Crosstalk among Oxidative Stress,Autophagy,and Apoptosis in the Protective Effects of Ginsenoside Rb1 on Brain Microvascular Endothelial Cells:A Mixed Computational and Experimental Study

Objective Brain microvascular endothelial cells (BMECs) were found to shift from their usually inactive state to an active state in ischemic stroke (IS) and cause neuronal damage. Ginsenoside Rb1 (GRb1),a component derived from medicinal plants,is known for its pharmacological benefits in IS,but its protective effects on BMECs have yet to be explored. This study aimed to investigate the potential protective effects of GRb1 on BMECs. Methods An in vitro oxygen-glucose deprivation/reperfusion (OGD/R) model was established to mimic ischemia-reperfusion (I/R) injury. Bulk RNA-sequencing data were analyzed by using the Human Autophagy Database and various bioinformatic tools,including gene set enrichment analysis (GSEA),Gene Ontology (GO) classification and enrichment analysis,Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis,protein-protein interaction network analysis,and molecular docking. Experimental validation was also performed to ensure the reliability of our findings. Results Rb1 had a protective effect on BMECs subjected to OGD/R injury. Specifically,GRb1 was found to modulate the interplay between oxidative stress,apoptosis,and autophagy in BMECs. Key targets such as sequestosome 1 (SQSTM1/p62),autophagy related 5 (ATG5),and hypoxia-inducible factor 1-alpha (HIF-1α) were identified,highlighting their potential roles in mediating the protective effects of GRb1 against IS-induced damage. Conclusion GRbl protects BMECs against OGD/R injury by influencing oxidative stress,apoptosis,and autophagy. The identification of SQSTM1/p62,ATG5,and HIF-1α as promising targets further supports the potential of GRb1 as a therapeutic agent for IS,providing a foundation for future research into its mechanisms and applications in IS treatment.

Ginsenoside Rb1 Attenuates Isoflurane/surgery-induced Cognitive Dysfunction via Inhibiting Neuroinflammation and Oxidative Stress

Objective Anesthetic isoflurane plus surgery has been reported to induce cognitive impairment. The underlying mechanism and targeted intervention remain largely to be determined. Ginsenoside Rb1 was reported to be neuroprotective. We therefore set out to determine whether ginsenoside Rb1 can attenuate isoflurane/surgery-induced cognitive dysfunction via inhibiting neuroinflammation and oxidative stress. Methods Five-months-old C57BL/6J female mice were treated with 1.4% isoflurane plus abdominal surgery for two hours. Sixty mg/kg ginsenoside Rb1 were given intraperitoneally from 7 days before surgery. Cognition of the mice were assessed by Barnes Maze. Levels of postsynaptic density-95 and synaptophysin in mice hippocampus were measured by Western blot. Levels of reactive oxygen species, tumor necrosis factor-α and interleukin-6 in mice hippocampus were measured by ELISA. Results Here we show for the first time that the ginsenoside Rb1 treatment attenuated the isoflurane/surgery-induced cognitive impairment. Moreover, ginsenoside Rb1 attenuated the isoflurane/surgery-induced synapse dysfunction. Finally, ginsenoside Rb1 mitigated the isoflurane/surgery-induced elevation levels of reactive oxygen species, tumor necrosis factor-α and interleukin-6 in the mice hippocampus. Conclusion These results suggest that ginsenoside Rb1 may attenuate the isoflurane/surgery-induced cognitive impairment by inhibiting neuroinflammation and oxidative stress pending future studies.

GPCR-Gs mediates the protective effects of ginsenoside Rb1 against oxygen-glucose deprivation/re-oxygenation-induced astrocyte injury

Objectives:To investigate whether the protective actions of ginsenoside Rb1(Rb1)on astrocytes are mediated through the G_(s)-type G-protein-coupled receptor(GPCR-G_(s)).Methods:Primary astrocyte cultures derived from neonatal mouse brain were used.Astrocyte injury was induced via oxygen-glucose deprivation/re-oxygenation(OGD/R).Cell morphology,viability,lactate dehydrogenase(LDH)leakage,apoptosis,glutamate uptake,and brain-derived neurotrophic factor(BDNF)secretion were assessed to gauge cell survival and functionality.Western blot was used to investigate the cyclic adenosine monophosphate(cAMP)and protein kinase B(Akt)signaling pathways.GPCR-G_(s)-specific inhibitors and molecular docking were used to identify target receptors.Results:Rb1 at concentrations ranging from 0.8 to 5μM did not significantly affect the viability,glutamate uptake,or BDNF secretion in normal astrocytes.OGD/R reduced astrocyte viability,increasing their LDH leakage and apoptosis rate.It also decreased glutamate uptake and BDNF secretion by these cells.Rb1 had protective effects of astrocytes challenged by OGD/R,by improving viability,reducing apoptosis,and enhancing glutamate uptake and BDNF secretion.Additionally,Rb1 activated the cAMP and Akt pathways in these cells.When the GPCR-G_(s) inhibitor NF449 was introduced,the protective effects of Rb1 completely disappeared,and its activation of cAMP and Akt signaling pathways was significantly inhibited.Conclusion:Rb1 protects against astrocytes from OGD/R-induced injury through GPCR-G_(s) mediation.

Regulatory effects of the combination of berberine and ginsenoside Rb1 on high-fat diet-induced nonalcoholic fatty liver disease via AMPK pathway and improved pharmacokinetics

Objective： We evaluated the protective effects of berberine （BBR） combined with ginsenoside Rb1 （G-Rb1） on high-fatdiet （HFD）-induced nonalcoholic fatty liver disease （NAFLD） in rats and futher investigated the underlying mechanisms.Methods： Rats were fed an HFD for 6 weeks and then randomly divided into four groups and treated with BBR （50mg/kg）, G-Rb1 （100 mg/kg）, BBR （50 mg/kg） ＋ G-Rb1 （100 mg/kg）, or fenofibrate （40 mg/kg）. Histological examinationof liver tissue was performed. In human hepatocellular carcinoma cells HepG2, protein expression of AMP-activatedprotein kinase （AMPK） and acetyl-CoA carboxylase was detected by western blotting, and the mRNA expression ofcarnitine palmitoyl transferase 1 and 3-hydroxy-3-methyl glutaryl coenzyme A reductase was detected by quantitativePCR. Pharmacokinetic assessments included analysis of bioavailability of BBR and G-Rb1 in vivo and G-Rb1 metabolismby intestinal bacteria in vitro. Results： Compared to the single-use group, BBR combined with G-Rb1 significantlyameliorated hepatic fat accumulation in HFD-induced obese rats, as demonstrated by reduced hepatic triglyceridecontent, and histological evaluation of liver sections. Activation of hepatic AMPK and phosphorylation of acetyl-CoAcarboxylase were significantly elevated in hepatocytes treated with both BBR and G-Rb1. Consistent with the activationof AMPK, the mRNA expression of carnitine palmitoyl transferase 1 was stimulated, while the mRNA expression of3-hydroxy-3-methyl glutaryl coenzyme A reductase was suppressed. Pharmacokinetic analysis revealed that BBRincreased the bioavailability of G-Rb1 in Sprague-Dawley rats. Additionally, BBR prevented degradation of G-Rb1 infecal solution in vitro. Conclusion： BBR combined with G-Rb1 improved NAFLD through the AMPK signaling pathway,and BBR enhanced G-Rb1 bioavailability via promoting the intestinal absorption of G-Rb1. This combination may be auseful therapeutic agent for NAFLD.

Protective Effects of Ginsenoside Rb1 on Septic Rats and Its Mechanism

This study aims to observe the protective effects of ginsenoside Rbl on liver and lung in rats with septic shock and reveal its mechanism. Rats were randomly divided into three groups： sham, cecal ligation and puncture （CLP）, and CLP with ginsenoside Rb1. Then, the survival rate, arterial blood pressure, TLR4 mRNA, and TNF-α levels were determined. The liver and lung tissues were stained with hematoxylin-eosin （HE）. The overall survival rate of the Rb1 group was significantly higher than that of the CLP group. Mean arterial blood pressure went down in both the CLP and Rb1 groups after CLP, and there was a significant difference both in the sham and Rb1 groups when compared with the CLP group. The Rb1 treatment group had markedly lower TLR4 mRNA expression and TNF-a levels than the CLP group. In the CLP group, pathology showed swelling, degeneration, necrosis, and neutrophii infiltration in the liver and alveolar epithelial cells. However, in the Rb1 group, there was mild degeneration and slight neutrophil infiltration, but no obvious necrosis. Rb1 may improve the survival rate, ameliorate arterial blood pressure, and protect the liver and lung in septic shock rats by downregulating the expression of TLR4 mRNA and inhibiting the production of TNF-α.

Ginsenoside Rb1 induces hepatic stellate cell ferroptosis to alleviate liver fibrosis via the BECN1/SLC7A11 axis

Liver fibrosis is primarily driven by the activation of hepatic stellate cells(HSCs),a process associated with ferroptosis.Ginsenoside Rb1(GRb1),a major active component extracted from Panax ginseng,inhibits HSC activation.However,the potential role of GRb1 in mediating HSC ferroptosis remains unclear.This study examined the effect of GRb1 on liver fibrosis both in vivo and in vitro,using CCl4-induced liver fibrosis mouse model and primary HSCs,LX-2 cells.The findings revealed that GRb1 effectively inactivated HSCs in vitro,reducing alpha-smooth muscle actin(a-SMA)and type I collagen(Col1A1)levels.Moreover,GRb1 significantly alleviated CCl4-induced liver fibrosis in vivo.From a mechanistic standpoint,the ferroptosis pathway appeared to be central to the antifibrotic effects of GRb1.Specifically,GRb1 promoted HSC ferroptosis both in vivo and in vitro,characterized by increased glutathione depletion,malondialdehyde production,iron overload,and accumulation of reactive oxygen species(ROS).Intriguingly,GRb1 increased Beclin 1(BECN1)levels and decreased the System Xc-key subunit SLC7A11.Further experiments showed that BECN1 silencing inhibited GRb1-induced effects on HSC ferroptosis and mitigated the reduction of SLC7A11 caused by GRb1.Moreover,BECN1 could directly interact with SLC7A11,initiating HSC ferroptosis.In conclusion,the suppression of BECN1 counteracted the effects of GRb1 on HSC inactivation both in vivo and in vitro.Overall,this study highlights the novel role of GRb1 in inducing HSC ferroptosis and promoting HSC inactivation,at least partly through its modulation of BECN1 and SLC7A11.

Ginsenoside Rb1 produces antidepressant-like effects in chronic social defeat stress model of depression through BDNF-TrkB sig⁃naling pathway

OBJECTIVE Ginsenoside Rb1(Rb1),an important bioactive ingredient of Panax ginseng,has potent neuroprotective effects.The objective of the study is to elucidate the impact of Rb1 treatment on chronic social defeat stress(CSDS)-induced depressive-like behaviors and its related mechanism.METHODS AND RE⁃SULTS The daily oral administration of Rb1(35 and 70 mg·kg-1)and imipramine(15 mg·kg-1)for 28 d significantly reversed the social avoidance behavior,anhedonia,and behavioral despair via CSDS exposure,as demonstrated by the consid⁃erable elevation in the time in the zone in social interaction test and consumption of sucrose solu⁃tion in sucrose preference test and decrease of immobility time in forced swim test.Moreover,Rb1 obviously restored the CSDS-induced decrease of BDNF-signaling pathway and hippo⁃campal neurogenesis.Rb1 significantly increased the hippocampal levels of ERK,AKT,and CREB phosphorylation and increased the number of DCX+cells in DG.Importantly,the antidepres⁃sant effects of Rb1 were completely blocked in mice by using K252a(the nonselective tyrosine kinase B inhibitor).CONCLUSION Rb1 exerts promising antidepressant-like effects in mice with CSDS-induced depression,and its effects was facilitated by enhancing the BDNF signaling cas⁃cade and up-regulation of hippocampal neuro⁃genesis.

Neuroprotective effects of ginsenoside Rb1 on hippocampal neuronal injury and neurite outgrowth

Ginsenoside Rb1 has been reported to exert anti-aging and anti-neurodegenerative effects. In the present study, we investigate whether ginsenoside Rb1 is involved in neurite outgrowth and neuroprotection against damage induced by amyloid beta（25–35） in cultured hippocampal neurons, and explore the underlying mechanisms. Ginsenoside Rb1 significantly increased neurite outgrowth in hippocampal neurons, and increased the expression of phosphorylated-Akt and phosphorylated extracellular signal-regulated kinase 1/2. These effects were abrogated by API-2 and PD98059, inhibitors of the signaling proteins Akt and MEK. Additionally, cultured hippocampal neurons were exposed to amyloid beta（25–35） for 30 minutes; ginsenoside Rb1 prevented apoptosis induced by amyloid beta（25–35）, and this effect was blocked by API-2 and PD98059. Furthermore, ginsenoside Rb1 significantly reversed the reduction in phosphorylated-Akt and phosphorylated extracellular signal-regulated kinase 1/2 levels induced by amyloid beta（25–35）, and API-2 neutralized the effect of ginsenoside Rb1. The present results indicate that ginsenoside Rb1 enhances neurite outgrowth and protects against neurotoxicity induced by amyloid beta（25–35） via a mechanism involving Akt and extracellular signal-regulated kinase 1/2 signaling.

Effect of the ginsenoside Rb1 on the spontaneous contraction of intestinal smooth muscle in mice

AIM: To investigate the effect and the possible mechanism of ginsenoside Rb1 on small intestinal smooth muscle motility in mice. METHODS: Intestinal smooth muscle strips were isolated from male ICR mice (5 wk old), and the effect of ginsenoside Rb1 on spontaneous contraction was recorded with an electrophysiolograph. The effect of ginsenoside Rb1 on ion channel currents, including the voltage-gated K + channel current (IK V ), calcium-activated potassium channel currents (IK Ca ), spontaneous transient outward currents and ATP-sensitive potassium channel current (IK ATP ), was recorded on freshly isolated single cells using the whole-cell patch clamp technique. RESULTS: Ginsenoside Rb1 dose-dependently inhibited the spontaneous contraction of intestinal smooth muscle by 21.15% ± 3.31%, 42.03% ± 8.23% and 67.23% ± 5.63% at concentrations of 25 μmol/L, 50 μmol/L and 100 μmol/L, respectively (n=5,P<0.05). The inhibitory effect of ginsenoside Rb1 on spontaneous contraction was significantly but incompletely blocked by 10 mmol/L tetraethylammonium or 0.5 mmol/L 4-aminopyridine, respectively (n=5, P<0.05). However, the inhibitory effect of ginsenoside Rb1 on spontaneous contraction was not affected by 10 μmol/L glibenclamide or 0.4 μmol/L tetrodotoxin. At the cell level, ginsenoside Rb1 increased outward potassium currents, and IK V was enhanced from 1137.71 ± 171.62 pA to 1449.73 ± 162.39 pA by 50 μmol/L Rb1 at +60 mV (n=6, P<0.05). Ginsenoside Rb1 increased IK Ca and enhanced the amplitudes of spontaneous transient outward currents from 582.77 ± 179.09 mV to 788.12 ± 278.34 mV (n=5, P<0.05). However, ginsenoside Rb1 (50 μmol/L) had no significant effect on IK ATP (n=3, P<0.05). CONCLUSION: These results suggest that ginsenoside Rb1 has an inhibitory effect on the spontaneous contraction of mouse intestinal smooth muscle mediated by the activation of IK V and IK Ca , but the K ATP channel was not involved in this effect.

Ginsenoside Rb1 protects dopaminergic neurons from inflammatory injury induced by intranigral lipopolysaccharide injection

Accumulating studies suggest that neuroinflammation characterized by microglial overactivation plays a pivotal role in the pathogenesis of Parkinson’s disease.As such,inhibition of microglial overactivation might be a promising treatment strategy to delay the onset or slow the progression of Parkinson’s disease.Ginsenoside Rbl,the most active ingredient of ginseng,reportedly exerts neuroprotective effects by suppressing inflammation in vitro.The present study aimed to evaluate the neuroprotective and anti-inflammatory effects of ginsenoside Rbl in a lipopolysaccharide-induced rat Parkinson’s disease model.Rats were divided into four groups.In the control group,sham-operated rats were intraperitoneally administered normal saline for 14 consecutive days.In the ginsenoside Rbl group,ginsenoside Rb1(20 mg/kg)was intraperitoneally injected for 14 consecutive days after sham surgery.In the lipopolysaccharide group,a single dose of lipopolysaccharide was unilaterally microinjected into the rat substantial nigra to establish the Parkinson’s disease model.Lipopolysaccharide-injected rats were treated with normal saline for 14 consecutive days.In the ginsenoside Rbl +lipopolysaccharide group,lipopolysaccharide was unilaterally microinjected into the rat substantial nigra.Subsequently,ginsenoside Rbl was intraperitoneally injected for 14 consecutive days.To investigate the therapeutic effects of ginsenoside Rbl,behavioral tests were performed on day 15 after lipopolysaccharide injection.We found that ginsenoside Rbl treatment remarkably reduced apomorphine-induced rotations in lipopolysaccharide-treated rats compared with the lipopolysaccharide group.To investigate the neurotoxicity of lipopolysaccharide and potential protective effect of ginsenoside Rbl,contents of dopamine and its metabolites in the striatum were measured by high-performance liquid chromatography.Compared with the lipopolysaccharide group,ginsenoside Rbl obviously attenuated the lipopolysaccharide-induced depletion of dopamine and its metabolites in the striatum.To further explore the neuroprotective effect of ginsenoside Rbl against lipopolysaccharide-induced neurotoxicity,immunohistochemistry and western blot assay of tyrosine hydroxylase were performed to evaluate dopaminergic neuron degeneration in the substantial nigra par compacta.The results showed that lipopolysaccharide injection caused a large loss of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra and a significant decrease in overall tyrosine hydroxylase expression.However,ginsenoside Rb1 noticeably reversed these changes.To investigate whether the neuroprotective effect of ginsenoside Rbl was associated with inhibition of lipopolysaccharide-induced microglial activation,we examined expression of the microglia marker Iba-1.Our results confirmed that lipopolysaccharide injection induced a significant increase in Iba-1 expression in the substantia nigra;however,ginsenoside Rbl effectively suppressed lipopolysaccharide-induced microglial overactivation.To elucidate the inhibitory mechanism of ginsenoside Rb1,we examined expression levels of inflammatory mediators(tumor necrosis factor-a,interleukin-1β,inducible nitric oxide synthase,and cyclooxygenase 2)and phosphorylation of nuclear factor kappa B signaling-related proteins(IκB,IKK)in the substantia nigra with enzyme-linked immunosorbent and western blot assays.Our results revealed that compared with the control group,phosphorylation and expression of inflammatory mediators IκB and IKK in the substantia nigra of lipopolysaccharide group rats were significantly increased;whereas,ginsenoside Rbl obviously reduced lipopolysaccharide-induced changes on the lesioned side of the substantial nigra par compacta.These findings confirm that ginsenoside Rbl can inhibit inflammation induced by lipopolysaccharide injection into the substantia nigra and protect dopaminergic neurons,which may be related to its inhibition of the nuclear factor kappa B signaling pathway.This study was approved by the Experimental Animal Ethics Committee of Shandong University of China in April 2016(approval No.KYLL-2016-0148).

High efficiency production of ginsenoside compound K by catalyzing ginsenoside Rb1 using snailase

The rare ginsenoside Compound K （C-K） is attracting more attention because of its good physiological activity and urgent need. There are many pathways to obtain ginsenoside C-K, including chemical and biological methods. Among these, the conversion of PPD-type ginsenosides by enzymatic hydrolysis is a trend due to its high efficiency and mild conditions. For effectively extracting from the other panaxadiol saponins, the conversion process for ginsenoside C-K was investigated using snailases in this study. The univariate experimental design and response surface methodology were used to determine the optimal hydrolysis conditions for the conversion of ginsenoside Rbl into ginsenoside C-K by snailases. The optimum conditions were as follows： pH 5,12, temperature 51 ℃, ratio of snailase/substrate 0.21, and reaction time 48 h. On the basis of these parameters, the addition of 1.0 mmol· L- 1 ferric ion was found to significantly improve the enzymolysis ofsnailases for the first time. With the above conditions, the maximum conversion rate reached 89.7%, suggesting that the process can obviously increase the yield of ginsenoside C-K. The bioassay tests indicated that the ginsenoside C-K showed anti-tumor activity in a series of tumor cell lines. Based on these results, we can conclude that the process of rare ginsenoside C- K production by enzymolysis with snailase is feasible, efficient, and suitable for the industrial production and application.

Inhibitory Effects of Ginsenoside Rb1 on Apoptosis Caused by HSV-1 in Human Glioma Cells

To investigate the inhibitory effects of Ginsenoside Rbl （GRbl） on apoptosis caused by Herpes Simplex Virus-1 （HSV-1） in Human Glioma Cells （U251）, U251 cells were infected by HSV-1 at a multiplicity of infection of 5 and GRbl, GRbl＋HSV-1, HSV-1 and control groups. MTT and cell apoptosis assays were used to detect the inhibitory effects of GRbl on the apoptosis of U251 cells that caused by HSV-1 infection for various concentrations of drug and virus treatments by MTT assay. We found that in the 400 μg/mL GRb 1 and 400 μg/mL GRbl＋HSV-1 groups, MTT values were higher than control group at all times （P〈0.05）. Moreover, the apoptosis rate in the 400 μg/mL GRbl＋HSV-1 group was lower than the HSV-1 group （P〈0. 05）. These results confirmed that, at appropriate concentrations, GRbl could inhibit nerve cell apoptosis in HSV-1 infections.

Ginsenoside Rb1 attenuates lipopolysaccharide-induced chronic neuroinflammation in mice by tuning glial cell polarization

Objective:To evaluate whether ginsenoside Rb1(Rb1) can attenuate lipopolysaccharide(LPS)-induced chronic neuroinflammation in mice and to explore its relationship with glial cell polarization.Methods:Intraperitoneal injection with an escalating dose of LPS was used to establish a chronic neuroinflammation model in mice.Once LPS was initiated,10 or 20 mg/kg Rbl,or sterile saline,was administered for 14 consecutive days.Open field test and beam walking test were used to monitor the changes in behavior.The concentration of cytokines in the serum and brain were used to monitor the systemic inflammation and neuroinflammation,respectively.Molecules specific to each glial cell phenotype were used to investigate glial cell polarization.Results:Mice in the LPS group had reduced spontaneous activities and impaired beam walking performance.Rbl obviously eased LPS-induced behavior distu rbances.Regarding the levels of serum cytokines,both tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) were significantly increased,while interleukin-10(IL-10) and transforming growth factor β(TGF-β) remarkably decreased after LPS treatment(all P <.001).Rb1 treatment significantly attenuated LPS-induced serum cytokine changes(all P <.05).The results of quantitative polymerase chain reaction and western blotting showed that the mRNA and protein expression levels of TNF-α and complement component 3(C3) in the brain were significantly increased after LPS treatment(all P<.01).Rbl treatment significantly inhibited LPS-induced inflammation in the brain(all P <.05).Glial cell polarization analysis showed that M1 and M2 microglia,and A1 astrocytes increased following LPS treatment,while A2 astrocytes decreased.Rb1 treatment reduced M1 and M2 microglia,and A1 astrocytes,and significantly increased A2 astrocytes.Conclusion:Rb1 can attenuate chronic neuroinflammation induced by LPS in mice,which may be partially attributable to its fine tuning of microglia and astrocyte polarization.Rb1 has potential value for treating neurodegenerative diseases.

Ginsenoside Rb1 improves energy metabolism after spinal cord injury

Mitochondrial damage caused by oxidative stress and energy deficiency induced by focal ischemia and hypoxia are important factors that aggravate diseases.Studies have shown that ginsenoside Rb1 has neurotrophic and neuroprotective effects.However,whether it influences energy metabolism after spinal cord injury remains unclear.In this study,we treated mouse and cell models of spinal cord injury with ginsenoside Rb1.We found that ginsenoside Rb1 remarkably inhibited neuronal oxidative stress,protected mitochondria,promoted neuronal metabolic reprogramming,increased glycolytic activity and ATP production,and promoted the survival of motor neurons in the anterior horn and the recovery of motor function in the hind limb.Because sirtuin 3 regulates glycolysis and oxidative stress,mouse and cell models of spinal cord injury were treated with the sirtuin 3 inhibitor 3-TYP.When Sirt3 expression was suppressed,we found that the therapeutic effects of ginsenoside Rb1 on spinal cord injury were remarkably inhibited.Therefore,ginsenoside Rb1 is considered a potential drug for the treatment of spinal cord injury,and its therapeutic effects are closely related to sirtuin 3.

Intranasal ginsenoside Rb1 protects pentyl⁃enetetrazole-induced epileptic mice

OBJECTIVE To evaluate whether ginsenoside Rb1 has antiepileptic effects on pen⁃tylenetetrazole(PTZ)-induced epileptic mice via intranasal therapeutic administration.METHODS Rb1 monoclonal antibody was used to observe the distribution of Rb120 mg·kg-1 in mouse brain tissues under different administration routes and to explore the feasibility of intranasal Rb1.PTZ was injected intraperitoneally into healthy ICR mice every 48 hours to construct a tonic-clonic epileptic model.Then Rb120 or 40 mg·kg-1 or valproate 300 mg·kg-1 or saline was administered intranasally for 30 d,and PTZ was continued every five days to imitate occa⁃sional convulsions in the clinic.Racine scale(RCS)and wireless electroencephalogram(EEG)monitoring were used to assess the presence and severity of seizure.Immunofluorescence(IF)was performed after drug treatment to evalu⁃ate the effect of Rb1 on brain neuron,microglia and astrocyte in epileptic mice.RESULTS Rb1 had specific binding with anti-Rb1 in the brain under different administration routes,and intrana⁃sal Rb1 was able to enter the brain and play a therapeutic role(P<0.01).PTZ-injured mice pre⁃sented body mass loss,higher seizure stage and shorter seizure latency.At the same time,epilep⁃tic waves,mainly spikes,were detected by wire⁃less EEG.Compared with PTZ group,intranasal Rb1 increased mice weight(P<0.01)and seizure latency(P<0.05),reduced seizure stage(P<0.01)and EEG spikes.In addition,Rb1 significantly reduced neuron loss(P<0.01)indicated by NeuN staining and decreased the number of acti⁃vated microglia(P<0.01)indicated by Iba-1 staining in the cortex and CA1 area of hippocam⁃pus.Moreover,Rb1 reduced the decrease of GLT-1 and GS expression(P<0.05)induced by PTZ.CONCLUTION Intranasal Rb1 has anti-epi⁃leptic effects on PTZ mice.Moreover,Intranasal Rb1 affects the functions of neurons,astrocytes and microglia through regulating the expression of GLT and GS in astrocytes,which may be related to its anti-epileptic effect.

Ginsenoside Rb1 attenuates adjuvant-induced arthritis in rats through inactivation of NF-κB signaling pathway

OBJECTIVE To investigate the anti-arthritic effect and mechanism of action of ginsenoside Rb1 on adju⁃vant-induced arthritis(AIA)in rats.METHODS Male SD rats were received 0.1 mL injections of FCA(10 g·L^-1)emulsion into the right hind metatarsal foot pad for arthritis induction.After that,rats were randomly divided into six groups,namely control group,untreated group,dexamethasone(DEX,2.5 mg·kg^-1)group,low(5 mg·kg^-1),medium(10 mg·kg^-1)and high(20 mg·kg^-1)doses of ginsenoside Rb1 groups,and treated intraperitoneally at the above dosage once a day for 2 weeks.After treatment,paw swelling and arthritis indexes were evaluated,the thymus and spleen index were calculated as well.HE staining were used to observe the joint histopathology in rats.Rat ELISA kits were used to determinate the TNF-α,IL-1βand IL-6 levels.Western blotting were used to detect the related protein expression of NF-κB signaling pathway in the tissues of inflamed joints.RESULTS Rb1 significantly decreased the paw swelling and arthritis index,Compared with AIA group.HE staining results revealed that medium and high doses of Rb1 significantly reduced synovial inflammatory cell infiltration,synovial lining hyperplasia and bone destruction,compared with AIA group.Elisa results showed that Rb1 significantly decreased the TNF-α,IL-1β and IL-6 levels(P<0.05,P<0.01).Western blotting results revealed that the expression of p-IκB and p-P65 were significantly reduced in 20 mg·kg^-1 of Rb1 group,compared with AIA group(P<0.05,P<0.01).CONCIUSION Rb1 manifests therapeutic anti-inflammatory effects on rats with AIA,poten⁃tially through a mechanism of inhibiting activation of the NF-κB.

EFFECTS OF GINSENOSIDE Rb1 AND Rg1 ON SYNAPTOSOMAL FREE CALCIUM LEVEL, ATPase AND CALMODULIN IN RAT HIPPOCAMPUS

Calcium homeostasis in synaptosomes is altered during aging. The intrasynaptomal free calcium concentration ([Ca2+]i) was determined in 3- and 24-month-old rats treated with or without Rbl and Rgl. As expected, the [Ca2-]i level increased with age. Treatment with Rbl and Rgl elicited an obvious decrease of [Ca2+]i content, especially in aged rates. In addition, Rbl and Rgl significantly stimulated the activity of Na+, K+-ATPase while Rbl inhibited the activity of Ca2+, Mg2--ATPase arid calmodulin. In view of the close relationship of [Ca2-]i level with aging, the changes of [Ca2+]i induced by Rbl and Rgl, as shown by our results, might provide an explanation of the mechanisms of their antiaging function.

Ginsenoside Rb1 improves brain,lung,and intestinal barrier damage in middle cerebral artery occlusion/reperfusion(MCAO/R)micevia the PPARγsignaling pathway

Ischemic stroke causes brain inflammation and multi-organ injury,which is closely associated with the peroxisome proliferator-activated receptor-gamma(PPARγ)signaling pathway.Recent studies have indicated that ginsenoside Rb1(GRb1)can protect the integrity of the blood-brain barrier after stroke.In the current study,a mouse model of middle cerebral artery occlusion/reperfusion(MCAO/R)was established to determine whether GRb1 can ameliorate brain/lung/intestinal barrier damage via the PPARγsignaling pathway.Staining(2,3,5-triphenyltetrazolium chloride,hematoxylin,and eosin)and Doppler ultrasonography were employed to detect pathological changes.Endothelial breakdown was investigated with the leakage of Evans Blue dye and the expression of TJs(tight junctions)and AJs(adherent junctions).Western blot and immunofluorescence were used to determine the levels of cell junction proteins,PPARγand NF-κB.Results showed that GRb1 significantly mitigated multi-organ injury and increased the expression of cerebral microvascular,pulmonary vascular,and intestinal epithelial connexins.In brain,lung,and intestinal tissues,GRb1 activated PPARγ,decreased the levels of phospho-NF-κB p65,and inhibited the production of proinflammatory cytokines,thereby maintaining barrier permeability.However,co-treatment with GRb1 and the PPARγantagonist GW9662 reversed the barrier-protective effect of GRb1.These findings indicated that GRb1 can improve stroke-induced brain/lung/intestinal barrier damagevia the PPARγpathway.

Effects of Ginsenoside Rb1 on Skeletal Muscle Insulin Resistance and Adenosine Monophosphate?activated Protein Kinase Signaling Pathway in Obese Mice

Objectives: The objective of the study is to observe the effects of ginsenoside Rb1 on indexes of body weight, body composition, blood lipid, skeletal muscle endurance, and insulin sensitivity in obese mice, probe into its pharmacological action, and further explore its effects on adenosine monophosphate-activated protein kinase(AMPK) signaling pathway in skeletal muscle. Materials and Methods: Eight-week-old C57 BL/6 J mice were fed with high-fat diet for 12 weeks to establish obese mouse model. The model-establishment obese mice were randomly divided into three groups including model control group, metformin group, and ginsenoside Rb1 group. In the normal control group, normal diet was administered. The intervention period was 8 weeks. Body weight and food intake of the mice were measured regularly every week. The treadmill test was performed at weeks 3 and 7, and the oral glucose tolerance test was carried out at weeks 4 and 8. Body composition of the mice was detected by applying NMR Animal Body Composition Analyzer at week 8. Four parameters of blood lipids and free fatty acid(FFA)levels were detected. The m RNA expression of AMPKα and proliferator-activated receptor gamma coactivator-1α(PGC-1α) in skeletal muscle was examined by real-time fluorescence quantitative polymerase chain reaction, and the influence of ginsenoside Rb1 on protein expression of AMPKα, p-AMPKα, and PGC-1α was observed by western blotting. Results: The body weight(since the 5 th week of drug administration)and food intake of the mice in the ginsenoside Rb1 group were significantly lower than those in the model control group(P < 0.05) in a time-dependent manner. Ginsenoside Rb1 could significantly reduce the levels of triglyceride and low-density lipoprotein cholesterol, while increase the high-density lipoprotein cholesterol level(P < 0.05). In addition, ginsenoside Rb1 could reduce the serum FFA level(P < 0.05).After the administration of ginsenoside Rb1 for 8 weeks, the body fat mass of obese mice decreased and the lean mass increased(P < 0.05).The skeletal muscle endurance and the oral glucose tolerance of the obese mice improved using ginsenoside Rb1. At the molecular level,ginsenoside Rb1 could up-regulate the mRNA and protein expression of AMPKα in skeletal muscle, and increase the content of p-AMPK protein significantly(P < 0.01). At the same time, the mRNA and protein level of PGC-1α was also un-regulated, correspondingly(P < 0.01).Conclusion: Ginsenoside Rb1 exerts effects on reducing body weight, decreasing blood lipid levels, enhancing the skeletal muscle endurance,and increasing the insulin sensitivity in obese mice by activating the related proteins in AMPK signaling pathway in skeletal muscle.

Effect of Ginsenoside-Rb_1 on Cardiomyocyte Apoptosis after Ischemia and Reper fusion in Rats

The effect of ginsenoside Rb 1 on cardiomyocyte apotosis after ischemia (30 min ) and reperfusion (6 h) in rats was observed. The ischemia/ reperfusion heart mo del was established by ligating left anterior descending branch of coronary arte ry in Wistar rats. The apoptotic cardiomyocytes were examined under transmission electron microscopy and counted by in situ nick end labeling (TUNEL) method and light microscopy. Results showed that (1) The apoptotic cardiomyocytes were found in ischemic regions in the ischemia/reperfusion group, but not in the sh am oper ating group under transmission electron microscopy; (2) The number of apoptotic cells were 134.45±45.61/field in the ischemia/reperfusion group, 0/field in the sham operating group and 51.65±13.71/field in the ginsenoside Rb 1 treated group. The differences were significant among the three groups ( P <0.01). It was concluded that myocardial ischemia reperfusion could induce cardiomyocyte a poptosis, and ginsenoside Rb 1 could significantly inhibit cardiomyocyte apopto sis induced by ischemia reperfusion in rats, indicating that ginsenoside Rb 1 could inhibit cardiomyocyte apoptosis induced by ischemia reperfusion, thus alleviating ischemia reperfusion injury.