Ophiopogonin D alleviates acute lung injury by regulating inflammation via the STAT3/A20/ASK1 axis.

BACKGROUND: Acute lung injury (ALI) is characterized by acute pulmonary inflammatory infiltration. Alveolar epithelial cells (AECs) release numerous pro-inflammatory cytokines, which result in the pathological changes seen in ALI. Ophiopogonin D (OD), extracted from the roots of Ophiopogon japonicus (Thunb.) Ker Gawl. (Liliaceae), reduces inflammation; however, the efficacy of OD in ALI has not been reported and the underlying molecular mechanisms remain unclear. PURPOSE: This study investigated the anti-inflammatory effects of OD, as well as the underlying mechanisms, in AECs and a mouse ALI model. METHODS: Lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α) were used to stimulate macrophages and A549 cells, and a mouse ALI model was established by intratracheal LPS administration. The anti-inflammatory effects and mechanisms of OD in the TNF-α-induced in vitro inflammation model was evaluated using real-time quantitative polymerase chain reaction qPCR), enzyme-linked immunosorbent assay (ELISA), western blotting, nuclear and cytoplasmic protein extraction, and immunofluorescence. The in vivo anti-inflammatory activity of OD was evaluated using hematoxylin and eosin staining, qPCR, ELISA, and western blotting. RESULTS: The bronchoalveolar lavage fluid and lung tissue of LPS-induced ALI mice exhibited increased TNF-α expression. TNF-α induced a significantly greater pro-inflammatory effect in AECs than LPS. OD reduced inflammation and mitogen-activated protein kinase (MAPK) and transcription factor p65 phosphorylation in vivo and in vitro and promoted signal transducer and activator of transcription 3 (STAT3) phosphorylation and A20 expression, thereby inducing apoptosis signal-regulating kinase 1 (ASK1) proteasomal degradation. CONCLUSION: OD exerts an anti-inflammatory effect by promoting STAT3-dependent A20 expression and ASK1 degradation. OD may therefore have therapeutic value in treating ALI and other TNF-α-related inflammatory diseases.

Two New Isospirostanol-Type Saponins from the Bulbs of Lilium Brownii and Their Anti-Hepatocarcinogenic Activity.

Bulbs of Lilium brownii, commonly known as "Bai-he" in China, serve both edible and medicinal purposes in clinical practice. In this study, two new isospirostanol-type saponins were isolated from L. brownii, and their structures were identified by spectroscopic method, and absolute configurations were elucidated by comprehensive analysis of spectral data obtained from combined acid hydrolysis. Two compounds were finally identified as 3-O-[α-L-rhamnopyranosyl-(1→2)-ß-D-glucopyranoside]-(22R,25R)-5α-spirosolane-3ß-ol (1) and 3-O-{α-L-rhamnopyranosyl-(1→2)-[ß-D-glucopyranosyl-(1→4)]-ß-D-glucopyranoside}-(22R,25R)-5α-spirosolane-3ß-ol (2), respectively. Further, we found that compound 2 significantly suppressed the proliferation of SMMC-7721 and HepG2 cells with IC50 values of 26.3±1.08 µM and 30.9±1.59 µM, whereas compound 1 didn't inhibit both of the two hepatocellular carcinoma. Subsequently, compound 2 effectively decreased the levels of interleukin-1ß and tumor necrosis factor-α and the expression of Bcl-2, and increased the expression of Bax and Caspase-3 proteins. Which indicated that the anti-hepatocellular carcinoma effect of compound 2 involves reducing the level of inflammation and inducing apoptosis.

Total synthesis and cytotoxicity evaluation of the spirostanol saponin gitonin.

The spirostanol saponin gitonin was efficiently synthesized in 12 steps (longest linear sequence) in 18.5% overall yield from the commercially available isopropyl ß-D-1-thiogalactopyranoside (IPTG) and tigogenin. A cascade two-step glycosylation and Schmidt's inverse procedure significantly facilitated the synthesis of gitonin and its derivatives. The cytotoxic activities of gitonin and its structural analogues were evaluated against A549, HepG2, and MCF-7, and most of them exhibited moderate to excellent inhibitory activity. Our study demonstrates that the removal of the ß-D-galactopyranosyl residue (attached at C-2 of the glucose unit) from gitonin would not decrease the inhibition activities; however, further cleavage of sugar units could seriously reduce the activities. A bioassay on these cancer cell lines also suggested that the presence of 2α-hydroxy on the aglycone weakened the cytotoxicity of the designed saponin.

Steroidal glycosides from Ornithogalum thyrsoides bulbs and their cytotoxicity toward HL-60 human promyelocytic leukemia cells and SBC-3 human small-cell lung cancer cells.

Ornithogalum thyrsoides Jacq belongs to the Asparagaceae family and is cultivated for ornamental purposes. The authors have previously reported several cholestane- and spirostan-type steroidal glycosides from O. thyrsoides. Conventional TLC analysis of the methanolic bulb extract of O. thyrsoides suggested the presence of unprecedented compounds; therefore, a detailed phytochemical investigation of the extract was performed and 35 steroidal glycosides (1-35), including 21 previously undescribed ones (1-21) were collected. The structures of 1-21 were determined mainly by analyses of their 1H and 13C NMR spectra with the aid of two-dimensional NMR spectroscopy. The isolated compounds were classified into three distinct groups: furostan-type (1, 2, 8-12, and 22), spirostan-type (3-7 and 23-26), and cholestane-type (13-21 and 27-35). Although the C/D-ring junction of the steroidal skeleton is typically trans-oriented, except for some cardiotonic and pregnane-type steroidal derivatives, 7 possess a cis C/D-ring junction. This is the first reported instance of such a configuration in spirostan-type steroidal derivatives, marking it as a finding of significant interest. Compounds 1-35 were evaluated for cytotoxicity against HL-60 human promyelocytic leukemia cells and SBC-3 human small-cell lung cancer cells. Compounds 3-6, 9, 17-21, 23-25, and 30-35 demonstrated cytotoxicity in a dose-dependent manner with IC50 values ranging from 0.000086 to 18 µM and from 0.00014 to 37 µM toward HL-60 and SBC-3 cells, respectively. Compound 19, which is obtained in a good yield and shows relatively potent cytotoxicity among the undescribed compounds, induces apoptosis in HL-60 cells, accompanied by arresting the cell cycle of HL-60 cells at the G2/M phase. In contrast, 19 causes oxidative stress-associated necrosis in SBC-3 cells. The cytotoxic mechanism of 19 is different between HL-60 and SBC-3 cells.

Aspidiatas C and D, two new spirostanol saponins from Aspidistra triradiata and their cytotoxic activities.

Aspidiatas C and D (1 and 2), two new spirostanol saponins, were isolated along with two known compounds, (25 R*)-spirost-5-en-3ß-yl α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-ß-D-glucopyranoside (3), (25 R*)-spirost-5-en-3ß-yl α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranoside (4) from the whole plant of Aspidistra triradiata collected in Vietnam. The chemical structures were determined by HRESIMS, 1D- and 2D-NMR analysis, and comparison with published data. Compound 3 exhibited potent cytotoxicity against MCF7, HepG2, SK-LU-1, and HT-29 human cancer cell lines with IC50 values ranging from 0.19 to 0.65 µM. Compounds 1, 2, and 4 displayed moderate cytotoxic effects with IC50 values ranging from 12.32 to 82.27 µM. Compounds 1-4 were isolated from the genus Aspidistra for the first time.

Gitogenin suppresses lung cancer progression by inducing apoptosis and autophagy initiation through the activation of AMPK signaling.

Lung cancer is a leading cause of tumor-associated death worldwide. Autophagy plays a key role in regulating lung cancer progression, and is a promising option for lung cancer treatment. Saponins are a group of naturally occurring plant glycosides, characterized by their strong foam-forming properties in aqueous solution, and exert various biological properties, such as anti-inflammation and anti-cancer. In the present study, we for the first time explored the effects of gitogenin (GIT), an important saponin derived from Tribulus longipetalus, on lung cancer progression both in vitro and in vivo. We found that GIT markedly reduced the proliferation and induced apoptosis in lung cancer cells through increasing the cleavage of Caspase-3 and poly (ADP-ribose) polymerases (PARPs). In addition, GIT-incubated lung cancer cells exhibited clear accumulation of autophagosome, which was essential for GIT-suppressed lung cancer. Mechanistically, GIT-induced autophagy initiation was mainly through activating AMP-activated protein kinase (AMPK) and blocking protein kinase B (AKT) signaling pathways, respectively. Moreover, the autophagic flux was disrupted in GIT-treated lung cancer cells, contributing to the accumulation of impaired autophagolysosomes. Importantly, we found that suppressing autophagy initiation could abolish GIT-induced cell death; however, autophagosomes accumulation sensitized lung cancer cells to cell death upon GIT treatment. More in vitro experiments showed that GIT led to reactive oxygen species (ROS) production in lung cancer cells, which was also involved in the modulation of apoptosis. The in vivo findings confirmed the effects of GIT against lung cancer progression with undetectable toxicity to organs. In conclusion, we provided new insights into the treatment of lung cancer, and GIT might be an effective strategy for future clinical application.

Ophiopogonin D'-induced mitophagy and mitochondrial damage are associated with dysregulation of the PINK1/Parkin signaling pathway in AC16 cells.

Shenmai injection (SMI) is a patented traditional Chinese medicine that is extracted from Panax ginseng and Ophiopogon japonicus and is commonly used to treat cardiovascular diseases and tumors. The O. japonicus extract Ophiopogonin D' (OPD') is highly cardiotoxic. Mitochondria are central to OPD'-induced cardiotoxicity, although the precise mechanisms remain unclear. Excessive mitophagy activation and mitochondrial dysfunction lead to apoptosis, and the PTEN-induced kinase 1(PINK1)/Parkin pathway is critical in regulating mitophagy and mitochondrial function. We investigated the role of the PINK1/Parkin pathway in OPD'-induced mitochondrial damage and cardiotoxicity in AC16 cells. Concentrations of 2 µM OPD' and above inhibited cardiomyocyte viability and increased lactate dehydrogenase (LDH) release in a concentration- and time-dependent manner. OPD' was toxic to cells and mitochondria and increased the rate of apoptosis, triggering pyknosis, decreasing mitochondrial membrane potential (MMP), and decreasing the protein expression of the biogenesis regulator peroxisome proliferator-activated receptor γ coactivator-1 alpha (PGC-1α). The increased ratio of microtubule-associated proteins 1 A/1B light chain 3B (LC3-II/LC3-I) in mitochondria indicated that OPD' induced mitophagy. OPD' significantly induced oxidative stress and apoptosis, including increased reactive oxygen species (ROS) generation and decreased nuclear factor erythroid-2 related factor 2 (Nrf2), heme oxygenase-1(HO-1), and B-cell lymphoma 2 (Bcl-2) protein expression. OPD' activated the PINK1/Parkin pathway and promoted PINK1/Parkin translocation to mitochondria. Inhibiting mitophagy attenuated OPD'-induced PINK1/Parkin pathway activation and preserved mitochondrial biogenesis, consequently mitigating OPD'-induced mitochondrial dysfunction and apoptosis. These findings suggest that OPD'-induced cardiomyocyte mitophagy and mitochondrial damage are at least partially mediated by dysregulation of the PINK1/Parkin pathway.

Anticancer Effect of Ruscogenin in B(a)P-Induced Lung Cancer in Mice via Modulation of Proinflammatory Cytokines and Mitochondrial Enzymes.

Lung cancer, one of the most often diagnosed malignancies, is the top cause of death in both men and women globally. In both developed and emerging countries, high incidences of cancer are becoming a huge health burden. Natural resources, including plants, have always been a possible source of lead compounds in the identification of optimal medications for cancer treatment, with natural resources accounting for around half of all anticancer drugs. Ruscogenin, a natural saponin, is a major component of Radix Ophiopogon japonicus with a well-established anticancer activity. In this study, the anticancer potential of ruscogenin against a B(a)P-challenged lung cancer model in mice was assessed. The mice were categorized into four groups: group I was as the control group, group II mice were challenged with B(a)P, group III rodents were treated with ruscogenin prior to challenge with B(a)P, and group IV rodents were treated with ruscogenin after B(a)P administration. Tumor incidence was calculated, and the following parameters were analyzed: body weight, lung weight, immunoglobulin (Ig) levels (IgG, IgA, and IgM), key marker enzymes, and proinflammatory cytokines in both treated and control mice. Lung tissues were analyzed via histopathological analysis. According to our results, all the markers that favor the growth of cancer were increased in the lung cancer group. After administration of ruscogenin, all the markers returned to their original levels, revealing the anticancer potential of ruscogenin.

Ruscogenin Alleviates Deep Venous Thrombosis and Pulmonary Embolism Induced by Inferior Vena Cava Stenosis Inhibiting MEK/ERK/Egr-1/TF Signaling Pathway in Mice.

BACKGROUND: Ruscogenin (RUS) has anti-inflammatory and antithrombotic effects, while its potential effects on deep venous thrombosis (DVT) and pulmonary embolism (PE) remain unclear. OBJECTIVE: We aimed to elucidate the effects of RUS on DVT and PE induced by the inferior vena cava stenosis (IVCS) model and investigate the underlying mechanism. METHODS: Male C57/BL6 mice were used to explore whether IVCS model could be complicated with deep venous thrombosis and pulmonary embolism. Then, effects of RUS on DVT and PE related inflammatory factors and coagulation were examined using H&E staining, ELISA, and real-time PCR. Western blot analysis was used to examine the effects of RUS on MEK/ERK/Egr-1/TF signaling pathway in PE. RESULTS: IVCS model induced DVT and complied with PE 48 h after surgery. Administration of RUS (0.01, 0.1, 1 mg/kg) inhibited DVT, decreased biomarker D-Dimer, cardiac troponin I, N-Terminal probrain natriuretic peptide in plasma to ameliorate PE induced by IVCS model. Meanwhile, RUS reduced tissue factor and fibrinogen content of lung tissue, inhibited P-selectin and C-reactive protein activity in plasma, and suppressed the expressions of interleukin-6 and interleukin-1ß in mice. Furthermore, RUS suppressed the phosphorylation of ERK1/2 and MEK1/2, decreasing the expressions of Egr-1 and TF in the lung. CONCLUSION: IVCS model contributed to the development of DVT and PE in mice and was associated with increased inflammation. RUS showed therapeutic effects by inhibiting inflammation as well as suppressing the activation of MEK/ERK/Egr-1/TF signaling pathway.

Ruscogenins Improve CD-Like Enteritis by Inhibiting Apoptosis of Intestinal Epithelial Cells and Activating Nrf2/NQO1 Pathway.

Interaction of intestinal barrier dysfunction and intestinal inflammation promotes the progression of Crohn's disease (CD). A more recent study has suggested that ruscogenins (RUS) can exert anti-inflammatory effects through activation of the Nrf2/NQO1 pathway. The current study is aimed at determining the functionalization of RUS on CD-like colitis. Wild-type (WT) mice induced with trinitrobenzene sulfonic acid (TNBS) exhibit a significant inflammation in their colon and are hence widely used for CD models. In the current study, the mice were treated with the Nrf-2 antagonist (ML385) or ruscogenin (RUS) whereas normal WT mice were kept as the negative control. Comparative analysis was then performed on the inflammation and barrier function of the colons. In vitro analysis of mouse colonic organoid systems revealed the influence of RUS on LPS-induced apoptosis, cytokine, and chemokine expressions in the intestinal epithelium. It was found that RUS ameliorates murine colitis through activation of the Nrf2/NQO1 pathway which was presented as a decrease in inflammation score and downregulated levels of cytokine and chemokine synthesis, as well as increased intestinal permeability. Further, it was noted that RUS alleviated LPS-induced apoptosis in the intestinal epithelium cells through upregulation of the Nrf2/NQO1 signaling pathway in the mouse colonic organoids. In addition, ruscogenin (RUS) attenuated the levels of Bax and C-caspase-3 through activation of the Nrf2/HO1 signaling pathway both in vivo and in vitro. Therefore, it was evident that RUS can be applied as a potential alternative therapeutic agent in CD based on its protective effects on the barrier function and anti-inflammatory activity.

Chemistry, Biosynthesis and Pharmacology of Sarsasapogenin: A Potential Natural Steroid Molecule for New Drug Design, Development and Therapy.

Sarsasapogenin is a natural steroidal sapogenin molecule obtained mainly from Anemarrhena asphodeloides Bunge. Among the various phytosteroids present, sarsasapogenin has emerged as a promising molecule due to the fact of its diverse pharmacological activities. In this review, the chemistry, biosynthesis and pharmacological potentials of sarsasapogenin are summarised. Between 1996 and the present, the relevant literature regarding sarsasapogenin was obtained from scientific databases including PubMed, ScienceDirect, Scopus, and Google Scholar. Overall, sarsasapogenin is a potent molecule with anti-inflammatory, anticancer, antidiabetic, anti-osteoclastogenic and neuroprotective activities. It is also a potential molecule in the treatment for precocious puberty. This review also discusses the metabolism, pharmacokinetics and possible structural modifications as well as obstacles and opportunities for sarsasapogenin to become a drug molecule in the near future. More comprehensive preclinical studies, clinical trials, drug delivery, formulations of effective doses in pharmacokinetics studies, evaluation of adverse effects and potential synergistic effects with other drugs need to be thoroughly investigated to make sarsasapogenin a potential molecule for future drug development.

Ophiopogonin­B targets PTP1B to inhibit the malignant progression of hepatocellular carcinoma by regulating the PI3K/AKT and AMPK signaling pathways.

Ophiopogonin­B (OP­B) is a bioactive component from the root of Ophiopogon japonicus, which can exert anticancer effects on multiple malignant tumors. The present study aimed to uncover the effects of OP­B on hepatocellular carcinoma (HCC) and the underlying mechanisms. An HCC­xenografted mouse model was established and subsequently treated with OP­B (15 and 75 mg/kg) to observe the effects of OP­B on HCC progression and protein tyrosine phosphatase 1B (PTP1B) expression in vivo. The HCC cell line MHCC97­H was transfected with either PTP1B overexpression (Ov)­PTP1B or empty vector control, and then exposed to different concentrations of OP­B. Subsequently, PTP1B expression, cell viability, proliferation, apoptosis, migration, invasion and angiogenesis were evaluated by western blotting, reverse transcription­quantitative PCR, Cell Counting Kit­8, colony formation, TUNEL staining, wound healing, Transwell and tube formation assays. The expression of phosphatidylinositol 3 kinase (PI3K)/AKT and adenosine 5'­monophosphate­activated protein kinase (AMPK) was also assessed by western blot assay. The results showed that OP­B inhibited tumor growth and the expression of Ki67, CD31, VEGFA and PTP1B in HCC xenograft model. The expression of PTP1B in HCC cells was also inhibited by OP­B in a concentration­dependent manner. Results from the in vitro studies revealed that OP­B suppressed cell proliferation, migration, invasion and angiogenesis, and promoted apoptosis of HCC cells. However, PTP1B overexpression reversed the effect of OP­B on HCC cells. PI3K/AKT was inactivated and AMPK was activated by OP­B exposure in HCC cells, and PTP1B overexpression blocked these effects. In conclusion, OP­B effectively inhibited the progression of HCC both in vivo and in vitro. These effects may depend on downregulating PTP1B expression, thereby inactivating the PI3K/AKT pathway and activating the AMPK pathway.

The anti-tumor effect of OP-B on ovarian cancer in vitro and in vivo, and its mechanism: An investigation using network pharmacology-based analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Maidong (Liliaceae) is used as a yin-nourishing medication for the treatment of cardiovascular disease, inflammation, and assistant cancer chemotherapy in the clinic. Ophiopogonin B (OP-B), a major saponin extracted from Maidong, is reported to have potential antitumor activities against various human cancers. However, the effects of OP-B on human ovarian cancer (OC) and the potential mechanisms of action are yet elusive. AIM OF THE STUDY: In this study, we aimed to explore the potential molecular mechanisms of OP-B in the treatment of OC using network pharmacology. In vivo and in vitro experiments were conducted to further verify the therapeutic effects of OP-B on OC. MATERIALS AND METHODS: To investigate the functions of OP-B against OC holistically, the related targets of OP-B and OC were each predicted based on four public databases. Subsequently, the identified PPI network was constructed to detect the hub potential targets. In addition, GO and KEGG enrichment analysis were applied by Metascape database. Furthermore, we simultaneously investigated the anticancer effects of OP-B on SKOV3 and A2780 human ovarian cancer cells using a cell viability assay, transwell assay, and an image-based cytometric assay. The quantitative real-time PCR and western-blot assay were used to validate the RNA and protein levels of target genes in OP-B treated OC cells. At last, SKOV3-bearing BALB/c nude mice were applied to observe the effectiveness and toxicity of OP-B. RESULTS: Through network pharmacological analysis, OP-B was found to play a critical role in OC via multiple targets and pathways, especially the STAT3 signaling pathways. In addition, in vitro experiments found OP-B suppressed SKOV3 and A2780 cells proliferation in a time and concentration dependent manner, and markedly impaired cancer cell migration. Flow cytometry analysis revealed that OP-B significantly increased early and late apoptosis, induced G2/M phase cell cycle arrest in SKOV3 cells and G0/G1 phase cell cycle arrest in A2780 cells. Moreover, OP-B administration down-regulated the expression of p-STAT3 protein, whereas the RNA expression and total protein levels of STAT3 were not altered. Finally, in vivo experiments confirmed the therapeutic effects of OP-B on OC in nude mice with low toxicity in heart, liver, lung, and kidney. CONCLUSION: OP-B could efficiently suppress OC cellular proliferation, migration and induce apoptosis, cell cycle arrest mainly via the regulation of STAT3 signaling pathway. This study provides a promising potential application for an alternative to chemotherapy in ovarian cancer.

Spirostane saponins with a rearranged A/B ring system isolated from the rhizomes of Ophiopogon japonicus.

In this study, the popular food and medicinal herb Ophiopogon japonicus was investigated alongside a 70% ethanol extract of its rhizomes, revealing twenty-three steroidal glycosides with four undescribed steroidal saponins, named ophiopogonols A-D. Among them, ophiopogonols A-B are two unusual spirostanols with a rearranged A/B ring system (5/7/6/5/5/6 ring system) that have not previously been identified in plants. The chemical structures of all isolated steroidal glycosides were elucidated by comprehensive analysis through chemical methods, HRESIMS, and NMR spectroscopy. Further, putative biosynthetic pathways for ophiopogonols A-B were proposed. In addition, based on traditional applications of O. japonicus, cytotoxic effects of the isolates were evaluated using human large cell lung carcinoma cells (NCI-H460 cells). Sprengerinin C displayed a remarkable cytotoxic effect with IC50 values of 2.1 ± 0.8 µM by inducing apoptosis and G2/M phase cycle arrest in the NCI-H460 cell line.

N6-methyladenosine modification of TGM2 mRNA contributes to the inhibitory activity of sarsasapogenin in rheumatoid arthritis fibroblast-like synoviocytes.

BACKGROUND: Developing alternative targets and drugs for rheumatoid arthritis (RA) treatment is currently an urgent issue. The relationship between TGM2 and the abnormal immune microenvironment in synovium tissues, as well as the specific role of TGM2 in RA are yet to be elucidated. Sarsasapogenin (Sar) is a sapogenin extracted from the Chinese medical herb Anemarrhena asphodeloides Bunge. and served as a representative anti-inflammatory drug capable of ameliorating inflammatory responses in several human diseases. However, the therapeutic effect of Sar on RA remains unknown. PURPOSE: This investigation aims to elucidate the role of TGM2 in RA and investigate whether Sar is a candidate drug to target TGM2 of fibroblast-like synoviocytes (FLS). METHODS: Bioinformatics analyses were applied for elucidating the role of N(6)-methyladenine (m6A) RNA methylation in RA and identifying the specific target regulated by m6A methylation in RA-FLS. Methylated RNA immunoprecipitation, CCK8 assay, Edu assay, flow cytometry, RT-qPCR and Western blot were utilized to investigate the function of Sar and TGM2 in RA-FLS. RESULTS: Bioinformatics analyses emphasized the importance of m6A RNA methylation in RA and identified an m6A methylation-mediated gene TGM2. Interestingly, both m6A RNA methylation and TGM2 expression in RA synovium tissues correlated with activated immuno-inflammatory phenotype and associated with clinical characteristics and therapy response of RA patients. TGM2 served as a promoter of RA-FLS proliferation by inducing DNA replication and cell cycle transition and inhibiting apoptosis through activating NF-κB signaling. Intriguingly, Sar could impair m6A methylation of TGM2 mRNA and downregulate TGM2 expression. Downregulated TGM2 contributed to the suppressive role of Sar in DNA replication and the stimulatory role of Sar in cell cycle arrest and apoptosis of RA-FLS. Mechanically, Sar inhibited the expression of key regulators in DNA replication, cell cycle, and apoptosis by impairing NF-κB signaling, thus abolishing FLS proliferation to ameliorate RA progression. CONCLUSIONS: This cross-validated work based on three independent datasets is detailedly delineated using cell lines and clinical samples, recognizing that TGM2 can be an attractive target and Sar might be a novel anti-RA drug.

Protective effect of sarsasapogenin in TNBS induced ulcerative colitis in rats associated with downregulation of pro-inflammatory mediators and oxidative stress.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory bowel condition considered by oxido-nitrosative stress and the release of pro-inflammatory cytokines that affects the mucosal lining of the colon. Sarsasapogenin (SG), as an active component, has been found in many plants, and it exhibits potential protective effects, such as anti-inflammatory, antioxidant, anti-psoriasis, anti-arthritis, anti-asthma, anti-depressant and anti-cancer. However, the effects of SG on UC remain unknown. OBJECTIVE: The purpose of this study was to investigate the effects of SG on 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced UC in rats. METHOD: Thirty Wistar rats were randomized into five groups: (i) Normal control, (ii) Disease control (TNBS), (iii) Sarsasapogenin (SG) (50 µg/rat), (iv) Fluticasone (FC) (50 µg/rat), (v) Sarsasapogenin + Fluticasone (SG + FC) (25 µg/rat). UC was induced in rats by trans-rectal instillation of TNBS (10 mg/kg). SG, FC and SG + FC were administered for 11 days and on the 8th day colitis was induced. Several molecular, biochemical and histological alterations were evaluated in the colon tissue. All treatment group results were compared to the TNBS group results. RESULT: The study results revealed that treatment of rats with SG and SG + FC combination significantly decreased the colon weight/length ratio, macroscopic inflammation score, lesions score, diarrhea score and adhesion score. Combination treatment in rats significantly reduced the production of biochemical parameters, proinflammatory cytokines, haematological parameters, serum IgE levels and restored the oxidative stress markers. SG and SG + FC treatment also considerably restored the histopathological changes induced by TNBS. CONCLUSION: Thus, SG and SG + FC combination could alter the disease progression and could be a hopeful therapeutic target for the management of UC by reducing its dose in combination with FC to elude the long term adverse effects of FC.

Ophiopogonin B induces reactive oxygen species­dependent apoptosis through the Hippo pathway in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a common malignant tumor in South China and is characterized by a high death rate. Ophiopogonin B (OP­B) is a bioactive component of Radix Ophiopogon japonicus, which is frequently used in traditional Chinese medicine to treat cancer. The present study aimed to examine the anti­cancer properties of OP­B on NPC cells. Cell viability and cell proliferation were measured using MTT and EdU assays. Flow cytometry was used to measure cell apoptosis, reactive oxygen species and mitochondrial membrane potential. Western blotting was used to investigate the expression of apoptosis and Hippo signaling pathway proteins. OP­B inhibited the proliferation of NPC cells by inducing apoptosis and disturbing the mitochondrial integrity. OP­B enhanced ROS accumulation. In addition, OP­B promoted the expression of mammalian STE20­like kinase 1, large tumor suppressor 1 and phosphorylated yes­associated protein (YAP) and suppressed the expression of YAP and transcriptional enhanced associate domain in NPC cells. OP­B increased the expression of forkhead box transcription factor O1 in the nuclear fraction. In conclusion, OP­B has therapeutic potential and feasibility in the development of novel YAP inhibitors for NPC.

Gracillin Isolated from Reineckia carnea Induces Apoptosis of A549 Cells via the Mitochondrial Pathway.

BACKGROUND: Reineckia carnea is commonly used to treat cough, pneumonia and other diseases in China. In our previous study, it was found that the ethanol extracts of Reineckia carnea have a strong inhibitory effect on the proliferation of human lung cancer A549 cells. Here, we isolated gracillin from ethanol extracts for the first time. PURPOSE: Clarify the antiproliferation effect of gracillin on A549 cells and further explore its mechanisms via the mitochondrial pathway. METHODS: Gracillin was isolated and purified by silica gel, D-101 macroporous resin and preparative RP-HPLC, then identified by NMR and HR-MS. The inhibitory effects of gracillin on the proliferation of A549 cells were detected by the MTS method. Its mechanisms were further explored by flow cytometry and Western blot. RESULTS: A steroid saponin, gracillin, was isolated and identified from Reineckia carnea for the first time. In a concentration-dependent and time-dependent manner, gracillin significantly inhibited the proliferation of A549 cells with an IC50 value at 2.54 µmol/L and induced morphological changes. The results of flow cytometry analysis showed that the apoptosis rate of A549 cells was significantly increased (p < 0.05), and the cells proportion was obviously arrested in S phase. The concentration of intracellular calcium was raised (p < 0.01), and the mitochondrial membrane potential was greatly decreased (p < 0.01). In addition, the expression levels of Bax, caspase-3, cleaved caspase-3, and cytochrome C were dramatically up-regulated while Bcl-2 was down-regulated (p < 0.05) in A549 cells. CONCLUSION: This study confirmed that gracillin has a significant antiproliferative effect on A549 cells. Gracillin could induce the apoptosis of A549 cells through the mitochondrial pathway, which might be associated with regulation of the concentration of intracellular calcium, the mitochondrial membrane potential and the expression levels of Bax, Bcl-2, caspase-3, cleaved caspase-3, and cytochrome C.

Ophiopogonin D alleviates diabetic myocardial injuries by regulating mitochondrial dynamics.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiopogonin D (OP-D) is a steroidal saponin extracted from Ophiopogon japonicus (Thunb.) Ker Gawl. (Liliaceae), that has been traditionally used to treat cough, sputum, and thirst in some Asian countries. Recently, various pharmacological roles of OP-D have been identified, including anti-inflammatory, cardioprotective, and anti-cancer effects. However, whether OP-D can prevent diabetic myocardial injury remains unknown. AIM OF THE STUDY: In this study, we aimed to observe the effects of OP-D on the diabetic myocardium. MATERIALS AND METHODS: Leptin receptor-deficient db/db mice were used as an animal model for type 2 diabetes. The effects of OP-D on blood glucose, blood lipids, myocardial ultrastructure, and mitochondrial function in mice were observed after four weeks of intragastric administration. Palmitic acid was used to stimulate cardiomyocytes to establish a myocardial lipotoxicity model. Cell apoptosis, mitochondrial morphology, and function were observed. RESULTS: Blood glucose and blood lipid levels were significantly increased in db/db mice, accompanied by myocardial mitochondrial injury and dysfunction. OP-D treatment reduced blood lipid levels in db/db mice and relieved mitochondrial injury and dysfunction. OP-D inhibited palmitic acid induced-mitochondrial fission and dysfunction, reduced endogenous apoptosis, and improved cell survival rate in H9C2 cardiomyocytes. Both in vivo and in vitro models showed increased phosphorylation of DRP1 at Ser-616, reduced phosphorylation of DRP1 at Ser-637, and reduced expression of fusion proteins MFN1/2 and OPA1. Meanwhile, immunofluorescence co-localization analysis revealed that palmitic acid stimulated the translocation of DRP1 protein from the cytoplasm to the mitochondria in H9C2 cardiomyocytes. The imbalance of mitochondrial dynamics, protein expression, and translocation of DRP1 were effectively reversed by OP-D treatment. In isolated mice ventricular myocytes, palmitic acid enhanced cytoplasmic Ca2+ levels and suppressed contractility in ventricular myocytes, accompanied by activation of calcineurin, a key regulator of DRP1 dephosphorylation at Ser-637. OP-D reversed the changes caused by palmitic acid. CONCLUSIONS: Our findings indicate that OP-D intervention could alleviate lipid accumulation and mitochondrial injury in diabetic mouse hearts and palmitic acid-stimulated cardiomyocytes. The cardioprotective effect of OP-D may be mediated by the regulation of mitochondrial dynamics.

Gracillin shows potent efficacy against colorectal cancer through inhibiting the STAT3 pathway.

Colorectal cancer (CRC) accounts for about 10% of all annually diagnosed cancers and cancer-related deaths worldwide. STAT3 plays a vital role in the occurrence and development of tumours. Gracillin has shown a significant antitumour activity in tumours, but its mechanism remains unknown. The human CRC cell lines HCT116, RKO, and SW480 and immunodeficient mice were used as models to study the effects of gracillin on cell proliferation, migration and apoptosis. These were evaluated by cell viability, colony formation, wound-healing migration and cell apoptosis assays. Luciferase reporter assay, and immunostaining and western blot analyses were used to explore the specific mechanism through which gracillin exerts its effects. Gracillin significantly reduces viability and migration and stimulates apoptosis in human CRC cells. It also significantly inhibits tumour growth with no apparent physiological toxicity in animal model experiments. Moreover, gracillin is found to inhibit STAT3 phosphorylation and STAT3 target gene products. In addition, gracillin inhibits IL6-induced nuclear translocation of P-STAT3. Gracillin shows potent efficacy against CRC by inhibiting the STAT3 pathway. It should be further explored as a unique STAT3 inhibitor for the treatment of CRC.