cyclin-d1 and Ischemic-Attack--Transient

Buyang Huanwu Decoction (BHD), a classic traditional Chinese medicine (TCM) formula, has been used for recovering neurological dysfunctions and treating post-stroke disability in China for 200 years. In the present study, we investigated the effects of BHD on inhibiting neuronal apoptosis, promoting proliferation and differentiation of neural stem cells (NSCs) and neurite formation and enhancing learning and memory functional recovery in an experimental rat ischemic stroke model. BHD significantly reduced infarct volume and decreased cell apoptosis in the ischemic brain. BHD enhanced neuronal cell viability in vitro. BHD dose-dependently promoted the proliferation of NSCs in ischemic rat brains in vivo. Moreover, BHD promoted neuronal and astrocyte differentiation in primary cultured NSCs in vitro. Water maze test revealed that BHD promoted the recovery of learning function but not memory functions in the transient ischemic rats. We then investigated the changes of the cellular signaling molecules by using two-dimension (2D) gel electrophoresis and focused on the PI3K/Akt/Bad and Jak2/Stat3/cyclin D1signaling pathway to uncover its underlying mechanisms for its neuroprotective and neurogenetic effects. BHD significantly upregulated the expression of p-PI3K, p-Akt, and p-Bad as well as the expression of p-Jak, p-Stat3, and cyclin D1 in vitro and in vivo. In addition, BHD upregulated Hes1 and downregulated cav-1 in vitro and in vivo. Taken together, these results suggest that BHD has neuroprotective effects and neurogenesis-promoting effects via activating PI3K/Akt/Bad and Jak2/Stat3/Cyclin D1 signaling pathways. Graphical Abstract Buyang Huanwu Decoction (BHD) activates the PI3K-AKT-BAD pathway in the ischemic brain for neuroprotection. BHD also activates JAK2/STAT3/Cyclin D1 signaling cascades for promoting neurogenesis in the hippocampus of post-ischemic brains. Moreover, BHD inhibits the expression of caveolin-1 and increases the expression of HES1 for promoting neuronal differentiation. The neuroprotective and neurogenesis-promoting effects in the hippocampus of post-ischemic brains promote learning ability.

Topics: 14-3-3 Proteins; Animals; Apoptosis; Astrocytes; Axons; bcl-Associated Death Protein; Caveolin 1; Cell Differentiation; Cell Proliferation; Cyclin D1; Down-Regulation; Drugs, Chinese Herbal; ErbB Receptors; Ischemic Attack, Transient; Ischemic Stroke; Janus Kinase 2; Male; Memory; Neural Stem Cells; Neuritis; Neurogenesis; Neuroprotection; Neuroprotective Agents; PC12 Cells; Phosphatidylinositol 3-Kinases; Proteomics; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Recovery of Function; Reperfusion Injury; Signal Transduction; STAT3 Transcription Factor; Transcription Factor HES-1; Up-Regulation; Xanthenes

Cyclin D1, a member of the G1 cyclin family, plays a critical role in the progression of the cell cycle. In the present study, we investigated chronological alterations in cyclin D1 immunoreactivity and its protein levels in the gerbil hippocampus after ischemia/reperfusion.. Chronological alterations in cyclin D1 immunoreactivity and its levels were examined in the gerbil hippocampus after ischemia/reperfusion using immunohistochemistry and western blot analysis.. Changes in cyclin D1 immunoreactivity in the ischemic hippocampus were distinct in pyramidal neurons of the CA1 region and granule cells of the dentate gyrus. Cyclin D1 immunoreactivity in pyramidal neurons of the CA1 region was increased up to 1 day after ischemia/reperfusion, although a transient decrease of cyclin D1 immunoreactivity was detected at 12 hour after ischemia/reperfusion. Thereafter, cyclin D1 immunoreactivity in the CA1 pyramidal neurons was very weak 2 days and disappeared nearly 4 and 7 days after ischemia/reperfusion. However, 4 days after ischemia/reperfusion, the cyclin D1 immunoreactivity in non-pyramidal neurons of the CA1 region was very strong. In the CA2/3 region, cyclin D1 immunoreactivity was higher than that in the CA1 region and not changed after ischemia/reperfusion. In the dentate gyrus, chronological change in cyclin D1 immunoreactivity was observed. Cells in the granule cell layer showed distinct change in cyclin D1 immunoreactivity after ischemia/reperfusion: the cyclin D1 immunoreactivity was lowest at 12 hours and strong 1 and 4 days after ischemia/reperfusion. In addition, change in cyclin D1 protein level was found in the ischemic hippocampus.. Our results indicate that cyclin D1 may play an important role in cellular events related with neuronal damage following ischemia/reperfusion.

Topics: Animals; CA1 Region, Hippocampal; Cell Death; Cyclin D1; Dentate Gyrus; Disease Models, Animal; Gerbillinae; Ischemic Attack, Transient; Male; Neurons; Prosencephalon; Pyramidal Cells; Reperfusion

Cerebral ischemia induces microglial and astroglial activation, which may play a crucial role in the development of ischemic neuronal damage. In this study, we examined the role of cell cycle proteins in glial proliferation in the hippocampus following 10min of global cerebral ischemia in the rat. Proliferating cells were identified with immunostaining for proliferating cell nuclear antigen (PCNA), and glial cells were visualized with immunostaining for microglial response factor-1 (microglia/macrophages) and glial fibrillary acidic protein (astrocytes). Expression of cyclin D1 and cyclin-dependent kinase-4 was also examined with double label immunohistochemistry. Proliferating cells in the CA1 region after ischemia consisted of microglia and much fewer astrocytes. Microglial activation and proliferation (7.6-fold increase in number after 7 days) were preceded by an increase in PCNA-positive microglia; 83% of microglia were PCNA-positive after 2 days. Astrocytes increased by 1.8-fold after 7 days, and only 6% of astrocytes became PCNA-positive by day 7. Cyclin D1 and cyclin-dependent kinase-4 immunoreactivity appeared in these glial cells in parallel with the expression of PCNA. The findings suggest that the accumulation of brain macrophages elicited by transient cerebral ischemia is caused predominantly by activation and proliferation of resident microglia through the upregulation of cell cycle proteins.

Topics: Analysis of Variance; Animals; Astrocytes; Cell Count; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Hippocampus; Immunohistochemistry; Ischemic Attack, Transient; Male; Microglia; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Time Factors

Mild hypothermia protects the brain against experimental ischemia, but the reasons are not well known. We examined whether the protective effects of mild hypothermia could be correlated with alterations in expression of Bcl-2, an anti-apoptotic protein in a rat model of transient global ischemia. Following 10 min of forebrain ischemia, hippocampal neurons were examined 72 h later for survival, expression of Bcl-2 family proteins and apoptosis. Intraischemic mild hypothermia was applied for 3 h (33 degrees C, isch-33) or normal body temperature was maintained (37 degrees C, isch-37). Survival of CA1 neurons was significantly improved in the isch-33 group compared to the isch-37 group (90 vs. 53% survival; P<0.01). The proportion of Bcl-2-positive cells among surviving CA1 neurons in the isch-33 group was increased compared to that of sham and isch-37 groups (P<0.01). Bax expression in CA1 was no different between sham and isch-33 groups, but was significantly decreased in isch-37 (P<0.05). TUNEL staining was positive in many isch-37 CA1 neurons, but absent in isch-33. Utilizing electron microscopy, more cells meeting criteria for apoptosis were observed in the isch-37 than isch-33. These data suggest that mild hypothermia attenuates apoptotic death, and that this protection may be related to increases in Bcl-2.

Topics: Animals; Body Temperature; Cyclin D1; Heating; Immunohistochemistry; In Situ Nick-End Labeling; Ischemic Attack, Transient; Male; Microscopy, Electron; Rats; Rats, Sprague-Dawley

Cyclin-dependent kinases (CDKs) are commonly known to regulate cell proliferation. However, previous reports suggest that in cultured postmitotic neurons, activation of CDKs is a signal for death rather than cell division. We determined whether CDK activation occurs in mature adult neurons during focal stroke in vivo and whether this signal was required for neuronal death after reperfusion injury. Cdk4/cyclin D1 levels and phosphorylation of its substrate retinoblastoma protein (pRb) increase after stroke. Deregulated levels of E2F1, a transcription factor regulated by pRb, are also observed. Administration of a CDK inhibitor blocks pRb phosphorylation and the increase in E2F1 levels and dramatically reduces neuronal death by 80%. These results indicate that CDKs are an important therapeutic target for the treatment of reperfusion injury after ischemia.

Topics: Animals; Apoptosis; Brain; Carrier Proteins; Cell Cycle Proteins; Cerebrovascular Circulation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Enzyme Inhibitors; Flavonoids; Ischemic Attack, Transient; Male; Neurons; Piperidines; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Retinoblastoma-Binding Protein 1; Transcription Factor DP1; Transcription Factors

Recent experiments suggest that apoptotic mechanisms are involved in neuronal cell death after ischemic injury. Although the exact mechanism that triggers activation of apoptotic machinery remains uncertain, in vitro studies revealed that forced expression of cell cycle-related proteins induced apoptosis. Thus, aberrant expression of such proteins might be related to ischemic neuronal death. In the present experiment, we investigated expression of cell cycle-related proteins, i.e., cyclin B1, cyclin D1, cdk4, and PCNA, in rat brain after transient MCA occlusion, and compared the temporal profile of the results with that of TUNEL study, which detects double strand breaks in DNA. There were no immunoreactivities for cyclin B1, cyclin D1, and PCNA in the brain with and without ischemia. As for cdk4, however, it became present at 1 and 3 days of reperfusion after 2 h of ischemia. On the other hand, TUNEL positive cells appeared as early as 3 h of reperfusion, which peaked at 1 and 3 days. These results indicate that aberrant expression of cdk4, but not cyclin B1, cyclin D1 or PCNA, actually takes place in the brain after MCA occlusion, but this is not the causative mechanism of apoptotic cell death in the brain with ischemia.

Topics: Animals; Apoptosis; Brain; Cell Cycle Proteins; Cyclin B; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; DNA Fragmentation; In Situ Nick-End Labeling; Ischemic Attack, Transient; Male; Middle Cerebral Artery; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins; Rats; Rats, Wistar; Reperfusion; Time Factors

An objective of therapeutic intervention after cerebral ischemia is to promote improved functional outcome. Improved outcome may be associated with a reduction of the volume of cerebral infarction and the promotion of cerebral plasticity. In the developing brain, neuronal growth is concomitant with expression of particular proteins, including microtubule-associated protein 2 (MAP-2), growth-associated protein 43 (GAP-43), and cyclin D1. In the present study we measured the expression of select proteins associated with neurite damage and plasticity (MAP-2 and GAP-43) as well as cell cycle (cyclin D1) after induction of focal cerebral ischemia in the rat.. Brains from rats (n=28) subjected to 2 hours of middle cerebral artery occlusion and 6 hours, 12 hours, and 2, 7, 14, 21, and 28 days (n=4 per time point) of reperfusion and control sham-operated (n=3) and normal (n=2) rats were processed by immunohistochemistry with antibodies raised against MAP-2, GAP-43, and cyclin D1. Double staining of these proteins for cellular colocalization was also performed.. Loss of immunoreactivity of both MAP-2 and GAP-43 was observed in most damaged neurons in the ischemic core. In contrast, MAP-2, GAP-43, and cyclin D1 were selectively increased in morphologically intact or altered neurons localized to the ischemic core at an early stage (eg, 6 hours) of reperfusion and in the boundary zone to the ischemic core (penumbra) during longer reperfusion times.. The selective expressions of the neuronal structural proteins (MAP-2 in dendrites and GAP-43 in axons) and the cyclin D1 cell cycle protein in neurons observed in the boundary zone to the ischemic core are suggestive of compensatory and repair mechanisms in ischemia-damaged neurons after transient focal cerebral ischemia.

Topics: Animals; Arterial Occlusive Diseases; Brain Chemistry; Cyclin D1; GAP-43 Protein; Immunoenzyme Techniques; Ischemic Attack, Transient; Male; Microtubule-Associated Proteins; Neuronal Plasticity; Neurons; Rats; Rats, Wistar

c-Jun, a transcriptional activator, as well as cyclin D1, a key regulator of the cell cycle, have been described in vitro as mediators of programmed neuronal death. After trophic factor deprivation, the activation of c-jun and cyclin D1 genes is considered as a necessary step within the cellular machinery that leads to cell death. We show here that both c-Jun and cyclin D1 proteins are present in neurones within the infarcted area after experimental cerebral ischaemia in the mouse. Since their presence was associated with DNA fragmentation revealed by the TUNEL procedure, we propose that c-Jun and cyclin D1 are involved in the process of neuronal death.

Topics: Animals; Apoptosis; Brain; Cell Death; Cyclin D1; Cyclins; DNA Fragmentation; Gene Expression Regulation; Genes, jun; Immunohistochemistry; Ischemic Attack, Transient; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Neurons; Oncogene Proteins; Organ Specificity; Proto-Oncogene Proteins c-jun; Tumor Suppressor Protein p53

Following 30 min of forebrain ischaemia in the rat, delayed neuronal death occurs in the CA1 sector of the hippocampus within two to three days, whereas neurons in other selectively vulnerable regions, such as the dorsolateral striatum, die within 6-12 h. In this study, we investigated cyclin D1 expression, which codes for a regulatory protein in cell cycle regulation, but it is also induced in sympathetic neurons undergoing programmed cell death. Cyclin D1 messenger RNA could not be detected by in situ hybridization techniques in brains of control rats, but was found at one and two days after ischaemia in regions of the dorsolateral striatum with neuronal degeneration. DNA fragmentation in this region, identified by the terminal transferase biotinylated-UTP nick end labelling (TUNEL) procedure, was observed from 6 h after ischaemia onward. In the hippocampus, increased levels of cyclin D1 messenger RNA were found at two and three days after ischaemia in the striatum pyramidale of the CA1 sector. This expression was associated with the occurrence of neuronal damage and TUNEL-stained neurons. By seven days cyclin D1 messenger RNA was found in hardly any brain structure. There was no temporospatial overlap of cyclin D1 expression with the expression of the immediate-early genes c-fos, c-jun, and mkp-1, a result which is clearly distinct from findings in sympathetic ganglion neurons undergoing programmed cell death. These results do not suggest a role for cyclin D1 in neuronal cell death following transient forebrain ischaemia. The similarity of the cyclin D1 expression profile with that of the microglia-specific CR3 complement receptor beta-subunit messenger RNA, and the results of combined in situ hybridization and microglia-specific immunohistochemistry suggest that microglia are the source of cyclin D1 messenger RNA in the postischaemic brain. Since cyclin D1 codes for a critical regulatory protein for progression of the G0 to G1 phase in the cell cycle and we did not observe prominent occurrence of DNA fragmentation in microglial cells in the hippocampus at time points when cyclin D1 messenger RNA was found, we suggest that cyclin D1 induction is involved in the onset of microglial cell proliferation.

Topics: Animals; Antibody Specificity; Arterial Occlusive Diseases; Astrocytes; Base Sequence; Biotin; Cell Death; Cyclin D1; Cyclins; Deoxyuracil Nucleotides; DNA Damage; Gene Expression Regulation; Genes, Immediate-Early; HSP70 Heat-Shock Proteins; In Situ Hybridization; Ischemic Attack, Transient; Male; Microglia; Neurons; Oncogene Proteins; Prosencephalon; Rats; Rats, Wistar; Receptors, Complement; RNA, Messenger; Staining and Labeling