This study investigates the effect of glycyrrhetinic acid(GA) combined with doxorubicin(DOX) on apoptosis in HepG2 cells and its possible mechanisms. HepG2 cells were cultured in vitro, and cell viability was assessed using the cell counting kit-8(CCK-8) method. Flow cytometry was used to measure apoptosis levels in HepG2 cells. The cells were divided into the following groups: control group(0 μmol·L~(-1)), DOX group(2 μmol·L~(-1)), GA group(150 μmol·L~(-1)), and DOX + GA combination group(2 μmol·L~(-1) DOX + 150 μmol·L~(-1) GA), with treatments given for 24 hours. The colocalization level between the endoplasmic reticulum(ER) and mitochondria was assessed by colocalization fluorescence imaging. Fluorescence probes were used to measure the Ca~(2+) content in the ER and mitochondria. The qRT-PCR and Western blot were used to determine the mRNA and protein expression of sirtuin-3(SIRT3). Co-immunoprecipitation(CO-IP) was applied to investigate the interactions between voltage-dependent anion channel 1(VDAC1) and SIRT3, as well as between VDAC1, glucose-regulated protein 75(GRP75), and inositol 1,4,5-trisphosphate receptor(IP3R). The results showed that the combination of DOX and GA promoted apoptosis in HepG2 liver cancer cells. The colocalization level between the ER and mitochondria was significantly reduced, the Ca~(2+) content in the ER was significantly increased, and the Ca~(2+) content in the mitochondria was significantly decreased. The relative expression of VDAC1, GRP75, and IP3R was significantly reduced, and interactions between VDAC1, GRP75, and IP3R were observed. SIRT3 mRNA and protein expression levels were significantly increased, and an interaction between SIRT3 and VDAC1 was detected. The acetylation level of VDAC1 was significantly decreased. In conclusion, GA combined with DOX induces apoptosis in HepG2 cells by mediating the deacetylation of VDAC1 through SIRT3, weakening the interactions among VDAC1, GRP75, and IP3R. This regulates the formation of endoplasmic reticulum-mitochondrial membrane domains(ERMMDs), affects Ca~(2+) transport between the ER and mitochondria, and ultimately triggers cell apoptosis.
Humans ; Apoptosis/drug effects* ; Hep G2 Cells ; Glycyrrhetinic Acid/pharmacology* ; Doxorubicin/pharmacology* ; Liver Neoplasms/genetics* ; Carcinoma, Hepatocellular/physiopathology* ; Mitochondria/metabolism* ; Endoplasmic Reticulum/metabolism* ; Cell Survival/drug effects* ; Membrane Proteins/genetics*

The 3-succinate-30-stearyl glycyrrhetinic acid(18-GA-Suc) was inserted into glycyrrhetinic acid(GA)-tanshinone Ⅱ_A(TSN)-salvianolic acid B(Sal B) liposome(GTS-lip) to prepare liver targeting compound liposome(Suc-GTS-lip) mediated by GA receptors. Next, pharmacokinetics and tissue distribution of Suc-GTS-lip and GTS-lip were compared by UPLC, and in vivo imaging tracking of Suc-GTS-lip was conducted. The authors investigated the effect of Suc-GTS-lip on the proliferation inhibition of hepatic stellate cells(HSC) and explored their molecular mechanism of improving liver fibrosis. Pharmacokinetic results showed that the AUC_(Sal B) decreased from(636.06±27.73) μg·h·mL~(-1) to(550.39±12.34) μg·h·mL~(-1), and the AUC_(TSN) decreased from(1.08±0.72) μg·h·mL~(-1) to(0.65±0.04) μg·h·mL~(-1), but the AUC_(GA) increased from(43.64±3.10) μg·h·mL~(-1) to(96.21±3.75) μg·h·mL~(-1). The results of tissue distribution showed that the AUC_(Sal B) and C_(max) of Sal B in the liver of the Suc-GTS-lip group were 10.21 and 4.44 times those of the GTS-lip group, respectively. The liver targeting efficiency of Sal B, TSN, and GA in the Suc-GTS-lip group was 40.66%, 3.06%, and 22.08%, respectively. In vivo imaging studies showed that the modified liposomes tended to accumulate in the liver. MTT results showed that Suc-GTS-lip could significantly inhibit the proliferation of HSC, and RT-PCR results showed that the expression of MMP-1 was significantly increased in all groups, but that of TIMP-1 and TIMP-2 was significantly decreased. The mRNA expressions of collagen-I and collagen-Ⅲ were significantly decreased in all groups. The experimental results showed that Suc-GTS-lip had liver targeting, and it could inhibit the proliferation of HSC and induce their apoptosis, which provided the experimental basis for the targeted treatment of liver fibrosis by Suc-GTS-lip.
Humans ; Liposomes ; Hepatic Stellate Cells ; Glycyrrhetinic Acid/pharmacology* ; Liver ; Liver Cirrhosis/genetics* ; Collagen/pharmacology*

Salvianolic acid B(Sal B), tanshinone Ⅱ_A(TSN Ⅱ_A), and glycyrrhetinic acid(GA) lipid emulsion(GTS-LE) was prepared by the high-speed dispersion method combined with ultrasonic emulsification.The preparation process of the emulsion was optimized by single-factor method and D-optimal method with appearance, centrifugal stability, and particle size of the emulsion as evalua-tion indexes, followed by verification.In vitro release of Sal B, TSN Ⅱ_A, and GA in GTS-LE was performed by reverse dialysis.In vivo pharmacokinetic evaluation was carried out in mice.The acute liver injury model was induced by acetaminophen.The effect of oral GTS-LE on the acute liver injury was investigated by serum liver function indexes and pathological changes in liver tissues of mice.The results showed that under the optimal preparation process, the average particle size of GTS-LE was(145.4±9.25) nm and the Zeta potential was(-33.6±1.45) mV.The drug-loading efficiencies of Sal B, TSN Ⅱ_A, and GA in GTS-LE were above 95%, and the drug release in vitro conformed to the Higuchi equation.The pharmacokinetic results showed that the C_(max) of Sal B, TSN Ⅱ_A, and GA in GTS-LE was 3.128, 2.7, and 2.85 times that of the GTS-S group, and AUC_(0-t) of Sal B, TSN Ⅱ_A, and GA in GTS-LE was 3.09, 2.23, and 1.9 times that of the GTS-S group.After intragastric administration of GTS-LE, the activities of alanine aminotransferase and aspartate aminotransferase were significantly inhibited, the content of malondialdehyde was reduced, and the structure of hepatocytes recovered to normal.In conclusion, GTS-LE can delay the release of Sal B and promote the release of TSN Ⅱ_A and GA.The encapsulation of three drug components in the emulsion can improve the oral bioavailability to varying degrees and can effectively prevent the acute liver injury caused by acetaminophen.
Abietanes/therapeutic use* ; Acetaminophen/therapeutic use* ; Alanine Transaminase/metabolism* ; Animals ; Antipyretics/therapeutic use* ; Aspartate Aminotransferases/metabolism* ; Benzofurans/therapeutic use* ; Chemical and Drug Induced Liver Injury/prevention & control* ; Depsides/therapeutic use* ; Emulsions ; Glycyrrhetinic Acid/therapeutic use* ; Liver/drug effects* ; Malondialdehyde ; Mice

Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; Glycyrrhetinic Acid/pharmacology* ; Humans

The optimal prescription of tanshinone Ⅱ_A(TSN)-glycyrrhetinic acid(GA) solid lipid nanoparticles(GT-SLNs) was explored and evaluated in vivo and in vitro, and its effect on acne after oral administration was investigated. The preparation processing and prescription were optimized and verified by single factor and response surface methodology. The in vitro release of GA and TSN in GT-SLNs was determined by ultra-performance liquid chromatography(UPLC). The effect of GT-SLNs on acne was investigated by the levels of sex hormones in mice, ear swelling model, and tissue changes in sebaceous glands, and the pharmacokinetics was evaluated. The 24-hour cumulative release rates of GA and TSN in SLNs were 65.87%±5.63% and 36.13%±2.31% respectively. After oral administration of GT-SLNs and the mixture of GA and TSN(GT-Mix), the AUC_(0-t) and AUC_(0-∞) of TSN in GT-SLNs were 1.98 times and 4.77 times those in the GT-Mix group, respectively, and the peak concentration of TSN in the GT-SLNs group was 17.2 times that in the GT-Mix group. After intragastric administration of GT-SLNs, the serum levels of testosterone(T) and the ratio of testosterone to estradiol(T/E2) in the GT-SLNs group significantly declined, and the sebaceous glands of mice were atrophied to a certain extent. The results demonstrated that obtained GT-SLNs with good encapsulation efficiency and uniform particle size could promote the release of GA and TSN. GT-SLNs displayed therapeutic efficacy on acne manifested by androgen increase, abnormal sebaceous gland secretion, and inflammatory damage.
Abietanes ; Acne Vulgaris/drug therapy* ; Animals ; Drug Carriers ; Glycyrrhetinic Acid ; Liposomes ; Mice ; Nanoparticles ; Particle Size ; Testosterone

In this study,a nano drug delivery system GA-DTX-NGO which could be used for liver tumor photothermal and chemotherapy was prepared and characterized,with docetaxel(DTX) as model drug,glycyrrhetinic acid(GA) as the target molecule,and nano graphene oxide(NGO) as the photosensitizer. Firstly,GA-NGO nanocomposites were synthesized by the amidation reaction,and then GA-DTX-NGO was prepared by ultrasonic dispersion method. The encapsulation efficiency and drug loading ratio were determined by high performance liquid chromatography(HPLC) and ultracentrifugation; the morphology was observed by transmission electron microscopy(TEM). The photothermal conversion test was carried out by laser irradiation at 808 nm and the drug release test in vitro was performed using reverse dialysis. Finally,the effect of GA-DTX-NGO on SMMC-7721 liver tumor cells proliferation was determined by using MTT assay. The results showed that GA-DTX-NGO had good water dispersibility,and TEM results showed a lamellar structure with about 200 nm in diameter. The encapsulation efficiency and drug loading ratio of GA-DTX-NGO were(98. 89 ± 0. 07) % and(64. 74±0. 26) %,respectively. GA-DTX-NGO had strong photothermal conversion performance under 808 nm of laser irradiation. The drug release test in vitro results showed GA-DTX-NGO had obvious sustained-release effects and temperature-dependent release characteristics. The results of cell assay showed that GA-DTX-NGO could effectively inhibit the proliferation of SMMC 7721 cells in a concentration-and time-dependent manner,and the inhibitory effect was enhanced after combination with the near-infrared therapy. In conclusion,the preparation process of GA-DTX-NGO nano drug delivery system is feasible,which could provide some theoretical basis for further study of photothermal and chemotherapy on liver tumor.
Antineoplastic Agents ; Drug Carriers ; Drug Delivery Systems ; Glycyrrhetinic Acid ; Graphite

OBJECTIVETo investigate the expression and effect of Connexin43 (Cx43) on tensile tension-stimulated osteogenic transcription factors of human periodontal ligament fibroblasts (hPDLFs).

METHODSAfter hPDLFs were treated with 5% elongation tension for 1 h, 2 h, 4 h, 8 h, and 24 h, we examined the expressions of Cx43, Osterix, and RUNX2 at the mRNA level. After Cx43 expression was suppressed by siRNA or 18α-GA, the changes The mRNA in hPDLFs of Osterix and RUNX2 were observed.

RESULTSThe expressions of Cx43, Osterix, and RUNX2 mRNA in hPDLFs increased in a time-dependent fashion following tensile strain (all P<0.05), with the highest level at 5% elongation for 24 h. After Cx43 expression was blocked by two different methods, the increasing expressions of Osterix and RUNX2 were inhibited.

CONCLUSIONS5% cyclic tension upregulates Cx43 expression and promotes the expression of Osterix and RUNX2 in a time-dependent manner. Cx43 may be involved in the osteogenic response of hPDLFs to mechanical tension.

Cells, Cultured ; Connexin 43 ; Fibroblasts ; Glycyrrhetinic Acid ; analogs & derivatives ; Humans ; Osteogenesis ; Periodontal Ligament ; RNA, Messenger ; RNA, Small Interfering ; Stress, Mechanical ; Transcription Factors ; Up-Regulation

OBJECTIVES: In this study, the destabilizing effect of glycyrrhetinic acid on pre-formed biofilms of Streptococcus mutans (S. mutans) was observed. METHODS: Alamar blue assay was used to determine the toxicity of glycyrrhetinic acid on pre-formed biofilms of S. mutans. Four different concentrations (0, 3.75, 7.5, 15 µg/ml) of glycyrrhetinic acid were tested. Changes in the biofilm architecture after exposure to glycyrrhetinic acid were analyzed by scanning electron microscopy (SEM). Moreover, the role of glycyrrhetinic acid in enhancing the antimicrobial activity of cetylpyridinium chloride (CPC), an antimicrobial agent commonly used in oral health care products, was evaluated. RESULTS: Glycyrrhetinic acid concentration of up to 15 µg/ml had little cytotoxic effect but significantly changed the biofilm architecture. SEM analysis revealed destabilized biofilm structure after the preformed biofilms were exposed to glycyrrhetinic acid. Supplementing 2.5 µg/ml CPC with 15 µg/ml glycyrrhetinic acid significantly enhanced the bactericidal effect of CPC on the pre-formed biofilms than that in the non-supplemented CPC treated control. This indicates that glycyrrhetinic acid enhanced the antimicrobial activity of CPC by modifying the structure, thus facilitating the penetration of CPC into the biofilm. CONCLUSIONS: Glycyrrhetinic acid could be a potential agent to effectively control S. mutans biofilms responsible for dental caries.
Biofilms* ; Cetylpyridinium ; Dental Caries ; Glycyrrhetinic Acid* ; Microscopy, Electron, Scanning ; Oral Health ; Streptococcus mutans* ; Streptococcus*

OBJECTIVETo explore the effect of 18-β glycyrrhetinic acid (GA) on the endoplasmic reticulum of nasal epithelial cells in allergic rhinitis (AR) model rats.

METHODSTotally 96 Wistar rats were randomly divided into the blank group, the AR model group, the loratadine group, the GA group, 24 in each group. AR models were established by peritoneally injecting ovalbumin (OVA). Morphological scoring was performed. GA at 21. 6 mg/kg was intragastrically administered to rats in the GA group. Nasal mucosal tissues were taken for electron microscopic examinations at the second, fourth, sixth, and tenth week after drug intervention.

RESULTSThe overlapping score was 2.10 ± 0.45 in the blank group, 5.10 ± 0.56 in the loratadine group, 5.10 ± 0.56 in the AR model group, 5.20 ± 0.78 in the GA group, showing statistical difference when compared with the blank group (P < 0.01). Results under transmission electron microscope showed that the number of the endoplasmic reticulum increased in the AR model group, with obvious cystic dilatation, a lot of vacuole formation, and degranulation. A large number of free ribosomes could be seen in cytoplasm. With persistent allergen exposure, changes mentioned above was progressively aggravated in the endoplasmic reticulum of nasal mucosal epithelium in the AR model group. But the dilation of endoplasmic reticulum, vacuole formation, and degranulation were relieved in the GA group, and got close to those of the blank group.

CONCLUSION18-β GA could improve the expansion, vacuolization, and degranulation of the endoplasmic reticulum of nasal epithelial cells in AR model rats.

Animals ; Anti-Inflammatory Agents ; pharmacology ; therapeutic use ; Endoplasmic Reticulum ; Epithelial Cells ; drug effects ; Glycyrrhetinic Acid ; pharmacology ; therapeutic use ; Nasal Mucosa ; drug effects ; Rats ; Rats, Wistar ; Rhinitis, Allergic ; drug therapy

A study on the microbial transformation of glycyrrhetinic acid (GA) was conducted by a fungus, Cunninghamella blakesleeana CGMCC 3.970 systematically. After incubation with the cell cultures of C. blakesleeana CGMCC 3.970 at 25 degrees C for 7 days on a rotary shaker operating at 135 r x min(-1), GA was converted into one major product and five minor products. The products were extracted and purified by solvent extraction, macroporous adsorbent resin, silica gel column chromatography, and semi-preparative RP-HPLC chromatography. Their structures were identified as 3-oxo-15α-hydroxy-18β-glycyrrhetinic acid(1), 3-oxo-15β-hydroxy-18β-glycyrrhetinic acid (2), 7β,15α-dihydroxy-18β-glycyrrhetinic acid (3), 3-oxo-7β, 15α-dihydroxy-18β-glycyrrhetinic acid (4), 7β-hydroxy-18β-glycyrrhetinic acid(5) and 15α-hydroxy-18β-glycyrrhetinic acid(6) by the analyses of MS, 1H-NMR and 13C-NMR spectroscopic data respectively. Among them, 2 was a new compound. These results suggest that C. blakesleeana CGMCC 3.970 has the capability of selective ketonization and hydroxylation for GA. [Key words] glycyrrhetinic acid; Cunninghamella blakesleeana CGMCC 3. 970; microbial transformation
Biotransformation ; Cunninghamella ; metabolism ; Glycyrrhetinic Acid ; analogs & derivatives ; chemistry ; metabolism ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization