18α￣glycyrrhetinic acid promoting neural stem cells proliferation through antioxidant in the subventricular zone of adult mice / 解剖学报

Objective To explore the effect of 18α-glycyrrhetinic acid (18α-GA) on the proliferation of adult mice neural stem cells (NSCs) and its underlying mechanism. Methods One hundred 6-month BALB/c mice were randomly divided into DMSO control group and 18α-GA group (mice were intraperitoneally injected with 40 mg/kg 18α-GA every day for 2 months). The proliferation capability, oxidative status and nuclear factor E2-related factor 2 (Nrf2) level of NSCs in the adult mice subventricular zone (SVZ) were measured through both in vivo and in vitro experiments, including Ki-67 staining, neurosphere formation assay, BrdU incorporation, CCK-8 assay, reactive oxygen species (ROS) detection, superoxide dismutase 1 (SOD1) determination, Real-time PCR and Western blotting. Results Elevated Ki-67 positive cells were observed in SVZ of mice with 18α-GA application. Meanwhile, ROS level attenuated but SOD1 mRNA and protein level increased significantly in the SVZ of 18α-GA group mice, the latter of which were (3. 17 ± 0. 073) and (2. 12±0. 02) times respectively than that of the control group (P<0. 05 and P<0. 001). Likewise, the similar changes were exhibited in vitro data. NSCs of 18α-GA group mice displayed higher proliferation potency confirmed by accelerated neurosphere formation and increased neurosphere number (P<0. 001), as well as higher BrdU positive ratio (P<0. 01) and NSCs vitality (P<0. 001). NSCs of mice with 18α-GA injection exhibited decreased ROS level by 18. 91%±4. 33% (P<0. 05) and enhanced SOD1 level, compared with those in NSCs of DMSO group mice. Furtherly, the Nrf2 expression in SVZ and NSCs of 18α-GA group mice was higher than that of the control group. Conclution 18α-GA administration plays a vital role in the maintainence and amelioration of adult mice NSCs proliferation through activating SOD1 and diminishing ROS aggregations.

Activation of Nrf2 by 18α-GA affects proliferation of adult neural stem cells / 中国病理生理杂志

AIM:To investigate the effect of nuclear factor E2-related factor 2 (Nrf2) activation by 18α-gly-cyrrhetinic acid (18α-GA) on the proliferation and self-renewal of adult neural stem cells (aNSCs), and to explore an ef-fective way of maintaining the viability of aNSCs .METHODS:NSCs were dissociated from subventricular zone of the mice at postnatal days 0, 60, and 300.The expression levels of Nrf2 in the NSCs at various ages were compared .After treatment with 18α-GA, the expression of Nrf2 was examined by real-time PCR and Western blot.shRNA lentiviral vector (LV) car-rying green fluorescent protein (GFP) gene was constructed to knock down Nrf2 expression.The knockdown efficiency in the aNSCs was detected by real-time PCR and Western blot .Subsequently , the aNSCs were divided into DMSO group , 18α-GA group, LV-GFP group and LV-Nrf2-shRNA group.BrdU incorporation assay , Tuj1 staining, CCK-8 assay, Hoechst 33342/PI staining and detection of reactive oxygen species ( ROS) were performed to analyze the proliferation , dif-ferentiation, viability, apoptosis and oxidative stress levels of the NSCs .RESULTS:The mRNA expression level of Nrf2 in adult and aged NSCs was significantly lower than that in newborn NSCs (P<0.01), while the ROS level of aNSCs was significantly higher (P<0.05).After treatment with 18α-GA, the expression level of Nrf2 in the aNSCs was significantly up-regulated as compared with DMSO group ( P<0.01).Increased number of BrdU +and Tuj1 +cells was observed in 18α-GA group, indicating that 18α-GA-treated cells had higher viability (P<0.05).Meanwhile, there were fewer apop-totic cells and lower ROS level in 18α-GA group than those in DMSO group (P<0.05).After knockdown of Nrf2 in aNSCs and then treated with 18α-GA, there were less BrdU +and Tuj1 +cells, as well as the aNSCs with lower viability in LV-Nrf2-shRNA group (P<0.05).Moreover, the ROS level was increased in LV-Nrf2-shRNA group as compared with LV-GFP group (P<0.05).CONCLUSION:Activation of Nrf2 by 18α-GA elevates the antioxidant capacity of aNSCs , thus ameliorating the cell proliferation and differentiation potentials .

Activation of Nrf2 by 18α-GA affects proliferation of adult neural stem cells / 中国病理生理杂志

AIM:To investigate the effect of nuclear factor E2-related factor 2 (Nrf2) activation by 18α-gly-cyrrhetinic acid (18α-GA) on the proliferation and self-renewal of adult neural stem cells (aNSCs), and to explore an ef-fective way of maintaining the viability of aNSCs .METHODS:NSCs were dissociated from subventricular zone of the mice at postnatal days 0, 60, and 300.The expression levels of Nrf2 in the NSCs at various ages were compared .After treatment with 18α-GA, the expression of Nrf2 was examined by real-time PCR and Western blot.shRNA lentiviral vector (LV) car-rying green fluorescent protein (GFP) gene was constructed to knock down Nrf2 expression.The knockdown efficiency in the aNSCs was detected by real-time PCR and Western blot .Subsequently , the aNSCs were divided into DMSO group , 18α-GA group, LV-GFP group and LV-Nrf2-shRNA group.BrdU incorporation assay , Tuj1 staining, CCK-8 assay, Hoechst 33342/PI staining and detection of reactive oxygen species ( ROS) were performed to analyze the proliferation , dif-ferentiation, viability, apoptosis and oxidative stress levels of the NSCs .RESULTS:The mRNA expression level of Nrf2 in adult and aged NSCs was significantly lower than that in newborn NSCs (P<0.01), while the ROS level of aNSCs was significantly higher (P<0.05).After treatment with 18α-GA, the expression level of Nrf2 in the aNSCs was significantly up-regulated as compared with DMSO group ( P<0.01).Increased number of BrdU +and Tuj1 +cells was observed in 18α-GA group, indicating that 18α-GA-treated cells had higher viability (P<0.05).Meanwhile, there were fewer apop-totic cells and lower ROS level in 18α-GA group than those in DMSO group (P<0.05).After knockdown of Nrf2 in aNSCs and then treated with 18α-GA, there were less BrdU +and Tuj1 +cells, as well as the aNSCs with lower viability in LV-Nrf2-shRNA group (P<0.05).Moreover, the ROS level was increased in LV-Nrf2-shRNA group as compared with LV-GFP group (P<0.05).CONCLUSION:Activation of Nrf2 by 18α-GA elevates the antioxidant capacity of aNSCs , thus ameliorating the cell proliferation and differentiation potentials .

Upregulation of proteasome activity by 18α-GA promotes proliferation of late-passage BMSCs in vitro / 中国病理生理杂志

AIM:To investigate the effect of 18 alpha-glycyrrhetinic acid (18α-GA) on delaying the senescent progress and promoting the proliferation in late-passage bone marrow mesenchymal stem cells ( BMSCs ) .METHODS:Late-passage BMSCs were incubated with 2.0 mg /L 18α-GA or the same volume of DMSO for 30 d, and the cells were harvested to determine the proteasome activity .The expression of senescence-related proteins p53, p21 and p16 was detec-ted by senescence-associated β-galactosidase ( SA-β-Gal) staining and Western blot .The cell proliferation , the expression level of cell cycle-related proteins and cell cycle distribution of the cells were measured by CCK -8 assay, BrdU incorpora-tion, Western blot and flow cytometry.RESULTS:Compared with DMSO group, the proteasome activity in 18α-GA group increased significantly by about 0.2 times (P<0.01).SA-β-Gal-positive cells in 18α-GA group decreased, and cell stai-ning was lighter.The contents of p53 and p21 in 18α-GA group were decreased (P<0.05).The results of CCK-8 assay showed that the A value in 18α-GA group was 0.3 times higher than that in DMSO group (P<0.01).BrdU incorporation showed the increased proliferation in 18α-GA group compared with DMSO group ( P<0.05).The cells in G1 phase in 18α-GA group decreased significantly compared with DMSO group , while the cells in S phase increased significantly ( P<0.05).The expression level of cyclin D1 in 18α-GA group was 2.8 times higher than that in DMSO group (P<0.01), and the CDK4 level was 1.4 times higher than that in DMSO group (P<0.05).CONCLUSION:Activation of the pro-teasome activity by 18α-GA delays the aging process in the BMSCs and promotes the cell proliferation via up -regulation of the cell cycle-related proteins .

Modulation of the respiratory rhythm by gap-junction blockers 18-α-glycyrrhetinic acid and 18-β-glycyrrhetinic acid.

Gap junction proteins are expressed in the pre-Bötzinger complex of the respiratory network but it remains under discussion how they modulate the respiratory rhythm generation. In the present study we tested whether the gap junction blockers 18-β-glycyrrhetinic acid (18-β- GA) and 18-α-glycyrrhetinic acid (18-α-GA) change either the phrenic nerve (PN) discharge frequency or amplitude. The PN discharge was recorded using the working heart-brainstem preparation of adult wistar rats (P22- P28) exposed to increasing concentrations of the gap junction blockers (0.1; 1; 10; 20 μM). With the two lower concentrations (0.1; 1 μM) of 18-β-GA, PN discharge frequency decreased to 46±15% (p=0.007) of the control value while it increased to 173±57% (p=0.16) with the two higher concentrations (10; 20 μM). Surprisingly, with 18-α-GA the PN discharge frequency was not significantly changed with any of the concentrations used. Enhancing the respiratory drive with 12% CO2, the PN discharge frequency increased tendencially with rising concentrations of 18-β-GA, but again no significant change was observed with 18-α-GA. PN-amplitudes were slightly reduced over the course of the experiments, while the frequency of the heartbeat was not significantly changed at any time with any concentration.

Effect of glycyrrhetinic acid C-18 epimerides on P-glycoprotein function and expression in Caco-2 cells / 中国药学杂志

OBJECTIVE: The aim of our work was to study the effects of C-18 epimers of glycyrrhetinic acid, 18α-glycyrrhetinic acid (α-GA) and 18β-glycyrrhetinic acid (β-GA) on the function and expression of P-gp in Caco-2 cell monolayers. METHODS: Caco-2 cells were cultured, the effects of P-gp function were analyzed by means of rhodamine accumulation test, real-time PCR was used to measure the expression of MDR1 mRNA and western blot was used to analysis P-gp expression in Caco-2 cell. RESULTS: In the rhodamine accumulation test, middle and high concentrations (1 and 10 μmol · L-1) α-GA and high concentration (10 μmol · L-1) β-GA decreased the cellular rhodamine concentration. At high concentrations (10 μmol · L-1), α-GA up-regulated the expression of MDR1 mRNA while β-GA has no effect on the expression of MDR1 mRNA at three test concentrations. α-GA up-regulated the expression of P-gp protein at high concentrations (10 μmol · L-1) while β-GA has no effect on the expression of P-gp protein at three test concentrations. CONCLUSION: Our findings provide experimental evidence that α-GA up-regulated both function and expression of P-gp while β-GA only up-regulate the function of P-gp.

Effect of glycyrrhetinic acid C-18 epimerides on membrane transport of P-gp substrate rhodamine 123 in Caco-2 cell monolayers / 中国药学杂志

OBJECTIVE: To study the effects of C-18 epimers of glycyrrhetinic acid, 18α-glycyrrhetinic acid(α-GA) and 18β-glycyrrhetinic acid(β-GA), on membrane transport of P-gp substrate rhodamine 123 in Caco-2 cell monolayers. METHODS: MTT assay was applied to determine the maximum non-cytotoxic dose of α-GA, β-GA and verapamil to ensure the activity of the cells during the test and to consult the maximum test concentration of each drug; appropriate Caco-2 monolayers were established in Transwell™ plates. ELISA Reader was applied to detect the concentration of Rho-123 in transfer fluid. The bi-directional transport of Rho-123 after being treated by instantaneous action and incubation with α-GA and β-GA for 72 h was studied. RESULTS: High concentration (10 μmol · L-1) of α-GA induced the efflux of Rho-123 both in the instantaneous action test and the incubation test. High concentration (10 μmol · L-1) of β-GA induced the efflux of Rho-123 only in the incubation test. The transport from AP to BL was not affected by any test drugs. CONCLUSION: The results of the transport experiments of P-gp substrates in Caco-2 monolayers indicated that α-GA and β-GA can induce the efflux function of P-gp and accelerate the excretion of P-gp substrates. It may be one of the mechanisms of that glycyrrhizin preparation enhances liver detoxification. Copyright 2012 by the Chinese Pharmaceutical Association.