Transcriptome-Wide Identification and Functional Analysis of PgSQE08-01 Gene in Ginsenoside Biosynthesis in Panax ginseng C.A.Mey.

Panax ginseng C.A.Mey.is an important plant species used in traditional Chinese medicine,whose primary active ingredient is a ginsenoside.Ginsenoside biosynthesis is not only regulated by transcription factors but also controlled by a variety of structural genes.Nonetheless,the molecular mechanism underlying ginsenoside biosynthesis has always been a topic in the discussion of ginseng secondary metabolites.Squalene epoxidase(SQE)is a key enzyme in the mevalonic acid pathway,which affects the biosynthesis of secondary metabolites such as terpenoid.Using ginseng transcriptome,expression,and ginsenoside content databases,this study employed bioinformatic methods to systematically analyze the genes encoding SQE in ginseng.We first selected six PgSQE candidates that were closely involved in ginsenoside biosynthesis and then identified PgSQE08-01 to be highly associated with ginsenoside biosynthesis.Next,we constructed the overexpression vector pCAMBIA3301-PgSQE08-01 and the RNAi vector pART27-PgSQE08-01 and transformed ginseng adventitious roots using Agrobacterium rhizogenes,to obtain positive hairy-root clones.Thereafter,quantitative reverse transcriptionpolymerase chain reaction and high-performance liquid chromatography were used to determine the expression of relevant genes and ginsenoside content,respectively.Then,we focused on the function of PgSQE08-01 gene,which was noted to be involved in ginsenoside biosynthesis.Thus,these findings not only provided a molecular basis for the identification of important functional genes in ginseng but also enriched genetic resources for the biosynthesis of ginsenosides using synthetic biology.

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.

Ocotillone-type Ginsenoside from Leaves of Panax ginseng

An ocotillone type ginsenoside, together with 2 known ginsenosides was isolated from leaves of Panax ginseng and identified as pseudoginsenoside RT 5 on the basis of chemical and physicochemical evidences. It has been so far the first example of ocotillone type ginsenoside discovered in Panax ginseng and its plausible biotransformation pathway also discussed.

Ginsenoside Rk3 modulates gut microbiota and regulates immune response of group 3 innate lymphoid cells to against colorectal tumorigenesis

The gut microbiota plays a pivotal role in the immunomodulatory and protumorigenic microenvironment of colorectal cancer(CRC).However,the effect of ginsenoside Rk3(Rk3)on CRC and gut microbiota remains unclear.Therefore,the purpose of this study is to explore the potential effect of Rk3 on CRC from the perspective of gut microbiota and immune regulation.Our results reveal that treatment with Rk3 significantly suppresses the formation of colon tumors,repairs intestinal barrier damage,and regulates the gut microbiota imbalance caused by CRC,including enrichment of probiotics such as Akkermansia muciniphila and Barnesiella intestinihominis,and clearance of pathogenic Desulfovibrio.Subsequent metabolomics data demonstrate that Rk3 can modulate the metabolism of amino acids and bile acids,particularly by upregulating glutamine,which has the potential to regulate the immune response.Furthermore,we elucidate the regulatory effects of Rk3 on chemokines and inflammatory factors associated with group 3 innate lymphoid cells(ILC3s)and T helper 17(Th17)signaling pathways,which inhibits the hyperactivation of the Janus kinase-signal transducer and activator of transcription 3(JAK-STAT3)signaling pathway.These results indicate that Rk3 modulates gut microbiota,regulates ILC3s immune response,and inhibits the JAK-STAT3 signaling pathway to suppress the development of colon tumors.More importantly,the results of fecal microbiota transplantation suggest that the inhibitory effect of Rk3 on colon tumors and its regulation of ILC3 immune responses are mediated by the gut microbiota.In summary,these findings emphasize that Rk3 can be utilized as a regulator of the gut microbiota for the prevention and treatment of CRC.

High-Performance Liquid Chromatographical Analysis of Ginsenosides in Panax ginseng,P.quinquef(?)lium and P.notoginseng

The compositions and contents of ginsenbsides in Panax ginseng,P.quinquefolium and P.notoginseng were determined and compared by reversed-phase High-Performance Liquid Chro- matography(HPLC).The method was performed on an Alltech Adsorbosphere HS C_(18) column,using 5×10^(-3)M NaH_2PO_4-H_3PO_4 buffer solution(pH 3.0)and acetonitrile-water(50:50)as gradient eluents. The baseline separation of ginsenosides Rb_1,Rb_2,Rb_1,Rc,Rd,Rf,Ro,and Re+Rg_1 was obtained in one analytical run.The ginsenosides are directly detected at 203 nm.The detection limit is 40μg at a signal to noise ratio of 3:1.The improved sample preparation and clean-up prior to injection with SEP-PAK C_(18)cartridge strongly reduced the front peaks caused by the impurities in the methanolic extracts of samples to afford a smooth baseline and clear background.The HPLC patterns of methanolic extracts mainly including the ginsenosides were found capable of serving as chemical fingerprints to differentiate the three species from each other.It was also found that there are no significant diffe- rences of the HPLC patterns between the wild Panax ginseng and the cultivated,the white and the red ginsengs,Chinese and Korean red ginsengs,and the tap roots of Panax ginseng collected in four consecutive months,only certain differences in contents of ginsenosides do exist.The contents of the nine major ginsenosides present in the rhizome,tap root and rootlet as well as the leaf of Panax quinquefolium were also determined and compared.

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.

Inhibiting effect of Endostar combined with ginsenoside Rg3 on breast cancer tumor growth in tumor-bearing mice

Objective: To study the inhibiting effect of Endostar combined with ginsenoside Rg3 on breast cancer tumor growth in tumor-bearing mice. Methods: Female mice were selected as experimental animals, and breast cancer tumor-bearing mouse models were established and then divided into group A, B, C and D that respectively received saline, recombinant human endostatin, ginsenosides Rg3 and recombinant human endostatin combined with Rg3 intervention; 7 d, 14 d and 21 d after intervention, tumor tissue volume was measured; 21 d after intervention, mice were killed, tumor tissue was collected, and m RNA contents of angiogenesis molecules, invasion molecules, autophagy marker molecules and autophagy signaling pathway molecules were detected. Results: At 7 d, 14 d and 21 d after intervention, tumor tissue volume of group B, C and D was lower than that of group A, and tumor tissue volume of group D was lower than that of group B and C; m RNA contents of VEGFA, VEGFB, VEGFC, MMP2, MMP9, p62, m TOR, PI3 K, Akt, JNK and Beclin-1 in tumor tissue of group B, C and D were significantly lower than those of group A, and LC3-II/LC3-I was significantly higher than that of group A; m RNA contents of VEGFA, VEGFB, VEGFC, MMP2, MMP9, p62, m TOR, PI3 K, Akt, JNK and Beclin-1 in tumor tissue of group D were significantly lower than those of group B and C, and LC3-II/LC3-I was higher than that of group B and C. Conclusions: Endostar combined with ginsenoside Rg3 has stronger inhibiting effect on breast cancer tumor growth in tumor-bearing mice than single drug, and it can inhibit angiogenesis and cell invasion, and enhance cell autophagy.

Ginsenoside Rg1 Attenuates Isoflurane-induced Caspase-3 Activation via Inhibiting Mitochondrial Dysfunction

Objective The inhalation anesthetic isoflurane has been shown to induce mitochondrial dysfunction and caspase activation, which may lead to learning and memory impairment. Ginsenoside Rgl is reported to be neuroprotective. We therefore set out to determine whether ginsenoside Rgl can attenuate isoflurane-induced caspase activation via inhibiting mitochondrial dysfunction. Methods We investigated the effects of ginsenoside Rgl at concentrations of 12.5, 25, and 50 μmol/L and pretreatment times of 12 h and 24 h on isoflurane-induced caspase-3 activation in H4 naive and stably transfected H4 human neuroglioma cells that express full-length human amyloid precursor protein （APP） （H4-APP cells）. For mitochondrial dysfunction, we assessed mitochondrial permeability transition pore （mPTP） and adenosine-5＇-triphosphate （ATP） levels. We employed Western blot analysis, chemiluminescence, and flowcytometry. Results Here we show that pretreatment with 50 μmol/L ginsenoside Rgl for 12 h attenuated isoflurane-induced caspase-3 activation and mitochondrial dysfunction in H4-APP cells, while pretreatment with 25 and 50 μmol/L ginsenoside Rgl for 24 h attenuated isoflurane-induced caspase-3 activation and mitochondrial dysfunction in both H4 naive and H4-APP cells. Conclusion These data suggest that ginsenoside Rgl may ameliorate isoflurane-induced caspase-3 activation by inhibiting mitochondrial dysfunction. Pending further studies, these findings might recommend the use of ginsenoside Rgl in preventing and treating isoflurane-induced neurotoxicity.

Neuroprotective effects of ginsenoside Rg1-induced neural stem cell transplantation on hypoxic-ischemic encephalopathy

Ginsenoside Rgl is the major pharmacologically active component of ginseng, and is reported to have various therapeutic actions. To determine whether it induces the differentiation of neural stem cells, and whether neural stem cell transplantation after induction has therapeutic effects on hypoxic-ischemic encephalopathy, we cultured neural stem cells in 10-80 ~tM ginsenoside Rgl. Immunohistochemistry revealed that of the concentrations tested, 20 mM ginsenoside Rgl had the greatest differentiation-inducing effect and was the concentration used for subsequent exper- iments. Whole-cell patch clamp showed that neural stem cells induced by 20 jaM ginsenoside Rgl were more mature than non-induced cells. We then established neonatal rat models of hypox- ic-ischemic encephalopathy using the suture method, and ginsenoside Rgl-induced neural stem cells were transplanted via intracerebroventricular injection. These tests confirmed that neural stem cells induced by ginsenoside had fewer pathological lesions and had a significantly better behavioral capacity than model rats that received saline. Transplanted neural stem cells expressed neuron-specific enolase, and were mainly distributed in the hippocampus and cerebral cortex. The present data suggest that ginsenoside Rgl-induced neural stem cells can promote the partial recovery of complicated brain functions in models of hypoxic-ischemic encephalopathy.

法舒地尔缓解β-淀粉样蛋白1-42诱导的SH-SY5Y细胞凋亡

背景:法舒地尔对阿尔茨海默病小鼠脑内的线粒体动力学有调节作用,并且可以抑制神经炎症,但能否调节线粒体自噬和NLRP3炎症小体进而减轻β-淀粉样蛋白毒性尚不清楚。目的:探究法舒地尔对β-淀粉样蛋白1-42诱导的人源神经母细胞瘤细胞株SH-SY5Y凋亡和线粒体自噬以及NLRP3炎症小体的调节作用。方法:将SH-SY5Y细胞接种于孔板内,细胞贴壁后分3组干预:对照组不加入任何药物,模型组加入20μmol/L β-淀粉样蛋白1-42,法舒地尔组同时加入20μmol/L β-淀粉样蛋白1-42与15 mg/L法舒地尔,干预24 h后,采用MTT法检测细胞活性,TUNEL染色检测细胞凋亡,qRT-PCR和Western blot检测凋亡相关蛋白表达,免疫荧光染色和Western blot检测线粒体自噬相关蛋白表达,免疫荧光染色和Western blot检测NLRP3炎症小体相关蛋白表达。结果与结论:(1)与对照组比较,模型组细胞活性降低、凋亡率升高(P<0.05);与模型组比较,法舒地尔组细胞活性升高、凋亡率降低(P<0.05);(2)qRT-PCR和Western blot检测结果显示,与对照组比较,模型组细胞Bax mRNA与蛋白表达升高(P<0.05),Bcl-2 mRNA与蛋白表达降低(P<0.05);与模型组比较,法舒地尔组细胞Bax mRNA与蛋白表达降低(P<0.05),Bcl-2 mRNA与蛋白表达升高(P<0.05);(3)免疫荧光染色和Western blot检测结果显示,与对照组相比,模型组细胞PINK1、帕金森病蛋白和LC3蛋白表达降低(P<0.05),p62蛋白表达升高(P<0.05);与模型组相比,法舒地尔组细胞PINK1、帕金森病蛋白和LC3蛋白表达升高(P<0.05),p62蛋白表达降低(P<0.05);(4)免疫荧光染色和Western blot检测结果显示,与对照组相比,模型组细胞NLRP3、ASC、Caspase-1和白细胞介素1β蛋白表达升高(P<0.05);与模型组相比,法舒地尔组细胞NLRP3、ASC、Caspase-1和白细胞介素1β蛋白表达降低(P<0.05);(5)结果表明,法舒地尔可以减轻β-淀粉样蛋白1-42诱导的SH-SY5Y细胞凋亡,其机制可能与激活线粒体自噬且抑制NLRP3炎症小体激活有关。

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.

Effectof Ginsenoside Re on Cardiomyocyte Apoptosis and Expression of Bcl-2/Bax Gene after Ischemia and Reperfusion in Rats

To observe the effectof ginsenoside Re on cardiomyocyte apoptosis and Bcl- 2 / Bax gene expression after ischemia (30 m in) and reperfusion (6 h) in rats and to elucidate the possible m echanism s of ginsenoside Re on inhibition of cardiom yocyte apoptosis,the ischem ia/ reperfusion heart m odel was established by ligating the left anterior descending branch of coronary artery in Wistar rats.The apoptotic cardiom yocytes were confirmed by transm ission electron m icroscopy and counted by in situ nick end labeling(TU NEL) method and lightm icroscopy.The m RNA and protein expression of Bcl- 2 and Bax genes were studied by in situ hybridization and im munohis- tochemical staining.Mean optical density (OD) value of the positive fields of m RNA and protein expression was quantitatively exam ined by im age analysis system.The results were as follows: (1) The apoptotic cardiomyocytes were found in ischemic fields in the ischem ia/ reperfusion group and weren't observed in the sham- operation group by transmission electron microscopy;(2 ) The num bers of the apoptotic cells were134.4 5± 4 5 .6 1/ field in the ischemia/ reperfusion group,and 90 .6 6± 19.2 2 / field in the ginsenoside Re- treated group.The differences was significant between two groups(P<0 .0 1) ;(3) Gene expression of Bcl- 2 and Bax were increased significantly in the is- chemia/ reperfusion group and ginsenoside Re- treated group when compared with the sham - opera- tion group.There was no significant difference in the gene expression of Bcl- 2 between the gin- senoside Re- treated group and ischemia/ reperfusion group(P>0 .0 5 ) ,but gene expression of Bax was decreased significantly in the ginsenoside Re- treated group as compared with the ischem ia/ reperfusion group(P<0 .0 1) .The ratio of Bcl- 2 / Bax was increased significantly in the ginseno- side Re- treated group when com pared with the ischem ia/ reperfusion group and sham- operation group.These findings suggest that m yocardial ischem ia- reperfusion can induce cardiom yocyte apoptosis,and ginsenoside Re can significantly inhibit cardiom yocyte apoptosis induced by ischemi- a- reperfusion in rats.It is concluded that ginsenoside Re inhibits cardiomyocyte apoptosis by in- hibiting expression of pro- apoptotic Bax gene and raising the ratio of Bcl- 2 / Bax.

Ginsenoside Rb1 attenuates activated microglia-induced neuronal damage

The microglia-mediated inflammatory reaction promotes neuronal damage under cerebral isch- emia/hypoxia conditions. We therefore speculated that inhibition of hypoxia-induced microglial activation may alleviate neuronal damage. To test this hypothesis, we co-cultured ginsenoside Rb 1, an active component of ginseng, and cortical neurons. Ginsenoside Rb l protected neuronal morphology and structure in a single hypoxic culture system and in a hypoxic co-culture system with microglia, and reduced neuronal apoptosis and caspase-3 production. The protective effect was observable prior to placing in co-culture. Additionally, ginsenoside Rbl inhibited levels of tumor necrosis factor-a in a co-culture system containing activated N9 microglial cells. Ginse-noside Rbl also significantly decreased nitric oxide and superoxide production induced by N9 microglia. Our findings indicate that ginsenoside Rbl attenuates damage to cerebral cortex neu-rons by downregulation of nitric oxide, superoxide, and tumor necrosis factor-a expression in hypoxia-activated microglia.

Synergistic effects of ginseng stem and leaf-extracted ginsenoside and choline on improving learning and memory in rats Association verification experiment in animals with multiple learning and memory disorders

BACKGROUND： Ginsenoside extracted from the stem and leaf of ginseng （GSL） and choline have both been shown to improve learning and memory functions; however, further studies are needed to understand the synergistic effects of a combination of both. OBJECTIVE： To verify the combined improved synergistic effects of GSL and choline on learning and memory disorders in rats. DESIGN： Control observation. SETTING： Taishan Medical College. MATERIALS： A total of 150 male Kunming mice weighing （204-2） g and 40 healthy male Wistar rats weighing （2204-20） g were provided by the Experimental Animal Department of Jilin University. Animal experimentation received confirmed consent from the local ethic committee. GSL was provided by the Department of Chemistry, Norman Bethune Medical University, and choline was provided by the Third Experiment Factory, Shanghai. METHODS： This study was performed at the Life Science Institute, Taishan Medical College from October 2006 to February 2007. ① Scopolamine-induced learning and memory disorders in rats： Forty rats were randomly divided into control group, model group, combination group （400 mg/kg GSL ＋ 200 mg/kg choline）, GSL （400 mg/kg） group, and choline （200 mg/kg） group, 8 rats/group. Rats were perfused and administrated in the morning, once a day for 14 successive days. Rats in the control group and model group were perfused with 20 mL/kg distilled water and underwent Morris water maze spatial resolution test 1 hour after perfusion on the 10m, 11m, and 12m days after administration. Rats also underwent passive step-down avoidance test 1 hour after reperfusion on the 13m and 14m days after administration. Thirty minutes prior to experimentation, rats in the remaining three groups were intraperitoneally （i.p） injected with 2 mg/kg scopolamine, and rats in the control group were i.p. injected with 2 mL/kg saline. ② Scopolamine-induced learning disorder and memory acquired disorder in mice： Fifty mice were randomly divided into control group, model group, combination group （400 mg/kg GSL ＋200 mg/kg choline）, GSL （400 mg/kg） group, and choline （200 mg/kg） group, with 10 mice/group. Mice were perfused and administrated in the morning, once a day for 9 successive days. Mice in the control group and model group were perfused with 20 mL/kg distilled water and underwent passive step down avoidance test 1 hour after reperfusion on the 8th and 9th day after administration. Twenty minutes prior to training, mice in the remaining three groups were i.p. injected with 2 mg/kg scopolamine, and mice in the control group were i.p. injected with 10 mL/kg saline. ③ Sodium nitrite-induced memory consolidation disorder in mice： Grouping, administration, and testing were the same as mentioned above. After training, mice in the remaining three groups were immediately subcutaneously injected with 120 mg/kg sodium nitrite, and mice in the control group were subcutaneously injected with 20 mL/kg saline. ④ Ethanol-induced memory reconsolidation disorder in mice： Grouping, administration, and testing were the same as mentioned above. At 24 hours after training and 20 minutes before retraining, mice in the remaining four groups were perfused with 10 mL/kg ethanol （0.3 volume fraction）, and mice in the control group were perfused with 10 mL/kg saline. MAIN OUTCOME MEASURES： Synergistic effects of GSL and choline on learning and memory deficits induced by scopolamine, sodium nitrite, and ethanol in experimental animals. RESULTS： All 40 rats and 150 mice were included in the final analysis. ① Synergistic effects of GSL and choline on learning and memory disorders induced by scopolamine in rats： During passive step-down avoidance and Morris water maze spatial resolution tests, the number of error responses and length of maze training in the model group were significantly greater than in the control group （P 〈 0.01）; while the number of error responses and length of maze training in the combination group were significantly less than in the model group, GSL group, and choline group （P 〈 0.05-0.01）. The Q value was 〉 1 after combining administration, which suggests that the combination of GSL and choline had synergistic effects. ② Synergistic effects of GSL and choline on learning disorder and memory-acquired disorder induced by scopolamine in mice： During passive step-down avoidance test, the number of error responses in the model group were significantly greater than in the control group （P 〈 0.01 ）; while the number of error responses in the combination group were significantly less than in the model group, GSL group, and choline group （P 〈 0.05-0.01）. The Q value was 〉 1 after combining administration, which suggests GSL and choline had synergistic effects. ③ Synergistic effects of GSL and choline on memory sodium nitrate-induced consolidation disorder in mice： During passive step down avoidance test, the number of error responses in the model group were significantly less than in the control group （P 〈 0.01 ）; while the number of error responses in the combination group were significantly less than in the model group, GSL group, and choline group （P 〈 0.05-0.01）. The Q value was 〉 1 after combined administration, which suggests GSL and choline had synergistic effects. ④ Synergistic effects of GSL and choline on ethanol-induced memory reconsolidation disorder in mice： During passive step down avoidance test, the number of error responses in the model group were significantly greater than in the control group （P 〈 0.01）; while the number of error responses in the combination group were significantly less than in the model group, GSL group, and choline group （P 〈 0.05-0.01）. The Q value was 〉 1 after combined administration, which suggests GSL and choline had synergistic effects. CONCLUSION： GSL and choline have synergistic effects on learning and memory functions.

Analysis of Low-polar Ginsenosides in Steamed Panax Ginseng at High-temperature by HPLC-ESI-MS/MS

A high performance liquid chromatography coupled with electrospray ionization-tandem mass spectrome try（HPLC-ESI-MS/MS） method was developed for the analysis and identification of ginsenosides in the extracts of raw Panax ginseng（RPG） and steamed Panax ginseng at high temperatures（SPGHT）. A total of 25 ginsenosides were extracted include of which 10 low-polar ginsenosides, such as ginsenosides F4, Rk3, Rh4, 20S-Rg3, 20R-Rg3 and so on, were identified according to their HPLC retention time and MS/MS data. The results indicated that the low polar ginsenosides were seldom found in RPG. For the exploration of the transformation pattern of the ginsenosides in steam processing, the standards of ginsenosides Re, Rg1, Rb1, Rc, Rb2, Rb3 and Rd were selected and hydrolyzed at a temperature of 120 oC. The results show that these polar ginsenosides can be converted to low-polar ginsenosides such as Rg2, Rg6, F4, Rk3 and Rg5 by hydrolyzing the sugar chains.

One Stone Four Birds:A Novel Liposomal Delivery System Multi-functionalized with Ginsenoside Rh2 for Tumor Targeting Therapy

Liposomes hold great potential in anti-cancer drug delivery and the targeting treatment of tumors.However,the clinical therapeutic efficacy of liposomes is still limited by the complexity of tumor microenvironment(TME)and the insufficient accumulation in tumor sites.Meanwhile,the application of cholesterol and polyethylene glycol(PEG),which are usually used to prolong the blood circulation and stabilize the structure of liposomes respectively,has been questioned due to various disadvantages.Herein,we developed a ginsenoside Rh2-based multifunctional liposome system(Rh2-lipo)to effectively address these challenges once for all.Different with the conventional’wooden’liposomes,Rh2-lipo is a much more brilliant carrier with multiple functions.In Rh2-lipo,both cholesterol and PEG were substituted by Rh2,which works as membrane stabilizer,long-circulating stealther,active targeting ligand,and chemotherapy adjuvant at the same time.Firstly,Rh2 could keep the stability of liposomes and avoid the shortcomings caused by cholesterol.Secondly,Rh2-lipo showed a specifically prolonged circulation behavior in the blood.Thirdly,the accumulation of the liposomes in the tumor was significantly enhanced by the interaction of glucose transporter of tumor cells with Rh2.Fourth,Rh2-lipo could remodel the structure and reverse the immunosuppressive environment in TME.When tested in a 4T1 breast carcinoma xenograft model,the paclitaxel-loaded Rh2-lipo realized high efficient tumor growth suppression.Therefore,Rh2-lipo not only innovatively challenges the position of cholesterol as a liposome component,but also provides another innovative potential system with multiple functions for anti-cancer drug delivery.

Protective effects of ginsenoside Rg1 against hydrogen peroxide-induced injury in human neuroblastoma cells

The active ingredient of ginseng,ginsenosides Rg1,has been shown to scavenge free radicals and improve antioxidant capacity.This study hypothesized that ginsenosides Rg1 has a protective role in human neuroblastoma cells injured by H2O2.Ginsenosides Rg1 at different concentrations（50 and 100 μM） was used to treat H2O2（150 μM）-injured SH-SY5 Y cells.Results demonstrated that ginsenoside Rg1 elevated the survival rate of SH-SY5 Y cells injured by H2O2,diminished the amount of leaked lactate dehydrogenase,and increased superoxide dismutase activity.Ginsenoside Rg1 effectively suppressed caspase-3 immunoreactivity,and contributed to heat shock protein 70 gene expression,in a dose-dependent manner.These results indicate that ginsenoside Rg1 has protective effects on SH-SY5 Y cells injured by H2O2 and that its mechanism of action is associated with anti-oxidation and the inhibition of apoptosis.

Effect of ginsenoside Rgl and Rhl on the anti-tumor activity of dendritic cell

AIM: To study the effect of ginsenoside Rgl and Rhl on the anti - tumor activity of dendritic cells (DC). METHODS: Effect of Rgl and Rhl on the production of IL- 12 p40 protein was detected by ELISA, and the IL- 12 p40 mRNA level of DC was monitored by RT- PCR. Anti - tumor activity of DC- LPAK was detemnined by neutral red staining assay. RESULTS: The results of ELLSA showed that Rgl and Rhl significantly enhanced the production of IL- 12 p40 of DC. Rgl at 1 mg/L and Rhl at 100 mg/L upregulated the IL- 12 p40 mRNA level. Rgl and Rhl enhanced the anti - tumor ability of DC, induced lyrnphokine and PHA activated killer (DC-LPAK) on human papillate tumor cell line. Each dose of Rgl can obviously accelerate the eytotoxity to L929 at the E: T ratio of 5 : 1 (P<0.05,0.01 ), while only Rhl 10 mg/L enhanced the eytotoxity ability of DC- LPAK (P < 0.05). CONCLUSION: Rgl and Rhl enhanced the production of IL-12 p40. This effect may be mediated by the increase in the mRNA level. As a result, Rg1 and Rhl oromote the ability of DC to stimulate the cytotoxitie aetieity of DC - LPAK.

Ginsenoside Rg1 protects against ischemia reperfusion-induced neurotoxicity through miR-144/Nrf2/ARE pathway

OBJECTIVE Ginsenoside Rg1(Rg1),a purified compound from Panax ginseng,has been well documented to be effective against ischemia/reperfusion(I/R) neurotoxicity.However,the underlying mechanism is stil obscure.METHODS The anti-I/R effect of Rg1 were investigated in vitro and in vivo,and the dynamics of nuclear accumulation and the transcriptional activity of NF-E2-related factor 2(Nrf2) determined by Western blotting and Dual Luciferase Reporter Assay,respectively.Nrf2 siRNA was employed to investigate Nrf2′s role in the protective effect of Rg1 against I/R.Furthermore,the role of miR-144,which could regulate post-translational Nrf2 levels,was investigated in the anti-I/R effect of Rg1 by injection of AAV-hypoxia-inducible factor miR-144-shRNA in the predicted ischemic penumbra.RESULTS It was found that the anti-I/R effect of Rg1 was related to its anti-oxidative capacity,which is mainly regulated by the Nrf2/antioxidant response element(ARE) pathway.Further study suggested that Rg1 contributes to the enhancement of the Nrf2/ARE pathway,as manifested by increasing the dynamic peak content of Nrf2,which prolonged the maintenance stage,and promoting the expression of ARE-target genes after oxygen glucose deprivation/reperfusion(OGD/R) in PC12 cells.Nrf2-siRNA application significantly reduced these changes.Furthermore,the enhancement of the Nrf2/ARE pathway by Rg1 was independent of disassociation from Keap1;rather it was a result of posttranslational regulations.It was found that Rg1 significantly reduced the expression of miR-144,which down-regulates Nrf2 production by targeting its 3′-untranslated region,after OGD/R.Knockdown of Nrf2 showed no effect on the expression of miR-144,indicating that miR-144 is an upstream regulator of Nrf2.Moreover,direct binding between Nrf2 and miR-144 in the PC12 cells was identified.Application of anti-miR-144 significantly reduced Rg1′s anti-OGD/R capacity.Final y,the role of miR-144 in Rg1′ s anti-I/R effect was tested by inhibiting miR-144 in the predicted ischemic penumbra when hypoxia-inducible-factor was activated.The results showed that loss of miR-144 abolished the anti-I/R effect of Rg1,which included reduced infarct volume,improved neurological scores,attenuated oxidative impairment,as well as activation of the Nrf2/ARE pathway.CONCLUSION Oxidative stress after I/R is alleviated by Rg1 through inhibition of miR-144 activity and subsequent promotion of the Nrf2/ARE pathway at the post-translational level.

Effects of Ginsenoside Rg1 on nuclear factor-kappa B activity in beta amyloid protein-treated neural cells

BACKGROUND： Modern pharmacological studies have shown that Ginsenoside Rgl is one of the active components of ginseng that promote intelligence in the nervous system. Ginsenoside Rgl can improve memory and learning in mouse models of β-amyloid protein （Aβ）-induced dementia. OBJECTIVE： To investigate whether effects of Ginsenoside Rgl against Aβ are associated with activity of nuclear factor-kappa B （NF-κB）. DESIGN, TIME AND SETTING： The randomized performed at the DME Center, Institute of Clinica controlled, cell biological experiment was Pharmacology, Guangzhou University of Chinese Medicine, China from July 2005 to May 2006. MATERIALS： Beta-amyloid fragment 25-35 （Aβ25-35） was supplied by the Neural Biochemical Laboratory, Xuanwu Hospital, Capital Medical University, China. Ginsenoside Rgl was obtained from National Institute for the Control of Pharmaceutical and Biological Products, China. Rabbit anti-rat NF-κB p65 antibody was purchased from Santa Cruz Biotechnology, USA. METHODS： Hippocampal neurons and cortical astrocytes of neonatal Sprague Dawley rats were harvested and treated with various concentrations （0, 5, 10, 20, and 40 μmol/L） of Aβ for 6, 12, and 24 hours to establish cellular models of Alzheimer＇s disease. Cellular models were pretreated with various concentrations of Ginsenoside Rgl （1,2, 4, 8, and 16 μmol/L）. According to cell morphology and activity, the following conditions were selected： 40 μmol/L Aβ for 24 hours, as well as 2, 4, and 8 μmol/L Ginsenoside Rg1. NF-κB activity was observed using immunofluorescence and cytochemical staining. MAIN OUTCOME MEASURES： Morphology and viability of hippocampal neurons and cortical astrocytes, and activities of NF-κB were measured. RESULTS： Hippocampal neuron activity was significantly greater in the normal and 2 and 4 μmol/L Ginsenoside Rgl groups compared with the model group （P 〈 0.05）. Astrocyte activity was significantly greater in the normal, 1,2, 4, 8, and 16 μmol/L Ginsenoside Rgl groups compared with the model group （P 〈 0.05）. NF-κB activity of hippocampal neurons was significantly greater in the normal, 2, 4, and 8 μmol/L Ginsenoside Rgl groups compared with the model group （P 〈 0.01）. NF-κB activity of astrocytes was significantly less in the normal, 2, 4, and 8 μmol/L Ginsenoside Rgl groups compared with the model group （P 〈 0.01 or P 〈 0.05）. No significant difference in NF-κB activity was determined between the 2 μmol/L Ginsenoside Rgl and normal groups （P 〉 0.05）. CONCLUSION： Ginsenoside Rgl protected neural cells by upregulating NF-κB activity in neurons and downregulating NF-κB activity in astrocytes. Ginsenoside Rgl （2 μmol/L） maintained cell activity and NF-κB activity at normal levels.