Transcriptomic profiling of adding cobalt chloride to improve dendrobine-type total alkaloid production.

Trichoderma longibrachiatum UN32 is known for its efficient production of dendrobine-type total alkaloids (DTTAs). This study aimed to determine the optimal medium composition for the UN32 strain using response surface methodology. Key factors, including glucose, beef extract, and CoCl2, were selected through the Plackett-Burman design. Subsequently, a factorial optimization approach was employed using the steepest ascent design, and 17 trial sets were completed via the Box-Behnken design. The optimal medium composition was found to consist of 29.4 g/L of glucose, 17.3 g/L of beef extract, and 0.28 mmol/L of CoCl2. This optimized medium resulted in an impressive 80.8% increase in mycelial dry weight (reaching 12.303 g/L) and a substantial 76.4% boost in DTTA production (reaching 541.63 ± 46.95 µg). Furthermore, the fermentation process was scaled up to a 5-L bioreactor, leading to a DTTA production approximately 1.95 times than the control. Transcriptome analysis of strain UN32 in response to CoCl2 supplementation revealed significant changes in the expression of critical genes associated with the TCA cycle and L-valine, L-leucine, and L-isoleucine biosynthesis changed. These alterations resulted in a heightened influx of acetyl-CoA into DTTA production. Additionally, the expression of genes related to antioxidant enzymes was modified to maintain homeostasis of reactive oxygen species (ROS). A potential mechanism for the accumulation of DTTAs based on ROS as a signal transduction was proposed. These findings provide valuable insights into the regulatory mechanisms of DTTA biosynthesis, potentially offering a method to enhance the production of secondary metabolites in the UN32 strain. KEY POINTS: • After the RSM optimization, there is a substantial increase of 80.8% in biomass production and a significant 76.4% rise in DTTA production. • Transcriptome analysis revealed that the inclusion of CoCl2 supplements resulted in an enhanced influx of acetyl-CoA. • Proposed a mechanism for the accumulation of DTTAs for the role of ROS as a signal transduction pathway.

Exploring the mechanism of dendrobine in treating metabolic associated fatty liver disease based on network pharmacology and experimental validation.

BACKGROUND: This study investigates the therapeutic mechanisms of dendrobine, a primary bioactive compound in Dendrobium nobile, for Metabolic Associated Fatty Liver Disease (MASLD) management. Utilizing network pharmacology combined with experimental validation, the clinical effectiveness of dendrobine in MASLD treatment was assessed and analyzed. RESULTS: The study demonstrates significant improvement in liver function among MASLD patients treated with Dendrobium nobile. Network pharmacology identified key targets such as Peroxisome Proliferator-Activated Receptor Gamma (PPARG), Interleukin 6 (IL6), Tumor Necrosis Factor (TNF), Interleukin 1 Beta (IL1B), and AKT Serine/Threonine Kinase 1 (AKT1), with molecular docking confirming their interactions. Additionally, dendrobine significantly reduced ALT and AST levels in palmitic acid-treated HepG2 cells, indicating hepatoprotective properties and amelioration of oxidative stress through decreased Malondialdehyde (MDA) levels and increased Superoxide Dismutase (SOD) levels. CONCLUSION: Dendrobine mitigates liver damage in MASLD through modulating inflammatory and immune responses and affecting lipid metabolism, potentially by downregulating inflammatory mediators like TNF, IL6, IL1B, and inhibiting AKT1 and Signal Transducer and Activator of Transcription 3 (STAT3). This study provides a theoretical basis for the application of dendrobine in MASLD treatment, highlighting its potential as a therapeutic agent.

Synthesis and in†vitro Anti-Inflammatory Activity of Novel Dendrobine Amide/Sulfonamide Derivatives.

A traditional Chinese medicine ingredient, dendrobine, has been demonstrated to have anti-inflammatory properties. However, due to its poor anti-inflammatory properties, its clinical use is limited. Consequently, we have designed and synthesized 32 new amide/sulfonamide dendrobine derivatives and screened their anti-inflammatory activities in vitro. Experiments showed that nitric oxide (NO) generation in lipopolysaccharide (LPS)-induced RAW264.7 cells was strongly reduced by derivative 14, with an IC50 of 2.96 µM. Western blot research revealed that 14 decreased the concentration-dependent expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (INOS). Molecular docking was used to predict the binding of the inflammation-associated proteins COX-2 and INOS to compound 14.

Dendrobine regulates STAT3 to attenuate mitochondrial dysfunction and senescence in vascular endothelial cells triggered by oxidized low-density lipoprotein.

Our previous studies have highlighted the potential therapeutic efficacy of dendrobine, an alkaloid, in atherosclerosis (AS), nevertheless, the underlying mechanism remains unclear. This study employs a combination of network pharmacology and in vitro experiments to explore the regulatory pathways involved. Through network pharmacology, the biological function for intersection targets between dendrobine and AS were identified. Molecular docking was conducted to investigate the interaction between the dominant target and dendrobine. Human umbilical vein endothelial cells (HUVECs) were treated with oxidized low-density lipoprotein (ox-LDL) to mimic AS, and the effects of dendrobine on cell viability, lipid deposition, mitochondrial function, and cellular senescence were evaluated. Subsequently, cells were treated with the mitophagy inhibitor Mdivi-1 and the STAT3 agonist colivelin to assess the role of mitophagy and STAT3 signaling in dendrobine regulation. Intersection targets were associated with biological processes, including reactive oxygen species production. Dendrobine attenuated the effects of ox-LDL treatment on HUVECs, mitigating changes in cell activity, lipid deposition, mitochondrial function, and cellular senescence. Both Mdivi-1 and colivelin treatments resulted in decreased cell viability and increased cellular senescence, with colivelin suppressing mitophagy. Cotreatment with Mdivi-1 and colivelin further aggravated cellular senescence and inhibited FoxO signaling. Together, this study indicated that dendrobine regulated the STAT3/FoxO signaling pathway, alleviating mitochondrial dysfunction and cellular senescence. This study contributes valuable insights to the potential clinical application of dendrobine.

Metabolic Pathway Engineering Improves Dendrobine Production in Dendrobium catenatum.

The sesquiterpene alkaloid dendrobine, widely recognized as the main active compound and a quality control standard of medicinal orchids in the Chinese Pharmacopoeia, demonstrates diverse biological functions. In this study, we engineered Dendrobium catenatum as a chassis plant for the production of dendrobine through the screening and pyramiding of key biosynthesis genes. Initially, previously predicted upstream key genes in the methyl-D-erythritol 4-phosphate (MEP) pathway for dendrobine synthesis, including 4-(Cytidine 5'-Diphospho)-2-C-Methyl-d-Erythritol Kinase (CMK), 1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase (DXR), 2-C-Methyl-d-Erythritol 4-Phosphate Cytidylyltransferase (MCT), and Strictosidine Synthase 1 (STR1), and a few downstream post-modification genes, including Cytochrome P450 94C1 (CYP94C1), Branched-Chain-Amino-Acid Aminotransferase 2 (BCAT2), and Methyltransferase-like Protein 23 (METTL23), were chosen due to their deduced roles in enhancing dendrobine production. The seven genes (SG) were then stacked and transiently expressed in the leaves of D. catenatum, resulting in a dendrobine yield that was two-fold higher compared to that of the empty vector control (EV). Further, RNA-seq analysis identified Copper Methylamine Oxidase (CMEAO) as a strong candidate with predicted functions in the post-modification processes of alkaloid biosynthesis. Overexpression of CMEAO increased dendrobine content by two-fold. Additionally, co-expression analysis of the differentially expressed genes (DEGs) by weighted gene co-expression network analysis (WGCNA) retrieved one regulatory transcription factor gene MYB61. Overexpression of MYB61 increased dendrobine levels by more than two-fold in D. catenatum. In short, this work provides an efficient strategy and prospective candidates for the genetic engineering of D. catenatum to produce dendrobine, thereby improving its medicinal value.

Dendrobine Alleviates Cellular Senescence and Osteoarthritis via the ROS/NF-κB Axis.

Osteoarthritis (OA) is a degenerative joint disease characterized by low-grade inflammation and cartilage degradation. Dendrobine (DEN) is reported to inhibit inflammation and oxidative stress in some diseases, but its role in chondrocyte senescence and OA progress has not yet been elucidated. Our study aimed to explore the protective effects of DEN on OA both in vitro and in vivo. We found that DEN inhibited extracellular matrix (ECM) degradation and promoted ECM synthesis. Meanwhile, DEN inhibited senescence-associated secretory phenotype (SASP) factors expression and senescence phenotype in IL-1ß-treated chondrocytes. Furthermore, DEN improved mitochondrial function and reduced the production of intracellular reactive oxygen species (ROS). Also, DEN suppressed IL-1ß-induced activation of the NF-κB pathway. Further, using NAC (ROS inhibitor), we found that DEN might inhibit NF-κB cascades by reducing ROS. Additionally, X-ray, micro-CT, and histological analyses in vivo demonstrated that DEN significantly alleviated cartilage inflammation, ECM degradation, and subchondral alterations in OA progression. In conclusion, DEN inhibits SASP factors expression and senescence phenotype in chondrocytes and alleviated the progression of OA via the ROS/NF-κB axis, which provides innovative strategies for the treatment of OA.

The Integration of the Metabolome and Transcriptome for Dendrobium nobile Lindl. in Response to Methyl Jasmonate.

Dendrobium nobile Lindl., as an endangered medicinal plant within the genus Dendrobium, is widely distributed in southwestern China and has important ecological and economic value. There are a variety of metabolites with pharmacological activity in D. nobile. The alkaloids and polysaccharides contained within D. nobile are very important active components, which mainly have antiviral, anti-tumor, and immunity improvement effects. However, the changes in the compounds and functional genes of D. nobile induced by methyl jasmonate (MeJA) are not clearly understood. In this study, the metabolome and transcriptome of D. nobile were analyzed after exposure to MeJA. A total of 377 differential metabolites were obtained through data analysis, of which 15 were related to polysaccharide pathways and 35 were related to terpenoids and alkaloids pathways. Additionally, the transcriptome sequencing results identified 3256 differentially expressed genes that were discovered in 11 groups. Compared with the control group, 1346 unigenes were differentially expressed in the samples treated with MeJA for 14 days (TF14). Moreover, the expression levels of differentially expressed genes were also significant at different growth and development stages. According to GO and KEGG annotations, 189 and 99 candidate genes were identified as being involved in terpenoid biosynthesis and polysaccharide biosynthesis, respectively. In addition, the co-expression analysis indicated that 238 and 313 transcription factors (TFs) may contribute to the regulation of terpenoid and polysaccharide biosynthesis, respectively. Through a heat map analysis, fourteen terpenoid synthetase genes, twenty-three cytochrome P450 oxidase genes, eight methyltransferase genes, and six aminotransferase genes were identified that may be related to dendrobine biosynthesis. Among them, one sesquiterpene synthase gene was found to be highly expressed after the treatment with MeJA and was positively correlated with the content of dendrobine. This study provides important and valuable metabolomics and transcriptomic information for the further understanding of D. nobile at the metabolic and molecular levels and provides candidate genes and possible intermediate compounds for the dendrobine biosynthesis pathway, which lays a certain foundation for further research on and application of Dendrobium.

Dendrobine protects HACAT cells from H2O2-induced oxidative stress and apoptosis damage via Nrf2/Keap1/ARE signaling pathway.

Skin offers protection, regulation, and sensation to the body. In collaboration with other stromal cells of the skin, keratinocytes, which differentiate from epidermis basal layers (low) to outer layers (high) leading to the stratum corneum, ensure that skin barrier function is achieved. Despite this, age-related inflammation and oxidative stress in the skin can negatively impact skin quality. Antioxidants can protect against skin damage, preventing skin aging or even reversing to some extent. Previous studies showed that Dendrobium Nobile (D. nobile) resists aging, prolongs life span, and attenuates oxidative damage and inflammation in various models. However, how D. nobile protects skin against aging or other damage is not well described yet. Therefore, in this study, a keratinocyte cell line (HACAT) was used to investigate the effect of dendrobine, the main active component of D. nobile, on oxidative damage in skin. We found that dendrobine reduced the level of intracellular reactive oxygen species by regulating the balance of antioxidant enzymes and oxidases, as well as decreased the cell apoptosis in H2O2-induced HACAT. Dendrobine also significantly activated the nuclear erythroid 2-related factor (Nrf2)/Keap1 signaling pathway. However, this antioxidant effect of dendrobine was abolished after Nrf2 gene being silenced. The results showed that dendrobine could resist the oxidative damage of skin cells, and its antioxidant function is related to the up-regulation of antioxidant enzymes as well as activation of Nrf2/Keap1 signaling pathway.

Metabolic characterization of a potent natural neuroprotective agent dendrobine in vitro and in rats.

Dendrobine is the main sesquiterpene alkaloid of Dendrobium nobile Lindl, which exhibits potent neuroprotective activity. However, its metabolism and disposition are little known. In this study, we investigated the metabolic characteristics of dendrobine in vitro and in rats. The metabolic stability and temporal profile of metabolites formation of dendrobine were assayed in human/rat liver microsomal and S9 fractions. Dendrobine metabolites were separated and identified mainly by UPLC-Q/Orbitrap MS. After oral administration of dendrobine (50 mg/kg) to rats, the accumulative excretion rate of dendrobine in feces, urine, and bile was 0.27%, 0.52%, and 0.031%, respectively, and low systematic exposure of dendrobine (AUC0-∞ = 629.2 ± 56.4 ng·h/mL) was observed. We demonstrated that the elimination of dendrobine was very rapid in liver microsomal incubation (the in vitro elimination t1/2 in rat and human liver microsomes was 1.35 and 5.61 min, respectively). Dendrobine underwent rapid and extensive metabolism; cytochrome P450, especially CYP3A4, CYP2B6, and CYP2C19, were mainly responsible for its metabolism. Aldehyde dehydrogenase, alcohol dehydrogenase and aldehyde oxidase were involved in the formation of carboxylic acid metabolites. By the aid of in-source fragmentation screening, hydrogen/deuterium exchange experiment, post-acquisition processing software, and available reference standards, 50 metabolites were identified and characterized in liver microsomal incubation and in rats. The major metabolic pathways of dendrobine were N-demethylation, N-oxidation, and dehydrogenation, followed by hydroxylation and glucuronidation. Collectively, the metabolic fate of dendrobine elucidated in this study not only yields benefits for its subsequent metabolism study but also facilitates to better understanding the mode of action of dendrobine and evaluating the pharmacologic efficiency of the high exposure metabolites.

Dendrobine modulates autophagy to alleviate ox-LDL-induced oxidative stress and senescence in HUVECs.

Dendrobine has potential advantages in suppressing atherosclerosis (AS). FK506-binding protein 1A (FKBP1A) is implicated in the regulation of autophagy, inflammation, and apoptosis. To reveal the mechanism by which dendrobine inhibits AS by modulating autophagy, oxidative stress, apoptosis, and senescence. An in vitro AS cell model was induced by culturing human umbilical vein endothelial cells (HUVECs) with oxidized low-density lipoprotein (ox-LDL). The cells were treated with dendrobine alone or in combination with short hairpin RNA (shRNA) targeting FKBP1A or together with 3-methyladenine (3MA), an autophagy inhibitor. Inflammatory cytokines levels tumor necrosis factor-α, interleukin-6 (IL-6), and IL-1ß were analyzed and oxidative stress levels were detected by the analysis of reactive oxygen species, malondialdehyde, and superoxide dismutase levels, followed by the analysis of apoptosis levels through terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Cell senescence was evaluated by senescence-associated ß-galactosidase and light chain 3 (LC3) levels were detected by immunofluorescence (IF) staining. The targeting relationship of dendrobine and FKBP1A was predicted by SwissTarget, PyMol, Autodock, and Open Babel software. Dendrobine reduced the levels of proinflammation factors, oxidative stress levels, apoptosis levels, and senescence phenotype in ox-LDL-induced HUVECs. Besides, cell viability has an opposite change. Furthermore, there was an increase in LC3 IF tensity, and LC3-II/I and Beclin1 expressions, and a decrease in p62 expression. However, these effects of dendrobine could be markedly destroyed by shRNA silencing FKBP1A and 3MA. Dendrobine can suppress inflammatory responses, oxidative stress, apoptosis, and senescence via FKBP1A-involved autophagy ox-LDL-treated HUVECs.

Recent advances and new insights in biosynthesis of dendrobine and sesquiterpenes.

Sesquiterpenes are one of the most diverse groups of secondary metabolites that have mainly been observed in terpenoids. It is a natural terpene containing 15 carbon atoms in the molecule and three isoprene units with chain, ring, and other skeleton structures. Sesquiterpenes have been shown to display multiple biological activities such as anti-inflammatory, anti-feedant, anti-microbial, anti-tumor, anti-malarial, and immunomodulatory properties; therefore, their therapeutic effects are essential. In order to overcome the problem of low-yielding sesquiterpene content in natural plants, regulating their biosynthetic pathways has become the focus of many researchers. In plant and microbial systems, many genetic engineering strategies have been used to elucidate biosynthetic pathways and high-level production of sesquiterpenes. Here, we will introduce the research progress and prospects of the biosynthesis of artemisinin, costunolide, parthenolide, and dendrobine. Furthermore, we explore the biosynthesis of dendrobine by evaluating whether the biosynthetic strategies of these sesquiterpene compounds can be applied to the formation of dendrobine and its intermediate compounds. KEY POINTS: • The development of synthetic biology has promoted the study of terpenoid metabolism and provided an engineering platform for the production of high-value terpenoid products. • Some possible intermediate compounds of dendrobine were screened out and the possible pathway of dendrobine biosynthesis was speculated. • The possible methods of dendrobine biosynthesis were explored and speculated.

[Correlation analysis and breeding application of agronomic traits and quality indexes in Dendrobium nobile].

The present study aimed to explore the correlation between agronomic traits and quality indexes of Dendrobium nobile and its application value in agricultural breeding. The cultivated strains of D. nobile in Hejiang-Chishui producing areas were extensively collected,and the main agronomic traits and quality indexes were measured. The agronomic traits with significant correlation with quality indexes were screened out by the correlation analysis,and then the parental lines and self-bred F_1 generation plants were furtherverified. Among 96 lines of D. nobile,the content of soluble polysaccharides showed a significant negative correlation with dendrobine( P < 0. 01),and no significant correlation with agronomic traits in stems and leaves. The content of dendrobine exhibited a significant positive correlation with the stem width-thickness ratio( at the largest cross section; P < 0. 01),and no significant correlation with other agronomic traits. Regression analysis further verified the positive correlation between dendrobine content and stem width-thickness ratio( R2> 0. 9). Two lines,JC-10 and JC-35,with significant differences in stem width-thickness ratio were screened out( P <0. 05). The corresponding F1 generation plants by self-pollination both showed that the dendrobine content was higher with greater stem width-thickness ratio( P < 0. 01). The experimental results suggested that within a certain range,the dendrobine content was higher in D. nobile with flatter stem. Therefore,in the breeding of D. nobile,this specific trait could be used for screening plants with high content of quality indexes such as dendrobine.

Pharmacokinetic study of dendrobine in rat plasma by ultra-performance liquid chromatography tandem mass spectrometry.

Dendrobine, considered as the major active alkaloid compound, has been used for the quality control and discrimination of Dendrobium which is documented in the Chinese Pharmacopoeia. In this work, a sensitive and simple ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for determination of dendrobine in rat plasma is developed. After addition of caulophyline as an internal standard (IS), protein precipitation by acetonitrile-methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C18 (2.1 ×100 mm, 1.7 µm) column with acetonitrile and 0.1% formic acid as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 264.2 → 70.0 for dendrobine and m/z 205.1 → 58.0 for IS. Calibration plots were linear throughout the range 2-1000 ng/mL for dendrobine in rat plasma. The RSDs of intra-day and inter-day precision were both <13%. The accuracy of the method was between 95.4 and 103.9%. The method was successfully applied to pharmacokinetic study of dendrobine after intravenous administration. Copyright © 2015 John Wiley & Sons, Ltd.

Dendrobine Ameliorates Alzheimer's Disease-like Pathology and Cognitive Decline in 3 × Tg-AD Mice.

Previous studies have shown that Dendrobium nobile Lindl. alkaloids (DNLAs) have neuroprotective effects in several Alzheimer's disease (AD) models. Dendrobine (DDB) is one of the monomer components with the highest content in DNLAs. However, the effects of DDB on cognitive impairments in AD remain unknown. In this study, we investigated the efficacy of DDB in 3 × Tg-AD mice to determine whether DDB was a key component of the anti-AD effect of DNLAs. Five-month mice were intragastrically administrated with DDB (10 and 20 mg/kg/d) or DNLAs (20 mg/kg/d) for seven consecutive months, and the effects of DDB and DNLAs were evaluated at twelve months. The results revealed that 3 × Tg-AD mice treated with DDB showed enhanced nesting ability. DDB also effectively rescued spatial learning and memory deficits in 3 × Tg-AD mice. Meanwhile, DDB treatment prevented the loss of dendritic spine density, with increased expression levels of synaptophysin, PSD95, and NCAM in the hippocampus. Finally, DDB ameliorated the increase in APP, sAPPß, CTF-ß, and ß-amyloid peptides, accompanied by the promotion of GSK phosphorylation at the Ser9 site, thereby reducing hyperphosphorylated tau levels. As the active component of DNLA, DDB can preserve cognitive function, alleviate neuronal and synaptic defects, and improve APP/tau pathology in 3 × Tg-AD mice.

Enhancing dendrobine production in Dendrobium nobile through mono-culturing of endophytic fungi, Trichoderma longibrachiatum (MD33) in a temporary immersion bioreactor system.

Introduction: Dendrobine, a valuable alkaloid found in Dendrobium nobile, possesses significant pharmaceutical potential. Methods: In this study, we explored innovative approaches to enhance dendrobine production by utilizing endophytic fungi in a Temporary Immersion Bioreactor System (TIBS, Nanjing BioFunction Co. Ltd., China) and traditional test bottles. Dendrobine was unequivocally identified and characterised in D. nobile co-culture seedlings through UHPLC analysis and LC-MS qTOF analysis, supported by reference standards. Results: The CGTB (control group) and EGTB (experimental group) 12-month-old D. nobile seedlings exhibited similar peak retention times at 7.6±0.1 minutes, with dendrobine identified as C16H25NO2 (molecular weight 264.195). The EGTB, co-cultured with Trichoderma longibrachiatum (MD33), displayed a 2.6-fold dendrobine increase (1804.23 ng/ml) compared to the CGTB (685.95 ng/ml). Furthermore, a bioanalytical approach was applied to investigate the mono-culture of T. longibrachiatum MD33 with or without D. nobile seedlings in test bottles. The newly developed UHPLC-MS method allowed for dendrobine identification at a retention time of 7.6±0.1 minutes for control and 7.6±0.1 minutes for co-culture. Additionally, we explored TIBS to enhance dendrobine production. Co-culturing D. nobile seedlings with Trichoderma longibrachiatum (MD33) in the TIBS system led to a substantial 9.7-fold dendrobine increase (4415.77 ng/ml) compared to the control (454.01 ng/ml) after just 7 days. The comparative analysis of dendrobine concentration between EGTB and EGTIBS highlighted the remarkable potential of TIBS for optimizing dendrobine production. Future research may focus on scaling up the TIBS approach for commercial dendrobine production and investigating the underlying mechanisms for enhanced dendrobine biosynthesis in D. nobile. The structural elucidation of dendrobine was achieved through 1H and 13C NMR spectroscopy, revealing a complex array of proton environments and distinct carbon environments, providing essential insights for the comprehensive characterization of the compound. Discussion: These findings hold promise for pharmaceutical and industrial applications of dendrobine and underline the role of endophytic fungi in enhancing secondary metabolite production in medicinal plants.

PPARα is one of the key targets for dendrobine to improve hepatic steatosis in NAFLD.

ETHNOPHARMACOLOGICAL RELEVANCE: Dendrobium nobile Lindl. (DNL) is a traditional Chinese ethnobotanical herb. Dendrobine (DNE) has been designated as a quality indicator for DNL in the Chinese Pharmacopoeia. DNE exhibits various pharmacological activities, including the reduction of blood lipids, regulation of blood sugar levels, as well as anti-inflammatory and antioxidant properties. AIM OF THE STUDY: The objective of this study is to explore the impact of DNE on lipid degeneration in nonalcoholic fatty liver disease (NAFLD) liver cells and elucidate its specific mechanism. The findings aim to offer theoretical support for the development of drugs related to DNL. MATERIALS AND METHODS: We utilized male C57BL/6J mice, aged 6 weeks old, to establish a NAFLD model. This model allowed us to assess the impact of DNE on liver pathology and lipid levels in NAFLD mice. We investigated the mechanism of DNE's regulation of lipid metabolism through RNA-seq analysis. Furthermore, a NAFLD model was established using HepG2 cells to further evaluate the impact of DNE on the pathological changes of NAFLD liver cells. The potential mechanism of DNE's improvement was rapidly elucidated using HT-qPCR technology. These results were subsequently validated using mouse liver samples. Following the in vitro activation or inhibition of PPARα function, we observed changes in DNE's ability to ameliorate pathological changes in NAFLD hepatocytes. This mechanism was further verified through RT-qPCR and Western blot analysis. RESULTS: DNE demonstrated a capacity to enhance serum TC, TG, and liver TG levels in mice, concurrently mitigating liver lipid degeneration. RNA-seq analysis unveiled that DNE primarily modulates the expression of genes related to metabolic pathways in mouse liver. Utilizing HT-qPCR technology, it was observed that DNE markedly regulates the expression of genes associated with the PPAR signaling pathway in liver cells. Consistency was observed in the in vivo data, where DNE significantly up-regulated the expression of PPARα mRNA and its protein level in mouse liver. Additionally, the expression of fatty acid metabolism-related genes (ACOX1, CPT2, HMGCS2, LPL), regulated by PPARα, was significantly elevated following DNE treatment. In vitro experiments further demonstrated that DNE notably ameliorated lipid deposition, peroxidation, and inflammation levels in NAFLD hepatocytes, particularly when administered in conjunction with fenofibrate. Notably, the PPARα inhibitor GW6471 attenuated these effects of DNE. CONCLUSIONS: In summary, DNE exerts its influence on the expression of genes associated with downstream fat metabolism by regulating PPARα. This regulatory mechanism enhances liver lipid metabolism, mitigates lipid degeneration in hepatocytes, and ultimately ameliorates the pathological changes in NAFLD hepatocytes.

Dendrobine rescues cognitive dysfunction in diabetic encephalopathy by inhibiting ferroptosis via activating Nrf2/GPX4 axis.

BACKGROUND: Ferroptosis playsa crucial role in the development of dementia and dendrobine (Den)possesseshypoglycemic and neuroprotective effects. However, the character of ferroptosis in diabetic encephalopathy (DE) and Den's therapeutic effect remains unclear. PURPOSE: This study aimed to verify the effects of Den on ferroptosis in treating DE and underlying mechanisms. STUDY DESIGN: Den's therapeutic effect was assessed in db/db mice and advanced glycation end products (AGEs)-induced HT22 cells. METHODS: After oral administration with Den orMetformin for 8-week, behavioral tests were used to assess cognitive capacity. Then, biochemical analysis was preformed to detect glucose and lipid metabolism levels; histological analysis and transmission electron microscope were applied to evaluate pathological injuries. Meanwhile, EdU staining and flow cytometry were applied to test cell apoptosis. Furthermore, mitochondrial dynamics, iron transport, and Nrf2/GPX4 axis related proteins were detected by western blot or immunofluorescence. RESULTS: Our results demonstrated that Den remarkably alleviated glucose and lipid metabolism disorders, as well as ameliorated mnemonic deficits of db/db mice. Meanwhile, Den could protect AGEs-induced HT22 cells from death and apoptosis. In addition, we noted that Den inhibited lipid peroxidation by restoring mitochondrial function and reducing reactive oxygen species production. Furthermore, ferroptosis was proven to exist in db/db mice brain and Den could inhibit it via activating Nrf2/GPX4 axis. CONCLUSION: These findings indicated that Den could rescue cognitive dysfunction in DE by inhibiting ferroptosis via activating Nrf2/GPX4 axis.

Age-dependent dendrobine biosynthesis in Dendrobium nobile: insights into endophytic fungal interactions.

Introduction: Dendrobium nobile (D. nobile), a valued Chinese herb known for its diverse pharmacological effects, owes much of its potency to the bioactive compound dendrobine. However, dendrobine content varies significantly with plant age, and the mechanisms governing this variation remain unclear. This study delves into the potential role of endophytic fungi in shaping host-microbe interactions and influencing plant metabolism. Methods: Using RNA-seq, we examined the transcriptomes of 1-year-old, 2-year-old, and 3-year-old D. nobile samples and through a comprehensive analysis of endophytic fungal communities and host gene expression in D. nobile stems of varying ages, we aim to identify associations between specific fungal taxa and host genes. Results: The results revealing 192 differentially expressed host genes. These genes exhibited a gradual decrease in expression levels as the plants aged, mirroring dendrobine content changes. They were enriched in 32 biological pathways, including phagosome, fatty acid degradation, alpha-linolenic acid metabolism, and plant hormone signal transduction. Furthermore, a significant shift in the composition of the fungal community within D. nobile stems was observed along the age gradient. Olipidium, Hannaella, and Plectospherella dominated in 1-year-old plants, while Strelitziana and Trichomerium prevailed in 2-year-old plants. Conversely, 3-year-old plants exhibited additional enrichment of endophytic fungi, including the genus Rhizopus. Two gene expression modules (mediumpurple3 and darkorange) correlated significantly with dominant endophytic fungi abundance and dendrobine accumulation. Key genes involved in dendrobine synthesis were found associated with plant hormone synthesis. Discussion: This study suggests that the interplay between different endophytic fungi and the hormone signaling system in D. nobile likely regulates dendrobine biosynthesis, with specific endophytes potentially triggering hormone signaling cascades that ultimately influence dendrobine synthesis.

Synergism Effect of Dendrobine on Cisplatin in Treatment of H1299 by Modulating the Balance of Treg/Th17.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for about 80% of lung cancers. Cisplatin is a broad-spectrum anti-cancer drug and is often used in combination with other drugs. Research suggests that dendrobine, a pyrrolizidine derivative alkaloid, exhibits antitumor activity in various cancers. This study explores the effect of dendrobine combined with cisplatin on NSCLC and its underlying molecular mechanism. METHODS: The effects of dendrobine combined with cisplatin on tumor progression were evaluated by xenograft model (in vivo) and clonogenic survival assay (in vitro) using H1299 cell line. Annexin V staining was used for detecting apoptotic cells. The population of T cells, B cells and other subpopulations in the peripheral blood was determined by flow cytometry. RESULTS: Dendrobine combined with cisplatin prolonged the survival of mice implanted with H1299 cells and reduced tumor volume compared with single drug application. However, dendrobine exhibited no effect on H1299 cells in clonal survival assays with or without cisplatin treatment and did not promote cisplatin-induced apoptosis in vitro. Importantly, dendrobine suppressed the regulatory T cells (Treg cells) and enhanced the T helper 17 cells (Th17 cells). Treatment of dendrobine significantly reduced Foxp3, and increased the level of IL-17 in serum. CONCLUSION: Dendrobine displayed a synergistic effect with cisplatin to exert anti-tumor effect in vivo, which might be achieved by modulating the balance of Treg/Th17 cells rather than regulating cell apoptosis.

Involvement of the Sch9/Rim15/Msn2 signaling pathway in the anti-aging activity of dendrobine from Dendrobium nobile Lindl. via modification of oxidative stress and autophagy.

BACKGROUND: Aging is an important pathogenic factor of age-related diseases and has brought huge health threat and economic burden to the society. Dendrobium nobile Lindl., a valuable herb in China, promotes longevity according to the record of ancient Chinese materia medica. This study aimed to discover the material basis of D. nobile as an anti-aging herb and elucidate its action mechanism. METHODS: K6001 yeast replicative lifespan assay was used to guide the isolation of D. nobile. The chronological lifespan assay of YOM36 yeast was further conducted to confirm the anti-aging activity of dendrobine. The mechanism in which dendrobine exerts anti-aging effect was determined by conducting anti-oxidative stress assay, quantitative real-time PCR, Western blot, measurements of anti-oxidant enzymes activities, determination of nuclear translocation of Rim15 and Msn2, and replicative lifespan assays of Δsod1, Δsod2, Δcat, Δgpx, Δatg2, Δatg32, and Δrim15 yeasts. RESULTS: Under the guidance of K6001 yeast replicative lifespan system, dendrobine with anti-aging effect was isolated from D. nobile. The replicative and chronological lifespans of yeast were extended upon dendrobine treatment. In the study of action mechanism, dendrobine improved the survival rate of yeast under oxidative stress, decreased the levels of reactive oxygen species and malondialdehyde, and enhanced the enzyme activities and gene expression of superoxide dismutase and catalase, but it failed to elongate the replicative lifespans of Δsod1, Δsod2, Δcat, and Δgpx yeast mutants. Meanwhile, dendrobine enhanced autophagy occurrence in yeast but had no effect on the replicative lifespans of Δatg2 and Δatg32 yeast mutants. Moreover, the inhibition of Sch9 phosphorylation and the promotion of nuclear translocation of Rim15 and Msn2 were observed after treatment with denrobine. However, the effect of dendrobine disappeared from the Δrim15 yeast mutant after lifespan extension, oxidative stress reduction, and autophagy enhancement. CONCLUSIONS: Dendrobine exerts anti-aging activity in yeast via the modification of oxidative stress and autophagy through the Sch9/Rim15/Msn2 signaling pathway. Our work provides a scientific basis for the exploitation of D. nobile as an anti-aging herb.