Inhibitory Effect Of Glycyrrhizin Research Articles

Glycyrrhizin induces autophagic cell death in MHCC97-H cell line in vitro

Objective To investigate the inhibitory effect of Glycyrrhizin in MHCC97-H cell line in vitro and explore the relevant mechanism. Methods MHCC97-H cells were cultured in vitro and treated with Glycyrrhizin in different concentrations and then cell viability was assayed at different time points. The concentration and time were selected with 50% cell viability. MHCC97-H cell plate clone formation assay and invasion-migration experiment were also performed to study the tumor-suppressor efficacy of Glycyrrhizin. Acridine orange staining was used to evaluate the formation of autophagic vacuoles. Meanwhile, 3-MA and Atg7-siRNA were both employed to avoid the autophagy activation in MHCC97-H cells and cell viability was reassessed. Western-blot was carried out to study the expression of autophagic proteins of LC3B, p-mTOR and p-ERK1/2. Results It showed Glycyrrhizin significantly inhibited MHCC97-H cell viability and the concentration and time at 50% cell viability were 2 mmol/L and 48 h respectively. Clone number in Glycyrrhizin group was significantly smaller than that in the control group (176.7±14.5 vs. 410.0±32.1). Invasion-migration rate was also lower in Glycyrrhizin group compared with the control group (41.0%±3.8% vs. 100%). Autophagic vacuoles was increased in MHCC97-H cells when treated with Glycyrrhizin and expression of LC3B-Ⅱ was enhanced and LC3B-Ⅱ/Ⅰ Ratio was increased, at the same time degradation of P62 was accelerated. Reduced p-mTOR in concurrence with upregulated p-ERK1/2 could be observed in MHCC97-H cells administered with Glycyrrhi-zin. Cell groups additionally treated with 3-MA or Atg7-siRNA exerted higher cell viability (64.3% vs. 45.9% and 67.7% vs. 47.1%, respectively). Conclusion Glycyrrhizin can induce excessive autophagy in hepatocellular carcinoma cells to cause autophagic cell death and exhibit great potential in clinical application. Key words: Hepatocellular carcinoma; Glycyrrhizin; Autophagy; MHCC97-H; Signaling pathway, mTOR, ERK1/2

CD34+CD31+ immature myeloid cells (IMC) as an inhibitor cell on the production of antimicrobial peptide: III. Translational studies of glycyrrhizin in humanized NOD-SCID IL-2rγ-/- mice (NOD-SCID mice) (89.30)

Abstract In the accompanying paper II, glycyrrhizin (GL) is shown to be inhibitory on the CD34+CD31+ IMC-mediated suppression of HBD-1 production by normal human epidermal keratinocytes (NHEK). In this study, the inhibitory effect of GL on CD34+CD31+ IMC-mediated suppression in HBD-1 production by NHEK was tested in human immune system mice (HIS mice) (X-irradiated NOD-SCID mice inoculated i.d. with NHEK and CD34+CD31+ IMC). Two days after the cell inoculation, inoculation site skin tissues were biopsied (8-mm diameter) from HIS mice treated with or without GL (Minophagen Pharmaceutical Co., Ltd., Tokyo, Japan) 6 and 24 hrs after the cell inoculation. Five biopsied tissue samples were homogenized together on ice in PBS. The concentrations of HBD-2 in the homogenates were measured by ELISA. In the results, 4.9 μg/ml of HBD-2 was detected in skin homogenates derived from NOD-SCID mice inoculated with NHEK, while HBD-2 was not detected in skin homogenates from HIS mice. When HIS mice were treated with 10 mg/kg of GL, HBD-2 was detected in IMC-inoculation site tissues of these mice. The amount of HBD-2 detected in this homogenate was corresponded to that detected in tissues of NOD-SCID mice inoculated with NHEK alone. These results indicate that GL improves the suppressed HBD-2 production in HIS mice. This study was supported by SHC #8610.

Inhibitory effect of glycyrrhizin on the neutrophil‐dependent increase of R5 HIV replication in cultures of macrophages

It has been described that polymorphonuclear neutrophils (PMNs) enhance the replication of CC‐chemokine receptor 5/macrophage‐tropic (R5) HIV in cultures of monocyte‐derived macrophages (MDMs). In this study, the inhibitory effect of glycyrrhizin (GL) on R5 HIV replication influenced by PMNs was investigated in MDM cultures. The replication of R5 HIV in MDMs was greatly enhanced when cells were co‐cultured with freshly isolated PMNs (syngeneic to MDMs). When GL was added to this culture, however, the viral replication enhanced by PMNs was completely inhibited. CCL2 and interleukin 10 (IL‐10) were produced in cultures of PMNs exposed to R5 HIV, and the replication of R5 HIV was greatly enhanced in MDM cultures supplemented with a mixture of recombinant CCL2 and IL‐10. However, CCL2 and IL‐10 were not produced by PMNs exposed to R5 HIV, when GL was added to the cultures. In the presence of GL, these soluble factors were not detected in co‐cultures of MDMs and PMNs exposed to R5 HIV. In addition, the replication of R5 HIV in MDMs stimulated with CCL2 and IL‐10 was not directly influenced by GL. These results indicated that GL suppresses the PMN‐dependent increase of R5 HIV replication in MDMs through inhibiting CCL2/IL‐10 production by PMNs stimulated with R5 HIV.

Inhibitory effect of glycyrrhizin on the production of CCL2 by various sources of immunosuppressive neutrophils (PMN‐II)

Three subtypes of PMN (PMN‐N, PMN‐I and PMN‐II) have been demonstrated. Among them, PMN‐II have been characterized as cells that participate in the increased susceptibility of burned hosts to bacterial translocation‐associated infectious complications. Since host‐mediated antibacterial effects of glycyrrhizin (GL) were demonstrated in these infections, in this study, the inhibitory effects of GL on the CCL2 production by various sources of PMN‐II were examined. CCL2 has been determined as an effector chemokine on the PMN‐II immunosuppressive activity. PMN‐II were prepared from peripheral blood of burn patients by Ficoll‐Hypaque and dextran sedimentations. Also, PMN‐II were induced from band PMN (isolated peripheral blood PMN from healthy volunteers 3 hrs after having exercised for 1 hr) by stimulation with SAC (0.0075%), GM‐CSF (0.1 ng/ml), VEGF (0.1 ng/ml) or terbutaline (β2‐AR agonist, 10−6 M). All of these PMN preparations were identified as PMN‐II by their abilities to produce CCL2 and IL‐10. In the results, the production of CCL2 by PMN‐II isolated from peripheral blood of severely burned patients was inhibited by 10 μg/ml of GL (Minophagen Pharmaceutical Co., Ltd., Tokyo, Japan). Also, the production of CCL2 by PMN‐II preparations, prepared in vitro from band PMN of exercised volunteers after stimulation with SAC, GM‐CSF, VEGF or terbutaline, was inhibited by GL (10 μg/ml). These results suggest that PMN‐II‐associated deficiency of the host antimicrobial resistance may be effectively intervened by GL.This study was supported by SHC NA #8840.

Inhibitory effect of glycyrrhizin on lipopolysaccharide and d-galactosamine-induced mouse liver injury

The effects of glycyrrhizin isolated from licorice root were investigated on acute hepatitis induced by lipopolysaccharide (LPS) and d-galactosamine in mice. Serum alanine aminotransferase (ALT) activity was markedly increased 6 h to 8 h after administration of LPS/ d-galactosamine. Levels in serum of cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and IL-12 reached a maximum by 2 h, whereas levels of IL-18, as well as of ALT, were maximal at 8 h. Glycyrrhizin (ED 50: 14.3 mg/kg) inhibited the increase in ALT levels when it was given to mice at 30 min before administration of LPS/ d-galactosamine. Inflammatory responses, including infiltration of neutrophils and macrophages in the liver injury, were modulated by glycyrrhizin. Increases in ALT levels were reduced by an administration of glycyrrhizin at 10 min and 60 min but not 3 h, even after LPS/ d-galactosamine treatment. However, glycyrrhizin had no effect on the production of TNF-α, IL-6, IL-10 and IL-12, whereas it significantly inhibited IL-18 production. Exogenous IL-18 further increased the elevation in ALT levels in mice treated with LPS/ d-galactosamine. Glycyrrhizin completely suppressed the effect of IL-18 of increasing ALT levels. IL-18 was detected by immunohistochemistry in inflammatory cells such neutrophils and macrophages in liver injury. Glycyrrhizin reduced the responsiveness of cells to IL-18 in the liver injury. These results suggest that glycyrrhizin inhibits the LPS/ d-galactosamine-induced liver injury through preventing inflammatory responses and IL-18 production. Furthermore, it seems that glycyrrhizin prevents IL-18-mediated inflammation in liver injury.

Inhibitory effect of glycyrrhizin on the expression of c-fos by an immunosuppressive subset of murine neutrophils (PMN-II) (44.30)

Abstract The root of Glycyrrhiza glabra (licorice) and its principle component (glycyrrhizin) have revealed various immunomodulatory properties that fight off attacking pathogens. In our previous studies, glycyrrhizin was shown to prevent infectious complications developed in mice with systemic inflammatory response syndrome (SIRS) through the inhibition of CCL2 production by PMN-II. c-fos (one of the nuclear transcription factors) is known as a CCL2 gene promoter. Therefore, in the present study, c-fos mRNA expression in PMN-II treated with or without glycyrrhizin was analyzed. For these studies, SIRS was induced by a flame burn injury (3rd degree, 25% TBSA burns). PMN-II were isolated from the peripheral blood of mice 18 hrs after burn injury by Ficoll-Hypague and dextran sedimentations, followed by the depletion of contaminating monocytes using magnetic beads coated with anti-F4/80 mAb. In the presence of Staphylococcus aureus antigen (0.0075%), PMN-II (1 x 106 cells/ml) were cultured with various doses of glycyrrhizin for 12 hrs, and the culture fluids obtained were assayed for CCL2. Also, the obtained cells were analyzed for c-fos mRNA by RT-PCR. In the results, 3.7 ng/ml of CCL2 was detected in the culture fluids of PMN-II, while PMN-II treated with glycyrrhizin did not produce CCL2. c-fos mRNA was expressed in PMN-II, but not in PMN from normal mice. When PMN-II were treated with 10 μg/ml of glycyrrhizin, c-fos mRNA expression was greatly decreased. These results suggest that c-fos expression in PMN-II may be controlled by glycyrrhizin when CCL2 production by these PMN is inhibited by the compound. This study was supported by SHC NA #8840.

Glycyrrhizin inhibits neutrophil-associated generation of alternatively activated macrophages

Patients with severe burn injuries are extremely susceptible to infection, and the host's antibacterial responses are frequently suppressed by alternatively activated macrophages (M2Mϕ), commonly demonstrated in these patients. An immunosuppressive subset of neutrophils (PMN-II), demonstrated in the peripheral blood of thermally injured patients, has been described as an inducer of M2Mϕ. In the present studies, the inhibitory effect of glycyrrhizin (GL) on M2Mϕ generation stimulated by PMN-II was examined. M2Mϕ were generated from resident Mϕ (R-Mϕ, lower chamber) after cultivation with PMN-II (upper chamber) in a dual-chamber transwell. However, M2Mϕ were not generated from R-Mϕ when the same transwell cultures were performed in the presence of GL. M2Mϕ were not generated from R-Mϕ after cultivation with PMN-II previously treated with GL, while R-Mϕ previously treated with GL converted to M2Mϕ after they were cultured with PMN-II in transwells. Interleukin-10 and CCL2 released from PMN-II were shown to be effector molecules responsible for the generation of M2Mϕ. However, these soluble factors were not produced by PMN-II treated with GL. These results indicate that GL inhibits PMN-II-stimulated M2Mϕ generation through the inhibition of CCL2/interleukin-10 production by PMN-II.

Inhibitory Effect of Glycyrrhizin on the Phosphorylation and DNA-Binding Abilities of High Mobility Group Proteins 1 and 2 in Vitro.

The physiological correlation between glycyrrhizin (GL) and high mobility group proteins I and 2 (HMG1/2) and the inhibitory effect of GL on their phosphorylation by three protein kinases (CK-I, CK-II and PKC) were investigated biochemically in vitro. It was found that GL binds directly to HMG1/2, because (i) HMG1/2 have a high affinity with a GL-affinity column; and (ii) GL induces the conformational changes in HMG1/2. Both purified HMG1/2 functioned as phosphate acceptors for these two protein kinases (CK-I and PKC), but not phosphorylated by CK-II. Phosphorylation of HMG1/2 by two protein kinases (CK-I and PKC) was completely inhibited by a glycyrrhetinic acid derivative (oGA) at one-tenth the concentration of GL. Also, the DNA-binding abilities of HNG1/2 were reduced by GL in a dose-dependent manner. These results show that the binding of GL to HMG1/2 results in the inhibition of their physiological activities (DNA-binding ability and phosphorylation by PKC or CK-I) in vitro. The GL-induced inhibition of the physiological activities of HMG1/2 may be involved in the anti-inflammatory effect of GL in vivo.

Inhibitory effect of glycyrrhizin on NF-kappaB binding activity in CCl4- plus ethanol-induced liver cirrhosis in rats.

Potenlini is an injectable compound whose active component is glycyrrhizin, which is extracted from licorice. Previous studies showed that it could reduce liver injury, improve alanine aminotransferase (ALT) levels and act as an antifibrotic agent. However, the mechanism of its action remains unclear. The aim of this study was to determine the molecular mechanism of its action by investigating the effects of potenlini on nuclear factor-kappaB (NF-kappaB) binding activity in an animal model of liver cirrhosis. Rats were randomly allocated into a normal control group, a model control group, and a potenlini group. Rats in the latter two groups were treated with CCl4 and ethanol solution in order to induce chronic liver injury. Rats in the potenlini group were given potenlini treatment at the same time. Serum ALT levels were significantly reduced in rats treated with potenlini compared with levels in rats of the model control group, which had dramatically increased ALT levels. Histologically, liver steatosis and fibrosis were severe in the rats of the model group, but were significantly improved in rats of the potenlini group. NF-kappaB binding activity was markedly increased in the liver specimens taken from the rats of the model control group in comparison with the binding of normal livers, but the binding levels were nearly normal in the livers of the potenlini group. The results suggest that potenlini can inhibit the NF-kappaB binding activity in CCl4 and ethanol-induced chronic liver injury, and that effect may be a possible mechanism by which potenlini protects the liver from hepatotoxin-induced liver injury and cirrhosis.

Inhibition of burn‐associated suppressor cell generation by glycyrrhizin through the induction of contrasuppressor T cells

The inhibitory effect of glycyrrhizin (GR), an anti-inflammatory Chinese herbal drug extracted from licorice roots, on the generation of suppressor T cells in thermally injured mice (TI-mice) was investigated. The burn-associated suppressor T cell (BTs cell) activity was demonstrated in splenic mononuclear cells (SMNC) from mice 2 to 8 days after thermal injury when suppressor cell activity was assayed in a one-way mixed lymphocyte reaction. However, when TI-mice were treated with GR, SMNC harvested 6 days after thermal injury showed no suppressor cell activity. This activity of GR demonstrated a dose-response effect, with a dose of 10 mg/kg exhibiting peak levels of the activity. Since GR had no direct inactivating activities against BTs cells in vitro, the inhibitory effect of SMNC, derived from TI-mice treated with GR, on the activity of BTs cells was examined in the same mixed lymphocyte reaction system, and the results showed that the SMNC from GR-treated mice 6 days after thermal injury counteracted the activity of BTs cells. The type of cell responsible for this inhibition of BTs cell activities was a CD3+, L3T4+, Vicia villosa lectin-adherent T cell with the same phenotypic properties previously exhibited by contrasuppressor cells. These results suggest that GR may regulate the generation of BTs cells through the induction of contrasuppressor cells. Since there are many reports describing septic infections due to the appearance of BTs cells in postburn patients, it may be possible to apply GR or blood preparations containing contrasuppressor cell populations induced by GR in healthy volunteers into immunosuppressed burn patients to avoid infections.

グリチルリチンによるＡ型肝炎ウイルス増殖の抑制

グリチルリチンによるＡ型肝炎ウイルス増殖の抑制

Inhibitory effect of glycyrrhizin on polypeptide phosporylation by polypeptide-dependent protein kinase (Kinase P) invitro

The anti-viral mechanism of glycyrrhizin (GL) has been investigated by considering in vitro effects on polypeptide phosphorylation. It was found that GL (i), at low doses, selectively inhibits protein phosphorylation by Kinase P, but has not significant effects on the activities of other kinases (Kinase A, Kinase C and histone kinase); (ii) binds directly to Kinase P and reduces kinase activity in a dose-dependent manner; and (iii) inhibits vesicular stomatitis virus (VSV)-associated kinase activity. These observations strongly suggest that direct binding of GL to the virus causes the direct inactivation of virus-associated kinase and the reduction of the viral infectivity.

Inhibitory effect of glycyrrhizin and caffeine on two-stage carcinogenesis in mice

Inhibitory effect of glycyrrhizin and caffeine on two-stage carcinogenesis in mice

Inhibitory effect of glycyrrhizin on the in vitro infectivity and cytopathic activity of the human immunodeficiency virus [HIV (HTLV-III/LAV)

Glycyrrhizin (GL), one of the plant extracts, was investigated for its antiviral action on the human immunodeficiency virus [HIV (HTLV-III/LAV)] in vitro, using cytopathic effect and plaque forming assay system in MT-4 cells (a HTLV-I-carrying cell line). Cloned Molt-4 cells (clone No. 8), which are sensitive to HIV and fuse to giant cells after infection, were also used as a parameter for cytopathic effect of HIV. GL completely inhibited HIV-induced plaque formation in MT-4 cells at a concentration of 0.6 mM, the 50% inhibitory dose being 0.15 mM. GL completely inhibited the cytopathic effect of HIV and the HIV-specific antigen expression in MT-4 cells at a concentration of 0.3 and 0.6 mM, respectively. Furthermore, GL inhibited giant cell formation of HIV-infected Molt-4 clone No. 8 cells. GL had no direct effect on the reverse transcriptase of HIV. Its mechanism of anti-HIV action remains to be elucidated.

The effects of digitonin and glycyrrhizin on liposomes

Digitonin is a strong hemolysin and glycyrrhizin has protective activity against the deterring effect of other hemolytic saponins. The interaction of these saponins with liposomes was studied as a function of cholesterol in membrane. In the case of multilamellar vesicles, which act as ideal osmometers, digitonin disrupted the barrier function of liposomes composed of phosphatidyl choline, dicetyl phosphate and cholesterol, however, did not influence on cholesterol-lacking liposomes. Glycyrrhizin had similar effect on liposomes irrespective of cholesterol in membrane. In the test with large unilamellar vesicles, digitonin increased the lysis with increasing cholesterol content in membrane, but glycyrrhizin showed no detectable change in cholesterol-containing liposomes. These results suggest that incorporation of cholesterol into liposomes increases the susceptibility to digitonin, resulting in lysis of liposomes, and that the inhibitory effect of glycyrrhizin against other hemolytic saponins is cholesterol-independent.