Unraveling the Mechanism of Curculiginis Rhizoma in Suppressing Cisplatin Resistance in Non-Small Cell Lung Cancer: An Experimental Study.

Introduction: Non-small cell lung cancer (NSCLC) stands as one of the most prevalent malignancies, and chemotherapy remains the primary treatment for advanced stages. However, the high expression of ABC binding cassette transporters, including MRP, P-gp, and LRP, along with multidrug resistance proteins, has been identified as a significant factor contributing to decreased chemotherapy drug sensitivity. This study aims to explore the impact and underlying mechanisms of Curculiginis Rhizoma [Hypoxidaceae; Curculigo orchioides Gaertn.] (CR) in combination with cisplatin on improving chemoresistance mediated by ABC binding cassette transporters and multidrug resistance proteins in NSCLC. Methods and Results: To unravel the relationship between JNK, MRP, P-gp, and LRP in NSCLC and gain insights into the regulatory mechanism of CR, this study employs an integrated approach encompassing bioinformatics, molecular docking, molecular dynamics, animal and cellular experiments. Bioinformatics analysis revealed a significant increase in the expression levels of JNK, MRP, P-gp, and LRP subtypes in multidrug-resistant non-small cell lung cancer. Subsequent animal experiments have shown that the combination of CR with cisplatin can improve the survival rate of lung cancer mice. Molecular docking and molecular dynamics analyses demonstrated favorable binding interactions between curculigoside and the aforementioned subtypes of JNK, MRP, P-gp, and LRP. In cellular experiments, the combination of cisplatin with both curculigoside and CR extract resulted in a notable decrease in cell viability and downregulation of the expression of JNK1, JNK2, MRP1, MRP2, MRP4, P-gp, and LRP1 in A549/cis cells. Conclusion: Remarkably, curculigoside exerted a significant downregulation effect on the expression levels of JNK1, MRP1, MRP2, MRP4, and LRP1. CR, particularly its main effective metabolite, curculigoside, has the potential to enhance the sensitivity of non-small cell lung cancer to cisplatin by regulating levels of JNK/MRP/LRP/P-gp and mitigating multidrug resistance.

Curculigoside Attenuates Endoplasmic Reticulum Stress-Induced Epithelial Cell and Fibroblast Senescence by Regulating the SIRT1-P300 Signaling Pathway.

The senescence of alveolar epithelial cells (AECs) and fibroblasts plays a pivotal role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a condition lacking specific therapeutic interventions. Curculigoside (CCG), a prominent bioactive constituent of Curculigo, exhibits anti-osteoporotic and antioxidant activities. Our investigation aimed to elucidate the anti-senescence and anti-fibrotic effects of CCG in experimental pulmonary fibrosis and delineate its underlying molecular mechanisms. Our findings demonstrate that CCG attenuates bleomycin-induced pulmonary fibrosis and lung senescence in murine models, concomitantly ameliorating lung function impairment. Immunofluorescence staining for senescence marker p21, alongside SPC or α-SMA, suggested that CCG's mitigation of lung senescence correlates closely with the deceleration of senescence in AECs and fibroblasts. In vitro, CCG mitigated H2O2-induced senescence in AECs and the natural senescence of primary mouse fibroblasts. Mechanistically, CCG can upregulate SIRT1 expression, downregulating P300 expression, enhancing Trim72 expression to facilitate P300 ubiquitination and degradation, reducing the acetylation levels of antioxidant enzymes, and upregulating their expression levels. These actions collectively inhibited endoplasmic reticulum stress (ERS) and alleviated senescence. Furthermore, the anti-senescence effects and mechanisms of CCG were validated in a D-galactose (D-gal)-induced progeroid model. This study provides novel insights into the mechanisms underlying the action of CCG in cellular senescence and chronic diseases, offering potential avenues for the development of innovative drugs or therapeutic strategies.

Curculigoside, a traditional Chinese medicine monomer, ameliorates oxidative stress in Alzheimer's disease mouse model via suppressing ferroptosis.

Alzheimer's disease (AD) is a neurodegenerative disorder where oxidative stress, induced by ferroptosis, has been linked to neuronal damage and cognitive deficits. The objective of this study is to investigate if the potential therapeutic agent, Curculigoside (CUR), could ameliorate AD by inhibiting ferroptosis. The potential therapeutic targets, such as GPX4 and SLC7A11, were identified using weighted gene co-expression network analysis (WGCNA). Concurrently, CUR was also screened against these potential targets using various analytical methods. For the in vivo studies, intragastric administration of CUR significantly ameliorated cognitive impairment in AD model mice induced by scopolamine and okadaic acid (OA). In vitro, CUR protected neuronal cells by altering the levels of ferroptosis-related specific markers in OA and scopolamine-induced neurotoxicity. The administration of CUR through intragastric route significantly reduced the levels of AD-promoting factors (such as Aß1-42, p-tau) and ferroptosis-promoting factors in the hippocampus and cortex of AD mice. Furthermore, CUR up-regulated the expression of GPX4 and decreased the expression of SLC7A11 in the ferroptosis signaling pathway, thereby increasing the ratio of glutathione (GSH)/oxidized glutathione (GSSG) in vivo and vitro. In conclusion, the cumulative results suggest that the natural compound CUR may serve as a promising therapeutic agent to ameliorate AD by inhibiting ferroptosis.

Curculigoside attenuates osteoporosis through regulating DNMT1 mediated osteoblast activity.

This work aims to study the function of curculigoside in osteoporosis and explore whether DNMT1 is closely involved in osteoblast activity. After OB-6 osteoblasts were treated with hydrogen peroxide (H2O2), a curculigoside treatment group was set up and a series of biological tests including MTT, flow cytometry, western blotting, ROS fluorescence intensity, mitochondrial membrane potential, and ELISA experiments were performed to verify the effect of curculigoside on the activity of osteoblasts. Then, alkaline phosphatase (ALP) activity, alizarin red staining, PCR, and western blotting assays were performed to detect the effects of curculigoside on osteoblast function. By constructing DNMT1 knockdown and overexpression OB-6 cell lines, the effect of DNMT1 on osteoblast function was verified. In addition, the expression level of Nrf2 in each group was detected to speculate the mechanism of DNMT1 in osteoporosis. The cell activity and level of bcl-2 and SOD were significantly increased; the cell apoptosis, ROS fluorescence intensity, mitochondrial membrane potential, MDA and level of caspase-3, Bax, and CAT was reduced in curculigoside treatment group compared with H2O2-induced OB-6 osteoblasts. Meanwhile, the ALP activity, number and area of bone mineralized nodules, and gene and protein expression of OSX and OPG were significantly elevated in curculigoside group. Moreover, DNMT1 knockdown had a similar promotion effect on osteoblast function as curculigoside, and DNMT1 overexpression could reverse the promotion effect of curculigoside on osteoblast function. Further mechanistic studies speculated that DNMT1 might play a role in osteoporosis by affecting Nrf2 methylation. Curculigoside enhances osteoblast activity through DNMT1 controls of Nrf2 methylation.

Curculigoside mitigates dextran sulfate sodium­induced colitis by activation of KEAP1­NRF2 interaction to inhibit oxidative damage and autophagy of intestinal epithelium barrier.

Curculigoside (CUR), a primary active ingredient of Curculigo orchioides Gaertn, serves an important role in the intervention of numerous diseases, including ulcerative colitis, rheumatoid arthritis, myocardial ischemia, etc. However its specific mechanisms of therapy have not been fully elucidated. The aim of the present study was to elucidate the mechanisms underlying the anti­oxidative stress and anti­ulcerative colitis (UC) effects of CUR. Mouse model of dextran sulfate sodium (DSS)­induced colitis, along with Caco2 and mouse intestine organoid in vitro models were used. The effect of CUR on mitigating the symptoms of chronic colitis was investigated. Through ELISA experiments, it was observed that CUR alleviated the inflammation status in mice with chronic colitis. This was evidenced by the downregulation of inflammatory cytokines such as TNF­α and IL­6 and ­1ß and decreased neutrophil infiltration along with downregulated myeloperoxidase activity. CUR helped in maintaining the barrier functions of intestinal epithelium. In vitro TNF­α stimulation of organoids and H2O2 stimulation of Caco2 cells demonstrated the capabilities of CUR to rescue cells from oxidative stress. There was activation of Nrf2 both in vivo and in vitro, accompanied by enhanced autophagy. Mechanistic studies of cells and Nrf2 knockout mice demonstrated that Nrf2 served a pivotal role in inhibition of UC by curculigoside via interaction with Kelch­like ECH­associated protein 1 (Keap1). In vitro and in vivo experiments confirmed that CUR activated Nrf2 via Keap1/Nrf2 interaction, resulting in decreased oxidative stress and promoted autophagy. These findings demonstrated that CUR could effectively mitigate colitis and may have clinical application in UC therapy.

Curculigoside rescues hippocampal synaptic deficits elicited by PTSD through activating cAMP-PKA signaling.

Chronic traumatic stress results in various psychiatric disorders, especially posttraumatic stress disorder (PTSD). Previous study demonstrated that curculigoside (CUR) a component of Rhizoma Curculiginis prevented fear extinction and stress-induced depression-like behaviors. However, its effects on PTSD and the mechanisms are still not completely clear. In this study, we observed typical PTSD-like phenotypes, synaptic deficit, and reduction of BDNF/TrkB signaling pathway in mice receiving modified single prolonged stress and electrical stimulation (SPS&S). By contrast, systemic administration of CUR blocked PTSD-like phenotypes and synaptic deficits, including reduction of BDNF/TrkB signaling pathway, GluA1 and Arc expression. Importantly, CUR reversed the impairment of PKA signaling pathway elicited by PTSD. We further confirmed that the effects of CUR on synaptic function were through PKA signaling pathway, as H-89, an inhibitor of PKA blocked the effect of CUR on behavioral changes and BDNF/TrkB signaling pathway. Thereafter, we verified that CUR on synaptic function were through PKA pathway using direct intracerebral injection of CUR and H-89. Direct intracerebral injection of CUR activated PKA/CREB/BDNF/TrkB, which was blocked by H-89. Additionally, the docking results showed high binding energies of CUR with A2AR, AC, PRKACA, and PRKAR1A, which might indicate that CUR functions through regulating PKA signaling pathway. In conclusion, CUR prevented the behavioral changes and hippocampal synaptic deficits elicited by PTSD through activating cAMP-PKA signaling.

Curculigoside Represses the Proliferation and Metastasis of Osteosarcoma via the JAK/STAT and NF-κB Signaling Pathways.

Curculigoside (Cur) is a natural component from Curculigo orchioides Gaertn, with various bioactivities. The function of Cur in the nervous system and osteoarthritis has been reported. However, its role in osteosarcoma (OS) needs to be investigated. Hence, we focus on probing the impact of Cur on OS. In vitro, cell counting kit 8 (CCK-8), flow cytometry and Transwell assay were used to investigate the effects of Cur on OS cell proliferation, apoptosis, migration and invasion. In vivo, we developed a xenograft model to figure out the effect of Cur on tumor growth in nude mice. Western blotting (WB) was conducted to compare the levels of Cur on apoptosis-related proteins (C-caspase-3, Bax, and Bcl-2), epithelial-mesenchymal transition (EMT)-related proteins (N-cadherin, Snail, and E-cadherin) and the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and nuclear factor-κB (NF-κB) pathways in vitro and in vivo. In-vitro data testified that Cur treatment markedly hampered OS cells' growth, migration and invasion and intensified their apoptosis compared to that of the control group. In vivo, Cur treatment notably hampered the growth of OS tumors in mice. In addition, both in vitro and in vivo experiments demonstrated that the phosphorylation of JAK2, STAT3, and NF-κB were inhibited through Cur treatment. Furthermore, the inhibition of Cur in OS cells was demonstrated by up-regulating the expression of JAK/STAT and NF-κB pathways protein levels. In summary, the data suggest that Cur curbs OS growth by down-regulating the JAK/STAT and NF-κB pathways, which is an underlying therapeutic option for OS treatment.

Curculigoside mitigates hepatic ischemia/reperfusion-induced oxidative stress, inflammation, and apoptosis via activation of the Nrf-2/HO-1 pathway.

Curculigoside has been shown to decrease oxidative stress and inflammatory reactions in many disorders, but its effects during hepatic ischemia-reperfusion injury (IRI) remain unknown. This research aims to determine the protective role and the potential mechanism of action of curculigoside in hepatic IRI. Here, a well-established rat model of partial warm IRI was constructed; serum ALT/AST and H&E staining were employed to assay the extent of liver injury; the superoxide dismutase, malondialdehyde, IL-6, and TNF-α contents were determined using the corresponding kits; the apoptosis index was evaluated by TUNEL staining; and the expression of Nrf-2, HO-1, and apoptosis-associated proteins was detected by qRT-PCR and Western blotting. The results showed that curculigoside pretreatment effectively mitigated hepatic IRI, as demonstrated by decreases in the levels of serum aminotransferases, hepatocellular necrosis and apoptosis, oxidative stress markers, infiltration of inflammatory cells, and secretion of proinflammatory cytokines. Mechanistically, the expression of Nrf-2 and HO-1 was greatly suppressed by hepatic IRI and reactivated by curculigoside. Furthermore, cotreatment with ML-385, an inhibitor of Nrf-2, counteracted the protective effect of curculigoside against hepatic IRI. The results of our study show that curculigoside plays a protective role in hepatic IRI by inhibiting oxidative stress, inflammation, and apoptosis and that its effects may be associated with activation of the Nrf-2/HO-1 pathway.

Curculigoside Ameliorates Bone Loss by Influencing Mesenchymal Stem Cell Fate in Aging Mice.

Senile osteoporosis is characterized by increased bone loss and fat accumulation in marrow. Curculigoside (CCG) is the major bioactive component of Curculigo orchioides, which has been used as anti-osteoporosis therapy for elder patients since antiquity. We aimed to investigate the underlying mechanisms by which CCG regulated the bone-fat balance in marrow of aging mice. In our study, CCG treatment was identified to interfere with the stem cell lineage commitment both in vivo and in vitro. In vivo, CCG promoted the transcriptional co-activator with PDZ-binding motif (TAZ) expression to reverse age-related bone loss and marrow adiposity. In vitro, proper concentration of CCG upregulated TAZ expression to increase osteogenesis and decrease adipogenesis of bone marrow mesenchymal stem cells (BMSCs). This regulating effect was discounted by TAZ knockdown or the use of MEK-ERK pathway inhibitor, UO126. Above all, our study confirmed the rescuing effects of CCG on the differential shift from adipogenesis to osteogenesis of BMSCs in aging mice and provided a scientific basis for the clinical use of CCG in senile osteoporosis.

Curculigoside attenuates oxidative stress and osteoclastogenesis via modulating Nrf2/NF-κB signaling pathway in RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Curculigo orchioides Gaertn is used for the treatment of impotence, atrophic debility of bones (osteoporosis), limb limpness, and arthritis of the lumbar and knee joints in traditional Chinese medicine and Ayurvedic medical system. Curculigoside (Cur) from Curculigo orchioides Gaertn has been shown to have regulatory effects on bone metabolism via anti-oxidative activities in rats and osteoblasts. However, little is known about the molecular pharmacological activity of Cur in osteoclastic bone resorption. AIM: The aim of this work is to investigate the inhibitory effect of Cur against osteoclasts (OCs) under the oxidative stress status, and explore the possible underlying mechanism. MATERIALS AND METHODS: OCs were induced from RAW264.7 cells using RANKL and H2O2. The number of OCs was measured by tartrate-resistant acid phosphatase (TRAP) staining. F-Actin and nuclear translocation of P65 and Nrf2 were stained with immunofluorescence assay and observed under a laser confocal microscope. The biochemical parameters of OCs were detected with an ELISA kit. The expression of Nrf2 and NF-κB pathway-related proteins was analyzed by Western Blot. RESULTS: Cur inhibited the TRAP activity, release of degrading products from bone slices and the expression of NFATc1, c-Fos, Cathepsin K (Ctsk) and matrix metallopeptidase 9 (MMP9) of OCs induced with RANKL and H2O2. In addition, Cur suppressed the ROS level and NADPH oxidase 1(NOX1) and NADPH oxidase 4 (NOX4) activities of OCS. More importantly, Cur enhanced the expression and nucleus translocation of Nrf2 and activities of its regulatory cytoprotective enzymes, and reduced the NF-κB expression and phosphorylation and nucleus translocation of p65 in OCs. Furthermore, the Nrf2 inhibitor ML385 and NF-κB inhibitor Bay11-7082 counteracted the effect of Cur in OCs. CONCLUSION: Cur mitigated oxidative stress and osteoclastogenesis by activating Nrf2 and inhibiting the NF-κB pathway, suggesting that Cur may prove to be a promising candidate for the treatment of osteoporosis. Our findings may also help partially explain the rationale behind the traditional use of Curculigo orchioides Gaertn.

Curculigoside promotes osteogenic differentiation of ADSCs to prevent ovariectomized-induced osteoporosis.

BACKGROUND: Curculigoside is a natural phenolic glycoside compound produced by Curculigo orchioides Gaertn. This study aimed to explore the effects of curculigoside in promoting the osteogenic differentiation of adipose-derived stem cells (ADSCs) as well as the underlying mechanism. METHODS: ADSCs were treated with curculigoside at different concentrations (0 µmol/L, 1 µmol/L, 2.5 µmol/L, 5 µmol/L, 10 µmol/L, and 20 µmol/L), and cell viability was assessed by CCK-8 assay. Then, the alkaline phosphatase (ALP) activity was determined, and alizarin red S (ARS) staining was performed to measure the extracellular mineralization of curculigoside. Information about protein-chemical interactions is provided by the search tool for interactions of chemicals (STITCH) database. Then, LY294002 was administered to explore the mechanism by which curculigoside promotes the osteogenic differentiation of ADSCs. Western blot assays were performed to assess changes in the expression of osteogenic-related markers and the phosphorylation of PI3K and AKT. Finally, we established an ovariectomized (OVX)-induced osteoporosis mouse model and administered curculigoside to explore the effects of curculigoside in preventing bone loss in vivo. RESULTS: The CCK-8 assay indicated that curculigoside did not induce cytotoxicity at a concentration of 5 µmol/L after 48 h. The ALP and ARS results revealed that the induced group had higher ALP activity and calcium deposition than the control group. Moreover, the curculigoside group exhibited increased biomineralization, ALP activity, and ARS staining compared to the induced and control groups, and these effects were partially inhibited by LY294002. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the target genes of curculigoside were mainly involved in the PI3K-Akt signaling pathway. PCR and western blot analysis showed that the expression of RUNX2, ALP, and Osterix was upregulated in curculigoside-treated ADSCs, but this effect was partially reversed by the PI3K inhibitor LY294002. Moreover, the curculigoside-treated group exhibited significantly increased phosphorylation of AKT to P-AKT compared with the osteogenic induction group. After treatment with curculigoside, the mice had a higher bone volume than the OVX mice, suggesting partial protection from cancellous bone loss. In addition, when LY294002 was added, the protective effects of curculigoside could be neutralized. CONCLUSIONS: Curculigoside could induce the osteogenic differentiation of ADSCs and prevent bone loss in an OVX model through the PI3K/Akt signaling pathway.

Prediction of Targets of Curculigoside A in Osteoporosis and Rheumatoid Arthritis Using Network Pharmacology and Experimental Verification.

PURPOSE: Network pharmacology is considered to be the next-generation drug development model that uses bioinformatics to predict and identify multiple drug targets and interactions in diseases. Here, network pharmacology was used to investigate the mechanism by which Curculigoside A (CA) acts in rheumatoid arthritis (RA) and osteoporosis. METHODS: First, TCMSP and SwissADME were applied to predict the druggability of CA. Then, potential targets were identified from overlapping data in SwissTarget and TargetNet, and targets were analyzed using Genemania and DAVID6.8 to obtain information about the GO and KEGG pathways. Ultimately, the drug-target-pathway network was identified after using Cytoscape 3.0 for visualization. Besides, qPCR was used to validate the predicted five major genes targets (EGFR, MAP2K1, MMP2, FGFR1, and MCL1). RESULTS: The results of TCMSP and SwissADME demonstrated that CA exhibits good druggability; 26 potential protein targets were classified by SwissTarget and TargetNet. The results of Genemania and DAVID6.8 indicated that CA probably caused anti-osteoporosis and anti-RA effects by regulating some biological pathways, especially nitrogen metabolism, estrogen signaling pathway, Rap1 signaling pathway, and PI3K/Akt signaling pathway. Besides, the result of Cytoscape 3.0 showed that the 26 targets participate in osteoporosis and RA-related pathways, metabolism, and other physiological processes. In vitro induced inflammation cell model experiments, the qPCR results showed that CA pretreatment significantly decreased the expression of EGFR, MAP2K1, MMP2, FGFR1, and MCL1 genes. CONCLUSION: These results suggested that network pharmacology may provide possible mechanism of how CA exerts therapeutic effects in osteoporosis and RA.

Curculigoside inhibits ferroptosis in ulcerative colitis through the induction of GPX4.

Curculigoside (CUR) is natural ingredient from Curculigo orchioides Gaertn with multiple biological activities. However, whether CUR protects from ulcerative colitis (UC) and underlying mechanisms are unclear. Herein, mice challenged with dextran sulfate sodium (DSS) were established and administrated with CUR for 7 days. Then histological pathologies and ferroptosis regulators were determined in vivo. The ferroptotic IEC-6 cells were prepared to investigate the underlying mechanism of CUR. Results showed that CUR inhibited the disease activity index, histological damage and cell death in mice with colitis. We also found that ferroptosis was induced in mice with colitis, as evidenced by iron overload, GSH depletion, ROS and MDA production, accompanied by decreased expression of SOD and GPX4. CUR treatment significantly reversed these alterations of ferroptotic features in DSS-induced mice. Furthermore, similar effects of CUR on ferroptosis were observed in IEC-6 cells under the combined treatment of H2O2 and iron chloride hexahydrate. Interestingly, we found that CUR could increase the selenium sensitivity and promote GPX4 transcription level in IEC-6 cells. Knockdown of GPX4 significantly blocked the protective effects of CUR on cell death, GSH and MDA contents as well as LDH activity in ferroptotic IEC-6 cells. Taken together, these findings suggest that CUR protects against ferroptosis in UC by the induction of GPX4, which presents a potential agent for UC treatment.

[Study on the effect of curculigoside on osteoporosis].

Objective: To investigate the regulation of curculigoside on osteogenic differentiation of MG63 and the protective effect on osteoporosis model mice. Methods: The effects of curculigoside on the survival rate of dexamethasone or H(2)O(2) treated MG63 were detected by methyl thiazolyl tetrazolium (MTT). The specimens were divided into six groups: blank control group, blank administration group, model group (dexamethasone or H(2)O(2) treatment group), low dose group (dexamethasone or H(2)O(2)+1.0 µmol/L curculigoside), medium dose group (dexamethasone or H(2)O(2)+2.5 µmol/L curculigoside) and high dose group (dexamethasone or H(2)O(2)+5.0 µmol/L curculigoside), the sample size of each group was 10. Western blotting was used to detect the expression of osteogenic differentiation-related proteins [type Ⅰ collagen, integrin ß1, osteoblast-specific transcription factor (Osterix), osteocalcin and osteopontin] in MG63 cells after 1, 7 and 14 days incubated with 0, 1.0, 2.5 and 5.0 µmol/L of curculigoside. The sample size for each group at each time point was six. The experimental mice were divided into 4 groups: blank group, model group (dexamethasone treatment group), curculigoside low-dose group (dexamethasone+5 mg/kg curculigoside) and high-dose group (dexamethasone+45 mg/kg curculigoside), twenty each. After treatment, the tibia of the mice in each group were subjected to sacral HE staining. The number of osteoclasts was counted, and the levels of oxidative related factors in serum were determined by enzyme-linked immunosorbent assay (ELISA). Results: The MTT results showed that compared with the blank control group [(100±3.7)%], the cell survival rate decreased to (44.1±5.7)% after treatment with dexamethasone, and the survival rate increased to (79.7±3.8)% after treatment with 5.0 µmol/L of curculigoside. The cell survival rate decreased to (59.1±4.7)% after H(2)O(2) treatment, and the survival rate increased to (80.8±3.5)% after treatment with 2.5 µmol/L of curculigoside. The results of Western blotting showed that the expression of type Ⅰ collagen and integrin ß1 in MG63 cells was significantly increased after 1.0, 2.5 and 5.0 µmol/L of curculigoside for 1, 7 and 14 days compared with 0 µmol/L of curculigo side for the same period. After increasing (P<0.05), the expression of Osterix and osteocalcin was significantly increased after 1 day of incubation (P<0.05). However, compared with 0 µmol/L curculigoside treatment, the expression of osteopontin in MG63 cells was not significantly different after incubation with 1.0, 2.5, 5.0 µmol/L of curculigoside for 7 and 14 days (P>0.05). Compared with the blank group, the number of tibia osteoclasts in the osteoporosis model group increased. In the low-dose and high-dose groups of curculigoside, the tibia cortex was more continuous and the number of osteoclasts decreased. Compared with the blank group, the activity of oxygen in the osteoporosis model group was significantly increased (P<0.05), and superoxide dimutase and catalase were significantly decreased (P<0.05). Conclusions: Curculigoside promotes the differentiation of MG63 cells by increasing the expression of osteoblast differentiation-related proteins, and has a certain therapeutic effect on dexamethasone-induced osteoporosis mice.

Integrated pathological cell fishing and network pharmacology approach to investigate main active components of Er-Xian decotion for treating osteoporosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Oxidative damage to osteoblasts was a key factor in the development of osteoporosis. Er-Xian Decotion (EXD) is widely used in China for the treatment of osteoporosis, which has a variety of antioxidant active ingredients. EXD may be an important source of protection against oxidative damage in osteoblasts, but the anti-osteoporotic active components of EXD is currently unclear. AIM OF THE STUDY: This work established an effective and reliable drug screening method to find main active ingredients in EXD (M-EXD) that can protect osteoblasts against oxidative stress and achieve anti-osteoporosis effects. MATERIALS AND METHODS: H2O2-induced osteoblast cell fishing with UHPLC-QTOF/MS was firstly used to discover the potential active components from EXD. Afterword, the EXD compound-osteoporosis target network was constructed using network pharmacology, thus potentially anti-osteoporosis ingredients were founded, and their combination were defined as the M-EXD. Finally, pharmacology effects of M-EXD was evaluated by ovariectomized rats, prednisolone induced-zebrafish and H2O2-induced osteoblasts. RESULTS: 40 candidate active ingredients in EXD were initially screened out via pathological cell fishing. According to network pharmacology result, M-EXD consisted of 13 ingredients since they had a close relationship with 65 osteoporosis-related targets. Pharmacological evaluation showed that M-EXD significantly ameliorated oxidative stress in H2O2-induced osteoblast model, evidently reversed the activity of ALP, ROS, GSH-px, NO and MDA compared with the model group. M-EXD showed better anti-oxidative activities than individual ingredients, presenting obvious synergetic effects. In osteoporosis rat and zebrafish models, M-EXD also demonstrated good anti-osteoporotic properties by mitigating the osteoporosis bone loss and increasing serum bone morphogenetic protein 2, and reversing osteocalcin expression in bone tissue. It significantly ameliorated oxidative stress in the in-vivo models. Moreover, M-EXD and EXD showed similar anti-osteoporotic and anti-oxidative properties, while the rest components of EXD had no satisfactory anti-osteoporotic efficacy. CONCLUSIONS: Our work successfully identified the main active components in EXD, which could represent the efficacy of EXD on treating osteoporosis, and meanwhile, it also provided an effective strategy to investigate active ingredients from natural medicines, which might be helpful for drug development and application.

Curculigoside facilitates fear extinction and prevents depression-like behaviors in a mouse learned helplessness model through increasing hippocampal BDNF.

Curculigoside (CUR) is the main active component of traditional Chinese medicine Curculigoorchioides Gaertn (Xianmao in Chinese), which exhibits a variety of pharmacological activities. In this study we investigated the effects of CUR on fear extinction and related depression-like behaviors in mice. In fear conditioning task, we found that administration of CUR (1.6, 8, 40 mg·kg-1·d-1, ip, for 7 days) did not affect memory consolidation, but CUR at higher doses (8, 40 mg·kg-1·d-1) significantly facilitated fear extinction, especially on D3 and D4. Moreover, CUR administration significantly ameliorated the fear conditioning-induced depression-like behaviors, likely through promoting fear extinction. We showed that CUR increased the expression of brain-derived neurotrophic factor (BDNF) and phosphorylation of tropomyosin receptor kinase B (TrkB) in the hippocampus, and activated protein kinase B (Akt)-mammalian target of the rapamycin (mTOR) signaling pathway. Administration of the selective TrkB agonist 7,8-dihydroxyflavone (7,8-DHF, 5 mg·kg-1·d-1, ip) also facilitated fear extinction, ameliorated depression-like behaviors. We established a mouse learned helplessness (LH) model to evaluate the antidepressant activity of CUR. The spatial memory was assessed in Morris water maze. We showed that LH-induced depression-like behaviors, including prolonged immobility times in forced swim and tail suspension tests as well as spatial memory impairments; LH also downregulated BDNF expression and the Akt-mTOR signaling pathway in the hippocampus. Administration of CUR (1.6, 8, 40 mg·kg-1·d-1, ip, for 14 days) or 7,8-DHF (5 mg·kg-1·d-1, ip, for 3 days) prevented LH-induced depression-like behaviors and promoted BDNF expression and the Akt-mTOR signaling pathway. In conclusion, CUR can accelerate the fear memory extinction and ameliorate depression-like behaviors in mice via promoting BDNF expression and activating the Akt-mTOR signaling pathway in the hippocampus.

Study on the effect of curculigoside on osteoporosis / 中华口腔医学杂志

Objective@#To investigate the regulation of curculigoside on osteogenic differentiation of MG63 and the protective effect on osteoporosis model mice.@*Methods@#The effects of curculigoside on the survival rate of dexamethasone or H2O2 treated MG63 were detected by methyl thiazolyl tetrazolium (MTT). The specimens were divided into six groups: blank control group, blank administration group, model group (dexamethasone or H2O2 treatment group), low dose group (dexamethasone or H2O2+1.0 μmol/L curculigoside), medium dose group (dexamethasone or H2O2+2.5 μmol/L curculigoside) and high dose group (dexamethasone or H2O2+5.0 μmol/L curculigoside), the sample size of each group was 10. Western blotting was used to detect the expression of osteogenic differentiation-related proteins [type Ⅰ collagen, integrin β1, osteoblast-specific transcription factor (Osterix), osteocalcin and osteopontin] in MG63 cells after 1, 7 and 14 days incubated with 0, 1.0, 2.5 and 5.0 μmol/L of curculigoside. The sample size for each group at each time point was six. The experimental mice were divided into 4 groups: blank group, model group (dexamethasone treatment group), curculigoside low-dose group (dexamethasone+5 mg/kg curculigoside) and high-dose group (dexamethasone+45 mg/kg curculigoside), twenty each. After treatment, the tibia of the mice in each group were subjected to sacral HE staining. The number of osteoclasts was counted, and the levels of oxidative related factors in serum were determined by enzyme-linked immunosorbent assay (ELISA).@*Results@#The MTT results showed that compared with the blank control group [(100±3.7)%], the cell survival rate decreased to (44.1±5.7)% after treatment with dexamethasone, and the survival rate increased to (79.7±3.8)% after treatment with 5.0 μmol/L of curculigoside. The cell survival rate decreased to (59.1±4.7)% after H2O2 treatment, and the survival rate increased to (80.8±3.5)% after treatment with 2.5 μmol/L of curculigoside. The results of Western blotting showed that the expression of type Ⅰ collagen and integrin β1 in MG63 cells was significantly increased after 1.0, 2.5 and 5.0 μmol/L of curculigoside for 1, 7 and 14 days compared with 0 μmol/L of curculigo side for the same period. After increasing (P<0.05), the expression of Osterix and osteocalcin was significantly increased after 1 day of incubation (P<0.05). However, compared with 0 μmol/L curculigoside treatment, the expression of osteopontin in MG63 cells was not significantly different after incubation with 1.0, 2.5, 5.0 μmol/L of curculigoside for 7 and 14 days (P>0.05). Compared with the blank group, the number of tibia osteoclasts in the osteoporosis model group increased. In the low-dose and high-dose groups of curculigoside, the tibia cortex was more continuous and the number of osteoclasts decreased. Compared with the blank group, the activity of oxygen in the osteoporosis model group was significantly increased (P<0.05), and superoxide dimutase and catalase were significantly decreased (P<0.05).@*Conclusions@#Curculigoside promotes the differentiation of MG63 cells by increasing the expression of osteoblast differentiation-related proteins, and has a certain therapeutic effect on dexamethasone-induced osteoporosis mice.

Pharmacokinetic and Metabolism Studies of Curculigoside C by UPLC-MS/MS and UPLC-QTOF-MS.

Pharmacokinetic and metabolism studies were carried out on curculigoside C (CC), a natural product with good antioxidant and neuroprotective effects, with the purpose of investigating the effects of the hydroxyl group at C-3' in curculigoside. A rapid and sensitive method with UPLC-MS was developed and fully validated for the first time in the pharmacokinetic analysis for quantification of CC in rat plasma. The assay was linear (R² > 0.9984) over the concentration range of 1⁻2500 ng/mL, with the lower limit of quantification (LLOQ) being 1 ng/mL. The intra-day and inter-day precision (expressed as relative standard deviation, RSD) ranged from 4.10% to 5.51% and 5.24% to 6.81%, respectively. The accuracy (relative error, RE) ranged from -3.28% to 0.56% and -5.83% to -1.44%, respectively. The recoveries ranged from 92.14% to 95.22%. This method was then applied to a pharmacokinetic study of rats after intragastric administration of 15, 30 and 60 mg/kg CC. The results revealed that CC exhibited rapid oral absorption (Tmax = 0.106 h, 0.111 h, and 0.111 h, respectively), high elimination (t1/2 = 2.022 h, 2.061 h, and 2.048 h, respectively) and low absolute bioavailability (2.01, 2.13, and 2.39%, respectively). Furthermore, an investigation on the metabolism of CC was performed by UPLC-QTOF-MSE. Twelve metabolites of CC from plasma, bile, urine and faeces of rats were confirmed. The main metabolic pathways of CC, which involve dehydration, glucosylation, desaturation, formylation, cysteine conjugation, demethylation and sulfonation, were profiled. In conclusion, this research has developed a sensitive quantitative method and demonstrated the metabolism of CC in vivo.

Curculigoside and polyphenol-rich ethyl acetate fraction of Molineria latifolia rhizome improved glucose uptake via potential mTOR/AKT activated GLUT4 translocation.

Adipose tissue is one of the major organs responsible for rapid restoration of postprandial glucose fluxes. Being the major isoform of glucose transporter in adipose tissue, regulations of insulin-dependent GLUT4 trafficking have always been of research interest. The present study aimed to examine the molecular mechanisms underlying the efficacy of curculigoside and polyphenol-rich ethyl acetate fraction (EAF) of Molineria latifolia rhizome in triggering glucose uptake. We assessed the adipogenic potential and glucose uptake stimulatory activity of curculigoside and EAF by employing a murine 3T3-L1 adipocyte model. The transcriptional and translational expressions of selected intermediates in the insulin signalling pathway were evaluated. While curculigoside neither promoted adipogenesis nor activated peroxisome proliferator activated receptor gamma, treatment with polyphenol-rich EAF resulted otherwise. However, both treatments enhanced insulin-stimulated uptake of glucose. This was coupled with increased availability of GLUT4 at the plasma membrane of the differentiated adipocytes although the total GLUT4 protein level was unaffected. In addition, the treatment increased the phosphorylation of both AKT and mTOR, which have been reported to be associated with GLUT4 translocation. The present findings proposed that curculigoside and EAF increased glucose transport activity of 3T3-L1 adipocytes via GLUT4 translocation as a result of potential mTOR/AKT activation. The more potent efficacy observed with EAF suggested potential synergistic and multi-targeted action.

Effects of curculigoside A on random skin flap survival in rats.

Necrosis in distal areas of random skin flaps remains a challenging issue. Curculigoside A (CA), one of the main bioactive phenolic compounds, has been reported to induce angiogenesis in vitro by increasing proliferation, tube formation, and migration. In addition, CA was shown to increase vascular endothelial growth factor (VEGF) expression. In this study, we investigated the potential use of CA as a novel candidate to enhance the viability of the ischemic skin flap. A modified McFarlane flap was used as a surgical model in Sprague-Dawley rats. Three groups of rats were treated as follows: the control group received 0.9% saline orally, while rats in the two treatment groups were administered 10 mg/kg or 20 mg/kg CA orally for 7 days, respectively. On day7, the mice were killed, and tissue samples were collected for hematoxylin and eosin staining and immunohistochemical examination, while laser Doppler imaging and oxide-gelatin angiography were performed to assess angiogenesis. Kits for the analysis of superoxide dismutase (SOD) and malondialdehyde (MDA) were used to verify the oxidative stress level. Treating animals with CA significantly increased the surviving portion of the flaps. VEGF and SOD expression and microvessel development were markedly increased, and the MDA level was reduced, in the CA treatment groups. Histological studies demonstrated that CA promoted angiogenesis and attenuated inflammatory cell numbers. These findings indicated that CA increases random skin flap survival.