Activation of autophagy by Citri Reticulatae Semen extract ameliorates amyloid-beta-induced cell death and cognition deficits in Alzheimer's disease.

JOURNAL/nrgr/04.03/01300535-202419110-00027/figure1/v/2024-03-08T184507Z/r/image-tiff Amyloid-beta-induced neuronal cell death contributes to cognitive decline in Alzheimer's disease. Citri Reticulatae Semen has diverse beneficial effects on neurodegenerative diseases, including Parkinson's and Huntington's diseases, however, the effect of Citri Reticulatae Semen on Alzheimer's disease remains unelucidated. In the current study, the anti-apoptotic and autophagic roles of Citri Reticulatae Semen extract on amyloid-beta-induced apoptosis in PC12 cells were first investigated. Citri Reticulatae Semen extract protected PC12 cells from amyloid-beta-induced apoptosis by attenuating the Bax/Bcl-2 ratio via activation of autophagy. In addition, Citri Reticulatae Semen extract was confirmed to bind amyloid-beta as revealed by biolayer interferometry in vitro, and suppress amyloid-beta-induced pathology such as paralysis, in a transgenic Caenorhabditis elegans in vivo model. Moreover, genetically defective Caenorhabditis elegans further confirmed that the neuroprotective effect of Citri Reticulatae Semen extract was autophagy-dependent. Most importantly, Citri Reticulatae Semen extract was confirmed to improve cognitive impairment, neuronal injury and amyloid-beta burden in 3×Tg Alzheimer's disease mice. As revealed by both in vitro and in vivo models, these results suggest that Citri Reticulatae Semen extract is a potential natural therapeutic agent for Alzheimer's disease via its neuroprotective autophagic effects.

Mutant p53R211* ameliorates inflammatory arthritis in AIA rats via inhibition of TBK1-IRF3 innate immune response.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune inflammation disease characterized by imbalance of immune homeostasis. p53 mutants are commonly described as the guardian of cancer cells by conferring them drug-resistance and immune evasion. Importantly, p53 mutations have also been identified in RA patients, and this prompts the investigation of its role in RA pathogenesis. METHODS: The cytotoxicity of disease-modifying anti-rheumatic drugs (DMARDs) against p53 wild-type (WT)/mutant-transfected RA fibroblast-like synoviocytes (RAFLSs) was evaluated by MTT assay. Adeno-associated virus (AAV) was employed to establish p53 WT/R211* adjuvant-induced arthritis (AIA) rat model. The arthritic condition of rats was assessed by various parameters such as micro-CT analysis. Knee joint samples were isolated for total RNA sequencing analysis. The expressions of cytokines and immune-related genes were examined by qPCR, ELISA assay and immunofluorescence. The mechanistic pathway was determined by immunoprecipitation and Western blotting in vitro and in vivo. RESULTS: Among p53 mutants, p53R213* exhibited remarkable DMARD-resistance in RAFLSs. However, AAV-induced p53R211* overexpression ameliorated inflammatory arthritis in AIA rats without Methotrexate (MTX)-resistance, and our results discovered the immunomodulatory effect of p53R211* via suppression of T-cell activation and T helper 17 cell (Th17) infiltration in rat joint, and finally downregulated expressions of pro-inflammatory cytokines. Total RNA sequencing analysis identified the correlation of p53R211* with immune-related pathways. Further mechanistic studies revealed that p53R213*/R211* instead of wild-type p53 interacted with TANK-binding kinase 1 (TBK1) and suppressed the innate immune TBK1-Interferon regulatory factor 3 (IRF3)-Stimulator of interferon genes (STING) cascade. CONCLUSIONS: This study unravels the role of p53R213* mutant in RA pathogenesis, and identifies TBK1 as a potential anti-inflammatory target.

N-Acetylcysteine overcomes epalrestat-mediated increase of toxic 4-hydroxy-2-nonenal and potentiates the anti-arthritic effect of epalrestat in AIA model.

Epalrestat, an aldose reductase inhibitor (ARI), has been clinically adopted in treating diabetic neuropathy in China and Japan. Apart from the involvement in diabetic complications, AR has been implicated in inflammation. Here, we seek to investigate the feasibility of clinically approved ARI, epalrestat, for the treatment of rheumatoid arthritis (RA). The mRNA level of AR was markedly upregulated in the peripheral blood mononuclear cells (PBMCs) of RA patients when compared to those of healthy donors. Besides, the disease activity of RA patients is positively correlated with AR expression. Epalrestat significantly suppressed lipopolysaccharide (LPS) induced TNF-α, IL-1ß, and IL-6 in the human RA fibroblast-like synoviocytes (RAFLSs). Unexpectedly, epalrestat treatment alone markedly exaggerated the disease severity in adjuvant induced arthritic (AIA) rats with elevated Th17 cell proportion and increased inflammatory markers, probably resulting from the increased levels of 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA). Interestingly, the combined treatment of epalrestat with N-Acetylcysteine (NAC), an anti-oxidant, to AIA rats dramatically suppressed the production of 4-HNE, MDA and inflammatory cytokines, and significantly improved the arthritic condition. Taken together, the anti-arthritic effect of epalrestat was diminished or even overridden by the excessive accumulation of toxic 4-HNE or other reactive aldehydes in AIA rats due to AR inhibition. Co-treatment with NAC significantly reversed epalrestat-induced upregulation of 4-HNE level and potentiated the anti-arthritic effect of epalrestat, suggesting that the combined therapy of epalrestat with NAC may sever as a potential approach in treating RA. Importantly, it could be regarded as a safe intervention for RA patients who need epalrestat for the treatment of diabetic complications.

Tricin promoted ATG-7 dependent autophagic degradation of α-synuclein and dopamine release for improving cognitive and motor deficits in Parkinson's disease.

Tricin, a natural nontoxic flavonoid distributed in grasses and euphorbia plants, has been reported to scavenge free radicals, possess anti-inflammatory and antioxidative effects. However, its autophagic effect on Parkinson's disease (PD) has not been elucidated. By adopting cellular and C. elegans models of PD, the autophagic effect of tricin was identified based on the level of autophagy markers (LC3-II and p62). Besides, the pharmacological effects on neurotransmitters (dopamine), inflammatory cytokines (IFN γ, TNFα, MCP-1, IL-10, IL-6 and IL-17A), histology (hematoxylin & eosin and Nissl staining) and behavioural pathology (open-field test, hindlimb clasping, Y-maze, Morris water-maze and nest building test) were also confirmed in the A53T-α-synuclein transgenic PD mouse model. Further experiments demonstrated that tricin induced autophagic flux and lowered the level of α-synuclein through AMPK-p70s6K- and ATG7-dependent mechanism. Compared to the existing clinical PD drugs, tricin mitigated pathogenesis and symptoms of PD with no observable side effects. In summary, tricin is proposed as a potential adjuvant remedy or nutraceutical for the prevention and treatment of PD.

Age-related self-DNA accumulation may accelerate arthritis in rats and in human rheumatoid arthritis.

The incidence of rheumatoid arthritis (RA) is increasing with age. DNA fragments is known to accumulate in certain autoimmune diseases, but the mechanistic relationship among ageing, DNA fragments and RA pathogenesis remain unexplored. Here we show that the accumulation of DNA fragments, increasing with age and regulated by the exonuclease TREX1, promotes abnormal activation of the immune system in an adjuvant-induced arthritis (AIA) rat model. Local overexpression of TREX1 suppresses synovial inflammation in rats, while conditional genomic deletion of TREX1 in AIA rats result in higher levels of circulating free (cf) DNA and hence abnormal immune activation, leading to more severe symptoms. The dysregulation of the heterodimeric transcription factor AP-1, formed by c-Jun and c-Fos, appear to regulate both TREX1 expression and SASP induction. Thus, our results confirm that DNA fragments are inflammatory mediators, and TREX1, downstream of AP-1, may serve as regulator of cellular immunity in health and in RA.

Licochalcone B, a Natural Autophagic Agent for Alleviating Oxidative Stress-Induced Cell Death in Neuronal Cells and Caenorhabditis elegans Models.

Autophagy has been implicated in the regulation of neuroinflammation and neurodegenerative disorders. Licochalcone B (LCB), a chalcone from Glycyrrhiza inflata, has been reported to have anti-cancer, anti-oxidation and anti-ß-amyloid fibrillation effects; however, its effect in autophagy remain un-investigated. In the current study, the potential neuro-protective role of LCB in terms of its anti-oxidative, anti-apoptotic, and autophagic properties upon oxidative stress-induced damage in neuronal cells was investigated. With the production of reactive oxygen species (ROS) as a hallmark of neuroinflammation and neurodegeneration, hydrogen peroxide (H2O2) was adopted to stimulate ROS-induced cell apoptosis in PC-12 cells. Our findings revealed that LCB reduced cell cytotoxicity and apoptosis of PC-12 cells upon H2O2-stimulation. Furthermore, LCB increased the level of the apoptosis-associated proteins caspase-3 and cleaved caspase-3 in H2O2-induced cells. LCB effectively attenuated the level of oxidative stress markers such as MDA, SOD, and ROS in H2O2-induced cells. Most importantly, LCB was confirmed to possess its anti-apoptotic effects in H2O2-induced cells through the induction of ATG7-dependent autophagy and the SIRT1/AMPK signaling pathway. As a novel autophagic inducer, LCB increased the level of autophagy-related proteins LC3-II and decreased p62 in both neuronal cells and Caenorhabditis elegans (C. elegans) models. These results suggested that LCB has potential neuroprotective effects on oxidative damage models via multiple protective pharmacological mechanisms.

The New Application of UHPLC-DAD-TOF/MS in Identification of Inhibitors on ß-Amyloid Fibrillation From Scutellaria baicalensis.

Literary evidence depicts that aggregated ß-amyloid (Aß) leads to the pathogenesis of Alzheimer's disease (AD). Although many traditional Chinese medicines (TCMs) are effective in treating neurodegenerative diseases, there is no effective way for identifying active compounds from their complicated chemical compositions. Instead of using a traditional herbal separation method with low efficiency, we herein apply UHPLC-DAD-TOF/MS for the accurate identification of the active compounds that inhibit the fibrillation of Aß (1-42), via an evaluation of the peak area of individual chemical components in chromatogram, after incubation with an Aß peptide. Using the neuroprotective herbal plant Scutellaria baicalensis (SB) as a study model, the inhibitory effect on Aß by its individual compounds, were validated using the thioflavin-T (ThT) fluorescence assay, biolayer interferometry analysis, dot immunoblotting and native gel electrophoresis after UHPLC-DAD-TOF/MS analysis. The viability of cells after Aß (1-42) incubation was further evaluated using both the tetrazolium dye (MTT) assay and flow cytometry analysis. Thirteen major chemical components in SB were identified by UHPLC-DAD-TOF/MS after incubation with Aß (1-42). The peak areas of two components from SB, baicalein and baicalin, were significantly reduced after incubation with Aß (1-42), compared to compounds alone, without incubation with Aß (1-42). Consistently, both compounds inhibited the formation of Aß (1-42) fibrils and increased the viability of cells after Aß (1-42) incubation. Based on the hypothesis that active chemical components have to possess binding affinity to Aß (1-42) to inhibit its fibrillation, a new application using UHPLC-DAD-TOF/MS for accurate identification of inhibitors from herbal plants on Aß (1-42) fibrillation was developed.

Quantitative Phosphoproteomic and Metabolomic Analyses Reveal GmMYB173 Optimizes Flavonoid Metabolism in Soybean under Salt Stress.

Salinity causes osmotic stress to crops and limits their productivity. To understand the mechanism underlying soybean salt tolerance, proteomics approach was used to identify phosphoproteins altered by NaCl treatment. Results revealed that 412 of the 4698 quantitatively analyzed phosphopeptides were significantly up-regulated on salt treatment, including a phosphopeptide covering the serine 59 in the transcription factor GmMYB173. Our data showed that GmMYB173 is one of the three MYB proteins differentially phosphorylated on salt treatment, and a substrate of the casein kinase-II. MYB recognition sites exist in the promoter of flavonoid synthase gene GmCHS5 and one was found to mediate its recognition by GmMYB173, an event facilitated by phosphorylation. Because GmCHS5 catalyzes the synthesis of chalcone, flavonoids derived from chalcone were monitored using metabolomics approach. Results revealed that 24 flavonoids of 6745 metabolites were significantly up-regulated after salt treatment. We further compared the salt tolerance and flavonoid accumulation in soybean transgenic roots expressing the 35S promoter driven cds and RNAi constructs of GmMYB173 and GmCHS5, as well as phospho-mimic (GmMYB173S59D ) and phospho-ablative (GmMYB173S59A ) mutants of GmMYB173 Overexpression of GmMYB173S59D and GmCHS5 resulted in the highest increase in salt tolerance and accumulation of cyaniding-3-arabinoside chloride, a dihydroxy B-ring flavonoid. The dihydroxy B-ring flavonoids are more effective as anti-oxidative agents when compared with monohydroxy B-ring flavonoids, such as formononetin. Hence the salt-triggered phosphorylation of GmMYB173, subsequent increase in its affinity to GmCHS5 promoter and the elevated transcription of GmCHS5 likely contribute to soybean salt tolerance by enhancing the accumulation of dihydroxy B-ring flavonoids.

Mechanisms of Soybean Roots' Tolerances to Salinity Revealed by Proteomic and Phosphoproteomic Comparisons Between Two Cultivars.

Understanding molecular mechanisms underlying plant salinity tolerance provides valuable knowledgebase for effective crop improvement through genetic engineering. Current proteomic technologies, which support reliable and high-throughput analyses, have been broadly used for exploring sophisticated molecular networks in plants. In the current study, we compared phosphoproteomic and proteomic changes in roots of different soybean seedlings of a salt-tolerant cultivar (Wenfeng07) and a salt-sensitive cultivar (Union85140) induced by salt stress. The root samples of Wenfeng07 and Union85140 at three-trifoliate stage were collected at 0 h, 0.5 h, 1 h, 4 h, 12 h, 24 h, and 48 h after been treated with 150 mm NaCl. LC-MS/MS based phosphoproteomic analysis of these samples identified a total of 2692 phosphoproteins and 5509 phosphorylation sites. Of these, 2344 phosphoproteins containing 3744 phosphorylation sites were quantitatively analyzed. Our results showed that 1163 phosphorylation sites were differentially phosphorylated in the two compared cultivars. Among them, 10 MYB/MYB transcription factor like proteins were identified with fluctuating phosphorylation modifications at different time points, indicating that their crucial roles in regulating flavonol accumulation might be mediated by phosphorylated modifications. In addition, the protein expression profiles of these two cultivars were compared using LC MS/MS based shotgun proteomic analysis, and expression pattern of all the 89 differentially expressed proteins were independently confirmed by qRT-PCR. Interestingly, the enzymes involved in chalcone metabolic pathway exhibited positive correlations with salt tolerance. We confirmed the functional relevance of chalcone synthase, chalcone isomerase, and cytochrome P450 monooxygenase genes using soybean composites and Arabidopsis thaliana mutants, and found that their salt tolerance were positively regulated by chalcone synthase, but was negatively regulated by chalcone isomerase and cytochrome P450 monooxygenase. A novel salt tolerance pathway involving chalcone metabolism, mostly mediated by phosphorylated MYB transcription factors, was proposed based on our findings. (The mass spectrometry raw data are available via ProteomeXchange with identifier PXD002856).

Application of Genetic Algorithm to Predict Optimal Sowing Region and Timing for Kentucky Bluegrass in China.

Temperature is a predominant environmental factor affecting grass germination and distribution. Various thermal-germination models for prediction of grass seed germination have been reported, in which the relationship between temperature and germination were defined with kernel functions, such as quadratic or quintic function. However, their prediction accuracies warrant further improvements. The purpose of this study is to evaluate the relative prediction accuracies of genetic algorithm (GA) models, which are automatically parameterized with observed germination data. The seeds of five P. pratensis (Kentucky bluegrass, KB) cultivars were germinated under 36 day/night temperature regimes ranging from 5/5 to 40/40 °C with 5 °C increments. Results showed that optimal germination percentages of all five tested KB cultivars were observed under a fluctuating temperature regime of 20/25 °C. Meanwhile, the constant temperature regimes (e.g., 5/5, 10/10, 15/15 °C, etc.) suppressed the germination of all five cultivars. Furthermore, the back propagation artificial neural network (BP-ANN) algorithm was integrated to optimize temperature-germination response models from these observed germination data. It was found that integrations of GA-BP-ANN (back propagation aided genetic algorithm artificial neural network) significantly reduced the Root Mean Square Error (RMSE) values from 0.21~0.23 to 0.02~0.09. In an effort to provide a more reliable prediction of optimum sowing time for the tested KB cultivars in various regions in the country, the optimized GA-BP-ANN models were applied to map spatial and temporal germination percentages of blue grass cultivars in China. Our results demonstrate that the GA-BP-ANN model is a convenient and reliable option for constructing thermal-germination response models since it automates model parameterization and has excellent prediction accuracy.