• Users Online: 2365
  • Home
  • Print this page
  • Email this page


 
 Table of Contents  
RESEARCH AND REPORT
Year : 2014  |  Volume : 9  |  Issue : 3  |  Page : 260-267

Polysaccharides from Angelica sinensis alleviate neuronal cell injury caused by oxidative stress


1 Department of Rehabilitation Medicine, Zhongnan Hospital and Cerebral Vascular Diseases Research Center, Zhongnan Hospital, Wuhan University, Wuhan, Hubei Province, China
2 School of Pharmaceutical Sciences, Wuhan University, Wuhan, Hubei Province, China

Date of Acceptance20-Jan-2014
Date of Web Publication24-Mar-2014

Correspondence Address:
Weijing Liao
Department of Rehabilitation Medicine, Zhongnan Hospital and Cerebral Vascular Diseases Research Center, Zhongnan Hospital, Wuhan University, 169# Donghu Road, Wuhan 430071, Hubei Province
China
Login to access the Email id

Source of Support: This study was supported by the National Natural Science Foundation of China, No. 81072917 and 81274048., Conflict of Interest: None


DOI: 10.4103/1673-5374.128218

Rights and Permissions
  Abstract 

Angelica sinensis has antioxidative and neuroprotective effects. In the present study, we aimed to determine the neuroprotective effect of polysaccharides isolated from Angelica sinensis. In a preliminary experiment, Angelica sinensis polysaccharides not only protected PC12 neuronal cells from H 2 O 2 -induced cytotoxicity, but also reduced apoptosis and intracellular reactive oxygen species levels, and increased the mitochondrial membrane potential induced by H 2 O 2 treatment. In a rat model of local cerebral ischemia, we further demonstrated that Angelica sinensis polysaccharides enhanced the antioxidant activity in cerebral cortical neurons, increased the number of microvessels, and improved blood flow after ischemia. Our findings highlight the protective role of polysaccharides isolated from Angelica sinensis against nerve cell injury and impairment caused by oxidative stress.

Keywords: nerve regeneration; cerebral ischemia; Angelica sinensis; polysaccharides; antioxidation; reactive oxygen species; mitochondrial membrane potential; apoptosis; microvessels; NSFC grant; neural regeneration


How to cite this article:
Lei T, Li H, Fang Z, Lin J, Wang S, Xiao L, Yang F, Liu X, Zhang J, Huang Z, Liao W. Polysaccharides from Angelica sinensis alleviate neuronal cell injury caused by oxidative stress. Neural Regen Res 2014;9:260-7

How to cite this URL:
Lei T, Li H, Fang Z, Lin J, Wang S, Xiao L, Yang F, Liu X, Zhang J, Huang Z, Liao W. Polysaccharides from Angelica sinensis alleviate neuronal cell injury caused by oxidative stress. Neural Regen Res [serial online] 2014 [cited 2019 Aug 21];9:260-7. Available from: http://www.nrronline.org/text.asp?2014/9/3/260/128218

Author contributions: Liao WJ was responsible for the funds, and also conceived and designed the study. Lei T, Li HF, Fang Z, Lin JB, Wang SS, Xiao LY, and Yang F wrote the manuscript, and provided and integrated data. Liu X, Zhang JJ, and Huang ZB analyzed data and revised the manuscript. All authors approved the final version of the manuscript.
Peer review: We chose a common method to identify the micromolecule, non-simple component polysaccharide extracted from Angelica sinensis. Both in vivo and in vitro experiments showed that, polysaccharide improved neuronal injury caused by oxidative stress and had potential effect in the studies addressing neuronal impairment in human brain.



  Introduction Top


Cerebral ischemia, induced by cerebral artery occlusion, dramatically decreases local cerebral blood flow, leading to cell death and functional deficits in the brain [1],[2],[3],[4] . Reactive oxygen radicals are involved in cerebral ischemia and reperfusion [5] . During reperfusion, reoxygenation from restored blood flow provides oxygen for enzymatic oxidation reactions in neurons and their subcellular organelles [6],[7] , including mitochondria [8],[9] . Reactive oxygen species are constantly produced in both cerebral ischemia and reperfusion processes [10] , and it has been well documented that in both processes reactive oxygen species cause cell death, both directly, through oxidative damage, and indirectly, by reactive oxygen species signaling pathways [11] . Although reperfusion may cause neuronal damage [12] , it is able to rescue cells and tissues. The rescue processes, which include preservation of surviving cells, restoration of impaired tissues and recuperation of brain functions, largely depend on restoration of local blood supply. Microvessels play an important role in providing blood supply to brain tissues [13] . But therapeutic trials of agents that protect against reperfusion injury have not yet shown consistent benefit [14],[15] , and there remains a need for the development of novel pharmacologic treatments.

According to traditional Chinese medicine, the roles of Angelica sinensis correlate with tonifying the blood and promoting its circulation [16] . Recent studies have shown that extracts of Angelica sinensis have antioxidative and neuroprotective effects [17],[18] . Injection of preparations from Angelica sinensis alleviate sciatic nerve crush injury and diabetic peripheral neuropathy [19],[20] , and recent evidence also suggests that a number of pharmacological effects of Angelica sinensis are closely associated with its polysaccharide fractions [21] . The polysaccharides from Angelica sinensis roots have immunomodulatory [22] , antitumor [23],[24] , and hematopoietic effects [16] ; however, the anti-oxidative function of Angelica sinensis polysaccharide (ASP) has rarely been addressed. The present study aims to determine whether ASPs exert protective effects against oxidative damage in vitro and, if so, whether antioxidant activity would also be observed in vivo, in rats with focal cerebral ischemia.


  Results Top


ASP inhibited H 2 O 2 -induced cytotoxicity in PC12 cells

We performed a 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay in the PC12 neuronal cell line. As expected, treatment with H 2 O 2 reduced cell viability to about 33% of that of untreated cells. However, pre-incubation with 0.1, 0.2, 0.4 or 0.8 mg/mL of the < 20 kDa ASP fraction increased the viability of H 2 O 2 -treated cells to 34%, 37%, 42% and 52% of that of untreated cells, respectively (P < 0.05; [Figure 1]A). When the cells were pretreated with the > 20 kDa ASP fraction at the same concentrations and under the same conditions, the viability of H 2 O 2 -treated PC12 cells remained unchanged (data not shown). These results demonstrate that the < 20 kDa ASP fraction is capable of protecting PC12 cells from H 2 O 2 -induced injury. Therefore, this fraction was used in the subsequent experiments.
Figure 1: Effect of pretreatment of Angelica sinensis polysaccharide (ASP) on 2O2-induced cytotoxicity, reactive oxygen species (ROS) accumulation, and reduction in mitochondrial membrane potential (MMP) in PC12 cells.
(A) Pretreatment of PC12 cells with ASP reduces H2O2-induced cytotoxicity (MTT assay). Relative cell viability is calculated as the percentage of viable cells in the experimental groups over that in the control group (no 2O2 or ASP; taken as 100%, not shown). (B)Pretreatment of PC12 cells with ASP inhibits 2O2-induced ROS accumulation. The level of intracellular ROS is presented as the fold change of fluorescence intensity in ASP-pretreated cells compared with control cells not exposed to 2O2 or ASP (taken as 1, not shown). (C)Pretreatment with ASP inhibits 2O2-in­duced reduction in MMP in PC12 cells. The mean rhodamine 123 fluorescence intensity of 1 × 104 cells was measured on a flow cytometer with the excitation wavelength at 488 nm and the emission wavelength at 520 nm. Mean fluorescence intensity of ASP-pretreated cells is expressed as a per­centage of that of control cells (no exposure to 2O2or ASP; taken as 100%, not shown). Data are expressed as mean ± SEM, and the experiment was repeated in triplicate. Data were analyzed by one-way analysis of variance and the least significant difference test for pairwise comparison. aP < 0.05, vs. 0 mg/mL ASP.


Click here to view


ASP reduced H 2 O 2 -induced reactive oxygen species accumulation in PC12 cells

Fluorescence results showed that treatment with H 2 O 2 significantly increased intracellular reactive oxygen species levels to 5.2-fold those of untreated cells. However, when the cells were pretreated with 0.1, 0.2, 0.4 or 0.8 mg/mL ASP, the level of H 2 O 2 -induced reactive oxygen species accumulation was reduced, respectively, to 3.6-, 3.0-, 2.5- and 1.8-fold that of control cells (P < 0.05), in a concentration-dependent manner ([Figure 1]B).

ASP inhibited H 2 O 2 -induced reduction of mitochondrial membrane potential (MMP) in PC12 cells

As mitochondria are a critical target of oxidative damage [25] , we tested the effect of ASP on H 2 O 2 -induced MMP changes using rhodamine 123, a fluorescent dye highly specific for mitochondria, which actively accumulates in living cells in direct proportion to the MMP [26] . MMP was significantly reduced to 75% of that of normal cells after exposure to H 2 O 2 alone. ASP pretreatment significantly inhibited this H 2 O 2 -induced decrease in MMP at all concentrations tested (0.1-0.8 mg/mL), resulting in MMP values of 84-91% of those of control cells (P < 0.05; [Figure 1]C).

ASP protected PC12 cells against H 2 O 2 -induced apoptosis

MMP is an early indicator of apoptosis [27] . We determined the effect of ASP on H 2 O 2 -induced apoptosis in PC12 cells using propidium iodide staining and flow cytometry. The apoptosis rate of control cells was 1.0% ([Figure 2]A) while that of the cells treated with H 2 O 2 alone was 10.9% ([Figure 2]B). When the cells were pre-incubated with 0.1, 0.2, 0.4 or 0.8 mg/mL ASP and then stressed with H 2 O 2 , apoptosis rates were reduced to 7.0%, 5.9%, 5.7% and 3.3%, respectively ([Figure 2]C-F). These data demonstrate that ASP is capable of protecting PC12 cells from H 2 O 2 -induced apoptosis.
Figure 2: Pretreatment with Angelica sinensis polysaccharide (ASP) prevented H2O2-induced apoptosis in PC12 cells, as measured using flow cytometry.
(A) Control cells; (B) cells exposed to H2O2 alone; (C-F) cells treated with 0.1 (C), 0.2 (D), 0.4 (E) or 0.8 (F) mg/mL ASP before incubation with H2O2.


Click here to view


Effect of ASP on antioxidant enzyme activity and lipid peroxidation level in cortical tissue of rats with focal cerebral ischemia

The above results clearly demonstrate the antioxidant activity of ASP in vitro. To examine the antioxidative effect of ASP in vivo, we determined the activities of antioxidant enzymes and the level of lipid peroxidation in a rat model of focal cerebral ischemia [28] . At 7 days after cerebral artery occlusion, the activities of the antioxidant enzymes superoxide dismutase and glutathione peroxidase were significantly lower in the cortical tissue of rats with cerebral ischemia than in that of control rats (P < 0.05). When middle cerebral artery-occluded rats were injected with ASP for 7 days, both superoxide dismutase and glutathione peroxidase activities were significantly greater than those in the model rats that were not injected with ASP (P < 0.05). Conversely, the level of malondialdehyde, a lipid oxidation product, was significantly greater in model rats compared with control rats (P < 0.05). When the rats with focal cerebral ischemia were injected with ASP, the level of malondialdehyde was lower than that in the model group (P < 0.05; [Table 1]).
Table 1: Effect of Angelica sinensis polysaccharides on antioxidant enzyme activity (U/mg) and malondialdehyde content (nmol/mg) in rats with middle cerebral artery occlusion

Click here to view


ASP increased the number of microvessels in the brain of rats with cerebral ischemia

Immunohistochemical staining showed that the number of microvessels in the brain of ASP-treated rats was much higher than that in the focal cerebral ischemia model rats, which in turn was higher than that in the control rats ([Figure 3]A-C).
Figure 3: Effect of Angelica sinensis polysaccharide (ASP) on CD31-positive cerebral microvessels and FITC-dextran-perfused cerebral microvessels in focal cerebral ischemia rats (× 400).
(A-C) CD31-positive cerebral microvessels (immunohistochemical staining). Control (A), model (B) and ASP-treated (C) groups. The number of microvessels (arrows) in rats treated with ASP was higher than that in the model group. The control group had fewest microvessels. (D-F) FITC-dextran-perfused cerebral microvessels (laser scanning confocal microscopy). Green fluorescence shows perfused microvessels, which were abundant in normal rats (D), sparse after focal cerebral ischemia (E), and abundant after treatment with ASP (F).


Click here to view


Under a laser scanning confocal microscope, the perfused microvessels in normal rat brain were abundant and homogeneously distributed. After focal cerebral ischemia, they became sparse and heterogeneously distributed. However, when ASP was administered to rats with focal cerebral ischemia, the density and distribution of perfused microvessels in the brain were noticeably protected ([Figure 3]D-F).


  Discussion Top


The polysaccharides from Angelica sinensis have a number of pharmacological activities. For example, a polysaccharide fraction from Angelica sinensis root has a strong immunomodulatory effect, facilitating the growth of murine peritoneal macrophages and regulating the expression of Th1 and Th2 related cytokines [22] . ASPs are also cardioprotective, limiting ischemia/reperfusion-induced myocardial injury [29] . Three polysaccharides isolated from Angelica sinensis are all found to have antioxidative activities in H 2 O 2 -injured macrophages [30] . Here, using PC12 cells, we found that the < 20 kDa ASP was capable of protecting cells from H 2 O 2 -induced injury, MMP reduction, and apoptosis. We further demonstrated the antioxidative effect of ASP on a rat model of middle cerebral artery occlusion; activities of antioxidant enzymes (superoxide dismutase and glutathione peroxidase) were significantly greater and the level of the lipid peroxidation product malondialdehyde was lower than in the model group, similarly to the effects of ASP previously shown in middle-aged women and in rabbits with cerebral ischemia reperfusion injury [31],[32] . Together, these data indicate that ASP not only protects PC12 neuronal cells from H 2 O 2 -induced oxidative and apoptotic injury, but also promotes recovery of middle cerebral artery-occluded rats from cerebral ischemia and reperfusion damage, suggesting that ASP has potential as a neuroprotective agent.

It has previously been shown that the polysaccharide from the brown marine alga Sargassum stenophyllum can inhibit the activity of heparin-binding vascular growth factors during microvessel formation [33] . The polysaccharides from Antrodia cinnamomea also have anti-angiogenic activity through immunomodulation [34] . Therefore, this type of polysaccharide is likely to have anticancer activity. Conversely, low molecular weight fucoidan stimulates therapeutic revascularization in critical hindlimb and ischemia migration [35] , and the polysaccharide isolated from Bletilla striata induces proliferation of endothelial cells and expression of vascular endothelial growth factor in vitro[36] . Interestingly, the hot-water extract of Angelica sinensis root has shown angiogenic potential by stimulating the expression of vascular endothelial growth factor in myocardium [37] . In the present study, we found that polysaccharides isolated from hot-water extract of Angelica sinensis root promoted recovery of microvessels from cerebral ischemia. This evidence demonstrates the angiogenesis-promoting potential of these polysaccharides. However, a crude extract of Angelica sinensis root also has an angiogenesis-inhibiting effect [38] . Other extracts, such as resveratrol from red grapes, have seemingly contradictory effects, promoting or inhibiting angiogenesis, depending on different disease contexts [39],[40] . Therefore, it will be of great interest to further investigate the underlying mechanisms of ASP in the promotion or inhibition of angiogenesis under specific disease conditions.

Brain cells die rapidly when they are deprived of blood supply [41] , and their high degree of specialization makes it difficult for the remaining live cells to assume the functions of the dead cells in the brain [42],[43] . Surgical decompression may be used within 48 hours from stroke onset to reduce fatality and improve clinical outcome, but many survivors are left with disabilities and depression [44] . Therefore, efforts have been made to find therapeutic strategies to tackle such problems. For example, recombinant tissue plasminogen activators are used to break down blood clots, indicating that intravenous thrombolysis is a promising approach to improve clinical outcome after acute ischemic stroke. However, this type of treatment is only effective and safe for patients within a few hours of onset of acute stroke [45] . Growth factors are also used in the recovery process after ischemic stroke because of their angiogenesis- and neurogenesis-promoting capacities, although further studies are still needed [3] . Recent research has revealed that ASPs promote blood production by stimulating secretion of interleukin-6, granulocyte-macrophage colony-stimulating factor and CD34 + cells [46],[47] . In the present study, we found that ASP significantly prevented the reduction in microvessel quantity after middle cerebral arterial occlusion in rats. Using laser scanning confocal microscopy, we further found that ASP improved the perfusion and distribution of microvessels in rats with middle cerebral arterial occlusion. These findings suggest that ASP not only helps increase the quantity of microvessels in rats with middle cerebral arterial occlusion, but also improves microvascular perfusion, indicating its potential to promote recovery from cerebral ischemia.

The limitation of this study is lack of the quantification on the microvessels and the investigation on the mechanism.

In conclusion, polysaccharides isolated from Angelica sinensis root inhibit the increase of intracellular reactive oxygen species and prevent the decrease in mitochondrial membrane potential in H 2 O 2 -treated PC12 cells, as well as protecting cells from H 2 O 2 -induced cytotoxicity and apoptosis. Importantly, we also showed that ASP has an antioxidant effect in vivo and increases the quantity and perfusion of microvessels in rats with middle cerebral arterial occlusion. This evidence reveals the potential of ASP in facilitating recovery from cerebral ischemia and reperfusion injury. Together, our results demonstrate the neuroprotective effects of ASPs and provide an insight into the potential of these and other polysaccharides as treatment for ischemic stroke.


  Materials and Methods Top


Design

A randomized, controlled in vitro and in vivo study.

Time and setting

The in vitro experiment was carried out at the School of Pharmaceutical Sciences, Wuhan University, China, and the animal experiment was performed at the Experimental Animal Center of Wuhan University, China from March 2012 to May 2013.

Materials

Cells0

The rat pheochromocytoma cell line PC12 was obtained from the American Type Culture Collection (ATCC).

Animals

Healthy male Sprague-Dawley rats, aged 3-4 months, weighing 200-250 g, were obtained from the Experimental Animal Center of Wuhan University, China (No. 4200500164). The animals were housed at 22°C and 55 ± 5% relative humidity with a regular 12-hour light/dark cycle, and standard diet and water available ad libitum. All animals were acclimatized to housing conditions for 1 week before the experiment. The protocols were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China [48] . A total of 55 rats were included in the study. The animals were randomly divided into three groups: control (n = 15), focal cerebral ischemia model (n = 20) and focal cerebral ischemia model with ASP treatment (n = 20). The latter group received ASP intraperitoneally (i.p.) after focal cerebral ischemia. An equivalent volume of normal saline was administered to the two other groups. Five rats in each of the model and ASP groups died during the experiment.

Drugs

ASP was extracted and isolated as described previously [49] . Sliced Angelica sinensis roots were purchased from Tongrentang Group (Beijing, China), identified by Professor Zebo Huang (Guangdong Pharmaceutical University, China), ground and passed through a 0.9 mm mesh sieve. The powder was extracted with 80% ethanol at 70°C, and, after removal of the solvent, the materials were immersed overnight in distilled water at room temperature. Water extraction was then performed at 80°C, and the extract was centrifuged at 1,000 r/min for 10 minutes. The supernatant was collected and concentrated under reduced pressure at 45°C, and the concentrated solution was precipitated with four volumes of ethanol. The precipitate was redissolved in distilled water and precipitated with ethanol another three times to further remove small molecules. After collection by filtration, the materials were redissolved in distilled water and potential protein contaminants were removed using the Sevag method. The polysaccharide solution was freeze-dried, redissolved in water and dialysed (20 kDa molecular weight cut-off). The dialysate and retentate were collected to obtain polysaccharides of < 20 kDa and > 20 kDa, respectively, by concentration under reduced pressure and then lyophilization.

Methods

PC12 cell culture

PC12 cells were maintained at 37°C in an atmosphere of 5% CO 2 in RPMI 1640 medium supplemented with 5% fetal bovine serum, 10% horse serum (Hyclone, Logan, Utah, USA), 100 U/mL penicillin and 100 mg/mL streptomycin [50] . The culture medium was replaced with fresh medium every other day, and the PC12 cells were passaged by trypsinization when the confluence reached about 80%.

PC12 cell viability determined by MTT assay

The viability of PC12 cells was determined using the MTT assay as previously described [51] . Briefly, 100 μL of cells (1 × 10 5 /mL) were incubated at 37°C in 96-well plates (Nest, Wuxi, Jiangsu Province, China) for 24 hours, and treated with 10 μL of polysaccharide at various concentrations (0.1-0.8 mg/mL) for 15 minutes. After the addition of 10 μL H 2 O 2 (final concentration, 150 μmol/L), the cells were further incubated for 24 hours at 37°C. To determine cell viability, 10 μL of 5 mg/mL MTT (Amersco, Solon, Ohio, USA) was added to each well and the plates were incubated at 37°C for 3 hours. The medium was then carefully removed, and the formazan crystals were dissolved in 150 μL of dimethyl sulfoxide by gentle shaking of the plate. Absorbance was determined with a spectrometer (Thermo Fisher, Waltham, MA, USA) at a wavelength of 570 nm.

Fluorescence detection of intracellular reactive oxygen species levels in PC12 cells

2 mL of PC12 cells (3 × 10 5 /mL) were seeded in 6-well plates and incubated at 37°C for 24 hours. After the medium was removed, the cells were washed twice with PBS and incubated with 200 μL of polysaccharide at various concentrations (0.1-0.8 mg/mL) in serum-free RPMI 1640 medium for 15 minutes. H 2 O 2 (20 μL, final concentration 150 μmol/L) containing dichloro-dihydro-fluorescein diacetate (DCFH-DA; final concentration 10 μmol/L; Beyotime, Nantong, Jiangsu Province, China) was added to each well, and the cells were incubated for 30 minutes. The cells were collected carefully and washed twice with ice cold PBS. The fluorescence intensity was measured on a flow cytometer (Epics Altra II, Beckman, Brea, CA, USA) with the excitation wavelength at 488 nm and the emission wavelength at 520 nm. The relative reactive oxygen species level (fold change) was expressed as the ratio of fluorescence intensity at 520 nm in an experimental group to that in the normal group [52].

MMP in PC12 cells determined by rhodamine 123

The MMP in PC12 cells was assessed using the fluorescent cationic dye rhodamine 123 as previously described [53]. The PC12 cells were treated with polysaccharide as above, and then incubated with 10 μmol/L of rhodamine 123 (Beyotime) for 30 minutes. The intensity of rhodamine 123 fluorescence was measured using flow cytometry. MMP was expressed as relative mean fluorescence intensity, which was the percentage of the mean fluorescence intensity (per 1 × 10 4 cells) of an experimental group over that of normal cells.

Detection of PC12 cell apoptosis using flow cytometry

Apoptotic cells were quantified using propidium iodide staining [54] . In brief, about 6 × 10 5 cells in 2 mL of medium were cultured in 6-well plates at 37°C for 24 hours. Then 200 μL of polysaccharides were added to the wells at the indicated concentrations (0.1-0.8 mg/mL) and incubated for 15 minutes. After addition of 200 μL of H 2 O 2 (final concentration 150 μmol/L), the cells were further incubated for 24 hours. The cells were then collected, resuspended in ice cold 70% ethanol, and placed at 4°C for 16 hours. The cells were collected by centrifugation, washed twice with PBS, resuspended in 1 mL of PBS containing 50 μg/mL propidium iodide (Sigma, St. Louis, MO, USA), and incubated at 4°C for 30 minutes. The red fluorescence for DNA was measured in about 1 × 10 4 cells using flow cytometry with the excitation wavelength set at 488 nm.

Preparation of focal cerebral ischemia model

Focal cerebral ischemia was introduced in the rats using the middle cerebral arterial occlusion model as previously described [55] . After the rats were anesthetized with chloral hydrate (350 mg/kg, i.p.), the right middle cerebral artery was occluded by inserting a nylon suture through the right common carotid artery into the internal carotid artery, up to about 17 mm from the bifurcation of the artery. The nylon suture was withdrawn after 2 hours. During the surgical procedure, body temperature was kept constant at 37.0 ± 0.5°C using a heat pad connected to a rectal probe. After revival from anesthesia, animals were returned to their home cages.

Injection of ASP

The polysaccharides were dissolved in 1 mL normal saline and a dose of 200 mg/kg i.p. was administered to animals in the ASP group at 2, 26, 50, 74, 98, 122 and 146 hours after middle cerebral artery occlusion, while an equal volume (1 mL) of normal saline was administered to animals in the model group.

Determination of antioxidant enzyme activity and lipid peroxidation level in cortex tissue of focal cerebral ischemic rats

Seven days after middle cerebral artery occlusion, six rats from each group were anesthetized and perfused transcardially with 250 mL of 0.9% saline, and the cortex around the penumbra was dissected. The cortical tissue (20 mg) was then homogenized in 2 mL of ice cold PBS for 10 minutes using a glass homogenizer and centrifuged at 12,000 × g for 10 minutes. The supernatant was collected to determine the activities of superoxide dismutase and glutathione peroxidase as well as the content of malondialdehyde, using colorimetry as previously described [56] . Protein content was determined using the Bradford method [57] . The Total Superoxide Dismutase Assay Kit, Cellular Glutathione Peroxidase Assay Kit, Lipid Peroxidation MDA Assay Kit and Bradford Protein Assay Kit were purchased from Beyotime.

Immunohistochemical visualization of microvessels

At 7 days after ischemia, six rats from each group were anesthetized and perfused transcardially with 250 mL of 0.9% saline and 250 mL of 4% paraformaldehyde. The brains were removed and immersed in 4% paraformaldehyde overnight, embedded in paraffin, and sectioned (6 μm thickness). Immunohistochemistry was performed using a streptavidin peroxidase kit (Maixin Bio, Fuzhou, Fujian Province, China). Goat anti-platelet endothelial cell adhesion molecule-1 (CD31) polyclonal antibody (1:100; Santa Cruz Biotechnology, Dallas, TX, USA) was used to detect blood vessels. The brain sections were incubated overnight with CD31 primary antibody at 4°C, and then with anti-goat IgG-TRITC antibody (1:400; Maixin Bio) at room temperature. For each animal, CD31-positive microvessels were quantified from 15 different fields of view (400 × magnification) around the marginal zone of the infarct region according to Weidner's counting methods [58].

Observation of microvessels by laser scanning confocal microscopy

Seven days after ischemia, the remaining rats were anesthetized with chloral hydrate and then injected with 2 mL of 50 mg/mL FITC-dextran (Sigma) into the left ventricle. After 1 minute, the brains were rapidly removed and placed in 4% paraformaldehyde at 4°C for 48 hours, before being transferred to PBS. Coronal sections (300 μm thickness) close to the optic chiasma were cut while the brains remained immersed in PBS. The cut coronal sections were analyzed using a laser scanning confocal microscope (Leica TCS-SP2-AOBS-MP, Wetzlar, Germany) at 488 nm (excitation wave). The areas of ischemic penumbra in the infarct cortex were located under 10 × magnification, and scanned along the z-axis with a 1 μm step under 40 × magnification with a 512 × 512 matrix in the x-y direction. The area analyzed each time was 750 × 750 μm 2[59].

Statistical analysis

Data are expressed as mean ± SEM and were analyzed by one-way analysis of variance with the least significance difference test for pairwise comparison. A value of P < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 15.0 for Windows (SPSS, Chicago, IL, USA).

 
  References Top

1.Go AS, Mozaffarian D, Roger VL, et al. Heart disease and stroke statistics--2013 update: a report from the American Heart Association. Circulation. 2013;127(1):e6-245.  Back to cited text no. 1
    
2.Jiang B, Wang WZ, Chen H, et al. Incidence and trends of stroke and its subtypes in China: results from three large cities. Stroke. 2006;37(1):63-68.   Back to cited text no. 2
    
3.Liu M, Wu B, Wang WZ, et al. Stroke in China: epidemiology, prevention, and management strategies. Lancet Neurol. 2007;6(5): 456-464.  Back to cited text no. 3
    
4.Chen H, Spagnoli F, Burris M, et al. Nanoerythropoietin is 10-times more effective than regular erythropoietin in neuroprotection in a neonatal rat model of hypoxia and ischemia. Stroke. 2012;43(3): 884-887.   Back to cited text no. 4
    
5.Muralikrishna Adibhatla R, Hatcher JF. Phospholipase A2, reactive oxygen species, and lipid peroxidation in cerebral ischemia. Free Radic Biol Med. 2006;40(3):376-387.   Back to cited text no. 5
    
6.Bigdeli MR. Preconditioning with prolonged normobaric hyperoxia induces ischemic tolerance partly by upregulation of antioxidant enzymes in rat brain tissue. Brain Res. 2009;1260:47-54.   Back to cited text no. 6
    
7.He X, Sandhu HK, Yang Y, et al. Neuroprotection against hypoxia/ischemia: ä-opioid receptor-mediated cellular/molecular events. Cell Mol Life Sci. 2013;70(13):2291-2303.   Back to cited text no. 7
    
8.Sims NR, Muyderman H. Mitochondria, oxidative metabolism and cell death in stroke. Biochim Biophys Acta. 2010;1802(1):80-91.  Back to cited text no. 8
    
9.Manzanero S, Santro T, Arumugam TV. Neuronal oxidative stress in acute ischemic stroke: sources and contribution to cell injury. Neurochem Int. 2013;62(5):712-718.   Back to cited text no. 9
    
10.Chen SD, Yang DI, Lin TK, et al. Roles of oxidative stress, apoptosis, PGC-1α and mitochondrial biogenesis in cerebral ischemia. Int J Mol Sci. 2011;12(10):7199-7215.   Back to cited text no. 10
    
11.Love S. Oxidative stress in brain ischemia. Brain Pathol. 1999;9(1): 119-131.  Back to cited text no. 11
    
12.Davis S, Campbell B, Christensen S, et al. Perfusion/Diffusion mismatch is valid and should be used for selecting delayed interventions. Transl Stroke Res. 2012;3(2):188-197.  Back to cited text no. 12
    
13.Fassbender JM, Whittemore SR, Hagg T. Targeting microvasc- ulature for neuroprotection after SCI. Neurotherapeutics. 2011;8(2): 240-251.   Back to cited text no. 13
    
14.Rhim T, Lee DY, Lee M. Drug delivery systems for the treatment of ischemic stroke. Pharm Res. 2013;30(10):2429-2444.  Back to cited text no. 14
    
15.Kandilis AN, Karidis NP, Kouraklis G, et al. Proteasome inhibitors: possible novel therapeutic strategy for ischemia-reperfusion injury? Expert Opin Investig Drugs. 2014;23(1):67-80.  Back to cited text no. 15
    
16.Liu PJ, Hsieh WT, Huang SH, et al. Hematopoietic effect of water-soluble polysaccharides from Angelica sinensis on mice with acute blood loss. Exp Hematol. 2010;38(6):437-445.  Back to cited text no. 16
    
17.Kuang X, Yao Y, Du JR, et al. Neuroprotective role of Z-ligustilide against forebrain ischemic injury in ICR mice. Brain Res. 2006; 1102(1):145-153.  Back to cited text no. 17
    
18.Xin J, Zhang J, Yang Y, et al. Radix Angelica Sinensis that contains the component Z-ligustilide promotes adult neurogenesis to mediate recovery from cognitive impairment. Curr Neurovasc Res. 2013;10(4):304-315.  Back to cited text no. 18
    
19.Cui Q, Zhang J, Zhang L, et al. Angelica injection improves functional recovery and motoneuron maintenance with increased expression of brain derived neurotrophic factor and nerve growth factor. Curr Neurovasc Res. 2009;6(2):117-123.  Back to cited text no. 19
    
20.Li R, Zhang J, Zhang L, et al. Angelica injection promotes peripheral nerve structure and function recovery with increased expressions of nerve growth factor and brain derived neurotrophic factor in diabetic rats. Curr Neurovasc Res. 2010;7(3):213-222.  Back to cited text no. 20
    
21.Jin ML, Zhao K, Huang QS, et al. Isolation, structure and bioactivities of the polysaccharides from Angelica sinensis (Oliv.) Diels: a review. Carbohydr Polym. 2012;89(3):713-722.  Back to cited text no. 21
    
22.Yang T, Jia M, Meng J, et al. Immunomodulatory activity of polysaccharide isolated from Angelica sinensis. Int J Biol Macromol. 2006;39(4-5):179-184.  Back to cited text no. 22
    
23.Cao W, Li XQ, Liu L, et al. Structure of an anti-tumor polysaccharide from Angelica sinensis (Oliv.) Diels. Carbohydr Polym. 2006; 66(2):149-159.  Back to cited text no. 23
    
24.Cao W, Li XQ, Wang X, et al. A novel polysaccharide, isolated from Angelica sinensis (Oliv.) Diels induces the apoptosis of cervical cancer HeLa cells through an intrinsic apoptotic pathway. Phytomedicine. 2010;17(8-9):598-605.  Back to cited text no. 24
    
25.Herrmann JM, Riemer J. Oxidation and reduction of cysteines in the intermembrane space of mitochondria: multiple facets of redox control. Antioxid Redox Signal. 2010;13(9):1323-1326.  Back to cited text no. 25
    
26.Ly JD, Grubb DR, Lawen A. The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update. Apoptosis. 2003;8(2):115-128.  Back to cited text no. 26
    
27.Kroemer G. Mitochondrial control of apoptosis: an introduction. Biochem Biophys Res Commun. 2003;304(3):433-435.  Back to cited text no. 27
    
28.Chauhan A, Sharma U, Jagannathan NR, et al. Rapamycin protects against middle cerebral artery occlusion induced focal cerebral ischemia in rats. Behav Brain Res. 2011;225(2):603-609.  Back to cited text no. 28
    
29.Zhang S, He B, Ge J, et al. Extraction, chemical analysis of Angelica sinensis polysaccharides and antioxidant activity of the polysaccharides in ischemia-reperfusion rats. Int J Biol Macromol. 2010; 47(4):546-550.   Back to cited text no. 29
    
30.Yang XB, Zhao Y, Lv Y. In vivo macrophage activation and physicochemical property of the different polysaccharide fractions purified from Angelica sinensis. Carbohydr Polym. 2008;71(3):372-379.  Back to cited text no. 30
    
31.Ai S, Fan X, Fan L, et al. Extraction and chemical characterization of Angelica sinensis polysaccharides and its antioxidant activity. Carbohydr Polym. 2013;94(2):731-736.  Back to cited text no. 31
    
32.Jiang J, Guo YJ, Niu AJ. Extraction, characterization of Angelica sinensis polysaccharides and modulatory effect of the polysaccharides and Tai Chi exercise on oxidative injury in middle-aged women subjects. Carbohydr Polym. 2009;77(2):384-388.  Back to cited text no. 32
    
33.Dias PF, Siqueira JM Jr, Vendruscolo LF, et al. Antiangiogenic and antitumoral properties of a polysaccharide isolated from the seaweed Sargassum stenophyllum. Cancer Chemother Pharmacol. 2005;56(4):436-446.  Back to cited text no. 33
    
34.Yang CM, Zhou YJ, Wang RJ, et al. Anti-angiogenic effects and mechanisms of polysaccharides from Antrodia cinnamomea with different molecular weights. J Ethnopharmacol. 2009;123(3):407-412.  Back to cited text no. 34
    
35.Luyt CE, Meddahi-Pellé A, Ho-Tin-Noe B, et al. Low-molecular-weight fucoidan promotes therapeutic revascularization in a rat model of critical hindlimb ischemia. J Pharmacol Exp Ther. 2003;305(1):24-30.  Back to cited text no. 35
    
36.Wang C, Sun J, Luo Y, et al. A polysaccharide isolated from the medicinal herb Bletilla striata induces endothelial cells proliferation and vascular endothelial growth factor expression in vitro. Biotechnol Lett. 2006;28(8):539-543.  Back to cited text no. 36
    
37.Meng H, Guo J, Sun JY, et al. Angiogenic effects of the extracts from Chinese herbs: Angelica and Chuanxiong. Am J Chin Med. 2008;36(3):541-554.  Back to cited text no. 37
    
38.Hui MK, Wu WK, Shin VY, et al. Polysaccharides from the root of Angelica sinensis protect bone marrow and gastrointestinal tissues against the cytotoxicity of cyclophosphamide in mice. Int J Med Sci. 2006;3(1):1-6.  Back to cited text no. 38
    
39.Bråkenhielm E, Cao R, Cao Y. Suppression of angiogenesis, tumor growth, and wound healing by resveratrol, a natural compound in red wine and grapes. FASEB J. 2001;15(10):1798-1800.  Back to cited text no. 39
    
40.Kaga S, Zhan L, Matsumoto M, et al. Resveratrol enhances neovascularization in the infarcted rat myocardium through the induction of thioredoxin-1, heme oxygenase-1 and vascular endothelial growth factor. J Mol Cell Cardiol. 2005;39(5):813-822.  Back to cited text no. 40
    
41.Guo C, Zhu Y, Weng Y, et al. Therapeutic time window and underlying therapeutic mechanism of breviscapine injection against cerebral ischemia/reperfusion injury in rats. J Ethnopharmacol. 2014;151(1):660-666.  Back to cited text no. 41
    
42.Reynolds MR, Grubb RL Jr, Clarke WR, et al. Investigating the mechanisms of perioperative ischemic stroke in the Carotid Occlusion Surgery Study. J Neurosurg. 2013;119(4):988-995.  Back to cited text no. 42
    
43.Winocur G, Thompson C, Hakim A, et al. The effects of high- and low-risk environments on cognitive function in rats following 2-vessel occlusion of the carotid arteries: a behavioral study. Behav Brain Res. 2013;252:144-156.  Back to cited text no. 43
    
44.Hofmeijer J, Kappelle LJ, Algra A, et al. Surgical decompression for space-occupying cerebral infarction (the Hemicraniectomy After Middle Cerebral Artery infarction with Life-threatening Edema Trial [HAMLET]): a multicentre, open, randomised trial. Lancet Neurol. 2009;8(4):326-333.  Back to cited text no. 44
    
45.Ahmed N, Wahlgren N, Grond M, et al. Implementation and outcome of thrombolysis with alteplase 3-4.5 h after an acute stroke: an updated analysis from SITS-ISTR. Lancet Neurol. 2010;9(9): 866-874.  Back to cited text no. 45
    
46.Liu PJ, Hsieh WT, Huang SH, et al. Hematopoietic effect of water-soluble polysaccharides from Angelica sinensis on mice with acute blood loss. Exp Hematol. 2010;38(6):437-445.  Back to cited text no. 46
    
47.Lee JG, Hsieh WT, Chen SU, et al. Hematopoietic and myeloprotective activities of an acidic Angelica sinensis polysaccharide on human CD34 + stem cells. J Ethnopharmacol. 2012;139(3):739-745.  Back to cited text no. 47
    
48.The Ministry of Science and Technology of People's Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.  Back to cited text no. 48
    
49.Zhang H, Pan N, Xiong S, et al. Inhibition of polyglutamine-mediated proteotoxicity by Astragalus membranaceus polysaccharide through the DAF-16/FOXO transcription factor in Caenorhabditis elegans. Biochem J. 2012;441(1):417-424.  Back to cited text no. 49
    
50.Lu Y, Li T, Zhao X, et al. Electrodeposited polypyrrole/carbon nanotubes composite films electrodes for neural interfaces. Biomaterials. 2010;31(19):5169-5181.  Back to cited text no. 50
    
51.Sharifi AM, Eslami H, Larijani B, et al. Involvement of caspase-8, -9, and -3 in high glucose-induced apoptosis in PC12 cells. Neurosci Lett. 2009;459(2):47-51.  Back to cited text no. 51
    
52.Cho ES, Jang YJ, Hwang MK, et al. Attenuation of oxidative neuronal cell death by coffee phenolic phytochemicals. Mutat Res. 2009; 661(1-2):18-24.  Back to cited text no. 52
    
53.Meng JL, Mei WY, Dong YF, et al. Heat shock protein 90 mediates cytoprotection by H 2 S against chemical hypoxia-induced injury in PC12 cells. Clin Exp Pharmacol Physiol. 2011;38(1):42-49.  Back to cited text no. 53
    
54.Ji BS, Gao Y. Protective effect of trihexyphenidyl on hydrogen peroxide-induced oxidative damage in PC12 cells. Neurosci Lett. 2008;437(1):50-54.  Back to cited text no. 54
    
55.Zhou Q, Zhang Q, Zhao X, et al. Cortical electrical stimulation alone enhances functional recovery and dendritic structures after focal cerebral ischemia in rats. Brain Res. 2010;1311:148-157.  Back to cited text no. 55
    
56.Li HF, Xu J, Liu YM, et al. Antioxidant and moisture-retention activities of the polysaccharide from Nostoc commune. Carbohydr Polym. 2011;83(4):1821-1827.  Back to cited text no. 56
    
57.Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254.  Back to cited text no. 57
    
58.Weidner N, Semple JP, Welch WR, et al. Tumor angiogenesis and metastasis--correlation in invasive breast carcinoma. N Engl J Med. 1991;324(1):1-8.  Back to cited text no. 58
    
59.Zhang ZG, Zhang L, Jiang Q, et al. VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain. J Clin Invest. 2000;106(7):829-838.  Back to cited text no. 59
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1]


This article has been cited by
1 Comparisons of the Effectiveness and Safety of Tuina, Acupuncture, Traction, and Chinese Herbs for Lumbar Disc Herniation: A Systematic Review and Network Meta-Analysis
Zhuomao Mo,Dong Li,Renwen Zhang,Minmin Chang,Binbin Yang,Shujie Tang
Evidence-Based Complementary and Alternative Medicine. 2019; 2019: 1
[Pubmed] | [DOI]
2 Angelica polysaccharide moderates hypoxia-evoked apoptosis and autophagy in rat neural stem cells by downregulation of BNIP3
Yongzhen Xue,Yongzhen Dongmei Li,Yongzhen Yige Zhang,Yongzhen Hang Gao,Hui Li
Artificial Cells, Nanomedicine, and Biotechnology. 2019; 47(1): 2492
[Pubmed] | [DOI]
3 Angelica polysaccharide alleviates oxidative response damage in HaCaT cells through up-regulation of miR-126
Xijun Zhang,Hong Xue,Ping Zhou,Li Liu,Jing Yu,Pengfei Dai,Manqing Qu
Experimental and Molecular Pathology. 2019; : 104281
[Pubmed] | [DOI]
4 Angelica polysaccharide mitigates lipopolysaccharide-evoked inflammatory injury by regulating microRNA-10a in neuronal cell line HT22
Yuni Zhou,Xiaoqian Guo,Weimei Chen,Jun Liu
Artificial Cells, Nanomedicine, and Biotechnology. 2019; 47(1): 3194
[Pubmed] | [DOI]
5 Polysaccharide from Angelica sinensis protects H9c2 cells against oxidative injury and endoplasmic reticulum stress by activating the ATF6 pathway
Xiaowei Niu,Jingjing Zhang,Chun Ling,Ming Bai,Yu Peng,Shaobo Sun,Yingdong Li,Zheng Zhang
Journal of International Medical Research. 2018; 46(5): 1717
[Pubmed] | [DOI]
6 Angelica sinensis polysaccharide inhibits proliferation, migration, and invasion by downregulating microRNA-675 in human neuroblastoma cell line SH-SY5Y
Jing Yang,Xiaojun Shao,Jian Jiang,Yan Sun,Lingzhen Wang,Lirong Sun
Cell Biology International. 2018;
[Pubmed] | [DOI]
7 Physicochemical Characterization and Functional Analysis of the Polysaccharide from the Edible Microalga Nostoc sphaeroides
Haifeng Li,Linnan Su,Sheng Chen,Libin Zhao,Hongyu Wang,Fei Ding,Hong Chen,Ruona Shi,Yulan Wang,Zebo Huang
Molecules. 2018; 23(3): 508
[Pubmed] | [DOI]
8 Panax notoginseng polysaccharide increases stress resistance and extends lifespan in Caenorhabditis elegans
Shiling Feng,Haoran Cheng,Zhou Xu,Ming Yuan,Yan Huang,Jinqiu Liao,Ruiwu Yang,Lijun Zhou,Chunbang Ding
Journal of Functional Foods. 2018; 45: 15
[Pubmed] | [DOI]
9 Angelica sinensis polysaccharide protects rat cardiomyocytes H9c2 from hypoxia-induced injury by down-regulation of microRNA-22
Hui Pan,Linlin Zhu
Biomedicine & Pharmacotherapy. 2018; 106: 225
[Pubmed] | [DOI]
10 Tanshinone IIA Inhibits Glutamate-Induced Oxidative Toxicity through Prevention of Mitochondrial Dysfunction and Suppression of MAPK Activation in SH-SY5Y Human Neuroblastoma Cells
Haifeng Li,Wenjing Han,Hongyu Wang,Fei Ding,Lingyun Xiao,Ruona Shi,Liping Ai,Zebo Huang
Oxidative Medicine and Cellular Longevity. 2017; 2017: 1
[Pubmed] | [DOI]
11 Angelica Sinensis Polysaccharide Prevents Hematopoietic Stem Cells Senescence in D-Galactose-Induced Aging Mouse Model
Xinyi Mu,Yanyan Zhang,Jing Li,Jieyu Xia,Xiongbin Chen,Pengwei Jing,Xiaoying Song,Lu Wang,Yaping Wang
Stem Cells International. 2017; 2017: 1
[Pubmed] | [DOI]
12 The Protective Effects of Dangguibohyul-tang (Dangguibuxuetang) against Disuse Muscle Atrophy in Rats
Bum Hoi Kim
Journal of Korean Medicine Rehabilitation. 2017; 27(4): 1
[Pubmed] | [DOI]
13 Polysaccharides of Dendrobium officinale Kimura & Migo protect gastric mucosal cell against oxidative damage-induced apoptosis in vitro and in vivo
Qiang Zeng,Chun-Hay Ko,Wing-Sum Siu,Long-Fei Li,Xiao-Qiang Han,Liu Yang,Clara Bik-San Lau,Jiang-Miao Hu,Ping-Chung Leung
Journal of Ethnopharmacology. 2017;
[Pubmed] | [DOI]
14 Neuroprotective effects of plant polysaccharides: A review of the mechanisms
Qing-Han Gao,Xueyan Fu,Rui Zhang,Zhisheng Wang,Muzhen Guo
International Journal of Biological Macromolecules. 2017;
[Pubmed] | [DOI]
15 Food-Derived Antioxidant Polysaccharides and Their Pharmacological Potential in Neurodegenerative Diseases
Haifeng Li,Fei Ding,Lingyun Xiao,Ruona Shi,Hongyu Wang,Wenjing Han,Zebo Huang
Nutrients. 2017; 9(7): 778
[Pubmed] | [DOI]
16 Broad spectrum targeting of tumor vasculature by medicinal plants: An updated review
Ashwaq H.S. Yehya,Muhammad Asif,Yi J. Tan,Sreenivasan Sasidharan,Amin M.S. Abdul Majid,Chern Ein Oon
Journal of Herbal Medicine. 2017;
[Pubmed] | [DOI]
17 Antioxidative mechanism of Lycium barbarum polysaccharides promotes repair and regeneration following cavernous nerve injury
Zhan-kui Zhao,Hong-lian Yu,Bo Liu,Hui Wang,Qiong Luo,Xie-gang Ding
Neural Regeneration Research. 2016; 11(8): 1312
[Pubmed] | [DOI]
18 Antioxidant and neuroprotective effects of Dictyophora indusiata polysaccharide in Caenorhabditis elegans
Ju Zhang,Ruona Shi,Haifeng Li,Yanxia Xiang,Lingyun Xiao,Minghua Hu,Fangli Ma,Chung Wah Ma,Zebo Huang
Journal of Ethnopharmacology. 2016;
[Pubmed] | [DOI]
19 Advances on Bioactive Polysaccharides from Medicinal Plants
Jian-Hua Xie,Ming-Liang Jin,Gordon A. Morris,Xue-Qiang Zha,Han-Qing Chen,Yang Yi,Jing-En Li,Zhi-Jun Wang,Jie Gao,Shao-Ping Nie,Peng Shang,Ming-Yong Xie
Critical Reviews in Food Science and Nutrition. 2016; 56(sup1): S60
[Pubmed] | [DOI]
20 Bioactive Peptides fromAngelica sinensisProtein Hydrolyzate Delay Senescence inCaenorhabditis elegansthrough Antioxidant Activities
Qiangqiang Wang,Yunxuan Huang,Chuixin Qin,Ming Liang,Xinliang Mao,Shuiming Li,Yongdong Zou,Weizhang Jia,Haifeng Li,Chung Wah Ma,Zebo Huang
Oxidative Medicine and Cellular Longevity. 2016; 2016: 1
[Pubmed] | [DOI]
21 Investigation of the effect of traditional Chinese medicine on pain and inflammation in chronic nonbacterial prostatitis in rats
Y.-J. Liu,G.-H. Song,G.T. Liu
Andrologia. 2016; : n/a
[Pubmed] | [DOI]
22 Effects of Sunphenon and Polyphenon 60 on proteolytic pathways, inflammatory cytokines and myogenic markers in H2O2-treated C2C12 cells
Allur Subramaniyan Sivakumar,Inho Hwang
Journal of Biosciences. 2015; 40(1): 53
[Pubmed] | [DOI]
23 Polysaccharides from Medicinal Herbs As Potential Therapeutics for Aging and Age-Related Neurodegeneration
Haifeng Li,Fangli Ma,Minghua Hu,Chung Wah Ma,Lingyun Xiao,Ju Zhang,Yanxia Xiang,Zebo Huang
Rejuvenation Research. 2014; 17(2): 201
[Pubmed] | [DOI]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Results
Discussion
Materials and Me...
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed2720    
    Printed47    
    Emailed0    
    PDF Downloaded497    
    Comments [Add]    
    Cited by others 23    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]