An-Gong-Niu-Huang Wan protects against cerebral ischemia induced apoptosis in rats: Up-regulation of Bcl-2 and down-regulation of Bax and caspase-3

Ethnopharmacological relevance

The An-Gong-Niu-Huang Wan (AGNH), a Chinese traditional medicine, has been used for treatment of cerebral diseases for centuries in China and other Asian countries, and is approved by the State Food and Drug Administration of China for the treatment of stroke. The aim of present study is to test the neuroprotective effects of AGNH on cerebral ischemia in rats and to explore the underlying mechanisms.

Materials and methods

75 Male Sprague-Dawley rats were randomly divided into 5 groups: sham, ischemia–reperfusion (I/R), and I/R plus 0.065 g/kg/d AGNH, 0.125 g/kg/d AGNH and 0.25 g/kg/d AGNH. Cerebral ischemia was induced by 1.5 h of middle cerebral artery occlusion (MCAO). Neurological functional deficits were evaluated according to Zea longa?s score, cerebral infarct area was measured by tetrazolium staining. Cell injury and apoptosis were assessed by Nissl staining and DNA fragmentation assay. The expression of Bax, Bcl-2 and caspase-3 were analyzed by Western blot.

Results

Rats subjected to MCAO exhibited worsened neurological score, infarct area, cell damage and apoptosis. These were all attenuated by AGNH (0.125 and 0.25 g/kg/d). Moreover, AGNH reversed cerebral ischemia induced decreases in Bcl-2 expression and increases in Bax and caspase-3 expression.

Conclusions

These results suggest that AGNH exerts neuroprotective effects, and the neuroprotection is likely to relate to depressed Bax/Bcl-2 ratio and caspase-3 level, leading to inhibition of apoptotic cell death.     

Neuroprotective effect of Artemisia absinthium L. on focal ischemia and reperfusion-induced cerebral injury

Ethnopharmacological relevance

Artemisia absinthium L. has long been used as traditional herbal medicine in China, Europe and Pakistan for the treatment of gastric pain, cardiac stimulation, to improve memory and for the restoration of declining mental function.

Aim of the study

The present study was designed to investigate the potential protective effects of Artemisia absinthium on cerebral oxidative stress and damage as well as behavioral disturbances induced by cerebral ischemia and reperfusion injury in rats.

Materials and methods

Focal ischemia and reperfusion were induced by middle cerebral artery occlusion (MCAO) for 90 min, followed by 24 h reperfusion. MCAO led to significant rise in infarct size and lipid peroxidation, and depletion in glutathione content, superoxide dismutase and catalase activity in brain. Further, behavioral deficits like motor incoordination and impairment of short-term memory were also significantly impaired by MCAO as compared with sham group.

Results

The brain oxidative stress and damage, and behavioral deficits were significantly attenuated by pre-treatment with the methanol extract of Artemisia absinthium (100 mg/kg and 200 mg/kg, p.o.).

Conclusion

These findings suggested that Artemisia absinthium is neuroprotective and may prove to be useful adjunct in the treatment of stroke.     

Neuroprotective effect of calycosin on cerebral ischemia and reperfusion injury in rats

Ethnopharmacological relevance

Radix Astragali has been commonly used as traditional herbal medicine in China for reinforcing vital energy, strengthening superficial resistance and promoting the discharge of pus and the growth of new tissue.

Aim of the study

The present study was to investigate the neuroprotective effect of calycosin isolated from the roots of Radix Astragali on cerebral ischemic/reperfusion injury.

Materials and methods

After 24 h of reperfusion following ischemia for 2 h induced by middle cerebral artery occlusion (MCAO), Sprague-Dawley rats were intragastrically administered different doses of calycosin (7.5, 15, 30 mg/kg, respectively). Neurological deficit, infarct volume, histopathology changes and some oxidative stress markers were evaluated after 24 h of reperfusion.

Results

Treatment with calycosin significantly ameliorated neurologic deficit and infarct volume after cerebral ischemia reperfusion. Calycosin also reduced the content of malondialdehyde (MDA), protein carbonyl and reactive oxygen species (ROS), and up-regulated the activities of superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px) in a dose-dependent manner. Moreover, calycosin can also inhibit the expression of 4-Hydroxy-2-nonenal (4-HNE).

Conclusion

These results suggest that calycosin has a neuroprotective effect against cerebral ischemia/reperfusion injury. The mechanism might be attributed to its antioxidant effects.     

Ginsenoside Rb1 regulates the expressions of brain-derived neurotrophic factor and caspase-3 and induces neurogenesis in rats with experimental cerebral ischemia

Aim of the study

Recent studies have revealed that ginsenoside Rb1 (GRb1) is neuroprotective for cerebral ischemia. However, the mechanism underlying of this function is unclear. We assessed whether this neuroprotective effect of GRb1 was mediated by the levels of brain-derived neurotrophic factor (BDNF), by the levels of caspase-3 proteins and by induced neurogenesis in rats following transient cerebral ischemia or not.

Materials and methods

Cerebral ischemia was prepared by a 2 h occlusion of the middle cerebral artery and reperfusion, followed by infusion of GRb1 (40 mg/kg) and saline (GRb1 and ischemia groups, respectively). All rats were sacrificed at 3 and 12 h, 1, 2, 3, 5, and 10 days after reperfusion. Normal and sham-operated rats were used in control group. Modified Neurological Severity Scores (mNSS) test and hematoxylin and eosin staining were respectively performed to evaluate neurological function and histological feature. Immunohistochemistry was used to identify intrinsic neurogenesis by nestin antibody. Western blotting was used to detect BDNF and caspase-3 protein content.

Results

GRb1 infusion after cerebral ischemia significantly promoted recoveries of neurological functions at 3 and 5 days after reperfusion compared to ischemic rats. The number of nestin-positive cells was apparently increased after GRb1 infusion compared to ischemia rats at given time. Moreover, BDNF was significantly increased in GRb1-treated rats compared to ischemia rats at different time points. In contrast, GRb1 infusion after the onset of reperfusion, caspase-3 at a given time was significantly reduced compared to ischemia rats, but still significantly increased compared to control rats.

Conclusions

Promotion of the neurogenesis and regulation of the expressions of BDNF and caspase-3 may be involved in GRb1-induced neuroprotection against cerebral ischemia.     

Cerebralcare Granule,a Chinese herb compound preparation,improves cerebral microcirculatory disorder and hippocampal CA1 neuron injury in gerbils after ischemia–reperfusion

Aim of the study

Cerebralcare Granule^® (CG) is a Chinese herb compound preparation that has been used for treatment of cerebrovascular related diseases. However, the effect of post-treatment with CG on ischemia and reperfusion (I/R) induced cerebral injury is so far unclear.

Materials and methods

In present study, cerebral global I/R was induced in Mongolian gerbils by clamping bilateral carotid arteries for 30 min followed by reperfusion for 5 days, and CG (0.4 g/kg or 0.8 g/kg) was administrated 3 h after the initiation of reperfusion.

Results

Post-treatment with CG for 5 days attenuated the I/R-induced production of hydrogen peroxide in, leukocyte adhesion to, and albumin leakage from cerebral microvessels, and, meanwhile, protected neuron from death, reduced the number of caspase-3- and Bax-positive cells, and increased Bcl-2-positive cells in hippocampal CA1 region.

Conclusion

The results suggest that CG given after initiation of reperfusion is able to ameliorate cerebral microvascular dysfunction and hippocampal CA1 neuron damage caused by I/R.     

Neuroprotective effects of Xiao-Xu-Ming decoction against ischemic neuronal injury in vivo and in vitro

Ethnopharmacological relevance

Xiao-Xu-Ming decoction (XXMD) has long been employed clinically to treat stroke in traditional Chinese Medicine.

Aim of the study

To investigate the neuroprotective effects of XXMD in vivo and in vitro stroke models and determine involved mechanisms.

Materials and methods

Two models (four-vessel occlusion in adult Wistar rats and oxygen–glucose deprivation primary cultured neurons) were employed to mimic ischemia-reperfusion damage, in vivo and in vitro, respectively. The effects of XXMD were investigated with respect to neuronal damage, activity of caspase-3 and expression of Bcl-2 in CA1 region of hippocampus after ischemia. The cognitive ability was measured 7 days after ischemia/reperfusion by using Morris water maze.

Results

Oral administration of XXMD significantly increased the density of neurons that survived in the CA1 region of hippocampus on the 3rd and 7th day after transient global ischemia was induced in a dose-dependent manner. XXMD ameliorated severe deficiencies in spatial cognitive performance induced by transient global ischemia. Inhibition of caspase-3 activity and up-regulation of Bcl-2 expression were induced in the high dose of XXMD-treated rats after ischemia. In oxygen–glucose deprivation model, both XXMD extract and drug-containing serum prepared from blood of high dose of XXMD-treated rats inhibited apoptotic neuronal death at 24 h after reoxygenation.

Conclusions

Our results clearly demonstrated that XXMD is neuroprotective and appears to influence deleterious pathological processes that are activated after the onset of ischemia.     

Physalin F from Physalis minima L. triggers apoptosis-based cytotoxic mechanism in T-47D cells through the activation caspase-3- and c-myc-dependent pathways

Ethnopharmacological relevance

Physalin F (a secosteroid derivative), is well recognized as a potent anticancer compound from Physalis minima L., a plant that is traditionally used to treat cancer. However, the exact molecular anticancer mechanism remains to be elucidated.

Aim of the study

We have recently reported the apoptosis-based cytotoxic effect of the chloroform extract of this plant. Here, we investigated the cytotoxicity and possible cell death mechanism elicited by the active constituent, physalin F on human breast T-47D carcinoma.

Materials and methods

Cytotoxic-guided fractionation of the chloroform extract of Physalis minima has led to the isolation of physalin F. The cytotoxicity activity was assayed using MTS assay. The effect of the compound to induce apoptosis was determined by biochemical and morphological observations through DeadEnd Colorimetric and annexin V assays, respectively, and RT-PCR analysis of mRNA expression of the apoptotic-associated genes.

Results

Cytotoxicity screening of physalin F displayed a remarkable dose-dependant inhibitory effect on T-47D cells with lower EC₅₀ value (3.60 μg/ml) than the crude extract. mRNA expression analysis revealed the co-regulation of c-myc- and caspase-3-apoptotic genes in the treated cells with the peak expression at 9 and 12 h of treatment, respectively. This apoptotic mechanism is reconfirmed by DNA fragmentation and phosphatidylserine externalization.

Conclusion

These findings indicate that physalin F may potentially act as a chemopreventive and/or chemotherapeutic agent by triggering apoptosis mechanism via the activation of caspase-3 and c-myc pathways in T-47D cells.     

A petrol ether extract of the roots of Onosma paniculatum induces cell death in a caspase dependent manner

Aim of the study

Traditional Chinese medicine (TCM) has become very popular in Western countries during the last years. Zicao, a remedy of TCM, has been traditionally used to treat cancer, and, its main constituents, naphthoquinones, have been reported to possess antitumor activity (4 and 20). Here, we prepared extracts of different polarities of Onosma paniculatum Bur. & Franch., a plant which is amongst others used as Zicao, but, much less investigated. The extracts were analyzed concerning their growth inhibitory and apoptosis-inducing activity in various tumor cells.

Materials and methods

Cell viability was measured by XTT viability and a growth inhibition assay. Effects on the cell cycle and caspase-3 were determined by flow cytometry.

Results

From three different extracts, a petrol ether extract showed significant growth inhibitory effect, cell cycle influence and caspase-3 dependent induction of apoptosis which was time and dose dependent.

Conclusion

To further determine the activity and mechanism of action of the petrol ether extract, we would like to isolate and identify the active principle and investigate the effects in more detail.     

Neuroprotective activity of Matricaria recutita Linn against global model of ischemia in rats

Ethnopharmacological relevance

Traditionally, the whole plant is used for various diseases, including neuronal disorders.

Aim of the study

To evaluate the neuroprotective effect of Matricaria recutita L. against global cerebral ischemia/reperfusion (I/R) injury-induced oxidative stress in rats.

Materials and methods

Neuroprotective activity was carried out by global cerebral ischemia on Sprague–Dawley rats by bilateral carotid artery (BCA) occlusion for 30 min followed by 60 min reperfusion. The antioxidant enzymatic and non-enzymatic levels were estimated along with cerebral infarction area and histopathological studies.

Results

The Matricaria recutita L. methanolic extract showed dose-dependent neuroprotective activity by significant decrease in lipid peroxidation (LPO) and increase in the superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and total thiol levels in extract treated groups as compared to ischemia/reperfusion group. Cerebral infarction area was significantly reduced in extract treated groups as compared to ischemia/reperfusion group.

Conclusion

The methanolic extract of Matricaria recutita L. showed potent neuroprotective activity against global cerebral ischemia/reperfusion injury-induced oxidative stress in rats.     

Protective effect of Sheng-Nao-Kang decoction on focal cerebral ischemia-reperfusion injury in rats

Ethnopharmacological relevance

Sheng-Nao-Kang decoction (SNK), a modified traditional Chinese medicine (TCM), has been used clinically for the treatment of acute and chronic cerebrovascular related diseases. To evaluate the protective effect of SNK on focal cerebral ischemia-reperfusion (I/R) injury in rats and investigate the underlying mechanisms.

Materials and methods

Focal cerebral I/R injury in rats was induced by middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion for 24 h. Adult male Sprague-Dawley (SD) rats were randomly divided into six kinds of groups: Sham group; I/R group; SNK-treated groups at doses of 0.7 g/kg, 1.4 g/kg and 2.8 g/kg; and nimodipine (NMP)-treated group. The recoveries of neurological function in rats were estimated by neurological defect scoring and 2,3,5-triphenyltetrazolium chloride (TTC) staining after 24 h reperfusion. Various biochemical indexes in serum were assayed by colorimetry, including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), inducible nitric oxide synthase (iNOS) and total nitric oxide synthase (TNOS). Histological structures of the brain in rats were observed by hematoxylin and eosin (H&E) staining. Immunohistochemistry was performed to detect the caspase-3 protein content in rats.

Results

SNK administration significantly reduced the neurological defect scores and lessened the cerebral infarction volume. The treatment of SNK lowered MDA content, up-regulated SOD and GSH-Px levels, down-regulated iNOS and TNOS levels in serum. Furthermore, histological examination indicated that dense neuropil and largely surviving neurons were seen in SNK-treated rats. SNK administration restrained the expression of caspase-3 positive protein significantly.

Conclusion

The results suggest that SNK demonstrates a strong and ameliorative effect on cerebral I/R damage in rats. The protective mechanisms of SNK are associated with its properties of anti-apoptosis and anti-oxidation as well as regulation of iNOS and TNOS.     

Effects of Kang-Jia-Wan,a Chinese medicinal herb officinal,on apoptosis induction in goiter of rats

Ethnopharmacological relevance

Kang-Jia-Wan (KJW), a traditional Chinese herbal medicine, is widely used to treat goiter in the clinics in China.

Aim

The mechanisms by which KJW treats goiter are unclear. It is known that insufficient apoptosis of thyrocytes is involved in the formation of goiter. Here, we investigated whether KJW could induce apoptosis in goiter of rats given methimazole (MMI).

Materials and methods

Fifty-six Wistar rats were randomly separated into four groups: normal, MMI, MMI + low-dose KJW and MMI + high-dose KJW. Except for the normal group (20 rats), all groups (each with 12 rats) were given 0.04% (w/v) MMI in their drinking water. One week later, the rats in MMI + low- and high-dose KJW groups were orally supplemented with KJW at 250 mg/kg d⁻¹ and 1000 mg/kg d⁻¹, respectively.

Results

After KJW treatment for 12 weeks, the relative thyroid weight (mg/100 g body weight) of the MMI + high-dose KJW group decreased significantly. Features of apoptosis were also apparent in thyroid tissues of rats given KJW treatment. Importantly, KJW markedly increased the caspase-3 and Fas protein expression, in a dose-dependent manner, in the thyroid specimens.

Conclusions

These results showed that KJW played a therapeutic role via apoptosis induction in the goitrous glands.     

Calycosin-7-O-β-d-glucoside regulates nitric oxide /caveolin-1/matrix metalloproteinases pathway and protects blood–brain barrier integrity in experimental cerebral ischemia–reperfusion injury

Ethnopharmacology relevance

Astragali Radix (AR) has been used for thousands years to treat ischemic stroke. Calycosin and its glycoside form calycosin-7-O-β-d-glucoside (CG) are two representative isoflavones in Astragali Radix. However, its neurological effects and related molecular mechanisms are largely unknown. The present study aims to evaluate the neuroprotective effects of CG on blood–brain barrier (BBB) integrity of ischemic brain tissue and explore the relevant signaling mechanisms.

Material and method

Male adult Sprague-Daweley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) plus 24 h or 14 days of reperfusion. CG (26.8 mg/kg) was intraperitoneally administered into the rats at 15 min before onset of ischemia. The neuroprotective effects of CG were evaluated by measuring infarct volume, histological damage and BBB permeability. Furthermore, the effects of CG on scavenging nitric oxide (NO), and modulating matrix metalloproteinases (MMPs) and caveolin-1 (cav-1) were investigated with in vitro cultured brain microvascular endothelial cells treated with NO donor or oxygen–glucose deprivation (OGD) and/or in vivo rat model of MCAO cerebral ischemia–reperfusion injury.

Results

CG treatment significantly reduced infarct volume, histological damage and BBB permeability in the in vivo MCAO ischemia–reperfusion rat model. CG treatment remarkably inhibited the expression and activities of MMPs, and secured the expression of cav-1 and tight junction proteins in the microvessels isolated from ischemic rat cortex. Furthermore, CG was revealed to scavenge NO, inhibit the activities of MMP-2 and MMP-9, and attenuate cell death in the in vitro cultured brain microvascular endothelial cells under OGD condition.

Conclusion

CG could protect BBB integrity in experimental cerebral ischemia–reperfusion injury via regulating NO/cav-1/MMPs pathway.     

n-Butanol soluble fraction of the water extract of Chinese toon fruit ameliorated focal brain ischemic insult in rats via inhibition of oxidative stress and inflammation

Aim of the study

Toona sinensis Roem. (Meliaceae; Toona sinensis; Chinese toon) is a type of arbor that is widely distributed in Asia. The fruits of Toona sinensis Roem has been traditionally recognized for treatment of cerebrovascular diseases. To evaluate the potential clinical use of the fruits of Toona sinensis Roem, we determined the dose dependence of the neuroprotective efficacy in a focal cerebral ischemic reperfusion model of rats and explored the underlying mechanisms.

Materials and methods

Rats were subjected to occlusion of the middle cerebral artery (MCAO) by a nylon filament and treated with different doses (20 mg/kg and 30 mg/kg) of n-butanol soluble fraction of the water extract of Chinese toon fruit or the vehicle for 1 week before induction of ischemia, s.i.d..

Results

n-Butanol soluble fraction of the water extract of Chinese toon fruit reduced in a dose-dependent manner the ischemia-induced cerebral infarct and edema volume and attenuated neurological deficits observed at 6 h point after ischemia. n-Butanol soluble fraction of the water extract of Chinese toon fruit reduced the levels of nitrate, nitrite, lipid peroxidation, cyclooxygenase-1, thromboxane in post-ischemic brain. n-Butanol soluble fraction of the water extract of Chinese toon fruit adjusted the elevation of the activity of glutathione peroxidase and superoxide dismutase in ischemic brain.

Conclusions

The present study was the first evidence of effectiveness of n-butanol soluble fraction of the water extract of Chinese toon fruit in the rat stroke models, as it reduced infarct volume, inhibited the oxidative stress and inflammation.     

Ginseng total saponins enhance neurogenesis after focal cerebral ischemia

Ethnopharmacological relevance

Ginseng, the root of Panax ginseng C.A. Meyer, is one of the most commonly used healing herbs for stroke and chronic debilitating conditions in China. Ginsenosides are the main active principles for ginseng's efficacy, but the mechanisms have not been fully clarified.

Aim of the study

To test the hypothesis whether or not the administration of Ginseng total saponins (GTS) can enhance neurogenesis after focal cerebral ischemia, and thereby improve neurological deficits.

Materials and methods

Male Wistar rats received intraperitoneal injections of GTS dissolved at a dose of 25 mg kg⁻¹ d⁻¹ or normal saline (NS) of same volume 3 days before the permanent middle cerebral artery occlusion (MCAO) model establishment until the animals were killed at the time points of 1 d, 3 d, 7 d and 14 d. The neurological function was assessed blindly. BrdU immunostaining and double staining were performed by following the 3-steps method.

Results

(A) GTS-treated rats have better neurological scores compared with those in NS group at 14 d time point (p < 0.05); (B) the number of BrdU⁺ cells and BrdU⁺/NeuN⁺ cells in GTS group were significantly higher than those in NS group in the ipsilateral subventricular zone and in the ipsilateral infarct area after MCAO, respectively (p < 0.05 or p < 0.01); (C) the increase of the number of BrdU⁺/NeuN⁺ cells highly correlated with the decrease of neurological scores. Coefficient correlation r = −0.828 (p < 0.01).

Conclusion

GTS can improve neurological deficits after focal cerebral ischemia by inducing endogenous neural stem cells activation and thereby enhance adult central nervous system regeneration.     

Xiao-Xu-Ming decoction preserves mitochondrial integrity and reduces apoptosis after focal cerebral ischemia and reperfusion via the mitochondrial p53 pathway

Ethnopharmacological relevance

Xiao-Xu-Ming decoction (XXMD) has been used to treat stroke and other neurological diseases for more than 1000 years. The purpose of this study was to investigate the effects of XXMD on mitochondrial damage and apoptosis after cerebral ischemia and reperfusion.

Materials and methods

Male Sprague-Dawley rats were randomly divided into 3 groups: sham, cerebral ischemia and reperfusion (I/R), and cerebral ischemia and reperfusion plus XXMD (60 g/kg/day) (XXMD60). Focal cerebral ischemia and reperfusion models were induced by middle cerebral artery occlusion. Cerebral ischemic injury was evaluated by hematoxylin and eosin staining. Ultrastructural features of mitochondria in the penumbra of the ischemic cortex were analyzed by transmission electron microscopy. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) staining and cleaved caspase 3 immunohistochemistry. Proteins in the mitochondrial p53 pathway were detected by western blot and immunofluorescence.

Results

The results showed that XXMD treatment markedly attenuated ischemic changes, preserved mitochondrial integrity, and significantly reduced apoptosis. In addition, we found that XXMD treatment reduced p53 and Bax levels and increased Bcl-2 levels in mitochondrial fractions. XXMD significantly blocked the release of cytochrome c and Smac/Diablo from mitochondria, and inhibited activation of caspase 9 and caspase 3 in cytoplasmic fractions. Increased expression of c-IAP1 was observed in the XXMD60 group.

Conclusions

The findings demonstrated that XXMD protected mitochondria from ischemic injury and inhibited apoptosis. The mitochondrial p53 pathway could be partially involved in the protective effects.     

Anti-diabetic effects of Panax notoginseng saponins and its major anti-hyperglycemic components

Ethnopharmacological relevance

Panax notoginseng (Burk) F.H. Chen (Araliaceae) is a well-known and commonly used traditional Chinese herb for treatment of various diseases, such as hemostasis, edema and odynolysis.

Aim of study

Our aim was to investigate the mechanisms of anti-hyperglycemic and anti-obese effects of Panax notoginseng saponins (PNS) in KK-Ay mice, and explore the components in PNS for such effects.

Materials and methods

KK-Ay mice received daily intraperitoneal injections of PNS 200 mg/kg or vehicle for 30 days while ginsenoside Re 14 mg/kg, Rd 15 mg/kg, Rg1 40 mg/kg, Rb1 60 mg/kg and notoginsenoside R1 6 mg/kg for 12 days. Fasting blood glucose levels (FBGL), glucose tolerance (GT), serum insulin, leptin levels, body weight changes, food intake, adipose tissues and blood fat levels were measured at different time points.

Results

The PNS group had significantly lower FBGL, improved GT and smaller body weight incremental percentage after the 30-day treatment. Additionally, Rb1 exhibited significant reduction of FBGL on day 12, and Re also exhibited a decreasing trend after the 12-day treatment.

Conclusions

PNS possess anti-hyperglycemic and anti-obese activities by improving insulin- and leptin sensitivity, and Rb1 is responsible for the anti-hyperglycemic effect among the five saponins in KK-Ay mice.     

Preconditioning with the traditional Chinese medicine Huang-Lian-Jie-Du-Tang initiates HIF-1α-dependent neuroprotection against cerebral ischemia in rats

Ethnopharmacological relevance

Huang-Lian-Jie-Du-Tang (HLJDT) is a classical heat-clearing and detoxicating formula of traditional Chinese medicine that is widely used to treat stroke. The present study was designed to investigate the effects of HLJDT preconditioning on neurons under oxygen and glucose deprivation (OGD) and rats subjected to middle cerebral artery occlusion (MCAO).

Materials and methods

A stroke model of rats was obtained through MCAO. Following HLJDT preconditioning, the cerebral infarction volume, cerebral water content, and neurological deficient score were determined. Cerebral cortical neurons cultured in vitro were preconditioned with HLJDT and then subjected to OGD treatment. The release of lactate dehydrogenase (LDH) from neurons was detected. The levels of hypoxia-inducible factor-1α (HIF-1α) and PI3K/Akt signaling were analyzed by western blotting, and the levels of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) in the supernatant of the neurons and the plasma of MCAO rats were measured through a radioimmunological assay. The apoptosis and proliferation of neurons were analyzed by immunohistochemistry.

Results

HLJDT preconditioning significantly reduced the cerebral infarction volume and cerebral water content and ameliorated the neurological deficient score of MCAO rats. In addition, HLJDT preconditioning protected neurons against OGD. Increased HIF-1α, EPO, and VEGF levels and the activation of PI3K/Akt signaling were observed as a result of HLJDT preconditioning. Furthermore, HLJDT preconditioning was found to inhibit ischemia-induced neuron apoptosis and to promote neuron proliferation under conditions of ischemia/reperfusion.

Conclusion

Both rats and neurons subjected to HLJDT preconditioning were able to resist ischemia/reperfusion or hypoxia injury through the inhibition of apoptosis and the enhancement of proliferation, and these effects were primarily dependent on the activation of the PI3K/Akt signaling pathway and HIF-1α.     

Matrine induces apoptosis in gastric carcinoma cells via alteration of Fas/FasL and activation of caspase-3

Aim of the study

Matrine, an alkaloid purified from the chinese herb Sophora flavescens Ait, is well known to possess activities including anti-inflammation, anti-fibrotic and anticancer. In this study, the mechanism of matrine inducing the apoptosis of gastric carcinoma cells was investigated.

Materials and Methods

Proliferation of SGC-7901 cells was examined by MTT assay. Cellular morphology was observed under transmission electron microscope. Flow cytometry (FCM) was used to observe the apoptosis of SGC-7901 cells by staining with annexinV-FITC/PI. The expression levels of Fas/FasL in SGC-7901 cells were monitored by FCM analysis using an indirect immunoflurenscence method. Activity of caspase-3 enzyme was measured by spectrofluorometry.

Results

MTT assay showed that matrine inhibited SGC-7901 cells proliferation in a dose-dependent and time-dependent manner. Apoptosis induction was demonstrated by morphological changes under electron microscope and FCM analysis. Florescence intensity levels of Fas and FasL were found to be equally up-regulated after matrine treatment, which were both correlated with apoptosis rate. The activity of caspase-3 enzyme increased in matrine groups, positively correlated with apoptosis rate.

Conclusions

Matrine could inhibit cell proliferation and induce apoptosis of SGC-7901 cells in vitro. The apoptosis induction appears to proceed by up-regulating Fas/FasL expression and activating caspase-3 enzyme.