Neuroprotective effects of salidroside on ageing hippocampal neurons and naturally ageing mice via the PI3K/Akt/TERT pathway.

Studies have found that salidroside, isolated from Rhodiola rosea L, has various pharmacological activities, but there have been no studies on the effects of salidroside on brain hippocampal senescence. The purpose of this study was to investigate the mechanistic role of salidroside in hippocampal neuron senescence and injury. In this study, long-term cultured primary rat hippocampal neurons and naturally aged C57 mice were treated with salidroside. The results showed that salidroside increased the viability and MAP2 expression, reduced ß-galactosidase (ß-gal) levels of rat primary hippocampal neurons. Salidroside also improved cognition dysfunction in ageing mice and alleviated neuronal degeneration in the ageing mice CA1 region. Moreover, salidroside decreased the levels of oxidative stress and p21, p16 protein expressions of hippocampal neurons and ageing mice. Salidroside promoted telomerase reverse transcriptase (TERT) protein expression via the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) pathway. In conclusion, our findings suggest that salidroside has the potential to be used as a therapeutic strategy for anti-ageing and ageing-related disease treatment.

Picroside II Exerts a Neuroprotective Effect by Inhibiting the Mitochondria Cytochrome C Signal Pathway Following Ischemia Reperfusion Injury in Rats.

Stroke is a common neurodegenerative disease in the wide world, and mitochondrial defects underlie the pathogenesis of ischemia, especially during reperfusion. Picroside II, the principal active component of Picrorhiza, is a traditional Chinese medicine. Our previous study demonstrated that the best therapeutic dose and time window were injection of picroside II at a dose of 10-20 mg/kg body weight following cerebral ischemia by 1.5-2.0 h. In this paper, the neuroprotective effect and the mechanism of picroside II were investigated, as well as its involvement in antioxidant and mitochondria cytochrome C (CytC) signal pathway following ischemia reperfusion (I/R) injury in rats. After 24 h of cerebral I/R, the neurobehavioral function was measured by modified neurological severity score test; the content of reactive oxygen species in brain tissue was measured by enzyme-linked immunosorbent assay; the cerebral infarction volume was detected by TTC staining; the morphology of brain tissue was observed by hematoxylin-eosin; the apoptotic cells were counted by terminal deoxynucleotidyl transferase dUTP nick end labeling assay; the ultrastructure of the cortical brain tissues was observation by transmission electron microscopy; the expressions of CytC and Caspase-3 were determined by immunohistochemical assay and Western blot. The results indicated that picroside II could scavenge ROS contents, decrease the cerebral infarction volume and apoptotic cells, protect the structure of mitochondria, down-regulate the expression of CytC and Caspase-3 in cerebral I/R rats. It can be concluded that picroside II exerts a neuroprotective effect by inhibiting the mitochondria CytC signal pathway following ischemia reperfusion injury in rats.

The Effect of Propofol on Mitochondrial Fission during Oxygen-Glucose Deprivation and Reperfusion Injury in Rat Hippocampal Neurons.

The neuroprotective role of propofol in transient global and focal cerebral ischemia reperfusion (I/R) animal model has recently been highlighted. However, no studies have conducted to explore the relationship between mitochondrial fission/fusion and I/R injury under the intervention of propofol. Moreover, neuroprotective mechanism of propofol is yet unclear. Culturing primary hippocampal cells were subjected to oxygen-glucose deprivation and re-oxygenation (OGD/R) model, as a model of cerebral I/R in vitro. Methods CCK-8 assay was used to test the effect of propofol on cell viability. We examined the effect of propofol on mitochondrial ultrastructure and mitochondrial fission evoked by OGD/R with transmission electron microscopy and immunofluorescence assay. To investigate possible neuroprotective mechanisms, the authors then examined whether propofol could inhibit calcium-overload, calcineurin (CaN) activation and the phosphorylation of dynamin-related protein 1 (Drp1) during the period of OGD/R, as well as the combination of Drp1-ser 637 and fission 1 (Fis1) protein by immunofluorescence assay, ELISA and double-labeling immunofluorescence analysis. Finally, the expression of Drp1-ser 637 and Fis1, apoptosis inducing factor (AIF) and cytochrome C (Cyt C) were detected by western blot. When added in culture media during OGD period, propofol (0.1µM-50µM) could alleviate neurons injury and protect mitochondrial ultrastructure, meanwhile inhibit mitochondrial fission. Furthermore, the concentration of intracellular free Ca2+, CaN activition and the phosphorylation of Drp1-ser637 were suppressed, as well as the translocation and combination of Drp1-ser 637 and Fis1. The authors also found that the expression of Cyt C, AIF, Drp1-ser637 and Fis1 were down-regulated. Notably, high dose of propofol (100µM-200µM) were confirmed to decrease the survival of neurons based on results of cell viability. Propofol could inhibit mitochondrial fission and mitochondrial apoptotic pathway evoked by OGD/R in rat hippocampal neurons, which may be via depressing calcium-overload.

[Effect of Picroside II on ERK1/2 Signal Pathway in Cerebral lschemic Injury Rats].

OBJECTIVE: To explore the neuroprotective effect and mechanism of picroside II on extracellular regulated protein kinases1/2 (ERK1/2) signal transduction pathway in cerebral ischemia injuryrats. METHODS The middle cerebral artery occlusion (MCAO) model was established by inserting a monofilament into middle cerebral artery. Totally 96 successfully modeled Wistar rats were divided into the modelgroup, the treatment (picroside II) group, the Lipopolysachcaride (LPS) group, and the U0126 group according to random digit table. Each group was further divided into 3 subgroups, i.e. 6, 12, and 24 h sub-groups. Picroside II (20 mg/kg) was peritoneally injected to rats in the treatment group 2 h after ischemia.LPS (20 mg/kg) and Picroside II (20 mg/kg) were peritoneally injected to rats in the LPS group 2 h after ischemia. U0126-EtOH (20 mg/kg)and Picroside II (20 mg/kg) were peritoneally injected to rats in the U0126group 2 h after ischemia. Equal volume of normal saline was peritoneally injected to rats in the control groupand the model group. The neurobehavioral function was evaluated by modified neurological severity score(mNSS) test. The structure of neurons was observed using hematoxylin-eosinstaining (HE) staining. Theapoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expression of phosphorylated extracellular signal-regulated protein kinase1,2 (pERK1,2) in cortex was detected using immunohistochemistry (IHC) and Western blot. RESULTS: After cerebral ischemia injury neurological impairment score increased, the damage of neuron in the cortical area was aggravated, apoptotic cells increased in the model group as time went by. The expression of pERK1/2 increased more significantly in the model group than in the control group (P <0.05). The damage of neuron in the cortical area was milder, while apoptotic cells decreased, the expression of pERK1f2 obviously decreased more in the treatment group and the U0126 group (P < 0.05). The early damage of neuron in the cortical area was more severe, apoptotic cells and the expression of pERK12 were comparatively higher in early stage of the LPS group, but the expression of pERK1/2 was somewhat decreased in late stage. CONCLUSIONS: Activating ERK12 pathway could mediate apoptosis and inflammatory reactions of neurons after cerebral ischemia injury. Picroside II could protect the nerve system possibly through reducing activation of ERKI2 pathway, inhibiting apoptosis of neurons and inflammation reaction.

Picroside II Inhibits the MEK-ERK1/2-COX2 Signal Pathway to Prevent Cerebral Ischemic Injury in Rats.

The objective of this study is to explore the neuroprotective effect and mechanism of picroside II on ERK1/2-COX2 signal transduction pathway after cerebral ischemic injury in rats. Focal cerebral ischemic models were established by inserting monofilament threads into the middle cerebral artery in 200 Wistar rats. Twenty four rats were randomly selected into control group, while the other rats were randomly divided into six groups: model group, picroside group, lipopolysaccharide (LPS) with picroside group, U0126 with picroside group, LPS group, and U0126 group with each group containing three subgroups with ischemia at 6, 12, and 24 h. Neurobehavioral function in the rats was evaluated by modified neurological severity score points (mNSS) test; structure of neurons was observed using hematoxylin-eosin (HE) staining; apoptotic cells were counted using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay; expressions of phosphorylated mitogen/extracellular signal-regulated kinase kinas1/2 (pMEK1/2), phosphorylated extracellular signal-regulated protein kinase1/2 (pERK1/2), and cyclooxygenase (COX2) in the cortex were determined using immunohistochemistry (IHC) and Western blot (WB); and real-time PCR was used to determine the level of COX2 mRNA. The neurological behavioral malfunction appeared in all rats with middle cerebral artery occlusion (MCAO). In the model group, neuron damage was extensive, while the neurobehavioral function score, apoptotic cell index, expression of pMEK1/2, pERK1/2, and COX2 and the level of COX2 mRNA increased significantly when compared to the control group. The peak COX2 mRNA level was in ischemia 12 h, prior to the peak in COX2 protein expression. In the picroside and U0126 groups, the neurological behavioral function was improved, and the number of apoptotic cells and the expression of pMEK1/2, pERK1/2, and COX2 decreased significantly when compared to the model group. In the LPS with picroside group, at ischemia 6 h neuron damage was extensive, and pMEK1/2, pERK1/2, and COX2 expression were much higher than in the model group. But at ischemia 12 and 24 h, the expression of pMEK1/2, pERK1/2, and COX2 decreased slightly, and the neurobehavioral function also improved slightly. In LPS group, neuron damage was extensive, pMEK1/2, pERK1/2, and COX2 expression was still at a high level, and COX2 mRNA peak arrived at ischemic 12 h. Picroside II downregulates COX2 expression after MCAO by inhibiting MEK-ERK1/2 in rats to protect neurons from apoptosis and inflammation.

Astragalus injection protects cerebral ischemic injury by inhibiting neuronal apoptosis and the expression of JNK3 after cerebral ischemia reperfusion in rats.

BACKGROUND: Astragalus is a widely used traditional Chinese medicine and has been proven beneficial for many aspects of human health. It is important to explore the neuroprotective effect and mechanism of astragalus injection in cerebral ischemia reperfusion injury. METHODS: The focal cerebral ischemic model with middle cerebral artery occlusion (MCAO) reperfusion was established by Longa's method in healthy adult male Wistar rats, and treated by injecting intraperitoneally astragalus injection (3 ml/kg). The neurobehavioral function of rats was evaluated by Longa's test. The cerebral blood flow (CBF) was measured by laser Doppler flowmetry and the cerebral infarct volume was calculated by tetrazolium chloride (TTC) stain. The shape and structure of neurons in parahippocampal area was observed by HE stain and the neuronal apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and flow cytometry. The expressions of c-jun N-terminal kinase 3 (JNK3) mRNA and protein were determined by RT-PCR and immunohistochemical assay and Western blotting respectively. RESULTS: After treatment with astragalus injection, the expressions of JNK3 mRNA and protein reduced significantly, the number of neuronal apoptosis minus, the cerebral infarct volume shrink, the neuronal shape-structure and animal neurobehavioral function improved significantly than those in model rats. CONCLUSIONS: It is suggested that astragalus injection could inhibit neuronal apoptosis, reduce infarct volume and improve neurobehavioral function by down-regulating the expression of JNK3 gene after cerebral ischemia reperfusion injury in rats.

The hypoglycemic effect of the kelp on diabetes mellitus model induced by alloxan in rats.

Hypoglycemic effects and the use of kelp in diabetes mellitus (DM) model rats induced by alloxan were investigated. Sixty healthy male rats were used to establish DM models by injecting alloxan intraperitoneally. Kelp powder was added to the general forage for the rats. The levels of fasting blood glucose (FBG) were determined by an automatic blood glucose device. Electrochemiluminescence immunoassay was applied to determine the serum levels of insulin. The serum levels of malondialdehyde (MDA) were measured by thiobarbituric acid assay and nitric oxide (NO) by nitrate reductase assay. The activities of superoxide dismutase (SOD) were determined by xanthinoxidase assay and glutathione peroxidase (GSH-Px) by chemical colorimetry. The shape and structure of islet cells were observed with Hematine-Eosin staining, and the expression of superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in islet cells were detected by immunohistochemical assay. The results showed that the serum levels of insulin after treatment with kelp powder increased significantly compared to those in the DM-model group, while the FBG in the medium-high dose treated groups decreased significantly compared to those in the DM-model group (P < 0.05). The levels of MDA and NO in the kelp powder groups were lower than those in the DM-model group, while the activities of SOD and GSH-Px were higher than those in the DM-model group, of which a significant difference existed between the medium-high dose treated groups and the DM-model group (P < 0.05). The shape and structure of islet cells improved with the up-expressing SOD and down-expressing iNOS in the medium-high dose treated groups compared to those in the DM-model group (P < 0.05). There were no significant differences between the medium and high dose treated groups, all above indexes (P > 0.05). It is suggested that kelp might aid recovery of the the islet cell secreting function and reduce the level of FBG by an antioxidant effect.

[Effect of tagalsin on p53 and Bcl-2 expression in hepatoma H(22) tumor-bearing mice].

OBJECTIVE: To explore the effect and mechanism of tagalsin on hepatoma cells. METHODS: The animal models were established by transplanting H(22) mouse hepatoma cells to mouse liver, and ten days later the mice were randomly divided into five groups: blank group, carmofur positive group and tagalsin groups, including low-dose, middle-dose and high-dose groups. Then medicine or oil was given to the mice by gastric gavage in consecutive 5 days with a 2-days interval as a course of treatment, two courses in all. All mice were killed at 24 hours after medication, and the survival period, ascites conditions, aggressive conditions intra- or extra-liver, weight changes, tumor volume and spleen index of the tumor-bearing mice were observed. Pathological changes of the tumors were examined. Apoptotic factors p53 and Bcl-2 protien and mRNA were detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). RESULTS: tagalsin inhibited the hepatoma growth effectively without influencing spleen index to some extent. The tumor inhibition rate of tagalsin low, middle and high dose groups were 17.9%, 63.1% and 71.8%, respectively. Immunohistochemical results showed that the p53 and Bcl-2 protein positive cell counts of the positive control and experimental groups were significantly lower than those of the blank group (P < 0.01). RT-PCR results showed that the p53 mRNA expression was significantly enhanced and Bcl-2 mRNA expression was decreased in the positive control groups and tagalsin treatment groups, especially in the high dose group, compared with those of the blank group (P < 0.05). CONCLUSIONS: tagalsin can inhibit the growth of mouse hepatoma cells significantly. The mechanism of its anti-tumor effect may work via up-regulating the wild type p53 gene expression and down-regulating Bcl-2 gene expression and thus regulating tumor cell apoptosis.

[Effect of picroside II on expressions of TLR4 and NFkappaB in rats with cerebral ischemia reperfusion injury].

OBJECTIVE: To explore the effects of picrodide II on the expressions of Toll-like receptor 4 (TLR4) and nuclear transcription factor kappaB (NFkappaB) in brain tissue of rat after cerebral ischemic reperfusion (I/R) injury. METHODS: Ten rats from 60 adult healthy female Wistar rats received sham-operation were set as the sham-operative group. Established as middle cerebral I/R model (MCAO/R) by thread tying method, the 30 successfully modeled rats were equally randomized into the negative control group, the positive control group and the treatment group. Besides, rats in the treatment group and the positive control group were respectively intervened with picrodide II (10 mg/kg) and salvianic acid A sodium (10 mg/kg) via caudal vein injection before I/R injury, while rats in the sham-operative group and the negative group were injected with equal volume of 0.1 mol/L PBS. Immunohistochemistry stain was used to determine the expressions of TLR4 and NFkappaB, and the apoptotic cells were counted by TUNEL-immunofluorescence assay. RESULTS: In the sham-operative group, the TLR4 and NFkappaB expressed weakly with few TUNEL positive cells scattering in the cortex, striatum and hippocampus. As compared with the sham-operative group, TLR4 and NFkappaB in the negative control group were significantly higher both in absorption A) value and cell number (P < 0.05). In the treatment group and the positive control group, the expressions of TLR4 and NFkappaB and the number of TUNEL positive cells were significantly lower than those in the negative control group (P < 0.05), but no significant difference was shown between the two treated groups (P > 0.05). CONCLUSIONS: Picroside II could down-regulate the expressions of TLR4 and NFkappaB, and inhibit the inflammatory response induced apoptosis in cerebral I/R injured rats.