Discovery of a Potent Antiosteoporotic Drug Molecular Scaffold Derived from Angelica sinensis and Its Bioinspired Total Synthesis.

Angelica sinensis, commonly known as Dong Quai in Europe and America and as Dang-gui in China, is a medicinal plant widely utilized for the prevention and treatment of osteoporosis. In this study, we report the discovery of a new category of phthalide from Angelica sinensis, namely falcarinphthalides A and B (1 and 2), which contains two fragments, (3R,8S)-falcarindiol (3) and (Z)-ligustilide (4). Falcarinphthalides A and B (1 and 2) represent two unprecedented carbon skeletons of phthalide in natural products, and their antiosteoporotic activities were evaluated. The structures of 1 and 2, including their absolute configurations, were established using extensive analysis of NMR spectra, chemical derivatization, and ECD/VCD calculations. Based on LC-HR-ESI-MS analysis and DFT calculations, a production mechanism for 1 and 2 involving enzyme-catalyzed Diels-Alder/retro-Diels-Alder reactions was proposed. Falcarinphthalide A (1), the most promising lead compound, exhibits potent in vitro antiosteoporotic activity by inhibiting NF-κB and c-Fos signaling-mediated osteoclastogenesis. Moreover, the bioinspired gram-scale total synthesis of 1, guided by intensive DFT study, has paved the way for further biological investigation. The discovery and gram-scale total synthesis of falcarinphthalide A (1) provide a compelling lead compound and a novel molecular scaffold for treating osteoporosis and other metabolic bone diseases.

Strategy and challenge of innovative drug research and development from clinically effective ingredients of traditional Chinese medicine / 中国中药杂志

Novel drug discovery from the active ingredients of traditional Chinese medicine is the most distinctive feature and advantageous field of China, which has provided an unprecedented opportunity. However, there are still problems such as unclear functional substance basis, action targets and mechanism, which greatly hinder the clinical transformation of active ingredients in traditional Chinese medicine. Based on the analysis of the current status and progress of innovative drug research and development in China, this paper aimed to explore the prospect and difficulties of the development of natural active ingredients from traditional Chinese medicine, and to explore the efficient discovery of trace active ingredients in traditional Chinese medicine, and obtain drug candidates with novel chemical structure, unique target/mechanism and independent intellectual property rights, in order to provide a new strategy and a new model for the development of natural medicine with Chinese characteristics.

Stapled anoplin peptide combined with photothermal therapy enhances oncolytic immunotherapy of triple-negative breast cancer / 中国中药杂志

This study constructed a nano-drug delivery system, A3@GMH, by co-delivering the stapled anoplin peptide(Ano-3, A3) with the light-harvesting material graphene oxide(GO), and evaluated its oncolytic immunotherapy effect on triple-negative breast cancer(TNBC). A3@GMH was prepared using an emulsion template method and its physicochemical properties were characterized. The in vivo and in vitro photothermal conversion abilities of A3@GMH were investigated using an infrared thermal imager. The oncoly-tic activity of A3@GMH against TNBC 4T1 cells was evaluated through cell counting kit-8(CCK-8), lactate dehydrogenase(LDH) release, live/dead cell staining, and super-resolution microscopy. The targeting properties of A3@GMH on 4T1 cells were assessed using a high-content imaging system and flow cytometry. In vitro and in vivo studies were conducted to investigate the antitumor mechanism of A3@GMH in combination with photothermal therapy(PTT) through inducing immunogenic cell death(ICD) in 4T1 cells. The results showed that the prepared A3@GMH exhibited distinct mesoporous and coated structures with an average particle size of(308.9±7.5) nm and a surface potential of(-6.79±0.58) mV. The encapsulation efficiency and drug loading of A3 were 23.9%±0.6% and 20.5%±0.5%, respectively. A3@GMH demonstrated excellent photothermal conversion ability and biological safety. A3@GMH actively mediated oncolytic features such as 4T1 cell lysis and LDH release, as well as ICD effects, and showed enhanced in vitro antitumor activity when combined with PTT. In vivo, A3@GMH efficiently induced ICD effects with two rounds of PTT, activated the host's antitumor immune response, and effectively suppressed tumor growth in 4T1 tumor-bearing mice, achieving an 88.9% tumor inhibition rate with no apparent toxic side effects. This study suggests that the combination of stapled anoplin peptide and PTT significantly enhances the oncolytic immunotherapy for TNBC and provides a basis for the innovative application of anti-tumor peptides derived from TCM in TNBC treatment.

A biomimetic nanoplatform for customized photothermal therapy of HNSCC evaluated on patient-derived xenograft models / 国际口腔科学杂志·英文版

Cancer cell membrane (CCM) derived nanotechnology functionalizes nanoparticles (NPs) to recognize homologous cells, exhibiting translational potential in accurate tumor therapy. However, these nanoplatforms are majorly generated from fixed cell lines and are typically evaluated in cell line-derived subcutaneous-xenografts (CDX), ignoring the tumor heterogeneity and differentiation from inter- and intra- individuals and microenvironments between heterotopic- and orthotopic-tumors, limiting the therapeutic efficiency of such nanoplatforms. Herein, various biomimetic nanoplatforms (CCM-modified gold@Carbon, i.e., Au@C-CCM) were fabricated by coating CCMs of head and neck squamous cell carcinoma (HNSCC) cell lines and patient-derived cells on the surface of Au@C NP. The generated Au@C-CCMs were evaluated on corresponding CDX, tongue orthotopic xenograft (TOX), immune-competent primary and distant tumor models, and patient-derived xenograft (PDX) models. The Au@C-CCM generates a photothermal conversion efficiency up to 44.2% for primary HNSCC therapy and induced immunotherapy to inhibit metastasis via photothermal therapy-induced immunogenic cell death. The homologous CCM endowed the nanoplatforms with optimal targeting properties for the highest therapeutic efficiency, far above those with mismatched CCMs, resulting in distinct tumor ablation and tumor growth inhibition in all four models. This work reinforces the feasibility of biomimetic NPs combining modular designed CMs and functional cores for customized treatment of HNSCC, can be further extended to other malignant tumors therapy.

A Phytomolecule Icariin Protects from Sarcopenia Partially by Suppressing Myosin Heavy Chain Degradation in Orchiectomized Rats.

Treatments are lacking for sarcopenia, which is an age-related disease characterized by loss of skeletal muscle mass, strength, and/or physical performance. Icariin is a phytomolecule from herbal Epimedium, a traditional Chinese medicine widely used to treat musculoskeletal disorders for thousands of years. Here the effects of icariin against sarcopenia are investigated and the underlying mechanism is elucidated. A classic rat model of bilaterally orchiectomized (ORX) is used to induce sarcopenia. After administration for 8 weeks, compared to the control group, the forelimb grip strength, the specific tetanic forces of the soleus (SOL) and extensor digitorum longus muscle (EDL) are higher, and the fiber cross-sectional areas (CSAs) of the gastrocnemius and tibialis anterior muscle are larger in the icariin group. In addition, icariin promotes mRNA and protein expressions of myosin heavy chain (MyHC) both in SOL and EDL. Mechanistically, icariin significantly suppresses the mRNA and protein expressions of FOXO3a, atrogin-1, and MuRF-1, which are related to the degradation of myosin heavy chain. Collectively, icariin protects from sarcopenia in ORX rats characterized by enhancing grip strength and skeletal muscle contraction, as well as increasing skeletal muscle CSA by inhibiting the ubiquitination degradation of the MyHC in skeletal muscle fibers.

Prevalence of diabetes mellitus among Uygur children in Hotan Prefecture of Xinjiang, China / 中国当代儿科杂志

OBJECTIVES@#To investigate the prevalence of diabetes mellitus (DM) among Uygur children in Hotan Prefecture of Xinjiang, China, as well as the factors influencing the development of DM.@*METHODS@#The cluster random sampling method was used to select 5 308 children, aged 4-18 years, from the middle and primary schools and kindergartens in Hotan Prefecture of Xinjiang. The survey methods included questionnaire survey and the measurement of height and weight. All subjects were tested for fasting fingertip blood glucose to investigate the prevalence of DM and impaired fasting glucose (IFG).@*RESULTS@#A total of 5 184 valid questionnaires were collected. Fourteen children (0.27%) were found to have DM, among whom 8 had type 1 DM, 2 had type 2 DM, and 4 had unclassified DM. Twenty-nine children (0.56%) were found to have IFG. There was no significant difference in the prevalence rate of DM and IFG between boys and girls (P>0.05). The prevalence rate of DM was 0.18% in the 4-<10 years group, 0.47% in the 10-<15 years group, and 0.07% in the 15-18 years group (P=0.072).The prevalence rate of IFG in the above three age groups was 0.18%, 0.94%, and 0.42%, respectively, with a significant difference among groups (P=0.007). The proportion of family history of DM and the proportion of overweight/obesity in children with DM were significantly higher than those in children without DM (P<0.05), while the proportion of children with DM who preferred coarse grains was significantly lower than that in children without DM (P<0.05).@*CONCLUSIONS@#The prevalence of DM and IFG in Uyghur children in Hotan Prefecture of Xinjiang is relatively low. There is no significant difference in the prevalence of DM among children of different genders or age groups, but the prevalence of IFG in children of different age groups is different. A family history of DM, overweight or obesity, and low intake of coarse grains might be associated with the development of DM.

PLGA/ß-TCP composite scaffold incorporating cucurbitacin B promotes bone regeneration by inducing angiogenesis.

OBJECTIVES: Vascularization is an essential step in successful bone tissue engineering. The induction of angiogenesis in bone tissue engineering can be enhanced through the delivery of therapeutic agents that stimulate vessel and bone formation. In this study, we show that cucurbitacin B (CuB), a tetracyclic terpene derived from Cucurbitaceae family plants, facilitates the induction of angiogenesis in vitro. METHODS: We incorporated CuB into a biodegradable poly (lactide-co-glycolide) (PLGA) and ß-tricalcium phosphate (ß-TCP) biomaterial scaffold (PT/CuB) Using 3D low-temperature rapid prototyping (LT-RP) technology. A rat skull defect model was used to verify whether the drug-incorporated scaffold has the effects of angiogenesis and osteogenesis in vivo for the regeneration of bone defect. Cytotoxicity assay was performed to determine the safe dose range of the CuB. Tube formation assay and western blot assay were used to analyze the angiogenesis effect of CuB. RESULTS: PT/CuB scaffold possessed well-designed bio-mimic structure and improved mechanical properties. CuB was linear release from the composite scaffold without affecting pH value. The results demonstrated that the PT/CuB scaffold significantly enhanced neovascularization and bone regeneration in a rat critical size calvarial defect model compared to the scaffold implants without CuB. Furthermore, CuB stimulated angiogenic signaling via up-regulating VEGFR2 and VEGFR-related signaling pathways. CONCLUSION: CuB can serve as promising candidate compound for promoting neovascularization and osteogenesis, especially in tissue engineering for repair of bone defects. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This study highlights the potential use of CuB as a therapeutic agent and strongly support its adoption as a component of composite scaffolds for tissue-engineering of bone repair.

An UHPLC-MS/MS method for simultaneous determination of ten sex steroid hormones in ovariectomy-induced osteoporosis rat and its application in discovery of sex steroid hormones regulatory components of Xian-Ling-Gu-Bao capsule.

Sex steroid hormones could directly affect the bone metabolism by regulating cell physiological functions. In female, it inevitably causes the abnormal levels of sex steroid hormones at post-menopause in vivo. Ovariectomized rats and mice are classic animal models of osteoporosis to better understand the action mechanism of anti-osteoporosis drugs. However, it is not clear whether Xian-Ling-Gu-Bao capsule (XLGB), a kidney-tonifying traditional Chinese medicine prescription, treat osteoporosis via regulating multiple sex steroid hormones. In the present study, a reliable method involving ultra high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/TQ-XS-MS) was developed for simultaneous quantitative analysis of ten sex steroid hormones (three estrogens, five androgens and two progestogens) in rat and mouse serum. The results of methodology were acceptable. The validated method was then successfully applied in the determination of the levels of sex steroid hormones in ovariectomy-induced osteoporosis rats, as well as drug (17ß-estradiol and XLGB) intervened rats. As a result, XLGB could not only significantly increase the level of 17ß-estradiol, but also improve the levels of progesterone, 17α-hydroxyprogesterone and androstenedione. Combined with molecular docking results and pharmacokinetic parameters, psoralen, isopsoralen and sweroside were considered as the key effective components of XLGB to activate adenylyl cyclase on promoting the biosynthesis of multiple sex steroid hormones. It is the first time to evaluate the regulatory effect of kidney-tonifying traditional Chinese medicine prescription on the levels of steroids in ovariectomy-induced osteoporosis rat, as well as the potential substance basis and mechanism of steroid hormone regulation.

Inhibition of AXL enhances chemosensitivity of human ovarian cancer cells to cisplatin via decreasing glycolysis.

Anexelekto (AXL), a member of the TYRO3-AXL-MER (TAM) family of receptor tyrosine kinases (RTK), is overexpressed in varieties of tumor tissues and promotes tumor development by regulating cell proliferation, migration and invasion. In this study, we investigated the role of AXL in regulating glycolysis in human ovarian cancer (OvCa) cells. We showed that the expression of AXL mRNA and protein was significantly higher in OvCa tissue than that in normal ovarian epithelial tissue. In human OvCa cell lines suppression of AXL significantly inhibited cell proliferation, and increased the sensitivity of OvCa cells to cisplatin, which also proved by nude mice tumor formation experiment. KEGG analysis showed that AXL was significantly enriched in the glycolysis pathways of cancer. Changes in AXL expression in OvCa cells affect tumor glycolysis. We demonstrated that the promotion effect of AXL on glycolysis was mediated by phosphorylating the M2 isoform of pyruvate kinase (PKM2) at Y105. AXL expression was significantly higher in cisplatin-resistant OvCa cells A2780/DDP compared with the parental A2780 cells. Inhibition of AXL decreased the level of glycolysis in A2780/DDP cells, and increased the cytotoxicity of cisplatin against A2780/DDP cells, suggesting that AXL-mediated glycolysis was associated with cisplatin resistance in OvCa. In conclusion, this study demonstrates for the first time that AXL is involved in the regulation of the Warburg effect. Our results not only highlight the clinical value of targeting AXL, but also provide theoretical basis for the combination of AXL inhibitor and cisplatin in the treatment of OvCa.

Bruceine D inhibits HIF-1α-mediated glucose metabolism in hepatocellular carcinoma by blocking ICAT/β-catenin interaction

@#Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths, characterized by highly hypoxic tumor microenvironment. Hypoxia-inducible factor-1α (HIF-1α) is a major regulator involved in cellular response to changes of oxygen levels, supporting the adaptation of tumor cells to hypoxia. Bruceine D (BD) is an isolated natural quassinoid with multiple anti-cancer effects. Here, we identified BD could significantly inhibit the HIF-1α expression and its subsequently mediated HCC cell metabolism. Using biophysical proteomics approaches, we identified inhibitor of β-catenin and T-cell factor (ICAT) as the functional target of BD. By targeting ICAT, BD disrupted the interaction of β-catenin and ICAT, and promoted β-catenin degradation, which in turn induced the decrease of HIF-1α expression. Furthermore, BD could inhibit HCC cells proliferation and tumor growth in vivo, and knockdown of ICAT substantially increased resistance to BD treatment in vitro. Our data highlight the potential of BD as a modulator of β-catenin/HIF-1α axis mediated HCC metabolism.

PKM2-c-Myc-Survivin Cascade Regulates the Cell Proliferation, Migration, and Tamoxifen Resistance in Breast Cancer.

The M2 isoform of pyruvate kinase (PKM2), as a key glycolytic enzyme, plays important roles in tumorigenesis and chemotherapeutic drug resistance. However, the intricate mechanism of PKM2 as a protein kinase regulating breast cancer progression and tamoxifen resistance needs to be further clarified. Here, we reported that PKM2 controls the expression of survivin by phosphorylating c-Myc at Ser-62. Functionally, PKM2 knockdown suppressed breast cancer cell proliferation and migration, which could be rescued by overexpression of survivin. Interestingly, we found that the level of PKM2 expression was upregulated in the tamoxifen resistant breast cancer cells MCF-7/TAMR, and knockdown of PKM2 sensitized the cells to 4-hydroxytamoxifen (4OH-T). In addition, the elevated level of PKM2 correlates with poor relapse-free survival in breast cancer patients treated with tamoxifen. Overall, our findings demonstrated that PKM2-c-Myc-survivin cascade regulated the proliferation, migration and tamoxifen resistance of breast cancer cells, suggesting that PKM2 represents a novel prognostic marker and an attractive target for breast cancer therapeutics, and that PKM2 inhibitor combined with tamoxifen may be a promising strategy to reverse tamoxifen resistance in breast cancer patients.

RSK2 protects human breast cancer cells under endoplasmic reticulum stress through activating AMPKα2-mediated autophagy.

Autophagy can protect stressed cancer cell by degradation of damaged proteins and organelles. However, the regulatory mechanisms behind this cellular process remain incompletely understood. Here, we demonstrate that RSK2 (p90 ribosomal S6 kinase 2) plays a critical role in ER stress-induced autophagy in breast cancer cells. We demonstrated that the promotive effect of RSK2 on autophagy resulted from directly binding of AMPKα2 in nucleus and phosphorylating it at Thr172 residue. IRE1α, an ER membrane-associated protein mediating unfolded protein response (UPR), is required for transducing the signal for activation of ERK1/2-RSK2 under ER stress. Suppression of autophagy by knockdown of RSK2 enhanced the sensitivity of breast cancer cells to ER stress both in vitro and in vivo. Furthermore, we demonstrated that inhibition of RSK2-mediated autophagy rendered breast cancer cells more sensitive to paclitaxel, a chemotherapeutic agent that induces ER stress-mediated cell death. This study identifies RSK2 as a novel controller of autophagy in tumor cells and suggests that targeting RSK2 can be exploited as an approach to reinforce the efficacy of ER stress-inducing agents against cancer.

Cinnamaldehyde Inhibits Inflammation of Human Synoviocyte Cells Through Regulation of Jak/Stat Pathway and Ameliorates Collagen-Induced Arthritis in Rats.

Cinnamaldehyde (Cin), a bioactive cinnamon essential oil from traditional Chinese medicine herb Cinnamomum cassia, has been reported to have multipharmacological activities including anti-inflammation. However, its role and molecular mechanism of anti-inflammatory activity in musculoskeletal tissues remains unclear. Here, we first investigated the effects and molecular mechanisms of Cin in human synoviocyte cells. Then in vivo therapeutic effect of Cin on collagen-induced arthritis (CIA) also studied. Cell Counting Kit CCK-8 assay was performed to evaluate the cell cytotoxicity. Proinflammatory cytokine expression was evaluated using quantitative polymerase chain reaction and ELISA. Protein expression was measured by western blotting. The in vivo effect of Cin (75 mg/kg per day) was evaluated in rats with CIA by gavage administration. Disease progression was assessed by clinical scoring, radiographic, and histologic examinations. Cin significantly inhibited interleukin (IL)-1ß-induced IL-6, IL-8, and tumor necrosis factor-α release from human synoviocyte cells. The molecular analysis revealed that Cin impaired IL-6-induced activation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 1 (STAT1), and STAT3 signaling pathway by inhibiting the phosphorylation of JAK2, STAT1, and STAT3, without affecting NF-κB pathway. Cin reduced collagen-induced swollen paw volume of arthritic rats. The anti-inflammation effects of Cin were associated with decreased severity of arthritis, joint swelling, and reduced bone erosion and destruction. Furthermore, serum IL-6 level was decreased when Cin administered therapeutically to CIA rats. Cin suppresses IL-1ß-induced inflammation in synoviocytes through the JAK/STAT pathway and alleviated collagen-induced arthritis in rats. These data indicated that Cin might be a potential traditional Chinese medicine-derived, disease-modifying, antirheumatic herbal drug. SIGNIFICANCE STATEMENT: In this study, we found that cinnamaldehyde (Cin) suppressed proinflammatory cytokines secretion in rheumatology arthritis synoviocyte cells by Janus kinase/signal transducer and activator of transcription pathway. The in vivo results showed that Cin ameliorated collagen-induced arthritis in rats. These findings indicate that Cin is a potential traditional Chinese medicine-derived, disease-modifying, antirheumatic herbal drug.

Magnesium and vitamin C supplementation attenuates steroid-associated osteonecrosis in a rat model.

Magnesium (Mg)-based biometal attracts clinical applications due to its biodegradability and beneficial biological effects on tissue regeneration, especially in orthopaedics, yet the underlying anabolic mechanisms in relevant clinical disorders are lacking. The present study investigated the effect of magnesium (Mg) and vitamin C (VC) supplementation for preventing steroid-associated osteonecrosis (SAON) in a rat experimental model. In SAON rats, 50 mg/kg Mg, or 100 mg/kg VC, or combination, or water control was orally supplemented daily for 2 or 6 weeks respectively. Osteonecrosis was evaluated by histology. Serum Mg, VC, and bone turnover markers were measured. Microfil-perfused samples prepared for angiography and trabecular architecture were evaluated by micro-CT. Primary bone marrow cells were isolated from each group to evaluate their potentials in osteoblastogenesis and osteoclastogenesis. The mechanisms were tested in vitro. Histological evaluation showed SAON lesions in steroid treated groups. Mg and VC supplementation synergistically reduced the apoptosis of osteocytes and osteoclast number, and increased osteoblast surface. VC supplementation significantly increased the bone formation marker PINP, and the combination significantly decreased the bone resorption marker CTX. TNFα expression and oxidative injury were decreased in bone marrow in Mg/VC/combination group. Mg significantly increased the blood perfusion in proximal tibia and decreased the leakage particles in distal tibia 2 weeks after SAON induction. VC significantly elevated the osteoblast differentiation potential of marrow cells and improved the trabecular architecture. The combination supplementation significantly inhibited osteoclast differentiation potential of marrow cells. In vitro study showed promoting osteoblast differentiation effect of VC, and anti-inflammation and promoting angiogenesis effect of Mg with underlying mechanisms. Mg and VC supplementation could synergistically alleviate SAON in rats, indicating great translational potentials of metallic minerals for preventing SAON.

Combating COVID-19 with integrated traditional Chinese and Western medicine in China

COVID-19, an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread throughout the world. China has achieved rapid containment of this highly infectious disease following the principles of early detection, early quarantine and early treatment with integrated traditional Chinese and Western medicine. The inclusion of traditional Chinese medicine (TCM) in the Chinese protocol is based on its successful historic experience in fighting against pestilence. Current findings have shown that the Chinese medicine can reduce the incidence of severe or critical events, improve clinical recovery and help alleviate symptoms such as cough or fever. To date there are over 133 ongoing registered clinical studies on TCM/integrated traditional Chinese and Western medicine. The three Chinese patent medicines (/ (Forsythiae and Honeysuckle Flower Pestilence-Clearing Granules/Capsules), (Honeysuckle Flower Cold-Relieving Granules) and (Stasis-Resolving & Toxin-Removing) were officially approved by the National Medical Products Administration to list COVID-19 as an additional indication. The pharmacological studies have suggested that Chinese medicine is effective for COVID-19 probably through its host-directed regulation and certain antiviral effects.

Regulation of Inflammatory Response in Human Osteoarthritic Chondrocytes by Novel Herbal Small Molecules.

In this study, 34 Traditional Chinese Medicine (TCM) compounds were screened for potential anabolic and anti-inflammatory properties on human osteoarthritic (OA) chondrocytes. The anabolic effects were assessed by measuring the glycosaminoglycan (GAG) relative to the DNA content using a 3D pellet culture model. The most chondrogenic compounds were tested in an inflammatory model consisting of 3 days of treatment with cytokines (IL-1ß/TNF-α) with or without supplementation of TCM compounds. The anti-inflammatory effects were assessed transcriptionally, biochemically and histologically. From the 34 compounds, Vanilic acid (VA), Epimedin A (Epi A) and C (Epi C), 2''-O-rhamnosylicariside II (2-O-rhs II), Icariin, Psoralidin (PS), Protocatechuicaldehyde (PCA), 4-Hydroxybenzoic acid (4-HBA) and 5-Hydroxymethylfurfural (5-HMF) showed the most profound anabolic effects. After induction of inflammation, pro-inflammatory and catabolic genes were upregulated, and GAG/DNA was decreased. VA, Epi C, PS, PCA, 4-HBA and 5-HMF exhibited anti-catabolic and anti-inflammatory effects and prevented the up-regulation of pro-inflammatory markers including metalloproteinases and cyclooxygenase 2. After two weeks of treatment with TCM compounds, the GAG/DNA ratio was restored compared with the negative control group. Immunohistochemistry and Safranin-O staining confirmed superior amounts of cartilaginous matrix in treated pellets. In conclusion, VA, Epi C, PS, PCA, 4-HBA and 5-HMF showed promising anabolic and anti-inflammatory effects.

Identification of a Novel Bcl-2 Inhibitor by Ligand-Based Screening and Investigation of Its Anti-cancer Effect on Human Breast Cancer Cells.

Bcl-2 family protein is an important factor in regulating apoptosis and is associated with cancer. The anti-apoptotic proteins of Bcl-2 family, such as Bcl-2, are overexpression in numerous tumors, and contribute to cancer formation, development, and therapy resistance. Therefore, Bcl-2 is a promising target for drug development, and several Bcl-2 inhibitors are currently undergoing clinical trials. In this study, we carried out a QSAR-based virtual screening approach to develop potential Bcl-2 inhibitors from the SPECS database. Surface plasmon resonance (SPR) binding assay was performed to examine the interaction between Bcl-2 protein and the screened inhibitors. After that, we measured the anti-tumor activities of the 8 candidate compounds, and found that compound M1 has significant cytotoxic effect on breast cancer cells. We further proved that compound M1 downregulated Bcl-2 expression and activated apoptosis by inducing mitochondrial dysfunction. In conclusion, we identified a novel Bcl-2 inhibitor by QSAR screening, which exerted significant cytotoxic activity in breast cancer cells through inducing mitochondria-mediated apoptosis.

Effect and mechanism of psoralidin on promoting osteogenesis and inhibiting adipogenesis.

BACKGROUND: Psoralidin (PL), a prenylated coumestrol, is isolated from Psoralea corylifolia L. (Fabaceae), which is frequently used for treatment of osteoporosis. PURPOSE: This study was designed to investigate the dual effects and potential mechanism of PL on promoting osteogenesis and inhibiting adipogenesis. METHODS: Bone marrow mesenchymal stem cells (BMSCs) were used to investigate the effect of PL on stimulating osteogenesis and inhibiting adipogenesis, while preosteoblast MC3T3-E1 cells and preadipocyte 3T3-L1 cells were employed to explore the potential mechanisms. Estradiol (E2) and ICI 182,780 (ICI) were used as the specific agonist and antagonist of classical estrogen receptors (ER), respectively, to interfere with classical ER signaling. Meanwhile, G-1 and G-15 were introduced as the selective agonist and antagonist of G protein coupled receptor 30 (GRP30, a membrane ER) to further clarify if membrane ER involved in PL mediating osteogenesis and adipogenesis RESULTS: PL not only promoted mineralization, but also inhibited adipocytes formation of BMSCs. In terms of osteogenesis, PL enhanced calcium nodule formation, alkaline phosphatase activity and osteocalcin levels in MC3T3-E1 cells. As for adipogenesis, PL decreased adipocyte formation in 3T3-L1 cells through down-regulating several mRNA expressions and protein synthesis of adipogenesis related factors. ICI completely blocked the effect of PL in promoting osteogenesis, but only partially suppressed its effect in inhibition of adipogenesis, while G-15 partially suppressed the effect of PL on promoting mineralization and inhibiting oil drop formation. Furthermore, during suppression of adipocyte differentiation, PL regulated protein kinase B / glycogen synthase kinase 3ß / ß-catenin signaling pathway. CONCLUSION: PL promoted osteogenesis via mediating classical ER pathway, and inhibited adipocytes formation by regulating combined classical and membrane ER pathways. PL might be a potential candidate for the treatment of postmenopausal osteoporosis by modulating the competitive relationship between osteogenesis and adipogenesis of bone marrow mesenchymal stem cells.

Eukaryotic elongation factor-2 kinase regulates the cross-talk between autophagy and pyroptosis in doxorubicin-treated human melanoma cells in vitro.

Eukaryotic elongation factor-2 kinase (eEF-2K), a negative regulator of protein synthesis, has been shown to play an important role in modulating autophagy and apoptosis in tumor cells under various stresses. In this study, we investigated the regulatory role of eEF-2K in pyroptosis (a new form of programmed necrosis) in doxorubicin-treated human melanoma cells. We found that doxorubicin (0.5-5 µmol/L) induced pyroptosis in melanoma cell lines SK-MEL-5, SK-MEL-28, and A-375 with high expression of DFNA5, but not in human breast cancer cell line MCF-7 with little expression of DFNA5. On the other hand, doxorubicin treatment activated autophagy in the melanoma cells; inhibition of autophagy by transfecting the cells with siRNA targeting Beclin1 or by pretreatment with chloroquine (20 µmol/L) significantly augmented pyroptosis, thus sensitizing the melanoma cells to doxorubicin. We further demonstrated that doxorubicin treatment activated eEF-2K in the melanoma cells, and silencing of eEF-2K blunted autophagic responses, but promoted doxorubicin-induced pyroptotic cell death. Taken together, the above results demonstrate that eEF-2K dictates the cross-talk between pyroptosis and autophagy in doxorubicin-treated human melanoma cells; suppression of eEF-2K results in inhibiting autophagy and augmenting pyroptosis, thus modulating the sensitivity of melanoma cells to doxorubicin, suggesting that targeting eEF-2K may reinforce the antitumor efficacy of doxorubicin, offering a new insight into tumor chemotherapy.

Tubeimoside-1, a triterpenoid saponin, induces cytoprotective autophagy in human breast cancer cells in vitro via Akt-mediated pathway.

Autophagy, a form of cellular self-digestion by lysosome, is associated with various disease processes including cancers, and modulating autophagy has shown promise in the treatment of various malignancies. A number of natural products display strong antitumor activity, yet their mechanisms of action remain unclear. To gain a better understanding of how traditional Chinese medicine agents exert antitumor effects, we screened 480 natural compounds for their effects on autophagy using a high content screening assay detecting GFP-LC3 puncta in HeLa cells. Tubeimoside-1 (TBMS1), a triterpenoid saponin extracted from Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae), was identified as a potent activator of autophagy. The activation of autophagy by TBMS1 was evidenced by increased LC3-II amount and GFP-LC3 dots, observation of autophagosomes under electron microscopy, and enhanced autophagic flux. To explore the mechanisms underlying TBMS1-activated autophagy, we performed cheminformatic analyses and surface plasmon resonance (SPR) binding assay that showed a higher likelihood of the binding between Akt protein and TBMS1. In three human breast cancer cell lines, we demonstrated that Akt-mTOR-eEF-2K pathway was involved in TBMS1-induced activation of autophagy, while Akt-mediated downregulations of Mcl-1, Bcl-xl, and Bcl-2 led to the activation of apoptosis of the breast cancer cells. Inhibition of autophagy enhanced the cytotoxic effect of TBMS1 via promoting apoptosis. Our results demonstrate the role and mechanism of TBMS1 in activating autophagy, suggesting that inhibition of cytoprotective autophagy may act as a therapeutic strategy to reinforce the activity of TBMS1 against cancers.