Ginsenoside Rh3 induces pyroptosis and ferroptosis through the Stat3/p53/NRF2 axis in colorectal cancer cells.

Ginsenoside Rh3 (GRh3) is a seminatural product obtained by chemical processing after isolation from Chinese herbal medicine that has strong antitumor activity against human tumors. However, its antitumor role remains to be elucidated. The aim of this study is to explore the mechanisms underlying the tumor suppressive activity of GRh3 from the perspective of pyroptosis and ferroptosis. GRh3 eliminates colorectal cancer (CRC) cells by activating gasdermin D (GSDMD)-dependent pyroptosis and suppressing solute carrier family 7 member 11 (SLC7A11), resulting in ferroptosis activation through the Stat3/p53/NRF2 axis. GRh3 suppresses nuclear factor erythroid 2-related factor 2 (NRF2) entry into the nucleus, leading to the decrease of heme oxygenase 1 (HO-1) expression, which in turn promotes NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and caspase-1 expression. Finally, caspase-1 activates GSDMD-dependent pyroptosis. Furthermore, GRh3 prevents NRF2 from entering the nucleus, which suppresses SLC7A11, causing the depletion of glutathione (GSH) and accumulation of iron, lipid reactive oxygen species (ROS) and malondialdehyde (MDA), and eventually leading to ferroptosis in CRC cells. In addition, GRh3 effectively inhibits the proliferation of CRC cells in vitro and in nude mouse models. Collectively, GRh3 triggers pyroptotic cell death and ferroptotic cell death in CRC cells via the Stat3/p53/NRF2 axis with minimal harm to normal cells, showing great anticancer potential.

Targeting Akt/CREB/BDNF signaling pathway by ginsenosides in neurodegenerative diseases: A mechanistic approach.

Neurodegenerative diseases (NDDs) are leading causes of death and morbidity in the elderly worldwide. From the mechanistic/pathological view, oxidative stress, inflammation, and apoptosis are responsible for the etiology of neuronal diseases, and play detrimental roles in neuronal cell death and neurodegenerative processes. The diverse pathophysiological pathways influencing NDDs necessitate the discovery of pivotal dysregulated signaling mediators. The current review describes essential functions of protein kinase B (Akt)/cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)/brain-derived neurotrophic factor (BDNF) pathway as possible therapeutic targets in the pathogenesis of NDDs. Consequently, finding new multi-target agents in regulating Akt/CREB/BDNF and thus associated downstream pathways is a critical factor in combating NDDs. Because of their neuroprotective properties, dietary phytochemicals have shown to be popular nutritional therapy methods. Ginsenosides, the most active ingredient of ginseng, and a secondary metabolite of steroid glycosides and triterpene saponins have been found to have a number of protective effects on the central nervous system (CNS). The protective roles of ginsenosides in CNS are potentially passing through Akt/CREB/BDNF pathway towards neuroprotective responses. In the present study, Akt/CREB/BDNF pathway is targeted by ginsenosides and associated nanoformulations towards potential neuroprotective effects.

Determination of ginsenoside Rh3 in rat plasma by LC-MS/MS and its application to a pharmacokinetic study.

Ginsenoside Rh3 (GRh3) is a bacterial metabolite of ginsenoside Rg5, which is the main component of hot-processed ginseng. A simple, efficient and sensitive method was developed and validated for the determination of GRh3 in rat plasma by LC-tandem mass spectrometry. After protein precipitation with methanol/acetonitrile (1:1, vol/vol) using propranolol as the internal standard, the target analytes were separated on an XDB C18 column, with methanol containing 0.1% formic acid and water containing 0.1% formic acid used as mobile phases for gradient elution. Mass spectrometry was performed in electrospray ion source-positive ion mode and multiple reaction monitoring mode, monitoring the transitions m/z 622.5 → 425.5 and m/z 260.1 → 116.1 for GRh3 and internal standard, respectively. The concentration range of GRh3 was 20-20,000 ng/mL and the correlation coefficient (r2 ) was greater than 0.99. The accuracy error and relative standard deviation were below 15%. The extraction recovery and matrix effect were 74.2% to 78.7% and 96.9% to 108.4%, respectively. Under different conditions, GRh3 was stable in the range of 1.8%-8.7%. This method has been successfully applied to study the pharmacokinetics of GRh3 with an oral dose of 10.0 mg/kg and an intravenous dose of 2.0 mg/kg in rats, respectively. The absolute bioavailability of GRh3 was 37.6%.

Transcriptome Analysis of the Anti-Proliferative Effects of Ginsenoside Rh3 on HCT116 Colorectal Cancer Cells.

The mechanism of ginsenoside Rh3 activity against cancer remains unclear. This study aimed to investigate the underlying mechanism. The effects of Rh3 on the cell proliferation, migration and invasion, and cycle and apoptosis were analyzed using CCK-8 assay, transwell migration assay and flow cytometry, respectively. The RNA transcriptome was sequenced and data were analyzed by R software. Protein expression and protein-protein interactions were determined by Western blotting and co-immunoprecipitation, respectively. The results showed Rh3 inhibited HCT116 cell proliferation, invasion, and migration, arrested cells at G1 phase; and increased apoptosis. Rh3 downregulated 314 genes and upregulated 371 genes. Gene Set Enrichment Analysis (GSEA) using The Kyoto Encyclopedia of Genes Genomics ranked DNA replication first, while GSEA using Gene Ontology ranked the initiation of DNA replication first. Compared with tumor data from The Cancer Genome Atlas (TCGA), most of genes related to DNA replication were oppositely regulated by Rh3. Furthermore, Rh3 down-regulated key protein expression related to DNA replication (Orc6, Cdt1, and Mcm2), but did not affect the loading of Mcm complexes onto ORC complexes nor the phosphorylation at ser139 of Mcm2. Therefore, Rh3 may inhibit colorectal cancer HCT116 cells by downregulation of genes related to DNA replication.

Ginsenoside Rh3 Inhibits Proliferation and Induces Apoptosis of Colorectal Cancer Cells.

BACKGROUND: Colorectal cancer is a common malignant tumor of the digestive tract, the morbidity rate of which is rising in recent years. Ginsenoside Rh3 was reported to have anticancer activity; however, the underlying mechanism still needs to be explored in depth. METHODS: Rabbit blood was used to test hemolytic effects of ginsenoside protopanaxadiol (PPD), Rh2, Rh3, and Rg3. Human colorectal cancer SW1116 cells were treated with different concentration of ginsenoside PPD, Rh2, Rh3, and Rg3 in vitro. MTT and TUNEL assay were used to examine cell proliferation and apoptosis. Semi quantitative RT-PCR, immunocytochemistry assay and flow cytometry assay were used to detect the expression of caspase3. RESULTS: The results showed that the inhibiting effects on SW1116 cells of PPD and Rh2 were stronger than those of Rh3 (p < 0.01), but Rh3 had better solubility and slighter hemolytic effects on blood cells than those ginsenosides. Ginsenoside Rh3 inhibited the proliferation of SW1116 cells at 60 µg/mL (p < 0.01), the inhibition effect was increased sharply when the dose of Rh3 was increased from 60 to 120 µg/mL, the inhibition rate was 62.1% at 120 µg/mL, the inhibition appeared at 9 h, and the peak activity occurred at 12 h and maintained until 48 h (p < 0.01). Compared to the control group, the ratio of apoptotic cells, the expression level of mRNA and protein of caspase3 increased in 120 µg/mL Rh3 treated group. CONCLUSION: As a potential anticancer medicine, ginsenoside Rh3 could inhibit the proliferation of colorectal cancer cells in a dose- and time-dependent manner and induce cell apoptosis through upregulating the expression of caspase3.

Bioinformatics study of the potential therapeutic effects of ginsenoside Rh3 in reversing insulin resistance.

Background: In recent years, the incidence of insulin resistance is increasing, and it can cause a variety of Metabolic syndrome. Ginsenosides have been clinically proven to improve fat metabolism and reduce insulin resistance, but their components and mechanism of action are still unclear. Objective: Ginsenoside, a bioactive compound derived from ginseng, exhibits significant potential in treating obesity, diabetes, and metabolic disorders. Despite evidence supporting its efficacy in ameliorating insulin resistance (IR) in obesity, the specific bioactive components and underlying mechanisms remain obscure. In this study, we endeavored to elucidate the potential molecular targets and pathways influenced by ginsenoside Rh3 (GRh3) to ameliorate IR in liver tissue. We employed a comprehensive approach that integrates system pharmacology and bioinformatics analysis. Materials and methods: Our methodology involved the identification of candidate targets for GRh3 and the profiling of differentially expressed genes (DEGs) related to IR in individuals with insulin resistance. The coalescence of candidate targets and DEGs facilitated the construction of a "GRh3-targets-disease" network for each tissue type, ultimately yielding 38 shared target genes. Subsequently, we conducted pathway enrichment analysis, established protein-protein interaction (PPI) networks, and identified hub targets among the GRh3 targets and IR-related DEGs. Additionally, we conducted animal experiments to corroborate the role of these hub targets in the context of GRh3. Results: Our investigation identified a total of 38 overlapping targets as potential candidates. Notably, our analysis revealed crucial hub targets such as EGFR, SRC, ESR1, MAPK1, and CASP3, alongside implicated signaling pathways, including those related to insulin resistance, the FoxO signaling pathway, the PPAR signaling pathway, and the IL-17 signaling pathway. This study establishes a robust foundation for the mechanisms underlying GRh3's efficacy in mitigating IR. Furthermore, these results suggest that GRh3 may serve as a representative compound within the ginsenoside family. Conclusion: This study elucidates the potential molecular targets and associated pathways through which GRh3 ameliorates IR, showcasing its multifaceted nature, spanning multiple targets, pathways, and mechanisms. These findings establish a robust foundation for subsequent experimental inquiries and clinical applications.

Ginsenoside Rh3 Inhibits Lung Cancer Metastasis by Targeting Extracellular Signal-Regulated Kinase: A Network Pharmacology Study.

Lung cancer has a high mortality rate and is very common. One of the main reasons for the poor prognosis of patients with lung cancer is the high incidence of metastasis. Ginsenoside Rh3, a rare ginsenoside extracted from Panax notoginseng, exhibits excellent anti-inflammatory and anti-tumor effects. Nonetheless, the inhibitory potential of Rh3 against lung cancer remains unknown. The target genes of Rh3 were screened by the PharmMapper database; the proliferation of lung cancer cells was detected by MTT assay; the migration and invasion of cells were detected by the Transwell method; and the expression of extracellular signal-regulated kinase (ERK) and EMT-related proteins in vivo and in vitro were detected by Western blotting. In addition, we established a lung metastasis model in nude mice using A549 cells to assess the effect of Rh3 on NSCLC tumor metastasis in vivo. Our findings suggest that Rh3 significantly inhibited lung cancer metastasis both in vivo and in vitro. It was determined by flow cytometry analysis that Rh3 notably inhibited cell proliferation by blocking the G1 phase. In addition, Rh3 inhibited metastasis in lung cancer cells and regulated the expression of metastasis-related proteins under hypoxia. Mechanistic studies suggested that Rh3 targeted ERK to inhibit lung cancer metastasis. The ERK inhibitor U0126 or siRNA-mediated knockdown of ERK had an enhanced effect on Rh3's ability to inhibit lung cancer metastasis. The studies revealed that the inhibitory effect of Rh3 on the metastatic ability of lung cancer cells may be supported by ERK-related signaling pathways.

Ginsenoside Rh3 activates Nrf2 signaling and protects endometrial cells from oxygen and glucose deprivation-reoxygenation.

Oxygen and glucose deprivation (OGD)-reoxygenation (OGDR) induces oxidative injury to endometrial cells in vitro. We tested the potential effect of ginsenoside Rh3 (GRh3) in the process. Our results show that GRh3 activated Nrf2 signaling in T-HESC cells and primary murine endometrial cells. GRh3 induced Nrf2 Ser-40 phosphorylation and Keap1-Nrf2 disassociation, causing Nrf2 protein stabilization and nuclear translocation, which led to transcription and expression of antioxidant response element-dependent genes (HO1, NQO1 and GCLC). In T-HESC cells and primary murine endometrial cells, GRh3 potently attenuated OGDR-induced reactive oxygen species production, lipid peroxidation and mitochondrial depolarization, as well as cell viability reduction and necrosis. Activation of Nrf2 is required for GRh3-induced anti-OGDR actions in endometrial cells. Nrf2 inhibition, by Nrf2 shRNA, knockout (through CRISPR-Cas9-editing) or S40T mutation, abolished GRh3-induced endometrial cell protection against OGDR. Additionally, forced activation of Nrf2, by Keap1 knockout, mimicked and nullified GRh3-induced anti-OGDR actions in T-HESC cells. Together, we conclude that GRh3 protects endometrial cells from OGDR via activation of Nrf2 signaling.

Inhibitory mechanism of ginsenoside Rh3 on granulocyte-macrophage colony-stimulating factor expression in UV-B-irradiated murine SP-1 keratinocytes.

BACKGROUND: Ultraviolet (UV) goes through the epidermis and promotes release of inflammatory cytokines in keratinocytes. Granulocyte-macrophage colony-stimulating factor (GM-CSF), one of the keratinocyte-derived cytokines, regulates proliferation and differentiation of melanocytes. Extracellular signal-regulated kinase (ERK1/2) and protein kinase C (PKC) signaling pathways regulate expression of GM-CSF. Based on these results, we found that ginsenoside Rh3 prevented GM-CSF production and release in UV-B-exposed SP-1 keratinocytes and that this inhibitory effect resulted from the reduction of PKCδ and ERK phosphorylation. METHODS: We investigated the mechanism by which ginsenoside Rh3 from Panax ginseng inhibited GM-CSF release from UV-B-irradiated keratinocytes. RESULTS: Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or UV-B induced release of GM-CSF in the SP-1 keratinocytes. To elucidate whether the change in GM-CSF expression could be related to PKC signaling, the cells were pretreated with H7, an inhibitor of PKC, and irradiated with UV-B. GM-CSF was decreased by H7 in a dose-dependent manner. When we analyzed which ginsenosides repressed GM-CSF expression among 15 ginsenosides, ginsenoside Rh3 showed the largest decline to 40% of GM-CSF expression in enzyme-linked immunosorbent assay. Western blot analysis showed that TPA enhanced the phosphorylation of PKCδ and ERK in the keratinocytes. When we examined the effect of ginsenoside Rh3, we identified that ginsenoside Rh3 inhibited the TPA-induced phosphorylation levels of PKCδ and ERK. CONCLUSION: In summary, we found that ginsenoside Rh3 impeded UV-B-induced GM-CSF production through repression of PKCδ and ERK phosphorylation in SP-1 keratinocytes.

Protective action of the ginsenoside Rh3 in a rat myocardial ischemia-reperfusion injury model by inhibition of apoptosis induced via p38 mitogen-activated protein kinase/caspase-3 signaling.

OBJECTIVE: To investigate the protective effects of the ginsenoside Rh3 on rats subjected to myocardial ischemia-reperfusion (MIR) via its impact on caspase-3 and the p38 mitogen-activated protein kinase (MAPK) pathway. METHODS: Fifteen male Sprague-Dawley rats were randomly categorized into the MIR group (MY group, n = 5), sham surgery group (SS group, n = 5), and ginsenoside Rh3 group (GR group, n = 5). RESULTS: The MY group exhibited the largest myocardial infarctions compared with the GR and SS groups. The GR group exhibited significantly higher cell viability of cardiomyocytes and significantly decreased apoptosis compared with the MY group. Fibrils of infarcted tissue in the GR group were disordered but less swollen, with a more organized fibril orientation than those in the MY group. The GR group showed reduced p-p38 MAPK protein and caspase-3 mRNA expression levels compared with the MY and SS groups. CONCLUSIONS: Rh3 significantly improved myocardial necrosis and caspase-3 levels in myocardial tissues by suppressing the p38 MAPK pathway, thereby inhibiting caspase-3 involvement in apoptosis. Thus, Rh3 was effective in inhibiting the escalated apoptotic pathway in myocardial infarction and can potentially serve as a useful therapeutic agent to rescue myocardial infarction.

Activation of Nrf2 by Ginsenoside Rh3 protects retinal pigment epithelium cells and retinal ganglion cells from UV.

Excessive Ultra-violet (UV) radiation shall induce damages to resident retinal pigment epithelium (RPE) cells (RPEs) and retinal ganglion cells (RGCs). Here we tested the potential activity of Ginsenoside Rh3 ("Rh3") against the process. In cultured human RPEs and RGCs, pretreatment with Rh3 inhibited UV-induced reactive oxygen species (ROS) production and following apoptotic/non-apoptotic cell death. Rh3 treatment in retinal cells induced nuclear-factor-E2-related factor 2 (Nrf2) activation, which was evidenced by Nrf2 protein stabilization and its nuclear translocation, along with transcription of antioxidant responsive element (ARE)-dependent genes (HO1, NOQ1 and GCLC). Nrf2 knockdown by targeted-shRNA almost abolished Rh3-induced retinal cell protection against UV. Further studies found that Rh3 induced microRNA-141 ("miR-141") expression, causing downregulation of its targeted gene Keap1 in RPEs and RGCs. On the other hand, Rh3-induced Nrf2 activation and retinal cell protection were largely attenuated by the miR-141's inhibitor, antagomiR-141. In vivo, intravitreal injection of Rh3 inhibited retinal dysfunction by light damage in mice. Rh3 intravitreal injection also induced miR-141 expression, Keap1 downregulation and Nrf2 activation in mouse retinas. We conclude that Rh3 protects retinal cells from UV via activating Nrf2 signaling.

Protective effect of ginsenoside Rh3 against anticancer drug-induced apoptosis in LLC-PK1 kidney cells.

BACKGROUND: Ginsenosides are active components of Panax ginseng that exert various health benefits including kidney protection effect. The medicinal activity of ginsenosides can be enhanced by modulating their stereospecificity by heat processing. Ginsenosides Rk2 and Rh3 represent positional isomers of the double bond at C-20(21) or C-20(22). METHODS: The present study investigated the kidney-protective effects of ginsenosides Rk2 and Rh3 against cisplatin, a platinum based anticancer drug, induced apoptotic damage in renal proximal LLC-PK1 cells. RESULTS: As a result, ginsenoside Rh3 shows a stronger protective effect than that shown by Rk2. Cisplatin-induced elevated protein levels of phosphorylated c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38, and cleaved caspase-3 decreased after cotreatment with ginsenoside Rh3. The increase in the percentage of apoptotic LLC-PK1 cells induced by cisplatin treatment also significantly reduced after cotreatment with ginsenoside Rh3. CONCLUSION: These results demonstrate that inhibition of the JNK and ERK mitogen-activated protein kinase signaling cascade plays a critical role in mediating the renoprotective effect of ginsenoside Rh3.

Anti-inflammatory mechanism of ginseng saponin metabolite Rh3 in lipopolysaccharide-stimulated microglia: critical role of 5'-adenosine monophosphate-activated protein kinase signaling pathway.

Ginsenoside Rh3 is a bacterial metabolite of Rg5, which is the main constituent of heat-processed ginseng. The present study was undertaken to examine the anti-inflammatory effect of ginsenoside Rh3 in lipopolysaccharide (LPS)-stimulated microglia. Rh3 inhibits the expressions of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6, at mRNA and protein levels, while Rh3 enhanced anti-inflammatory hemeoxygenase-1 expression. Moreover, Rh3 inhibited nuclear factor-κB (NF-κB) by upregulation of sirtuin 1 (SIRT1) and enhanced Nrf2 DNA-binding activities. Analysis of signaling pathways revealed that Rh3 enhanced the phosphorylation of 5'-adenosine monophosphate-activated protein kinase (AMPK) and inhibited Akt and janus kinase 1 (JAK1)/signal transducer and activator of transcription 1 (STAT1) induced by LPS. By treatment of BV2 cells with AICAR (a pharmacological activator of AMPK), we found that AMPK is an upstream regulator of phosphatidylinositol 3-kinase (PI3K)/Akt and JAK1/STAT1. Furthermore, AMPK knockdown experiments demonstrated the anti-inflammatory role of AMPK in LPS/Rh3-treated BV2 microglia. Our data collectively suggest that Rh3 exerts an anti-inflammatory effect in microglia by modulating AMPK and its downstream signaling pathways.

Inhibitory mechanism of Korean Red Ginseng on GM-CSF expression in UVB-irradiated keratinocytes.

BACKGROUND: UV-irradiated keratinocytes secrete various proinflammatory cytokines. UV-induced skin damage is mediated by growth factors and proinflammatory cytokines such as granulocyte macrophage colony stimulating factor (GM-CSF). In a previous study, we found that the saponin of Korean Red Ginseng (SKRG) decreased the expression of GM-CSF in UVB-irradiated SP-1 keratinocytes. In this study, we attempted to find the inhibitory mechanism of SKRG on UVB-induced GM-CSF expression in SP-1 keratinocytes. METHODS: We investigated the inhibitory mechanism of SKRG and ginsenosides from Panax ginseng on UVB-induced GM-CSF expression in SP-1 keratinocytes. RESULTS: Treatment with SKRG decreased the expression of GM-CSF mRNA and protein induced by irradiation of UVB in SP-1 keratinocytes. The phosphorylation of ERK was induced by UVB at 10 min, and decreased with SKRG treatment in SP-1 keratinocytes. In addition, treatment with SKRG inhibited the UVB-induced phosphorylation of epidermal growth factor receptor (EGFR), which is known to be an upstream signal of ERK. From these results, we found that the inhibition of GM-CSF expression by SKRG was derived from the decreased phosphorylation of EGFR. To identify the specific compound composing SKRG, we tested fifteen kinds of ginsenosides. Among these compounds, ginsenoside-Rh3 decreased the expression of GM-CSF protein and mRNA in SP-1 keratinocytes. CONCLUSION: Taken together, we found that treatment with SKRG decreased the phosphorylation of EGFR and ERK in UVB-irradiated SP-1 keratinocytes and subsequently inhibited the expression of GM-CSF. Furthermore, we identified ginsenoside-Rh3 as the active saponin in Korean Red Ginseng.