Energy stress induced cardiac autophagy detection via a chronic and dynamic cardiomyocytes-based biosensing platform.

Hypoglycemia is a common complication which occurs during the treatment of diabetes, closely associated with cardiovascular events. A sudden decrease in blood glucose increases the risk of arrhythmia, which can lead to sudden cardiac death. This event is usually accompanied by abnormal electrophysiological activities in cardiomyocytes. However, traditional models do not efficiently reflect real-time cardiomyocyte electrophysiological changes under various glucose deprivation conditions in a large-scale and high-throughput manner. Therefore, we need to develop a new biosensing platform to aid in related scientific research. In this study, a cardiomyocyte-based biosensor was developed for real-time, noninvasive monitoring of the electrophysiological responses of cardiomyocytes under different glucose concentrations. The findings show that low-glucose conditions result in abnormal electrophysiology in cardiomyocytes, but autophagy enables cells to survive this adversity. Inhibition of autophagy exacerbates electrophysiological abnormalities, and long-term glucose starvation causes irreversible damage to cardiomyocytes. The proposed chronic and dynamic cardiomyocyte-based biosensing platform provides a new tool for understanding the effects of hypoglycemia on the in vitro cardiomyocyte-based heart model, revealing that autophagy has the potential to be an alternative treatment for diabetes and hypoglycemia.

Scalable Drug-Mimicking Nanoplasmonic Therapy for Bradyarrhythmia in Cardiomyocytes.

Bradyarrhythmia poses a serious threat to human health, with chronic progression causing heart failure and acute onset leading to sudden death. In this study, we develop a scalable drug-mimicking nanoplasmonic therapeutic strategy by introducing gold nanorod (Au NR) mediated near-infrared (NIR) photothermal effects. An integrated sensing and regulation platform is established for in situ synchronized NIR laser regulation and electrophysiological property recording. The Au NR plasmonic regulation enables the restoration of normal cardiomyocyte rhythm from the bradyarrhythmia. By regulating the aspect ratio and concentration of Au NRs, as well as the intensity and time of NIR irradiation, we precisely optimized the plasmonic photothermal effect to explore effective therapeutic strategies. Furthermore, mRNA sequencing revealed a significant increase in the number of differentially expressed genes (DEGs) involved in the electrophysiological activities of cardiomyocytes following photothermal therapy. Au NR-mediated plasmonic photothermal therapy, as an efficient and noninvasive approach to bradyarrhythmia, holds profound implications for cardiology research.

A smart tablet-phone-based high-performance pancreatic cancer cell biosensing system for drug screening.

Exploring more efficient pancreatic cancer drug screening platforms is of significant importance for accelerating the drug development process. In this study, we developed a high-sensitivity bioluminescence system based on smartphones and smart tablets, and constructed a pancreatic cancer drug screening platform (PCDSP) by combining the pancreatic cancer cell sensing model (PCCSM) on the multiwell plates (MTP). A smart tablet was used as the light source and a smartphone as the colorimetric sensing device. The smartphone dynamically controls the color and brightness displayed on the smart tablet to achieve lower LOD and wider detection ranges. We constructed PCCSM for 24 h, 48 h, and 72 h , and performed colorimetric experiments using both PCDSP and a commercial plate reader (CPR). The results showed that the PCDSP had a lower LOD than that of CPR. Moreover, PCDSP even exhibited a lower LOD for 24 h PCCSM testing compared to CPR for 48 h PCCSM testing, effectively shortening the drug evaluation process. Additionally, the PCDSP offers higher portability and efficiency compared with CPR, making it a promising platform for efficient pancreatic cancer drug screening.

Elevating intracellular action potential recording in cardiomyocytes: A precision-enhanced and biosafe single-pulse electroporation system.

Action potentials play a pivotal role in diverse cardiovascular physiological mechanisms. A comprehensive understanding of these intricate mechanisms necessitates a high-fidelity intracellular electrophysiological investigative approach. The amalgamation of micro-/nano-electrode arrays and electroporation confers substantial advantages in terms of high-resolution intracellular recording capabilities. Nonetheless, electroporation systems typically lack precise control, and commonly employed electroporation modes, involving tailored sequences, may escalate cellular damage and perturbation of normal physiological functions due to the multiple or higher-intensity electrical pulses. In this study, we developed an innovative electrophysiological biosensing system customized to facilitate precise single-pulse electroporation. This advancement serves to achieve optimal and uninterrupted intracellular action potential recording within cardiomyocytes. The refinement of the single-pulse electroporation technique is realized through the integration of the electroporation and assessment biosensing system, thereby ensuring a consistent and reliable means of achieving stable intracellular access. Our investigation has unveiled that the optimized single-pulse electroporation technique not only maintains robust biosafety standards but also enables the continuous capture of intracellular electrophysiological signals across an expansive three-day period. The universality of this biosensing system, adaptable to various micro/nano devices, furnishes real-time analysis and feedback concerning electroporation efficacy, guaranteeing the sustained, secure, and high-fidelity acquisition of intracellular data, thereby propelling the field of cardiovascular electrophysiological research.

Dynamic and Label-Free Sensing of Cardiomyocyte Responses to Nanosized Vesicles for Cardiac Oxidative Stress Injury Therapy.

Cardiac oxidative stress is a significant phenotype of myocardial infarction disease, a leading cause of global health threat. There is an urgent need to develop innovative therapies. Nanosized extracellular vesicle (nEV)-based therapy shows promise, yet real-time monitoring of cardiomyocyte responses to nEVs remains a challenge. In this study, a dynamic and label-free cardiomyocyte biosensing system using microelectrode arrays (MEAs) was constructed. Cardiomyocytes were cultured on MEA devices for electrophysiological signal detection and treated with nEVs from E. coli, gardenia, HEK293 cells, and mesenchymal stem cells (MSC), respectively. E. coli-nEVs and gardenia-nEVs induced severe paroxysmal fibrillation, revealing distinct biochemical communication compared to MSC-nEVs. Principal component analysis identified variations and correlations between nEV types. MSC-nEVs enhanced recovery without inducing arrhythmias in a H2O2-induced oxidative stress injury model. This study establishes a fundamental platform for assessing biochemical communication between nEVs and cardiomyocytes, offering new avenues for understanding nEVs' functions in the cardiovascular system.

Leverage of nuclease-deficient CasX for preventing pathological angiogenesis.

Gene editing with a CRISPR/Cas system is a novel potential strategy for treating human diseases. Pharmacological inhibition of phosphoinositide 3-kinase (PI3K) δ suppresses retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Here we show that an innovative system of adeno-associated virus (AAV)-mediated CRISPR/nuclease-deficient (d)CasX fused with the Krueppel-associated box (KRAB) domain is leveraged to block (81.2% ± 6.5%) in vitro expression of p110δ, the catalytic subunit of PI3Kδ, encoded by Pik3cd. This CRISPR/dCasX-KRAB (4, 269 bp) system is small enough to be fit into a single AAV vector. We then document that recombinant AAV serotype (rAAV)1 efficiently transduces vascular endothelial cells from pathologic retinal vessels, which show high expression of p110δ; furthermore, we demonstrate that blockade of retinal p110δ expression by intravitreally injected rAAV1-CRISPR/dCasX-KRAB targeting the Pik3cd promoter prevents (32.1% ± 5.3%) retinal p110δ expression as well as pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy. These data establish a strong foundation for treating pathological angiogenesis by AAV-mediated CRISPR interference with p110δ expression.

Deciphering the metabolic profile and anti-colorectal cancer mechanism of Capilliposide A using ultra performance liquid chromatography mass spectrometry combined with non-targeted metabolomics studies.

Colorectal cancer is a highly prevalent malignancy that threatens human health worldwide. Despite the availability of chemotherapy as a primary treatment option, individuals with CRC undergoing frequent chemotherapy are susceptible to developing drug resistance, which can result in poor treatment outcomes. Consequently, there is an urgent need to discover new bioactive compounds for the treatment of CRC. Capilliposide A is a triterpenoid saponin that is extracted from Lysimachia capillipes Hemsl. Although it has been reported that LC-A exhibits good bioactivity, its metabolic profile and potential mechanism underlying its anti-CRC effects remain unknown. In this study, the metabolic products of LC-A in rat plasma, feces, and urine were identified using an LC-MS platform. In addition, LC-MS-based metabolomics was employed to investigate the mechanism of LC-A against CRC. The results showed that LC-A significantly inhibited CRC cell proliferation, attenuated tumor growth, and alleviated metabolic abnormalities in CRC-bearing mice. Furthermore, the levels of p-cresol sulfate and phenylacetylglycine in CRC model plasma decreased, with an increment in sphingosine 1-phosphate, D-tryptophan, and L-2-aminoadipic acid. These metabolite levels can be reversed by LC-A treatment. These metabolite alterations were related to the sphingolipid and amino acid metabolic pathways, demonstrating that LC-A anti-CRC effects were regulated through the modulation of underlying metabolism. Additionally, seven metabolites of LC-A were characterized in rat feces, plasma, and urine. This study offers a scientific foundation for elucidating the metabolism of LC-A and its treatment of colorectal cancer.

Capilliposide B inhibits the migration of prostate cancer by inducing autophagy through the ROS/AMPK/mTOR pathway.

Capilliposide B (CPS-B), a novel oleanane triterpenoid saponin derived from Lysimachia capillipes Hemsl, is a potent anticancer agent. However, its anticancer mechanism remains elusive. In the present study, we demonstrated the potent anti-tumor activity and molecular mechanism of CPS-B both in vitro and in vivo. Proteomic analysis using isobaric tags for relative and absolute quantitation techniques suggested that CPS-B modulated autophagy in prostate cancer (PC). Moreover, Western blotting showed that both autophagy and epithelial-mesenchymal transition occurred place after CPS-B treatment in vivo, which was also proven in PC-3 cancer cells. We deduced that CPS-B inhibited migration by inducing autophagy. We examined the accumulation of reactive oxygen species (ROS) in cells, and in downstream pathways, LKB1 and AMPK were activated while mTOR was inhibited. Transwell experiment results showed that CPS-B inhibited the metastasis of PC-3 cells and that this effect was significantly attenuated after pretreatment with chloroquine, indicating that CPS-B inhibited metastasis via autophagy induction. Altogether, these data suggest that CPS-B is a potential therapeutic agent for cancer treatment that acts by inhibiting migration through the ROS/AMPK/mTOR signaling pathway.

NFκB-Mediated Expression of Phosphoinositide 3-Kinase δ Is Critical for Mesenchymal Transition in Retinal Pigment Epithelial Cells.

Epithelial mesenchymal transition (EMT) plays a vital role in a variety of human diseases including proliferative vitreoretinopathy (PVR), in which retinal pigment epithelial (RPE) cells play a key part. Transcriptomic analysis showed that the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway was up-regulated in human RPE cells upon treatment with transforming growth factor (TGF)-ß2, a multifunctional cytokine associated with clinical PVR. Stimulation of human RPE cells with TGF-ß2 induced expression of p110δ (the catalytic subunit of PI3Kδ) and activation of NFκB/p65. CRISPR-Cas9-mediated depletion of p110δ or NFκB/p65 suppressed TGF-ß2-induced fibronectin expression and activation of Akt as well as migration of these cells. Intriguingly, abrogating expression of NFκB/p65 also blocked TGF-ß2-induced expression of p110δ, and luciferase reporter assay indicated that TGF-ß2 induced NFκB/p65 binding to the promoter of the PIK3CD that encodes p110δ. These data reveal that NFκB/p65-mediated expression of PI3Kδ is essential in human RPE cells for TGF-ß2-induced EMT, uncovering hindrance of TGF-ß2-induced expression of p110δ as a novel approach to inhibit PVR.

Biomimetic integrated gustatory and olfactory sensing array based on HL-1 cardiomyocyte facilitating drug screening for tachycardia treatment.

The ectopic co-expression of taste and olfactory receptors in cardiomyocytes provides not only possibilities for the construction of biomimetic gustatory and olfactory sensors but also promising novel therapeutic targets for tachycardia treatment. Here, bitter taste and olfactory receptors endogenously expressed in HL-1 cells were verified by RT-PCR and immunofluorescence staining. Then HL-1 cardiomyocyte-based integrated gustatory and olfactory sensing array coupling with the microelectrode array (MEA) was first constructed for drugs screening and evaluation for tachycardia treatment. The MEA sensor detected the extracellular field potentials and reflected the systolic-diastolic properties of cardiomyocytes in real time in a label-free and non-invasive way. The in vitro tachycardia model was constructed using isoproterenol as the stimulator. The proposed sensing array facilitated potential drug screening for tachycardia treatment, such as salicin, artemisinin, xanthotoxin, and azelaic acid which all activated specific receptors on HL-1 cells. IC50 values for four potential drugs were calculated to be 0.0036 µM, 309.8 µM, 14.68 µM, and 0.102 µM, respectively. Visualization analysis with heatmaps and PCA cluster showed that different taste and odorous drugs could be easily distinguished. The mean inter-class Euclidean distance between different bitter drugs was 1.681, which was smaller than the distance between bitter and odorous drugs of 2.764. And the inter-class distance was significantly higher than the mean intra-class Euclidean distance of 1.172. In summary, this study not only indicates a new path for constructing novel integrated gustatory and olfactory sensors but also provides a powerful tool for the quantitative evaluation of potential drugs for tachycardia treatment.

Guangsangon E triggers mitochondria dysfunction and mitophagy in triple-negative breast cancer and leads to non-apoptotic cell death.

Guangsangon E (GSE) is a natural product separated from Morus alba L. It has been reported to treat lung cancer through autophagy. However, whether GSE is effective in repressing triple-negative breast cancer (TNBC) cells is yet to be elucidated. In the present study, GSE inhibited cell growth of MDA-MB-231, MDA-MB-453, and MDA-MB-468 cells. Moreover, GSE induced mitochondrial dysfunction, including membrane potential loss, mitochondria fission, and reactive oxygen species accumulation, and finally led to mitophagy-related non-apoptotic cell death. In the xenograft tumor nude mice, GSE treatment significantly reduced the size and weight of MDA-MB-231 tumors. The tumor inhibition rates of GSE treatment were 49.68% (low-dose) and 48.73% (high-dose). In summary, GSE is a potential anticancer drug available for treating TNBC with apoptosis resistance.

Idelalisib inhibits experimental proliferative vitroretinopathy.

Proliferative vitreoretinopathy (PVR) is a fibrotic eye disease that develops after rhegmatogenous retinal detachment surgery and open-globe traumatic injury. Idelalisib is a specific inhibitor of phosphoinositide 3-kinase (PI3K) δ. While PI3Kδ is primarily expressed in leukocytes, its expression is also considerably high in retinal pigment epithelial (RPE) cells, which play a crucial part in the PVR pathogenesis. Herein we show that GeoMx Digital Spatial Profiling uncovered strong expression of fibronectin in RPE cells within epiretinal membranes from patients with PVR, and that idelalisib (10 µM) inhibited Akt activation, fibronectin expression and collagen gel contraction induced by transforming growth factor (TGF)-ß2 in human RPE cells. Furthermore, we discovered that idelalisib at a vitreal concentration of 10 µM, a non-toxic dose to the retina, prevented experimental PVR induced by intravitreally injected RPE cells in rabbits assessed by experienced ophthalmologists using an indirect ophthalmoscope plus a + 30 D fundus lens, electroretinography, optical coherence tomography and histological analysis. These data suggested idelalisib could be harnessed for preventing patients from PVR.

Comparative Proteomic and Metabolomic Analyses of Plasma Reveal the Novel Biomarker Panels for Thyroid Dysfunction.

BACKGROUND: Thyroid dysfunction, including hypothyroidism (THO) and hyperthyroidism (THE), commonly arise from pathological processes in the thyroid gland. The current diagnosis of thyroid dysfunction varies because of the age and sex of the patients. The aim of this study was to explore novel candidate biomarker panels for hypothyroidism and hyperthyroidism screening with mass spectrometry and bioinformatics. METHODS: Plasma samples were collected from 15 THE patients, 9 THO patients, and 15 healthy controls. Data Independent Acquisition(DIA)-based proteomic and untargeted metabolomic analyses were performed to identify novel biomarker panels for THO and THE patients. Finally, three candidate biomarkers were verified by ELISA in 34 samples. RESULTS: A total of 2738 proteins and 6103 metabolites were identified, and 173 proteins and 2487 metabolites were found to be differentially expressed among the THE, THO and control groups. The results of the ensemble feature selection, K-means clustering and least absolute shrinkage and selection operator (LASSO) regression model showed that two proteins (C4-A and C3/C5 convertase) combined with two metabolites (L-arginine and L-proline), and proteins (APOL1 and ITIH4) combined with metabolites (cortisol, and cortisone) identified by plasma proteomics and metabolomics could help distinguish THO and THE patients from healthy controls, respectively. CONCLUSIONS: This study identified and verified two pairs of biomarker panels that can be used to distinguish THE and THO patients regardless of age and sex. Consequently, our findings represent a comprehensive analysis of thyroid dysfunction plasma, which is significant for clinical diagnosis.

Mitochondrial proteomic analysis reveals the regulation of energy metabolism and reactive oxygen species production in Clematis terniflora DC. leaves under high-level UV-B radiation followed by dark treatment.

Clematis terniflora DC. is an important medicinal plant from the family Ranunculaceae. A previous study has shown that active ingredients in C. terniflora, such as flavonoids and coumarins, are increased under ultraviolet B radiation (UV-B) and dark treatment and that the numbers of genes related to the tricarboxylic acid cycle and mitochondrial electron transport chain (mETC) are changed. To uncover the mechanism of the response to UV-B radiation and dark treatment in C. terniflora, mitochondrial proteomics was performed. The results showed that proteins related to photorespiration, mitochondrial membrane permeability, the tricarboxylic acid cycle, and the mETC mainly showed differential expression profiles. Moreover, the increase in alternative oxidase indicated that another oxygen-consuming respiratory pathway in plant mitochondria was induced to minimize mitochondrial reactive oxygen species production. These results suggested that respiration and mitochondrial membrane permeability were deeply influenced to avoid energy consumption and maintain energy balance under UV-B radiation and dark treatment in C. terniflora leaf mitochondria. Furthermore, oxidative phosphorylation was able to regulate intracellular oxygen balance to resist oxidative stress. This study improves understanding of the function of mitochondria in response to UV-B radiation and dark treatment in C. terniflora. SIGNIFICANCE: C. terniflora was an important traditional Chinese medicine for anti-inflammatory. Previous study showed that the contents of coumarins which were the main active ingredient in C. terniflora were induced by UV-B radiation and dark treatment. In the present study, to uncover the regulatory mechanism of metabolic changes in C. terniflora, mitochondrial proteomics analysis of leaves was performed. The results showed that photorespiration and oxidative phosphorylation pathways were influenced under UV-B radiation and dark treatment. Mitochondria in C. terniflora leaf played a crucial role in energy mechanism and regulation of cellular oxidation-reduction to maintain cell homeostasis under UV-B radiation followed with dark treatment.

A Prognostic Signature Constructed by CTHRC1 and LRFN4 in Stomach Adenocarcinoma.

BACKGROUND: Stomach adenocarcinoma (STAD) is the most common histological type of stomach cancer, which causes a considerable number of deaths worldwide. This study aimed to identify its potential biomarkers with the notion of revealing the underlying molecular mechanisms. METHODS: Gene expression profile microarray data were downloaded from the Gene Expression Omnibus (GEO) database. The "limma" R package was used to screen the differentially expressed genes (DEGs) between STAD and matched normal tissues. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used for function enrichment analyses of DEGs. The STAD dataset from The Cancer Genome Atlas (TCGA) database was used to identify a prognostic gene signature, which was verified in another STAD dataset from the GEO database. CIBERSORT algorithm was used to characterize the 22 human immune cell compositions. The expression of LRFN4 and CTHRC1 in tissues was determined by quantitative real-time PCR from the patients recruited to the present study. RESULTS: Three public datasets including 90 STAD patients and 43 healthy controls were analyzed, from which 44 genes were differentially expressed in all three datasets. These genes were implicated in biological processes including cell adhesion, wound healing, and extracellular matrix organization. Five out of 44 genes showed significant survival differences. Among them, CTHRC1 and LRFN4 were selected for construction of prognostic signature by univariate Cox regression and stepwise multivariate Cox regression in the TCGA-STAD dataset. The fidelity of the signature was evaluated in another independent dataset and showed a good classification effect. The infiltration levels of multiple immune cells between high-risk and low-risk groups had significant differences, as well as two immune checkpoints. TIM-3 and PD-L2 were highly correlated with the risk score. Multiple signaling pathways differed between the two groups of patients. At the same time, the expression level of LRFN4 and CTHRC1 in tissues analyzed by quantitative real-time PCR were consistent with the in silico findings. CONCLUSION: The present study constructed the prognostic signature by expression of CTHRC1 and LRFN4 for the first time via comprehensive bioinformatics analysis, which provided the potential therapeutic targets of STAD for clinical treatment.

Chalcomoracin prevents vitreous-induced activation of AKT and migration of retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells are the major cell type in the epi- or sub-retinal membranes in the pathogenesis of proliferative vitreoretinopathy (PVR), which is a blinding fibrotic eye disease and still short of effective medicine. The purpose of this study is to demonstrate whether Chalocomoracin (CMR), a novel purified compound from fungus-infected mulberry leaves, is able to inhibit vitreous-induced signalling events and cellular responses intrinsic to PVR. Our studies have revealed that the CMR IC50 for ARPE-19 cells is 35.5 µmol/L at 72 hours, and that 5 µmol/L CMR inhibits vitreous-induced Akt activation and p53 suppression; in addition, we have discovered that this chemical effectively blocks vitreous-stimulated proliferation, migration and contraction of ARPE-19 cells, suggesting that CMR is a promising PVR prophylactic.

DHOK Exerts Anti-Cancer Effect Through Autophagy Inhibition in Colorectal Cancer.

DHOK (14,15ß-dihydroxyklaineanone) is a novel diterpene isolated from roots of Eurycoma longifolia Jack, a traditional herb widely applied in Southeast Asia. It is reported that DHOK has cytotoxic effect on cancer cells, but its anti-cancer mechanism has still been not clear. In our study, we first observed that DHOK inhibits cell proliferation of colorectal cancer cells in a time- and dose-dependent manner. Next, we performed transcriptome sequencing to identify the targets of DHOK and found that autophagy-related signaling pathways are involved under DHOK treatment. Indeed, in DHOK-treated cells, the level of autophagosome marker LC3 and the formation of GFP-LC3 puncta were decreased, indicating the reduction of autophagy. Moreover, confocal microscopy results revealed the lysosomal activity and the formation of autolysosomes are also inhibited. Our western blotting results demonstrated the activation of mammalian target of rapamycin (mTOR) signaling pathway by DHOK, which may be attributed to the enhancement of ERK and AKT activity. Functionally, activation of autophagy attenuated DHOK-caused cell death, indicating that autophagy serves as cell survival. In xenograft mouse model, our results also showed that DHOK activates the mTOR signaling pathway, decreases autophagy level and inhibits the tumorigenesis of colon cancer. Taken together, we revealed the molecular mechanism of DHOK against cancer and our results also demonstrate great potential of DHOK in the treatment of colorectal cancer.

Phosphoproteomics Reveals Regulation of Secondary Metabolites in Mahonia bealei Exposed to Ultraviolet-B Radiation.

Mahonia bealei (M. bealei) is a traditional Chinese medicine containing a high alkaloid content used to treat various diseases. Generally, only dried root and stem are used as medicines, considering that the alkaloid content in M. bealei leaves is lower than in the stems and roots. Some previous research found that alkaloid and flavonoid contents in the M. bealei leaves may increase when exposed to ultraviolet B (UV-B) radiation. However, the underlying mechanism of action is still unclear. In this study, we used titanium dioxide material enrichment and mass-based label-free quantitative proteomics techniques to explore the effect and mechanism of M. bealei leaves when exposed to UV-B treatment. Our data suggest that UV-B radiation increases the ATP content, photosynthetic pigment content, and some enzymatic/nonenzymatic indicators in the leaves of M. bealei. Moreover, phosphoproteomics suggests phosphoproteins related to mitogen-activated protein kinase (MAPK) signal transduction and the plant hormone brassinosteroid signaling pathway as well as phosphoproteins related to photosynthesis, glycolysis, the tricarboxylic acid cycle, and the amino acid synthesis/metabolism pathway are all affected by UV-B radiation. These results suggest that the UV-B radiation activates the oxidative stress response, MAPK signal transduction pathway, and photosynthetic energy metabolism pathway, which may lead to the accumulation of secondary metabolites in M. bealei leaves.

A novel Diels-Alder adduct of mulberry leaves exerts anticancer effect through autophagy-mediated cell death.

Guangsangon E (GSE) is a novel Diels-Alder adduct isolated from leaves of Morus alba L, a traditional Chinese medicine widely applied in respiratory diseases. It is reported that GSE has cytotoxic effect on cancer cells. In our research, we investigated its anticancer effect on respiratory cancer and revealed that GSE induces autophagy and apoptosis in lung and nasopharyngeal cancer cells. We first observed that GSE inhibits cell proliferation and induces apoptosis in A549 and CNE1 cells. Meanwhile, the upregulation of autophagosome marker LC3 and increased formation of GFP-LC3 puncta demonstrates the induction of autophagy in GSE-treated cells. Moreover, GSE increases the autophagy flux by enhancing lysosomal activity and the fusion of autophagosomes and lysosomes. Next, we investigated that endoplasmic reticulum (ER) stress is involved in autophagy induction by GSE. GSE activates the ER stress through reactive oxygen species (ROS) accumulation, which can be blocked by ROS scavenger NAC. Finally, inhibition of autophagy attenuates GSE-caused cell death, termed as "autophagy-mediated cell death." Taken together, we revealed the molecular mechanism of GSE against respiratory cancer, which demonstrates great potential of GSE in the treatment of representative cancer.

Combined ultraviolet and darkness regulation of medicinal metabolites in Mahonia bealei revealed by proteomics and metabolomics.

Roots of Mahonia bealei have been used as traditional Chinese medicine with antibacterial, antioxidant and anti-inflammatory properties due to its high alkaloid content. Previously, we reported that alkaloid and flavonoid contents in the M. bealei leaves could be increased by the combined ultraviolet B and dark treatment (UV+D). To explore the underlying metabolic pathways and networks, proteomic and metabolomic analyses of the M. bealei leaves were conducted. Proteins related to tricarboxylic acid cycle, transport and signaling varied greatly under the UV + D. Among them, calmodulin involved in calcium signaling and ATP-binding cassette transporter involved in transport of berberine were increased. Significantly changed metabolites were overrepresented in phenylalanine metabolism, nitrogen metabolism, phenylpropanoid, flavonoid and alkaloid biosynthesis. In addition, the levels of salicylic acid and gibberellin decreased in the UV group and increased in the UV + D group. These results indicate that multi-hormone crosstalk may regulate the biosynthesis of flavonoids and alkaloids to alleviate oxidative stress caused by the UV + D treatment. Furthermore, protoberberine alkaloids may be induced through calcium signaling crosstalk with reaction oxygen species and transported to leaves. SIGNIFICANCE: Mahonia bealei root and stem, not leaf, were used as traditional medicine for a long history because of the high contents of active components. In the present study, UV-B combined with dark treatments induced the production of alkaloids and flavonoids in the M. bealei leaf, especially protoberberine alkaloids such as berberine. Multi-omics analyses indicated that multi-hormone crosstalk, enhanced tricarboxylic acid cycle and active calcium signaling were involved. The study informs a strategy for utilization of the leaves, and improves understanding of the functions of secondary metabolites in M. bealei.