injection (TLJNI), a traditional Chinese compound drug removing toxin to dredge brain collaterals, and then further study the effects of different kinds of conditioned mediums (CMECs-CM) of cerebral microvascular endothelial cells on ischemia and ischemia/reperfusion cerebral cortex cells, and to probe into the drug pharmacological mechanisms of CMECs in modulating the neurons. 

Methods: Three kinds of CMECs (normal, ischemic and ischemic/reperfusional) were all treated by TLJNI previously, and then the three pairs of CMECs-CM without serum were collected respectively for LDH assay. Rat cerebral cortex neurons were also primarily cultured and then divided into similar three groups (normal, ischemic and ischemic/reperfusional). The neuron responses caused by CMECs-CM at different concentrations were observed by using LDH transudation rate assay. 

Results: The LDH release values of ischemic and ischemic/reperfusional CMECs with TLJNI treatment were obviously reduced (P＜0.01) compared with the same kinds of CMECs untreated. For ischemic neurons, both conditioned medium of ischemic CMECs (Is-CM) and conditioned medium of ischemic CMECs with drug treatment (IsT-CM) in high concentration of 100% increased the LDH transudation rate (P＜0.01), while in low concentration of 10%, IsT-CM reduced the transudation rate (P＜0.05). For ischemia/reperfusion neurons, all kinds of CMECs-CM reduced the transudation rate respectively (P＜0.05 or P＜0.01). As far as each group concentration was concerned, 10% or 50% showed relatively stronger effects, and both conditioned medium of normal CMECs (N-CM) group and conditioned medium of ischemic/reperfusional CMECs (Rp-CM) group had statistical significance (P＜0.05 or P＜0.01). For normal neurons, all kinds of CMECs-CM increased the transudation rate respectively (P＜0.05 or P＜0.01). As far as each group concentration was concerned, only conditioned medium of normal CMECs (N-CM)had statistical significance (P＜0.05 or P＜0.01). 

Conclusion: The study shows that TLJNI is capable of preventing the damage of CMECs from both ischemia and ischemia/reperfusion states. Chinese drug can restrain the brain ischemia and ischemia/reperfusion damage by the media that CMECs modulate the neurons, demonstrating the pharmacological mechanisms of TLJNI. This work also indicates that there exist some active substances against ischemia/reperfusion injury secreted from CMECs-CM with TLJNI treatment.

Key words: cerebral microvascular endothelial cells, neurons, cerebral ischemia, rats, Chinese herbal drugs

CLC Number: 

  • R743

Figure 1

Standard curve of lactate dehydrogenase"

Table 1

LDH release value of different CMECs-CM ($\bar{x}±S$)"

CMECs-CMnLDH release (OD value)
CM without drug treatmentCM with TLJNI treatment
Normal CMECs-CM60.150±0.0100.170±0.010
Ischemia CMECs-CM60.070±0.004△△ 0.030±0.003*
Ischemia/reperfusion CMECs-CM60.240±0.010▲▲ 0.090±0.003*

Table 2

LDH leakage rate of ischemia neurons intervened by different CMECs-CM ($\bar{x}±S$)"

Groupn LDH leakage rate (%)
Untreated control625.9±4.8
Drug-treated657.4±12.4**
10% Is-CM625.2±1.8△△
50% Is-CM620.4±0.5△△
100% Is-CM643.1±15.0**
10% IsT-CM616.8±1.8*△△
50% IsT-CM624.7±1.1△△
100% IsT-CM639.3±0.6**△

Table 3

LDH leakage rates of normal and ischemia/reperfusion neurons intervened by different CMECs-CM ($\bar{x}±S$,%)"

GroupnNormal neuronsI/R neurons
Control without CM612.9±2.351.3±9.5
10% N-CM614.4±0.631.9±3.1*
50% N-CM620.5±2.1** △△ 25.4±1.9** △
100% N-CM616.6±0.9*△ 35.8±1.5*△△
10% NTCM619.1±1.3**27.2±3.6**
50% NT-CM622.2±2.5**34.0±6.8*
100% NT-CM622.8±3.3**30.0±3.4*
10% Rp-CM618.0±2.0**25.2±5.3**
50% Rp-CM616.5±3.0*27.0±2.2**
100% Rp-CM617.5±1.4**33.0±2.6*△
10% RpT-CM614.9±0.530.7±5.7*
50% RpT-CM617.9±4.4*25.0±6.3**
100% RpT-CM617.1±1.2*34.6±5.4*
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