Effect of Salvianolic Acid B on Mitochondrial Function of Cerebral Ischemia in Mice

TSINGHUA SCIENCE AND TECHNOLOGY ISSNll1007-0214ll16/19llpp528-533 Volume 14, Number 4, August 2009

Effect of Salvianolic Acid B on Mitochondrial Function of Cerebral Ischemia in Mice* JIANG Yufeng (ߠံ‫)׬‬1,**, LUO Xuechun (৥༲ҝ)2,3, WANG Ximei (ฆឌਚ)1, FANG Lei (ֺ त)1, HUANG Qifu (ܻ୳‫)׾‬1 1. Department of Pathology, Beijing University of Chinese Medicine, Beijing 100029, China; 2. Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China; 3. State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing 100084, China Abstract: The effects of salvianolic acid B (SalB) on the mitochondrial membrane potential (MMP), calcium, and apoptosis of neurons with cerebral ischemia in mice were investigated using an acute cerebral ischemia model established by ligating the bilateral common carotid arteries in mice. The MMP, the intracellular calcium concentration, and the apoptosis rate of cortical neurons were measured at 6 min, 12 min, 18 min, 24 min, and 30 min after cerebral ischemia by a flow cytometer. The experiments show that SalB increases the MMP and reduces the intracellular calcium and the apoptosis rate at different stages of the cerebral ischemia in mice. The results show that the protective mechanism of SalB on cerebral ischemia enhances the MMP and maintains intracellular calcium homeostasis. Key words: brain ischemia; mitochondrial membrane potential; calcium; apoptosis; salvianolic acid B (SalB); NIH mice

Introduction The brain tissue requires a very high oxygen consumption rate. Once brain ischemia persists for over 6 min, cellulay energy stores are exhausted and abnormal signal transductions occur, which initiates mitochondrial dysfunction. Those changes cause progressive damage to high level brain functions which is comprehensive and irreversible. As shown by epidemiological studies, cerebral ischemia belongs to the ischemic cerebrovascular disease class which includes cerebral thrombosis, cerebral infarction, and cerebral vasospasm. The incidence of cerebrovascular disease has reached 75%. Therefore, studies on the pathological changes and prevention of ischemic brain injury play Received: 2007-11-27; revised: 2009-02-23

* Supported by the National Natural Science Foundation of China (No. 30472281)

** To whom correspondence should be addressed. E-mail: [email protected]; Tel: 86-10-64286965

an important role in the medical field. Danshen is one Chinese medicinal herb used widely for prevention and treatment of ischemic diseases. Modern studies of traditional Chinese medicine have shown that[1,2] Salvianolic acid B (SalB), which has the basic structure of Danshensu (ȕ-3, 4-dihydroxybenyl lactic acid), has a strong antioxidation effect and scavenges free radicals because of the phenol hydroxyl group molecular structure. Therefore, SalB is an effective compound for the treatment of ischemic encephalopathy[3,4] and is listed as an experimental control article in the Pharmacopoeia of the People’s Republic of China[5]. The mechanisms for the pathophysiological changes for brain injuries related to ischemic cerebrovascular disease are very complicated, but the dysbolismus of energy is considered to be the initiating agent for the ischemic neurone injury. Nuclear magnetic resonance (NMR was used to show that the phosphocreatine (PCr) content decreased while the inorganic phosphate

JIANG Yufeng (ߠံ‫ )׬‬et al.ġEffect of Salvianolic Acid B on Mitochondrial Function of Cerebral …

content increased 8 min after ischemia of mice in vivo[6]. This indicates that the severity of the ischemia in the brain leads to reduced energy synthesis. That preliminary work led to a model for acute cerebral ischemia in mice. This paper presents measurements of the mitochondrial membrane potential (MMP), intracellular calcium concentration, and cellular apoptosis rate in the cerebral cortex at 6 min, 12 min, 18 min, 24 min, and 30 min after cerebral ischemia as measured using a flow cytometer. This study of the mechanism by which SalB affects the cerebral cortex provides empirical evidence for the clinical application of SalB for cerebral ischemia at an early stage.

1

Materials and Methods

1.1

Model

NIH mice (male mice of SPF grade, 28-32 g provided by the Experimental Animal Center of the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) were randomly divided into four groups, i.e., a Sham-operated group (Sham), a cerebral Ischemia group (Ischemia), a SalB-treated group (SalB, National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China; Batch No. 111302-200504), and a Nim-collated group (Nim, Bayer, Germany; Batch No. BXBT7J1). The SalB-treated group mice were injected with SalB (22.5 mgŽkg1) in the tail vein 30 min before the experiment and then anesthetized with chloral hydrate (350 mgŽkg1, ip). An incision was made at the median neck and the bilateral common carotid arteries were exposed and carefully separated from the vagus nerves. A loop of thread was passed under each artery. As sodium nitroprusside (3.25 mgŽkg1, ip, Shuanghe Modern Medicine Technology Limited Company, Beijing, China; Batch No. 060516) was injected, the thread was tightened rapidly to occlude the blood flow in the bilateral common carotid arteries. The Nim-collated group received Nim (0.03 mgŽkg1) injected into the tail vein 30 min before the experiment. Mice in the Sham-operated group and the Ischemia group were injected with normal saline (5 mLŽkg1) into the tail vein 30 min before the experiment. In these three groups, the empirical method was the same as the SalB-treated group, except for not injecting the sodium nitroprusside and not ligating the bilateral

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common carotid arteries in the Sham-operated group. The mice were killed with pallium obtained to measure the indexes 6 min, 12 min, 18 min, 24 min, and 30 min after the cerebral Ischemia. 1.2

Measurement of mitochodrial membrance potential

Cerebral cortex was obtained[7] and made into a 5% homogenate by adding ice-cold phosphate- buffered saline (PBS, 0.01 molŽL1, pH 7.4), which was then filtered through a 200-mesh filter. Then, a single-cell suspension was obtained and centrifuged at 600g for 5 min. The liquid supernatant was removed and the cell suspension was washed with PBS. About 100 ȝL Rhodamine 123 (Rh 123, USA; 5 ȝmolŽL1) was added to the cell suspension and incubated at 37ć for 20 min in an incubator, then washed twice with PBS. The fluorescence of the specimen was analyzed by flow cytometry (FACSCalibur, Becton Dickinson Company, USA). The excitation light wavelength was 488 nm and the emitting light wavelength was 530 nm. CELLQuest was used to analyze the data obtained from 1×104 cells. 1.3 Measurement of intracellular calcium concentration Fluo-3 (Sigma, USA, 5 ȝmol·L1) was added into collected cells and incubated at 37ć for 40 min in an incubator, with the collected cells then washed three times with PBS to remove the unconjugated colourant. The emittance of 1×104 cells obtained from flow cytometry (excitation wavelength of 488 nm and emitting wavelength of 525 nm) was analyzed with CELLQuest to calculate the average fluorescence profiles. The intracellular calcium concentration was indicated by the Fluo-3 fluorescence intensity after combining with Ca2+[8]. 1.4

Measurement of cellular apoptosis rate

According to the method used by Tao et al.[9], collected cells were fixed in ice-cold alcohol (70%) at 4ć for 18 h and washed twice with PBS. A cell suspension was obtained and mixed with RNase (Sigma, USA; 20 mg·L1) at 37ć for 30 min. The suspension was then mixed with propidium iodide (PI, Sigma, USA)  staining solution (50 mg·L1) and shaken. The cell

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suspension was kept from light at 4ć for 1 h. The emissions of 1×104 cells obtained using flow cytometry (excitation wavelength of 488 nm and emitting wavelength of 570 nm) were analyzed with Modifit LT to calculate the cellular DNA and the apoptosis rate of each sample. 1.5

Tsinghua Science and Technology, August 2009, 14(4): 528-533

relative to the Sham group after 18 min (P < 0.01). The calcium fluorescence intensity decreased after the cerebral ischemia in both the SalB and Nim groups with the calcium fluorescence intensities in both groups, significantly less than that in the Ischemia group (P < 0.01) after 24 min.

Statistical analysis

The data was evaluated using the one-way analysis of variance (ANOVA) in the SPSS 11.5 software package. All values are given as mean ± SEM. Differences between individual groups were compared using the one-factor analysis of variance and considered significant at P < 0.05.

2

Results

2.1

Effect of SalB on the MMP during cerebral ischemia in mice

As shown in Fig. 1, the MMP fluorescence intensity decreased gradually in the Ischemia group, with a significant difference (P < 0.01) relative to the Sham group 24 min after the cerebral ischemia. The MMP fluorescence intensity increased in various phases after the cerebral ischemia in both the SalB-treated and Nim-collated groups. The MMP fluorescence intensity in these two groups was significantly more than that in the Ischemia group 30 min after the cerebral ischemia (P < 0.01).

Fig. 1 Effect of SalB on the MMP of cerebral ischemia in mice (n=10, bP < 0.05, cP < 0.01 vs Sham group; e P < 0.05, fP < 0.01 vs Ischemia group)

2.2

Effect of SalB on the calcium during cerebral ischemia in mice

Figure 2 shows that the intracellular calcium fluorescence intensity increased in the Ischemia group after the cerebral ischemia with a significant difference

Fig. 2 Effect of SalB on calcium concentration after the cerebral ischemia in mice (n=10, bP < 0.05, cP < 0.01 vs Sham group; eP < 0.05, fP < 0.01 vs Ischemia group)

2.3

Effect of SalB on cellular apoptosis rate during cerebral ischemia in mice

Figure 3 shows that the cellular apoptosis rate increased in the Ischemia group after the cerebral ischemia with a significant difference relative to the Sham group after 24 min (P < 0.01). The cellular apoptosis rate decreased after the cerebral ischemia in both the SalB and Nim groups with significant differences relative to the Ischemia group at 30 min (P < 0.01).

Fig. 3 Effect of SalB on cellular apoptosis during cerebral ischemia in mice (n=10, bP < 0.05, cP < 0.01 vs Sham group; eP < 0.05, fP < 0.01 vs Ischemia group)

3

Discussion

The mitochondria, which generate ATP by oxidative phosphorylation, are an important organelle in cells. MMP, which is the potential difference caused by the difference in the ion concentrations on the two sides of the mitochonaria membrane, is a sensitive indicator

JIANG Yufeng (ߠံ‫ )׬‬et al.ġEffect of Salvianolic Acid B on Mitochondrial Function of Cerebral …

reflecting the mitochondrial function. Recent research[10,11] showed that the proper MMP is required to maintain mitochondrial function and that high energy phosphate compounds help regulate the MMP. Thus, the maintenance of the MMP depends on the mitochondrial function, which in turn depends on the MMP, so the mitochondria and MMP are closely related. The mitochondrial endomembrane has a negative charge due to the electronegative glycoprotein on the endomembrane. The transmembrane potential difference with a negative interior potential and a positive exterior potential is caused by many protons outside the endomembrane. Rh123, which is positively charged, is a cation fluorescent dye. After Rh123 enters the mitochondria, it is attracted to the mitochondrial endomembrane, so its fluorescence specifically marks the mitochondrion endomembrane. The fluorescence intensity then indicates changes of the MMP[12]. The experimental results show that the reduction of Rh 123 attracted by the mitochondria of nerve cells after cerebral ischemia reduced the MMP early in the cerebral ischemia. As the ischemia progressed, the MMP was significently reduced. The dysfunction of the ATP caused by the cerebral ischemia was associated with the change of the MMP[13,14]. In the past few years, studies on cellular apoptosis have shifted emphasis from caryon to mitochondrion. Hengartner[15] indicated that the loss of MMP was an important apoptotic indicator. Many experimental studies have indicated that the permeability transition pore (PTP) opens between the mitochondrial adventitia and the endomembrane due to pathological factors, such as lack of energy, production of active oxygen, calcium overload, and mitochondrial membrane potential decrease[16,17]. Following the increased mitochondrial membrane permeability, cytochrome C and apoptosis inducing factor (AIF) are released from the mitochondrial matrix. They separately activate different Caspase and are released to the kytoplasm by PTP or transfered to the nucleus. The apoptosis cascade reaction, which eventually leads to cell death, is initiated[18,19]. Thus, the PTP opening is an important mechanism which regulates cell apoptosis through the mitochondria. In the experiment, the collected cells were mixed with RNase and PI stain. The percentage of cellular apoptosis was calculated from 1×104 cells obtained by flow cytometry. The results showed that

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the cellular apoptosis rate increased significantly with the extension of the ischemia time. Thus, the    mitochondria are not only an important cell organ for energy generation, but also a participant in many important pathological processes. The latest research demonstrates that the pathological mechanisms of various diseases are related to mitochondrial damage[20-22]. The mitochondria are important for the regulation of intracellular calcium homeostasis, which is involved not only in intracellular signal transduction in the cell, but also in apoptosis which is accompanied by increasing calcium[23-26]. The calcium content in the mice brains in the early stages of cerebral ischemia was observed by adding a calcium-specific fluorochrome (Fluo-3) into the cell suspension, prepared from the brain tissue homogenate to indicate the intracellular calcium. The fluorescence intensity indicated changes of the intracellular calcium concentration. The results show that, at the early stage of cerebral ischemia as the ATP is decreasing, the mitochondria can not maintain the dynamic intracellular calcium balance in this physiological state. Thus, the intracellular calcium concentration increases and is positively correlated with ischemia time. SalB, a water soluble monomer isolated from Salvia miltiorrhiza[4,27], easily passes the blood brain barrier and has commendable therapeutic efficacy in ischemic cerebrovascular disease. However, there are no known reports about the mechanism of SalB on the mitochondrial function during cerebral ischemia. These experiments used an acute cerebral ischemia model in NIH mice established by depressurization and ligation of the bilateral common carotid arteries. The continuous pathophysiological changes in the mitochondrial function of neurons in various phases of the early stage of cerebral ischemia were then observed. The results demonstrate that 6 min after cerebral ischemia, the MMP fluorescence intensity is gradually decreasing, while the intracellular calcium fluorescence intensity and cellular apoptosis rates are increasing. As the ischemia time increases, the changes in these indexes increase further. SalB enhances the MMP flourescence intensity and reduces the intracellular calcium concentration and the cellular apoptosis rate in various phases of the early stages of cerebral ischemia. Thirty minutes after cerebral ischemia, there were significant differences relative to the Ischemia group. Thus, the data

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suggests that SalB has protective effects on the nerve cells during cerebral ischemia. SalB enhances the MMP and maintains the dynamic balance of the intracellular calcium, thereby reducing the apoptosis rate.

4

Conclusions

The experimental data suggests that, at the early stage of acute cerebral ischemia, SalB directly acts on nerve cells after passing the blood brain barrier to enhance the MMP and decrease the intracellular calcium concentration and cellular apoptosis rate, so as to change the pathological processes caused by cerebral ischemia. This research explored the pathophysiology changes in the mitochodrial functions at various times in the early stages of cerebral ischemia to explain the mechanism for the prophylactic effect of SalB on cerebral ischemia. This study provides empirical evidence for the clinical application of SalB for acute cerebral ischemia in the early stages. References [1] Du G H, Zhang J T. Development of the water-soluble ingredients in Salvia miltiorrhizaq-Salvianolic acids. Bas. Medi. Sci. ˂ Cli., 2000, 20(5): 394-398. (in Chinese) [2] Du G H, Zhang J T. The general situation and progress of the modern research of red sage root (radix salviae miltiorrhizae). Herald of Medicine, 2004, 23(6): 355-360. (in Chinese) [3] Liu Y, Li L, Liu Y, et al. Antioxidant activiy and protection effect of salvianolic acids on DNA damage.Chin. J. Public. Heal.,2007, 23(4): 448-449. (in Chinese) [4] Chen Y H, Du G H, Zhang J T. Salvianolic acid B protects brain against injuries caused by ischemia-reperfusion in rats. Acta Pharmacol. Sin., 2000, 21(5): 463-466. [5] National Pharmacopoeia Committee. Pharmacopoeia of the People’s Republic of China, PartI. Beijing: Chemical Industry Press, 2005: 52-53. (in Chinese) [6] Jiang Y F, Pan Y S, Huang Q F, et al. The effect of herbs on cerebral energy metabolism in cerebral ischemia- reperfusion mice. Chin. Med. J., 2001, 114(8): 881-883. [7] Du Y J, Sun G J. Effect of moxibustion on mitochondrial membrane potentials and neuronal apoptosis in aged rats. Acupuncture Research, 2005, 30(4): 212-214. (in Chinese)

Tsinghua Science and Technology, August 2009, 14(4): 528-533 Traditional Chinese Medicine, 2004, 27(3): 53-56. (in Chinese) [9] Tao Y M, Mo X M, Peng W, et al. Effect of deep hypothermic circulatory arrest on cortex neuron apoptosis of rats. Modern Medical Journal, 2004, 32(3): 187-189. (in Chinese) [10] Zhan R Z, Fujihara H, Baba H, et al. Ischemic preconditioning is capable of inducing mitochondrial tolerance in the rat brain. Anesthesiology, 2002, 97(4): 896-901. [11] Wang Y S, Xu L, Wang J J, et al. Protective effects of Egb761 against glutamate-induced neurotoxicity in rat retinal neuronal cultures. Chin. Ophthal. Res., 2006, 24(1): 24-26. (in Chinese) [12] Mathur A, Hong Y, Kemp B K, et al. Evaluation of fluorescent dyes for the detection of mitochondrial membrane potential changes in cultured cardiomyocytes. Cardiovasc Res., 2000, 46(1): 126-138. [13] Luo X C, Jiang Y F, Zhang R Q. In vivo dynamic studies of brain metabolism. Tsinghua Science and Technology, 2005, 10(4): 496-498. [14] Cuzzocrea S, Riley D P, Caputi A P, et al. Antioxidant therapy: A new pharmacological approach in shock, inflammation, and ischemia/reperfusion injury. Pharmacol. Rev., 2001, 53(1): 135-159. [15] Hengartner M O. The biochemistry of apoptosis. Nature, 2000, 407(6805): 770-776. [16] Finkel E. The mitochondrion: Is it central to apoptosis? Science, 2001, 292(5517): 624-626. [17] Li M, Xia T, Jiang C S, et al. Cadmium directly induced the opening of membrane permeability pore of mitochondria which possibly involved in cadmium-triggered apoptosis. Toxicology, 2003, 194(1-2): 19-33. [18] Crompton M. On the involvement of mitochondrial intermembrane junctional complexes in apoptosis. Curr. Med. Chem., 2003, 10(16): 1473-1484. [19] Crompton M. The mitochondrial permeability transition pore and its role in cell death. J. Biochem., 1999, 341(Pt 2): 233-249. [20] Lindholm D, Eriksson O, Korhonen L. Mitochondrial proteins in neuronal degeneration. Biochem. Biophys. Res. Commun., 2004, 321(4): 753-758. [21] Panov A V, Andreeva L, Greenamyre J T. Quantitative evaluation of the effects of mitochondrial permeability

[8] Zhang W S, Zhu L Q, Zhang L H, et al. Protective effects

transition pore modifiers on accumulation of calcium

of danshensu on the mitochondria in the nerve cells injured

phosphate: Comparison of rat liver and brain mitochondria.

by hypoxia/hypoglycemia. Journal of Beijing University of

Arch. Biochem. Biophys., 2004, 424(1): 44-52.

JIANG Yufeng (ߠံ‫ )׬‬et al.ġEffect of Salvianolic Acid B on Mitochondrial Function of Cerebral … [22] Duchen M R. Mitochondria and calcium: From cell signalling to cell death. J. Physiol., 2000, 529(Pt 1): 57-68. [23] James A M, Murphy M P. How mitochondrial damage affects cell function. J. Biomed. Sci., 2002, 9(6 Pt 1): 475-487. [24] Verkhratsky A. Physiology and pathophysiology of the calcium store in the endoplasmic reticulum of neurons.

533

[26] Groenendyk J, Lynch J, Michalak M. Calreticulin, Ca2ˇ, and calcineurin-signaling from the endoplasmic reticulum. Mol. Cells, 2004, 17(3): 383-389. [27] Jiang Y F, Wang Q H, Liu Z Q, et al. Effects of salvianolic acid B on cerebral energy charge and activity of ATPase in mice with cerebral ischemia. China Journal of Chinese Materia Medica, 2007, 32(18): 1903-1906. (in Chinese)

Physiol. Rev., 2005, 85: 201-279. [25] Szewczyk A, Wojtczak L. Mitochondria as a pharma-      cological target. Pharmacol. Rev., 2002, 54(1): 101-127.

Tsinghua University Unveils Research Center for Japanese Studies At an inauguration ceremony on April 10, 2009, Tsinghua University unveiled its Research Center for Japanese Studies, an inter-college research institute which aims to promote mutual understanding as well as academic and student exchanges and cooperation between China and Japan through cross-disciplinary research. The Research Center for Japanese Studies will have Co-Chairmen, one from China and one from Japan. Former Vice-Minister of Japan’s Ministry of International Trade and Industry Fukukawa Shinji was announced at the inauguration as the Center’s Chairman from Japan and Tsinghua Vice President Xie Weihe becomes the Center’s Chairman from China. Tsinghua University Professor Qu Delin becomes the Center’s first Director. The Chairman of the Foreign Affairs Committee of the NPC Standing Committee and China’s former Minister of Foreign Affairs Li Zhaoxing, Vice Chairman of the Chinese Peoples’ Association for Friendship with Foreign Countries and Vice Chairman of the China-Japan Friendship Association Jing Dunquan, China’s Former Ambassador to Japan Yang Zhenya, Former Chinese Minister of Culture Liu Deyou, Chairman of the Japan Business Federation and Chairman and CEO of Canon, Inc. Fujio Mitarai, the Japanese Ambassador to China Miyamoto Yuji, the Former Vice-Minister of Japan’s Ministry of International Trade and Industry Fukukawa Shinji, the Chairman of the Japan-China Friendship Center Murakami Tatumi, and the Chairman of Japan-China Economic Association Kiyokawa Yuji all were in attendance at the ceremony. Tsinghua University President Gu Binglin, Tsinghua University Council Chairman Hu Heping, and Tsinghua University Vice President Xie Weihe also attended the event. Tsinghua President Gu, Mr. Jing Dunquan, Mr. Fujio Mitarai, and Mr. Miyamoto Yuji together inaugurated the Center. Tsinghua President Gu Binglin addressed the inauguration audience. He noted that exchanges and cooperation have been further promoted between Tsinghua and Japan in recent years, and he expressed the hope that the Center would develop into a multi-disciplinary platform for high-level research. (From http://news.tsinghua.edu.cn, 2009-04-13)