The Protection And Mechanism Of Lyophilized Powder Of Catalpol And Puerarin On Neurovascular Unit Under Ischemic Penumbra Condition

1. BackgroundDrug deficiency for cerebral ischemia is a worldwide problem always plaguing medical workers. Preventing cell apoptosis or necrosis in the penumbra during the acute stage and avoiding expanding of infarction are recognized as an ideal treatment strategy for stroke. Lyophilized powder of catalpol and puerarin(C-P), a drug developed by our group, is composed of bioactive ingredients catalpol in rehmannia and puerarin in pueraria. Our preliminary research indicated that C-P could exert protective effects on the brain of rats and mice with permanent ischemia in non-acute stage, as well as on the ischemia-reperfusion injured rats. However, the neuroprotective effects of C-P on middle cerebral artery occlusion-induced stroke in the acute stage have not been understood. The overall protective effects of C-P on neurovascular unit(NVU) in the ischemic penumbra and its involved mechanism also remain unclear.This study was supported by the Natural Science Foundation of China(No. 81473549), the Special Fund for Ministry of Education Doctoral Program(No. 20110182110012), the Fundamental Research Funds for the Central Universities(XDJK2014D023), and the Chongqing Municipal Health Bureau in Major Medical Research Projects(No. 2010[60]2010-1-4).2. ObjectivesThis study aims to observe the neuroprotective effects of C-P on acute ischemic rats, as well clarify its overall protective effects on NVU in the ischemic penumbra and the involved mechanism, so that providing preclinical evidence for C-P as a candidate drug for stroke.3. Methods and Results3.1 C-P protects acute ischemic stroke rats from brain injuryMethods: Suture method was used to obstruct the middle cerebral artery of rats to prepare stroke model. Two hours after the recovery from anesthesia, ischemic rats were scored by modified neurological severity scores(m NSS). Rats got 4 or more scores were regarded as successful ischemic models. Sixty-four model rats were randomly divided into four groups: a model control group and three C-P groups(65.4 mg·kg-1, 32.7 mg·kg-1, and 16.4 mg·kg-1), with 16 in each. Another 16 sham operated rats were set as sham control. After grouped, the rats were treated with C-P or saline for one day(two times).Twenty-four hours after ischemia, rats were scored by m NSS again. The differences of neural function between each group were analyzed. Besides, 8 rats were taken from each group and sacrificed after being anesthetized. The brain was removed and incised and stained by TTC. The stained brains in each group were photographed and compared by the percentage of infarct area. Moreover, another 8 rats were taken from each group and anesthetized. After being preserved in 4% paraformaldehyde and fixed, the brains in each group were sliced and were made HE staining. The damage of ischemic brain tissues were identified by microscopy.Results: Compared with the sham operated rats, the ischemic rats showed increased m NSS scores(p < 0.01), displayed large-area of necrosis in ischemic brain tissues, and presented highly loose mesh-like structures in part of the cortex, with ambiguous structures of cells and significantly increased red necrotic neurons. However, compared with the model control rats, the rats treated with C-P(65.4 mg·kg-1, 32.7 mg·kg-1, and 16.4 mg·kg-1) had significantly reduced m NSS scores and infarct volume percentages(p < 0.01, p < 0.05). The necrosis in the infarct tissues of C-P treated rats was also ameliorated, and the red necrotic neurons were decreased.3.2 The mechanism of C-P protecting acute ischemic rats from penumbra brain damageMethods: The methods of model preparation and treatment were as same as that in “3.1”. Twenty-four hours after the ischemia, 6 rats were taken from each group and their brains were frozen and sliced. The sliced sections were labeled by TUNEL plus specific protein and were observed under a fluorescence microscope. The cell growth morphology of cortex neurons, astrocytes, and microvascular endothelial cells in penumbra areas were observed and their apoptosis rates were evaluated. Additionally, the sliced brain sections were labeled with Bax, Bcl-2, Caspase3, Cleaved caspase-3,and Cyt-C by immunohistochemistry and were observed for the penumbra cortex under a microscope. What’s more, another 6 rats were taken from each group and the brains of which were obtained after anesthetizing. The cortex tissues in penumbra areas were homogenated and analyzed with the expressions of Bax, Bcl-2, Caspase3, Cleaved caspase-3, and Cyt-C by Western blot.Results: Compared with sham operated rats, the ischemic rats showed increased apoptosis rates of neurons, astrocytes, and microvascular endothelial cells in ischemic penumbra cortex(p < 0.01). The cell growth morphology in ischemic rats was severely damaged. The pro-apoptotic proteins(Bax, Caspase3, Cleaved caspase-3, and Cyt-C) in ischemic rats were significantly increased(p < 0.01) and the anti-apoptotic protein Bcl-2 had significantly reduced expression(p < 0.01). However, after treated with C-P(65.4 mg·kg-1, 32.7 mg·kg-1, and 16.4 mg·kg-1), the ischemic rats displayed decreased apoptosis rates for the three cells(p < 0.01), ameliorated growth morphology, and reduced pro-apoptotic proteins(p<0.01,p<0.05). The ischemic rats treated with 65.4 mg·kg-1 of C-P showed up-regulated Bcl-2 expression(p < 0.01).3.3 The establishment of penumbra injured NVU model and the protective effects of C-P on itMethods: The primary culturing of neurons, astrocytes and microvascular endothelial cells, and the establishment of NVU model were both conducted according to our previous methods. The co-cultured NVU system was damaged by penumbra conditioned medium for 24 h. And the success of penumbra injured NVU model was evaluated as follows: the cell growth morphology, cell amount, axon length, and transendothelial cell resistance value in the penumbra injured NVU model were significantly worse or lower than that in the normal control(P < 0.05). Then, the established NVU models were divided into control group, penumbra injured(PI) group, and PI + C-P groups(49.0 μg·m L-1, 24.5 μg·m L-1, and 12.25 μg·m L-1). Twenty-four hours after the treatment of penumbra conditioned medium, the growth morphology of cells in different groups was observed by microscopy. The numbers of astrocytes and microvascular endothelial cells were detected by trypan blue staining. The length of neuron axons was analyzed using image analysis software. The transendothelial cell resistance value was measured by cell resistance meter.Results: High purity of neurons, astrocytes, and microvascular endothelial cells were obtained, which prompted the successful establishment of the NVU. Compared with cultured alone, the cells co-cultured got better growth: the neurons were round and three-dimensional and the axons of the neurons were thicker and longer and formed a dense network; the microvascular endothelial cells and astrocytes contacted each other more closely and formed a dense monolayer; and the numbers of microvascular endothelial cells and astrocytes, the length of neuron axons, and the transendothelial cell resistance value were significantly increased(p < 0.05, p < 0.01). However, after the penumbra injury, the NVU showed poor stereoscopic neurons, with small cell bodies and axons, and with decreased axonal network density. Also, the cell bodies of microvascular endothelial cells and astrocytes shrinked, and some of the cells detached. The number of the survival microvascular endothelial cells and astrocytes, the length of the neuron axon, and the transendothelial resistance value were all significantly decreased(p < 0.01). Compared with the PI group, the C-P groups(49.0 μg·m L-1, 24.5 μg·m L-1, and 12.25 μg·m L-1) showed significantly improved cell morphology: the neurons had no obvious contraction and the axon network was still dense; the microvascular endothelial cells and astrocytes were slightly contracted and the two kinds of cells contacted closely; the numbers of microvascular endothelial cells and astrocytes, the length of neuron axons, and the transendothelial cell resistance value were significantly increased(p < 0.01). The neuron axons in C-P 49.0 μg·m L-1 and 24.5 μg·m L-1 groups was significantly lengthened(p < 0.01).3.4 The mechanism of C-P protecting NVU cells from penumbra injuryMethods: The establishment and identification of penumbra injured NVU model were as same as in “3.3”. The established models were randomly divided into seven groups: normal control group(cultured under normal condition), PI group(cultured under penumbra-conditioned medium), C-P groups(49.0 μg·m L-1, 24.5 μg·m L-1, and 12.25 μg·m L-1, C-P was added in the medium when changing with penumbra-conditioned medium), MG132 group(10 μM, MG132 was added when changing with penumbra-conditioned medium), and C-P + MG132 group(49.0 μg·m L-1+ 10 μM, CP and MG132 was added when changing with penumbra-conditioned medium). After treated for 24 h, the penumbra injured NVU cells were assessed with their apoptosis rates using flow cytometer. The total protein in the penumbra injured NVU cells was extracted and quantitated. And the expressions of Bax, Bcl-2, Caspase3, Cleaved caspase-3, Caspase-9, Cleaved caspase-9, Cyt-C, and XIAP were determined by western blot.Results: Compared with the cells in normal control, the neurons, astrocytes, and endothelial cells in the penumbra injured NVU model were found with significantly increased apoptosis(p < 0.01), and more expressions of Bax, Cyt-C, Cleaved caspase-9, and Cleaved caspase-3(p < 0.01), but with less expressions of Bcl-2 and XIAP(p < 0.01). However, C-P at the doses of 49.0 μg·m L-1 and 24.5 μg·m L-1 inhibited the apoptosis(p < 0.01), lowered the expressions of Cyt-C, Cleaved caspase-9, and Cleaved caspase-3(p < 0.01), and promoted the expressions of Bcl-2 and XIAP(P < 0.01). 49.0 μg·m L-1 of C-P lowered the expression of Bax(p < 0.01). In addition, compared with the C-P 49.0 μg·m L-1 group, the MG132 + C-P group showed increased expressions of XIAP and Cleaved caspase-3(p < 0.01, p < 0.05).4. Conclusions4.1 C-P could lessen the morphological changes of injured cells in ischemic areas, inhibit the cell apoptosis, reduce the infarct volume, and improve the neural function so that exerting its protective effects on acute ischemic rats.4.2 C-P exerting protective effects on acute ischemic rats mainly through inhibiting the apoptosis of penumbra injured NVU cell, which was related to the mitochondrial apoptosis pathway.4.3 C-P regulated the mitochondrial apoptosis in penumbra injured NVU system mainly relying on promoting the expression of XIAP.4.4 The results in vitro in this study was consistent with the in vivo results, indicating that the NVU model in vitro can be used to explore the mechanism of cell injury in penumbra NVU and to evaluate the protective effects of relative drugs.