Ginsenoside Rg1 promotes ߭amyloid peptide degradation through inhibition of the ERK/PPARγ phosphorylation pathway in an Alzheimer's disease neuronal model.

ß-Amyloid peptide (Aß) deposition in the brain is an important pathological change in Alzheimer's disease (AD). Insulin-degrading enzyme (IDE), which is regulated transcriptionally by peroxisome proliferator-activated receptor γ (PPARγ), is able to proteolyze Aß. One of the members of the MAPK family, ERK, is able to mediate the phosphorylation of PPARγ at Ser112, thereby inhibiting its transcriptional activity. Ginsenoside Rg1 is one of the active ingredients in the natural medicine ginseng and has inhibitory effects on Aß production. The present study was designed to investigate whether ginsenoside Rg1 is able to affect the regulation of PPARγ based on the expression of its target gene, IDE, and whether it is able to promote Aß degradation via inhibition of the ERK/PPARγ phosphorylation pathway. In the present study, primary cultured rat hippocampal neurons were treated with Aß1-42, ginsenoside Rg1 and the ERK inhibitor PD98059, and subsequently TUNEL staining was used to detect the level of neuronal apoptosis. ELISA was subsequently employed to detect the intra- and extracellular Aß1-42 levels, immunofluorescence staining and western blotting were used to detect the translocation of ERK from the cytoplasm to the nucleus, immunofluorescence double staining was used to detect the co-expression of ERK and PPARγ, and finally, western blotting was used to detect the phosphorylation of PPARγ at Ser112 and IDE expression. The results demonstrated that ginsenoside Rg1 or PD98059 were able to inhibit primary cultured hippocampal neuron apoptosis induced by Aß1-42 treatment, reduce the levels of intra- and extraneuronal Aß1-42 and inhibit the translocation of ERK from the cytoplasm to the nucleus. Furthermore, administration of ginsenoside Rg1 or PD98059 resulted in attenuated co-expression of ERK and PPARγ, inhibition of phosphorylation of PPARγ at Ser112 mediated by ERK and an increase in IDE expression. In addition, the effects when PD98059 to inhibit ERK followed by treatment with ginsenoside Rg1 were found to be more pronounced than those when using PD98059 alone. In conclusion, ginsenoside Rg1 was demonstrated to exert neuroprotective effects on AD via inhibition of the ERK/PPARγ phosphorylation pathway, which led to an increase in IDE expression, the promotion of Aß degradation and the decrease of neuronal apoptosis. These results could provide a theoretical basis for the clinical application of ginsenoside Rg1 in AD.

Sestrin2 attenuates depressive-like behaviors and neuroinflammation in CUMS mice through inhibiting ferroptosis.

Sestrin2 (SESN2) is a stress-inducible protein and acts as a neuroprotective regulator. The present study aimed to explore the antidepressant activity of SESN2 and its relevant mechanism. Depression mouse model was established by chronic unpredictable mild stress (CUMS) for a successive 5 weeks. Behaviors tests were conducted to examine depressive-like behaviors including sugar preference test, tail suspension test and open field test. The expression of SESN2 and ferroptosis-related proteins was examined by western blot. The production of cytokines was measured by ELISA. Iron deposition was assessed using Prussian blue staining and Fe 2+ content was measured using commercial kits. Lipid peroxidation was evaluated by thiobarbituric acid reactive substances assay. BV-2 cells were treated with LPS to induce microglial activation, which was evaluated by the iba-1 level adopting immunofluorescence assay. The ferroptosis inducer Erastin was adopted for the pretreatment in BV-2 cells to conduct a rescue experiment. SESN2 was downregulated in CUMS-induced mice, and SESN2 overexpression dramatically ameliorated CUMS-induced depression-like behaviors. Meanwhile, SESN2 reduced the production of pro-inflammatory cytokines and iba-1 level in hippocampus of CUMS mice, as well as reducing iron deposition and lipid peroxidation, demonstrating that SESN2 reduced microglial activation, neuroinflammation and ferroptosis in CUMS mice. Similarly, SESN2 also restricted iba-1 level, pro-inflammatory cytokines production, and ferroptosis in LPS-induced BV-2 cells, which was partly reversed by additional treatment of Erastin. These findings suggest that SESN2 possesses potent antidepressant property through inhibiting ferroptosis and neuroinflammation.

Ginsenoside Rg1 reduces β­amyloid levels by inhibiting CDΚ5­induced PPARγ phosphorylation in a neuron model of Alzheimer's disease.

The accumulation of β­amyloid peptides (Aβ) in the brain is a hallmark of Alzheimer's disease (AD). Studies have indicated that ginsenoside Rg1, a primary component of ginseng (Panax ginseng), reduces brain Aβ levels in an AD model through peroxisome proliferator­activated receptor γ (PPARγ), thereby regulating the expression of insulin­degrading enzyme (Ide) and β­amyloid cleavage enzyme 1 (Bace1), which are PPARγ target genes. However, the effects of ginsenoside Rg1 on PPARγ remain unclear. Since cyclin­dependent kinase 5 (CDK5) mediates PPARγ phosphorylation in adipose tissue, this study aimed to investigate whether ginsenoside Rg1 regulates PPARγ target genes and reduces Aβ levels by inhibiting PPARγ phosphorylation through the CDK5 pathway. In the present study, a model of AD was established by treating primary cultured rat hippocampal neurons with Aβ1­42. The cells were pretreatment with ginsenoside Rg1 and roscovitine, a CDK5­inhibitor, prior to the treatment with Aβ1­42. Neuronal apoptosis was detected using TUNEL staining. PPARγ phosphorylation and protein expression levels of PPARγ, CDK5, IDE, BACE1, amyloid precursor protein (APP) and Aβ1­42 were measured by western blotting. The mRNA expression levels of PPARγ, CDK5, IDE, BACE1 and APP were assessed using reverse transcription­quantitative PCR. The results of the present study demonstrated that in an AD model induced by Aβ1­42, ginsenoside Rg1 significantly decreased CDK5 expression, inhibited PPARγ phosphorylation at serine 273, elevated IDE expression, downregulated BACE1 and APP expression, decreased Aβ1­42 levels and attenuated neuronal apoptosis. The CDK5 inhibitor, roscovitine, demonstrated similar effects. These results suggest that ginsenoside Rg1 has neuroprotective properties and has potential for use in the treatment of AD.

CDK5 Participates in Amyloid-ß Production by Regulating PPARγ Phosphorylation in Primary Rat Hippocampal Neurons.

Cyclin-dependent kinase 5 (CDK5) in adipose tissue mediates peroxisome proliferator-activated receptor γ (PPARγ) phosphorylation at Ser273 to inhibit its activity, causing PPARγ target gene expression changes. Among these, insulin-degrading enzyme (IDE) degrades amyloid-ß peptide (Aß), the core pathological product of Alzheimer's disease (AD), whereas ß-amyloid cleavage enzyme 1 (BACE1) hydrolyzes amyloid-ß protein precursor (AßPP). Therefore, we speculated that CDK5 activity in the brain might participate in Aß production, thereby functioning as a key molecule in AD pathogenesis. To confirm this hypothesis, we transduced primary rat hippocampal neurons using CDK5-expressing lentiviral vectors. CDK5 overexpression increased PPARγ Ser273 phosphorylation, decreased IDE expression, increased BACE1 and AßPP expression, increased Aß levels, and induced neuronal apoptosis. The CDK5 inhibitor roscovitine effectively reversed these CDK5 overexpression-mediated effects. Moreover, silencing of the Cdk5 gene via CDK5 shRNA-expressing lentiviral vectors in primary hippocampal neurons did not exert any protective effect against normal neuronal apoptosis, nor were significant effects observed on Aß levels, PPARγ phosphorylation, or PPARγ target gene expression in the cells. However, Cdk5 gene silencing exhibited a neuroprotective effect in the Aß-induced AD neuron model by effectively inhibiting the Aß-induced neuronal apoptosis, PPARγ phosphorylation, PPARγ expression downregulation, and PPARγ target gene expression changes, and reducing Aß levels. In conclusion, this study demonstrated that CDK5 played an important role in the pathogenesis of AD. Specifically, CDK5 participated in Aß production by regulating PPARγ phosphorylation. Targeted therapy against CDK5 could effectively reduce and reverse the neurotoxic effects of Aß and may represent a novel approach for AD treatment.

Pioglitazone Reduces ß Amyloid Levels via Inhibition of PPARγ Phosphorylation in a Neuronal Model of Alzheimer's Disease.

It has been demonstrated that peroxisome proliferator-activated receptor γ (PPARγ) can regulate the transcription of its target gene, insulin-degrading enzyme (IDE), and thus enhance the expression of the IDE protein. The protein can degrade ß amyloid (Aß), a core pathological product of Alzheimer's disease (AD). PPARγ can also regulate the transcription of other target gene, ß-amyloid cleavage enzyme 1 (BACE1), and thus inhibit the expression of the BACE1 protein. BACE1 can hydrolyze amyloid precursor protein (APP), the precursor of Aß. In adipose tissue, PPARγ agonists can inhibit the phosphorylation of PPARγ by inhibiting cyclin-dependent kinase 5 (CDK5), which in turn affects the expression of target genes regulated by PPARγ. PPARγ agonists may also exert inhibitory effects on the phosphorylation of PPARγ in the brain, thereby affecting the expression of the aforementioned PPARγ target genes and reducing Aß levels. The present study confirmed this hypothesis by showing that PPARγ agonist pioglitazone attenuated the neuronal apoptosis of primary rat hippocampal neurons induced by Aß1-42, downregulated CDK5 expression, weakened the binding of CDK5 to PPARγ, reduced PPARγ phosphorylation, increased the expression of PPARγ and IDE, decreased the expression of BACE1, reduced APP production, and downregulated intraneuronal Aß1-42 levels. These effects were inhibited by the PPARγ antagonist GW9662. After CDK5 silencing with CDK5 shRNA, the above effect of pioglitazone was not observed, except when upregulating the expression of PPARγ in Aß1-42 treated neurons. In conclusion, this study demonstrated that pioglitazone could inhibit the phosphorylation of PPARγ in vitro by inhibiting CDK5 expression, which in turn affected the expression of PPARγ target genes Ide and Bace1, thereby promoting Aß degradation and reducing Aß production. This reduced Aß levels in the brain, thereby exerting neuroprotective effects in an AD model.

Ginsenoside Rg1 inhibits high-voltage-activated calcium channel currents in hippocampal neurons of beta-amyloid peptide-exposed rat brain slices.

OBJECTIVE: To examine whether ginsenoside Rg1 (Rg1) inhibits the high-voltage-activated calcium currents (ICa,HVA) via mitogen-activated protein kinase (MAPK) in hippocampal neurons in rat brain slices exposed to beta-amyloid peptide 25-35 (Aß25-35). METHODS: An experimental Alzheimer disease (AD) model was prepared by exposure of rat brain slices to Aß25-35 (10 µmol/L). After treatment with Rg1 (20 µmol/L), the ICa,HVA elicited in hippocampal neurons in these rat brain slices upon depolarization from-40 to 40 mV for 200 ms was recorded by a whole-cell patch clamp to analyze the changes in the peak current density, I-V curve, activation-V curve, and inactivation-V curve. RESULTS: Exposure of rat brain slices to Aß led to a significant increase in ICa,HVA, enhancement of the voltage sensitivity of channel activation, and reduction of the voltage sensitivity of channel inactivation in neurons in the hippocampus of rat brain slices. Rg1 treatment significantly inhibited these changes. These effects of Rg1 could be effectively inhibited by the MAPK inhibitor PD98059. CONCLUSION: Rg1 can inhibit Ica,HVA via MAPK in hippocampal neurons in Aß-exposed rat brain slices.

Ginsenoside Rg1 decreases Aß(1-42) level by upregulating PPARγ and IDE expression in the hippocampus of a rat model of Alzheimer's disease.

BACKGROUND AND PURPOSE: The present study was designed to examine the effects of ginsenoside Rg1 on expression of peroxisome proliferator-activated receptor γ (PPARγ) and insulin-degrading enzyme (IDE) in the hippocampus of rat model of Alzheimer's disease (AD) to determine how ginsenoside Rg1 (Rg1) decreases Aß levels in AD. EXPERIMENTAL APPROACH: Experimental AD was induced in rats by a bilateral injection of 10 µg soluble beta-amyloid peptide 1-42 (Aß(1-42)) into the CA1 region of the hippocampus, and the rats were treated with Rg1 (10 mg·kg(-1), intraperitoneally) for 28 days. The Morris water maze was used to test spatial learning and memory performance. Hematoxylin-eosin staining was performed to analyze the hippocampal histopathological damage. Immunohistochemistry, western blotting, and real-time PCR were used to detect Aß(1-42), PPARγ, and insulin-degrading enzyme (IDE) expression in the hippocampus. KEY RESULTS: Injection of soluble Aß(1-42) into the hippocampus led to significant dysfunction of learning and memory, hippocampal histopathological abnormalities and increased Aß(1-42) levels in the hippocampus. Rg1 treatment significantly improved learning and memory function, attenuated hippocampal histopathological abnormalities, reduced Aß(1-42) levels and increased PPARγ and IDE expression in the hippocampus; these effects of Rg1 could be effectively inhibited by GW9662, a PPARγ antagonist. CONCLUSIONS AND IMPLICATIONS: Given that PPARγ can upregulate IDE expression and IDE can degrade Aß(1-42), these results indicate that Rg1 can increase IDE expression in the hippocampus by upregulating PPARγ, leading to decreased Aß levels, attenuated hippocampal histopathological abnormalities and improved learning and memory in a rat model of AD.

Cellular and molecular mechanisms underlying the action of ginsenoside Rg1 against Alzheimer's disease.

Ginsenoside Rg1 inhibits oxidation, aging and cell apoptosis, and improves cognitive function. In this study, we pretreated rat brain tissue sections with ginsenoside Rg1, and established brain slice models of Alzheimer's disease induced by okadaic acid. The results revealed that ginsenoside Rg1 pretreatment suppressed the increase in phosphorylated Tau protein expression induced by incubation with okadaic acid, and reduced brain-derived neurotrophic factor expression. These results suggest that ginsenoside Rg1 upregulates brain-derived neurotrophic factor expression and inhibits Tau protein phosphorylation in brain slices from a rat model of Alzheimer's disease.

Scavenging effect of Naoerkang on amyloid beta-peptide deposition in the hippocampus in a rat model of Alzheimer's disease.

OBJECTIVE: To observe the effect of a Chinese medicine compound, Naoerkang (NEK), on amyloid-beta peptide (1-42; Aß(1-42)) and matrix metalloproteinase-9 (MMP-9) expressions in the hippocampus of Alzheimer's disease (AD) model rats. METHODS: A total of 48 male Sprague Dawley (SD) rats were randomly divided into normal control, untreated, and piracetam groups, and low-dose, medium-dose, and high-dose NEK groups, with 8 rats in each group. The 5-µL aggregated Aß(1-42) (2 µg/µL) were injected into both CA1 areas of the hippocampus in the rats to establish an AD model, whereas the normal control was treated with the same dose of normal saline. The rats in the NEK groups were treated with a high, medium, or low dose of NEK [60 g/(kg·d), 30 g/(kg·d), and 15 g/(kg·d)], respectively, intragastrically for 28 days; piracetam (0.375 g/kg, intragastrically) was consecutively administered in the piracetam group; and normal saline was applied in the normal control and untreated groups. A Y-maze test was used for behavioral study to test the learning and memory abilities. Aß(1-42) and MMP-9 expressions in the hippocampus was determined immunohistochemically, and the results were analyzed by image acquisition and an analysis system. RESULTS: Aggregated Aß(1-42) induced obvious learning and memory dysfunction, as well as up-regulation of Aß(1-42) expression in the hippocampus. Compared with those in the normal control group, the learning and memory abilities of rats in the untreated group significantly decreased (P<0.01), and the expression of Aß(1-42) was significantly increased (P<0.01). Twenty-eight days after different treatments, compared with those in the untreated group, the learning and memory abilities of AD model rats in the piracetam, low-dose, medium-dose and high-dose NEK groups were significantly improved (P<0.01 or P<0.05), and the expression of Aß(1-42) in the hippocampus decreased (P<0.01 or P<0.05), and MMP-9 increased (P<0.01 or P<0.05), especially in the high-dose NEK group. CONCLUSION: NEK might play a role of anti-dementia by increasing the expression of MMP-9 in the hippocampus of AD model rats, resulting in the reduction of the quantity of Aß(1-42) and improvement in learning and memory ability in AD model rats.

[Effects of gensenoside Rg1 on tau protein phosphorylation induced by okadaic acid in rat brain slices].

OBJECTIVE: To investigate the effects of gensenoside Rg1 on expressions of phosphorylated tau protein (P-tau), protein phosphatase 2A (PP2A) and tau protein in Alzheimer's disease-like tau phosphorylation rat brain slices, and to explore the mechanisms of gensenoside Rg1 in inhibiting tau phosphorylation. METHODS: Brains of 5-week-old Wistar rats were cut into slices which were 400 µm thick. These brain slices were randomly divided into blank control group, untreated group, low-dose ginsenoside Rg1 group, medium-dose ginsenoside Rg1 group and high-dose ginsenoside Rg1 group, with 10 slices in each group. All brain slices were cultured with artificial cerebrospinal fluid (ACSF). And brain slices in the ginsenoside R1 groups were administered with ginsenoside Rg1 (60, 120 and 240 µmol/L respectively) in ACSF for 2 h firstly. After 2-hour culture, okadaic acid (OA) was administered into ACSF of the untreated group and the ginsenoside Rg1 groups separately for 3 h to induce tau phosphorylation to prepare AD models. The concentration of OA in each group was 1 µmol/L. There was no any intervention for the brain slices in the blank control group. Expressions of P-tau, PP2A and tau proteins in the brain slices were determined by immunohistochemical method, and the results were analyzed by image acquisition and analysis system. RESULTS: Compared with the blank control group, the expression of P-tau protein was significantly increased (P<0.01) and the expressions of tau and PP2A proteins were decreased (P<0.01, P<0.05) in the untreated group. Compared with the untreated group, the expression of P-tau was significantly decreased (P<0.01) and the expressions of tau and PP2A proteins were increased (P<0.01, P<0.05) in the ginsenoside Rg1 groups, especially in the high-dose ginsenoside Rg1 group. CONCLUSION: Ginsenoside Rg1 can promote dephosphorylation of P-tau by increasing the expression of PP2A in Alzheimer's disease-like tau phosphorylation rat brain slices, so as to inhibit tau phosphorylation.

[Experimental research on effect of gensenoside Rg1 on expressions of P-Tau and caspase-3 in brain slices from AD model rats].

OBJECTIVE: To observe the effect of ginsenoside Rg1 on the expressions of phosphory protein Tau (P-Tau) and caspase-3 in brain slices from AD model rats. METHOD: The brains of 5-week-old Wista rats were cut into slices which were 400 microm thick. These brain slices were divided into five groups: normal contral group, untreated group, low-dose, medium-dose and high-dose ginsenoside Rg1 groups (60, 120, 240 micromol x L(-1)). And there were 10 slices in each group. These brain slices were cultured with artificial cerebrospinal fluid. After the brain slices in ginsenoside Rg1 groups were administration with ginsenoside Rg1 for 2 h preventively, brain slices in untreated group and ginsenoside Rg1 groups were administrated with okadaic acid (OA) for 3 h to induce hyperphosphorylation of Tau protein to prepare AD models. And the effects of ginsenoside Rg1 on the expressions of P-Tau and caspase-3 in brain slices from AD model rats in each group were observed with immunohistochemistry and image analysis technology. RESULT: The levels of the expressions of P-Tau and caspase-3 in the untreated group were significantly higher than those in the normal control group (P < 0.01). Compared with untreated group, the levels of the expressions of P-Tau and caspase-3 in ginsenoside Rg1 groups were significantly low (P < 0.01 or P < 0.05). CONCLUSION: Ginsenoside Rg1 could inhibit the expression of P-Tau to slow the formation of neurofibrillary tangles and could inhibit the expression of caspase-3 to inhibit neuronal apoptosis to protect the nerve cells, so as to play the role of anti-dementia.

[Effects of Naoerkang on expressions of beta-amyloid peptide 1-42 and neprilysin in hippocampus in a rat model of Alzheimer's disease].

OBJECTIVE: To investigate the effects of Naoerkang (NEK), a compound traditional Chinese herbal medicine, on the expressions of beta-amyloid peptide 1-42 (Abeta(1-42)) and neprilysin (NEP) in hippocampal tissues in a rat model of Alzheimer's disease (AD). METHODS: Forty-eight male SD rats were randomly divided into normal control group, untreated group, piracetam group, low-dose NEK group, medium-dose NEK group, and high-dose NEK group, with 8 rats in each group. Five microliters of Abeta(1-42) (2 microg/microL) were injected into CA1 area of hippocampus in rat to establish AD model whereas the normal control rats were injected with same volume of normal saline for comparison. The rats in the NEK groups were treated respectively with high-, medium- and low-dose [60, 30, 15 g/(kg.d)] NEK for 28 days consecutively; piracetam [0.375 g/(kg.d)] was intragastrically administered to rats in the piracetam group; and normal saline was applied in the control and untreated groups. Y-maze test was used for behavioral study to test the learning and memory abilities of rats in different groups. The expressions of Abeta(1-42) and NEP in hippocampus were determined by immunohistochemical method, and the results were analyzed by image acquisition and analysis system. RESULTS: Injection of Abeta(1-42) could induce learning and memory dysfunction and up-regulate Abeta(1-42) expression in hippocampal tissue in rats of the untreated group. Compared with the normal control group, the abilities of learning and memory of rats in the untreated group were significantly decreased (P<0.01) and the expression of Abeta(1-42) was significantly increased (P<0.01) after model establishment. After 28-day administration of NEK and piracetam, the abilities of learning and memory of AD rats in piracetam and low-dose, medium-dose and high-dose NEK groups were significantly improved as compared with the untreated group (P<0.01 or P<0.05); the expression of Abeta(1-42) in hippocampal tissues was decreased (P<0.01 or P<0.05) and the expression of NEP was increased (P<0.01 or P<0.05), especially in the high-dose NEK group. CONCLUSION: NEK can play the role of anti-dementia by increasing the expression of NEP in hippocampal tissues of AD rats so as to reduce the quantity of AAbeta(1-42) and by improving the ability of learning and memory of rats with AD.

[Effect of ginsenoside Rg1 on expressions of phosphory protein tau and N-methyl-D-aspartate receptor subunits NR1 and NR2B in rat brain slice model of Alzheimer's disease].

OBJECTIVE: To investigate the effect of ginsenoside Rg1 on the expressions of phosphory protein Tau (P-Tau), N-methyl-D-aspartate receptor subunit 1 (NR1) and N-methyl-D-aspartate receptor subunit 2B(NR2B) in rat brain slice model of Alzheimer's disease. METHOD: Brains of 5-week-old Wistar rats were cut into slices which were 400 microm thick. These brain slices were randomly divided into normal control group, untreated group, low-dose ginsenoside Rg1 group, medium-dose ginsenoside Rg1 group and high-dose ginsenoside Rg1 group, with 10 slices in each group. All brain slices were cultured with artificial cerebrospinal fluid (ACSF) that was aerated via polyethylene tubing attached to a source of 95% O2, 5% CO2 at (32.0 +/- 0.5) degrees C. And brain slices in the ginsenoside R1 groups were administrated with the ginsenoside Rg1 (60, 120 and 240 micromol x L(-1) respectively) in ACSF for 2 h firstly. Then okadaic acid (OA) was administrated into ACSF of untreated group and ginsenoside Rg1 groups separately for 3 h to induce Tau phosphorylation to prepare AD models. The concentration of OA in each group was 1 micromol x L(-1). And there was no any intervention for the brain slices in the normal control group. The expressions of P-Tau, NR1 and NR2B in brain slices in each group were determined by immunohistochemical method, and the results were analyzed by image acquisition and analysis system. RESULT: Compared with the normal control group, the expression of P-Tau was significantly increased (P < 0.05 or P < 0.01) and the expressions of NR1 and NR2B were decreased (P < 0.01) in untreated group. Compared with the untreated group, the expression of P-Tau was significantly decreased (P < 0.01 or P < 0.05) and the expressions of NR1 and NR2B were increased (P < 0.01 or P < 0.05) in ginsenoside Rg1 groups, especially in high-dose ginsenoside Rg1 group. CONCLUSION: Ginsenoside Rg1 can play the role of anti-dementia by inhibiting the expression of P-Tau so as to slow the formation of neurofibrillary tangles and increasing the expression of NR1 and NR2B so as to improve learning and memory abilities in rat brain slice model of Alzheimer's disease.