Advancements in Research on Genetic Kidney Diseases Using Human-Induced Pluripotent Stem Cell-Derived Kidney Organoids.

Genetic or hereditary kidney disease stands as a pivotal cause of chronic kidney disease (CKD). The proliferation and widespread utilization of DNA testing in clinical settings have notably eased the diagnosis of genetic kidney diseases, which were once elusive but are now increasingly identified in cases previously deemed CKD of unknown etiology. However, despite these diagnostic strides, research into disease pathogenesis and novel drug development faces significant hurdles, chiefly due to the dearth of appropriate animal models and the challenges posed by limited patient cohorts in clinical studies. Conversely, the advent and utilization of human-induced pluripotent stem cells (hiPSCs) offer a promising avenue for genetic kidney disease research. Particularly, the development of hiPSC-derived kidney organoid systems presents a novel platform for investigating various forms of genetic kidney diseases. Moreover, the integration of the CRISPR/Cas9 technique into this system holds immense potential for efficient research on genetic kidney diseases. This review aims to explore the applications of in vitro kidney organoids generated from hiPSCs in the study of diverse genetic kidney diseases. Additionally, it will delve into the limitations of this research platform and outline future perspectives for advancing research in this crucial area.

Maslinic acid alleviates alcoholic liver injury in mice and regulates intestinal microbiota via the gut-liver axis.

BACKGROUND: Maslinic acid (MA), a pentacyclic triterpene acid, is widely distributed in natural plants and mainly found in the fruit and leaves of olives and hawthorn. MA has been reported as having many health-promoting functions, such as anticancer, anti-inflammation and neuroprotective activities. According to previous study, hawthorn extract has certain hepatoprotective effects. However, the detailed mechanism is still unclear, especially the effect of MA on gut microbiota. RESULTS: Our study reveals that MA effectively counteracts alcohol-induced liver injury and oxidative stress. It mitigates alcohol-induced intestinal barrier damage, reverses increased permeability and reduces translocation of lipopolysaccharide (LPS). This prevents LPS/Toll-like receptor 4 activation, leading to decreased TNF-α and IL-1ß production. Furthermore, MA rebalances gut microbiota by reversing harmful bacterial abundance and enhancing beneficial bacteria post-alcohol consumption. CONCLUSION: MA, through modulation of gut microbiota, alleviates alcohol-induced liver injury via the gut-liver axis. These findings support the potential use of MA as a functional food ingredient for preventing or treating alcoholic liver disease. © 2024 Society of Chemical Industry.

Ginkgolic acids induce liver injury in mice through cell cycle arrest and immune stress under specific condition.

Ginkgo biloba L. is a valuable plant, which can be used for medicine, food and ornamental purposes. Despite the above benefits, the components of ginkgolic acids (GA) in ginkgo are considered to cause allergies, embryotoxicity, liver damage and some other adverse reactions. However, the mechanism of GA induced liver injury is still unclear. In this study, we developed an acute liver injury model induced by GA in mice, and investigated the mechanism of GA induced liver injury from the perspectives of oxidative stress, steatosis, apoptosis, and immune response. Intraperitoneal injection of GA (400 mg/kg) can cause liver damage. The levels of serum transaminase, oxidation and triglycerides were increased, liver fibrosis, hepatocyte apoptosis, G2/M phase arrest of the hepatic cell cycle and monocyte infiltration in the liver were detected in GA-treated mice. Flow cytometry analysis of cells separated from the spleen showed that the proportion of Th1 and Th17 cells were increased, and the proportion of Th2 cells were decreased in GA-treated mice. The rise in Th1/Th2 ratio and Th17 cell ratio usually cause inflammatory problems. At the same time, cleaved Caspase-8 and Caspase-3 were detected in hepatocytes, indicating that GA may induce apoptosis through FADD pathway. Although GA is capable of causing the above problems, the inflammation and damage in liver tissue are not severe and there are certain individual differences. Our study reveals the potential hepatotoxicity of GA in ginkgo and its mechanism of action, providing a new perspective for the intervention and prevention of ginkgo toxicity.

Single-cell RNA sequencing revealed the role of the Th17 pathway in the development of anti- human leukocyte antigen antibodies in a highly sensitized mouse model.

Background: The aim of this study is to investigate the specific pathway involved in human leukocyte antigen (HLA) sensitization using single-cell RNA-sequencing analysis and an allo-sensitized mouse model developed with an HLA.A2 transgenic mouse. Methods: For sensitization, wild-type C57BL/6 mouse received two skin grafts from C57BL/6-Tg(HLA-A2.1)1Enge/J mouse (allogeneic mouse, ALLO). For syngeneic control (SYN), skin grafts were transferred from C57BL/6 to C57BL/6. We performed single-cell RNA-sequencing analysis on splenocytes isolated from ALLO and SYN and compared the gene expression between them. Results: We generated 9,190 and 8,890 single-cell transcriptomes from ALLO and SYN, respectively. Five major cell types (B cells, T cells, natural killer cells, macrophages, and neutrophils) and their transcriptome data were annotated according to the representative differentially expressed genes of each cell cluster. The percentage of B cells was higher in ALLO than it was in SYN. Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that the highly expressed genes in the B cells from ALLO were mainly associated with antigen processing and presentation pathways, allograft rejection, and the Th17 cell differentiation pathway. Upregulated genes in the T cells of ALLO were involved in the interleukin (IL)-17 signaling pathway. The ratio of Th17 cluster and Treg cluster was increased in the ALLO. On flow cytometry, the percentage of Th17 (IL-17+/CD4+ T) cells was higher and regulatory T cells (FOXP3+/CD4+ T) was lower in the ALLO compared to those in the SYN. Conclusion: Our results indicate that not only the B cell lineage but also the Th17 cells and their cytokine (IL-17) are involved in the sensitization to HLA.

Combined Use of Tocilizumab and Mesenchymal Stem Cells Attenuate the Development of an Anti-HLA-A2.1 Antibody in a Highly Sensitized Mouse Model.

Sensitization to HLA can result in allograft loss for kidney transplantation (KT) patients. Therefore, it is required to develop an appropriate desensitization (DSZ) technique to remove HLA-donor-specific anti-HLA antibody (DSA) before KT. The aim of this research was to investigate whether combined use of the IL-6 receptor-blocking antibody, tocilizumab (TCZ), and bone-marrow-derived mesenchymal stem cells (BM-MSCs) could attenuate humoral immune responses in an allo-sensitized mouse model developed using HLA.A2 transgenic mice. Wild-type C57BL/6 mice were sensitized with skin allografts from C57BL/6-Tg (HLA-A2.1)1Enge/J mice and treated with TCZ, BM-MSC, or both TCZ and BM-MSC. We compared HLA.A2-specific IgG levels and subsets of T cells and B cells using flow cytometry among groups. HLA.A2-specific IgG level was decreased in all treated groups in comparison with that in the allo-sensitized control (Allo-CONT) group. Its decrease was the most significant in the TCZ + BM-MSC group. Regarding the B cell subset, combined use of TCZ and BM-MSC increased proportions of pre-pro B cells but decreased proportions of mature B cells in BM (p < 0.05 vs. control). In the spleen, an increase in transitional memory was observed with a significant decrease in marginal, follicular, and long-lived plasma B cells (p < 0.05 vs. control) in the TCZ + BM-MSC group. In T cell subsets, Th2 and Th17 cells were significantly decreased, but Treg cells were significantly increased in the TCZ+BM-MSC group compared to those in the Allo-CONT group in the spleen. Regarding RNA levels, IL-10 and Foxp3 showed increased expression, whereas IL-23 and IFN-γ showed decreased expression in the TCZ + BM-MSC group. In conclusion, combined use of TCZ and BM-MSC can inhibit B cell maturation and up-regulate Treg cells, finally resulting in the reduction of HLA.A2-specific IgG in a highly sensitized mouse model. This study suggests that the combined use of TCZ and BM-MSC can be proposed as a novel strategy in a desensitization protocol for highly sensitized patients.

Carotenoid Cleavage Dioxygenase 1 and Its Application for the Production of C13-Apocarotenoids in Microbial Cell Factories: A Review.

C13-apocarotenoids are naturally derived from the C9-C10 (C9'-C10') double-bond cleavage of carotenoids by carotenoid cleavage dioxygenases (CCDs). As high-value flavors and fragrances in the food and cosmetic industries, the sustainable production of C13-apocarotenoids is emerging in microbial cell factories by the carotenoid cleavage dioxygenase 1 (CCD1) subfamily. However, the commercialization of microbial-based C13-apocarotenoids is still limited by the poor performance of CCD1, which severely constrains its conversion efficiency from precursor carotenoids. This review focuses on the classification of CCDs and their cleavage modes for carotenoids to generate corresponding apocarotenoids. We then emphatically discuss the advances for C13-apocarotenoid biosynthesis in microbial cell factories with various strategies, including optimization of CCD1 expression, improvement of CCD1's catalytic activity and substrate specificity, strengthening of substrate channeling, and development of oleaginous microbial hosts, which have been verified to increase the conversion rate from carotenoids. Lastly, the current challenges and future directions will be discussed to enhance CCDs' application for C13-apocarotenoids biomanufacturing.

Modeling of FAN1-Deficient Kidney Disease Using a Human Induced Pluripotent Stem Cell-Derived Kidney Organoid System.

Karyomegalic interstitial nephritis (KIN) is a genetic kidney disease caused by mutations in the FANCD2/FANCI-Associated Nuclease 1 (FAN1) gene on 15q13.3, which results in karyomegaly and fibrosis of kidney cells through the incomplete repair of DNA damage. The aim of this study was to explore the possibility of using a human induced pluripotent stem cell (hiPSC)-derived kidney organoid system for modeling FAN1-deficient kidney disease, also known as KIN. We generated kidney organoids using WTC-11 (wild-type) hiPSCs and FAN1-mutant hiPSCs which include KIN patient-derived hiPSCs and FAN1-edited hiPSCs (WTC-11 FAN1+/-), created using the CRISPR/Cas9 system in WTC-11-hiPSCs. Kidney organoids from each group were treated with 20 nM of mitomycin C (MMC) for 24 or 48 h, and the expression levels of Ki67 and H2A histone family member X (H2A.X) were analyzed to detect DNA damage and assess the viability of cells within the kidney organoids. Both WTC-11-hiPSCs and FAN1-mutant hiPSCs were successfully differentiated into kidney organoids without structural deformities. MMC treatment for 48 h significantly increased the expression of DNA damage markers, while cell viability in both FAN1-mutant kidney organoids was decreased. However, these findings were observed in WTC-11-kidney organoids. These results suggest that FAN1-mutant kidney organoids can recapitulate the phenotype of FAN1-deficient kidney disease.

Generation of a human induced pluripotent stem cell line from a patient with dent disease.

Dent disease, an X-linked tubular disorder, is a rare condition that leads to low-molecular-weight proteinuria, hypercalciuria, kidney stones, and chronic kidney disease. Here, we successfully established a human induced pluripotent stem cells (hiPSC) line from peripheral blood mononuclear cells of 10-year-old male with Dent disease 1 caused by the mutation of Chloride Voltage-Gated Channel 5 gene. This hiPSCs displayed features similar to human embryonic stem cells, including pluripotency-associated markers expression, normal karyotype, and the ability to differentiate into cells representing all three germ layers. The implications of this research extend to the potential development of novel treatments for Dent disease.

Hydroxytyrosol attenuates ethanol-induced liver injury by ameliorating steatosis, oxidative stress and hepatic inflammation by interfering STAT3/iNOS pathway.

Objective: Hydroxytyrosol (HT) is a polyphenol with a wide range of biological activities. Excessive drinking can lead to oxidative stress and inflammation in the liver, which usually develop into alcohol liver disease (ALD). At present, there is no specific drug to treat ALD. In this paper, the protection effect of HT on ALD and the underline mechanism were studied.Methods: HepG2 cells were exposed to ethanol in vitro and C57BL/6J mice were fed with a Lieber-DeCarli ethanol liquid diet in vivo.Results: triglyceride (TG) level in serum and the expression of fatty acid synthase (FASN) were reduced significantly by the treatment with HT The acetaldehyde dehydrogenase (ALDH) activity was increased, the serum level of malondialdehyde (MDA) was decreased, catalase (CAT) and glutathione (GSH) were increased, suggesting that HT may reduce its oxidative damage to the body by promoting alcohol metabolism. Furthermore, according to the mRNA levels of tnf-α, il-6 and il-1ß, HT inhibited ethanol-induced inflammation significantly. The anti-inflammatory mechanism of HT may be related to suppress the STAT3/iNOS pathway.Dissussion: Our study showed that HT could ameliorate ethanol-induced hepatic steatosis, oxidative stress and inflammation and provide a new candidate for the prevention and treatment of ALD.

Study on Synergistic Anti-Inflammatory Effect of Typical Functional Components of Extracts of Ginkgo Biloba Leaves.

There are some differences in the anti-inflammatory activities of four typical components in EGB (extracts of ginkgo biloba leaves), and there is also a synergistic relationship. The order of inhibiting the NO-release ability of single functional components is OA > GF > OPC > G. Ginkgolide (G), proanthocyanidins (OPC), and organic acids (OA) all have synergistic effects on ginkgo flavonoids (GF). GF:OA (1:9) is the lowest interaction index among all complexes, showing the strongest synergy. The anti-inflammatory mechanism of the compound affects the expression of p-JNK, p-P38, and p-ERK1/2 proteins by inhibiting the expression of iNOS and COX2 genes on NFKB and MAPK pathways. This also provides a research basis for the development of anti-inflammatory deep-processing products of EGB.

Modeling of Fabry disease nephropathy using patient derived human induced pluripotent stem cells and kidney organoid system.

OBJECTIVES: To explore the possibility of kidney organoids generated using patient derived human induced pluripotent stem cells (hiPSC) for modeling of Fabry disease nephropathy (FDN). METHODS: First, we generated hiPSC line using peripheral blood mononuclear cells (PBMCs) from two male FD-patients with different types of GLA mutation: a classic type mutation (CMC-Fb-001) and a non-classic type (CMC-Fb-003) mutation. Second, we generated kidney organoids using wild-type (WT) hiPSC (WTC-11) and mutant hiPSCs (CMC-Fb-001 and CMC-Fb-003). We then compared alpha-galactosidase A (α-GalA) activity, deposition of globotriaosylceremide (Gb-3), and zebra body formation under electromicroscopy (EM). RESULTS: Both FD patients derived hiPSCs had the same mutations as those detected in PBMCs of patients, showing typical pluripotency markers, normal karyotyping, and successful tri-lineage differentiation. Kidney organoids generated using WT-hiPSC and both FD patients derived hiPSCs expressed typical nephron markers without structural deformity. Activity of α-GalA was decreased and deposition of Gb-3 was increased in FD patients derived hiPSCs and kidney organoids in comparison with WT, with such changes being far more significant in CMC-Fb-001 than in CMC-Fb-003. In EM finding, multi-lammelated inclusion body was detected in both CMC-Fb-001 and CMC-Fb-003 kidney organoids, but not in WT. CONCLUSIONS: Kidney organoids generated using hiPSCs from male FD patients might recapitulate the disease phenotype and represent the severity of FD according to the GLA mutation type.

CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman's Disease Phenotype in a Patient-Derived Kidney Organoid System.

The aim of this study is to explore the possibility of modeling Gitelman's disease (GIT) with human-induced pluripotent stem cell (hiPSC)-derived kidney organoids and to test whether gene correction using CRISPR/Cas9 can rescue the disease phenotype of GIT. To model GIT, we used the hiPSC line CMCi002 (CMC-GIT-001), generated using PBMCs from GIT patients with SLC12A3 gene mutation. Using the CRISPR-Cas9 system, we corrected CMC-GIT-001 mutations and hence generated CMC-GIT-001corr. Both hiPSCs were differentiated into kidney organoids, and we analyzed the GIT phenotype. The number of matured kidney organoids from the CMC-GIT-001corr group was significantly higher, 3.3-fold, than that of the CMC-GIT-001 group (12.2 ± 0.7/cm2 vs. 3.7 ± 0.2/cm2, p < 0.05). In qRT-PCR, performed using harvested kidney organoids, relative sodium chloride cotransporter (NCCT) mRNA levels (normalized to each iPSC) were increased in the CMC-GIT-001corr group compared with the CMC-GIT-001 group (4.1 ± 0.8 vs. 2.5 ± 0.2, p < 0.05). Consistently, immunoblot analysis revealed increased levels of NCCT protein, in addition to other tubular proteins markers, such as LTL and ECAD, in the CMC-GIT-001corr group compared to the CMC-GIT-001 group. Furthermore, we found that increased immunoreactivity of NCCT in the CMC-GIT-001corr group was colocalized with ECAD (a distal tubule marker) using confocal microscopy. Kidney organoids from GIT patient-derived iPSC recapitulated the Gitelman's disease phenotype, and correction of SLC12A3 mutation utilizing CRISPR-Cas9 technology provided therapeutic insight.

CRISPR/Cas9-mediated A4GALT suppression rescues Fabry disease phenotypes in a kidney organoid model.

The objective of this study was to investigate whether CRISPR/Cas9-mediated suppression of A4GALT could rescue phenotype of Fabry disease nephropathy (FDN) using human induced pluripotent stem cells (hiPSCs) derived kidney organoid system. We generated FDN patient-derived hiPSC (CMC-Fb-002) and FD-specific hiPSCs (GLA-KO) by knock-out (KO) of GLA in wild-type (WT) hiPSCs using CRISPR/Cas9. We then performed A4GALT KO in both CMC-Fb-002 and GLA-KO to make Fb-002-A4GALT-KO and GLA/A4GALT-KO, respectively. Using these hiPSCs, we generated kidney organoids and compared alpha-galactosidase-A enzyme (α-GalA) activity, globotriaosylceramide (Gb-3) deposition, and zebra body formation under electron microscopy (EM). We also compared mRNA expression levels using RNA-seq and qPCR. Generated hiPSCs showed typical pluripotency markers without chromosomal disruption. Expression levels of GLA in CMC-Fb-002 and GLA-KO and expression levels of A4GALT in Fb-002-A4GALT-KO and GLA/A4GALT-KO were successfully decreased compared to those in WT-hiPSCs, respectively. Generated kidney organoids using these hiPSCs expressed typical nephron markers. In CMC-Fb-002 and GLA-KO organoids, α-GalA activity was significantly decreased along with increased deposition of Gb-3 in comparison with WT organoids. Intralysosomal inclusion body was also detected under EM. However, these disease phenotypes were rescued by KO of A4GALT in both GLA/A4GALT-KO and Fb-002-A4GALT-KO kidney organoids. RNA-seq showed increased expression levels of genes related to FDN progression in both GLA-mutant organoids compared to those in WT. Such increases were rescued in GLA/A4GALT-KO or Fb-002-A4GALT-KO organoids. CRISPR/Cas9 mediated suppression of A4GALT could rescue FDN phenotype. Hence, it can be proposed as a therapeutic approach to treat FDN.

Highly Efficient Biotransformation of Notoginsenoside R1 into Ginsenoside Rg1 by Dictyoglomus thermophilum ß-xylosidase Xln-DT.

Notoginsenoside R1 and ginsenoside Rg1 are the main active ingredients of Panax notoginseng, exhibiting anti-fatigue, anti-tumor, anti-inflammatory, and other activities. In a previous study, a GH39 ß-xylosidase Xln-DT was responsible for the bioconversion of saponin, a natural active substance with a xylose group, with high selectivity for cleaving the outer xylose moiety of notoginsenoside R1 at the C-6 position, producing ginsenoside Rg1 with potent anti-fatigue activity. The optimal bioconversion temperature, pH, and enzyme dosage were obtained by optimizing the transformation conditions. Under optimal conditions (pH 6.0, 75°C, enzyme dosage 1.0 U/ml), 1.0 g/l of notoginsenoside R1 was converted into 0.86 g/l of ginsenoside Rg1 within 30 min, with a molar conversion rate of approximately 100%. Furthermore, the in vivo anti-fatigue activity of notoginsenoside R1 and ginsenoside Rg1 were compared using a suitable rat model. Compared with the control group, the forced swimming time to exhaustion was prolonged in mice by 17.3% in the Rg1 high group (20 mg/kg·d). Additionally, the levels of hepatic glycogen (69.9-83.3% increase) and muscle glycogen (36.9-93.6% increase) were increased. In the Rg1 group, hemoglobin levels were also distinctly increased by treatment concentrations. Our findings indicate that treatment with ginsenoside Rg1 enhances the anti-fatigue effects. In this study, we reveal a GH39 ß-xylosidase displaying excellent hydrolytic activity to produce ginsenoside Rg1 in the pharmaceutical and food industries.

Discovery of TGFBR1 (ALK5) as a potential drug target of quercetin glycoside derivatives (QGDs) by reverse molecular docking and molecular dynamics simulation.

Quercetin glycoside derivatives (QGDs) are a class of common compounds with a wide range of biological activities, such as antitumor activities. However, their molecular targets associated with biological activities have not been investigated. In this study, four common QGDs with mutual bioconversion were selected, and studied in the large-scale reverse docking experiments. Network pharmacology analysis showed that most of the four QGDs can bind several potential protein targets that were closely related to breast cancer disease. Among them, a druggable protein, transforming growth factor beta receptor I (TGFBR1/ALK5) was screened via high docking scores for the four QGDs. This protein has been proven to be an important target for the treatment of breast cancer by regulating the proliferation and migration of cancer cells in the past. Subsequently, the molecular dynamics (MD) simulation and MM/GBSA calculation demonstrated that all QGDs could thermodynamically bind with TGFBR1, indicating that TGFBR1 might be one of the potential protein targets of QGDs. Finally, the cytotoxicity test and wound-healing migration assay displayed that isoquercetin, which can perform best in MD experiment, might be a promising agent in the treatment of breast cancer metastasis.

Extracts of Waste from Poplar Wood Processing Alleviate Experimental Dextran Sulfate-Induced Colitis by Ameliorating Oxidative Stress, Inhibiting the Th1/Th17 Response and Inducing Apoptosis in Inflammatory Lymphocytes.

As a fast-growing tree, poplar is widely planted and typically used for wood processing in China. During poplar wood processing, a large amount of poplar sawdust (PS) and poplar leaves (PL) are produced and abandoned. To make full use of poplar resources and clarify the use of poplar as a feed additive, the active ingredients in PS and PL were extracted and isolated, and the anti-inflammatory effects of the extracts on mice with dextran sulfate sodium (DSS)-induced colitis were investigated. In vitro anti-inflammatory experiments showed that the ethyl acetate extract of PS and PL (PSE and PLE, respectively) could significantly inhibit the proliferation of concanavalin A (Con A)-activated lymphocytes. Salicortin, tremulacin and salireposide were identified in both PSE and PLE. Oral administration of PSE and PLE rescued DSS-induced colonic shortening, repaired tissue damage, and decreased the disease activity index (DAI). The antioxidant capacity, including the increased activities of glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD and catalase (CAT) and decreased activity of myeloperoxidase (MPO), in the colons of mice with colitis was enhanced through the activation of ERK after treatment with PSE and PLE. The ratio of Th1 to Th17 cells, which can lead to inflammation in the spleen, was significantly decreased by the administration of PSE and PLE, while the phosphorylation of related transcription factors (p65, STAT1, and STAT3) was inhibited. Furthermore, PSE and PLE could induce apoptosis in Con A-activated lymphocytes, which may be associated with the increase in p-TBK1, as the molecular docking results also indicated that salireposide in PSE and PLE could interact with the TBK1 protein. Overall, our study provides a promising feed additive for improving intestinal inflammation in animals and a method for the full utilization of poplar resources.

Isovitexin Inhibits Ginkgolic Acids-Induced Inflammation Through Downregulating SHP2 Activation.

It has been reported that Celtis sinensis Pers. is employed as a folk medicine for the treatment of inflammatory diseases. But the mechanism supporting its use as anti-inflammatory remains unclear. To investigate the anti-inflammatory of Celtis sinensis Pers. ICR mice were provided Celtis sinensis Pers. leaf extract (CLE) at 100, 200 mg/kg after ginkgolic acids (GA) sensitization. Our data showed that CLE and the main flavonoid isovitexin in CLE could ameliorate GA-induced contact dermatitis in mice. Ear swelling, inflammatory cell infiltration and splenomegaly were inhibited significantly by isovitexin, while the weight loss of mice in the isovitexin-treated group was much better than that in the dexamethasone-treated group (positive control drug). It has been reported in previous research that GA-induced inflammation is closely related to the T cell response. Therefore, T cells were the focus of the anti-inflammatory effect of isovitexin in this paper. The in vivo results showed that isovitexin (10, 20 mg/kg) inhibited the expression of proinflammatory cytokines (TNF-α, IFN-γ, IL-2 and IL-17A) in lymph nodes, inhibited the secretion of cytokines into the serum from mice with contact dermatitis and promoted the expression of apoptosis-related proteins. In vitro, isovitexin also induced apoptosis and inhibited proinflammatory cytokine expression in Con A-activated T cells. Further study showed that the MAPK and STAT signaling pathways and the phosphorylation of SHP2 were inhibited by isovitexin. Both molecular docking and biological experiments indicated that SHP2 may be an anti-inflammatory target of isovitexin in T cells. Taken together, isovitexin can serve as a potential natural agent for the treatment or prevention of GA-induced inflammatory problems.

Efficient extraction of bioactive flavonoids from Celtis sinensis leaves using deep eutectic solvent as green media.

In recent years, deep eutectic solvent (DES) has attracted comprehensive attention on the extraction of natural products, and is regarded as an alternative to traditional organic solvents for the environmental advantages. Twenty-six DESs were compared for their extraction yield of total flavonoids and isovitexin (the main flavonoid in Celtis sinensis) from Celtis sinensis. The results show that the extraction yields of total flavonoids by betaine/glycolic acid (DES8), ethylamine hydrochloride/1,2-propanediol (DES12) and tetrapropylammonium bromide/lactic acid (DES17) are the highest, while the extraction yields of isovitexin by ethylene glycol/malonic acid (DES23), ethylene glycol/glycolic acid (DES24) and 1,2-propanediol/glycolic acid (DES26) are the highest. The extraction conditions using the above six DESs were further optimized systematically. Under optimum conditions, the extraction rates of total flavonoids and isovitexin can be increased up to 95.39 and 10.58 mg g-1, respectively, which were significantly higher than that of methanol extraction. In order to exclude the effect of DESs on the bioactivity of Celtis sinensis extract, the macroporous resin D-101 was used to purify the total flavonoids from DESs extract, and the recovery rates of flavonoids from the above six kinds of DESs were all over 80%. Next, the anti-inflammatory activity of DES extracts was compared using a lymphocyte transformation experiment. The result showed that the inhibition rate of the DES24 extract on the proliferation of Con A-activated T cells was up to 72% with an IC50 value of 124.8 µg mL-1. None of the DESs extracted exhibited cytotoxicity on normal T cells. The mechanism of the anti-inflammatory activity against Con A-activated T cells may be that DES24 flavonoids extract induced the apoptosis of inflammatory T cells, and activated the expression of pro-apoptotic protein. Taken together, DES has showed significant advantages on the extraction of natural products for the relatively mild extraction condition, high yield and environmental-friendliness.

Bioassay-guided isolation of anti-inflammatory constituents from Celtis sinensis leaves.

Ginkgo acids (GAs) in ginkgo products usually lead to allergies or liver toxicity. In this study, the GA-induced toxicity was attenuated and Con A-induced T-lymphocyte proliferation was inhibited by extracts of Celtis sinensis leaves (ECSL). So, the active ingredients in ECSL were studied to solve the problems caused by GAs. First, the eight components of MeOH extracts were determined by HPLC-DAD/LC-MS. Then, the 12 active ingredients were separated based on the anti-inflammatory activity. Lymphocyte conversion showed that the inhibition rates of apigenin, quercetin, and isovitexin at 100 µM on Con A-activated proliferation of T cells were up to 82.46%, 62.86% and 42.76%, respectively. The inhibition rate on the LPS-induced NO release in RAW 264.7 cells of quercetin, apigenin, isovitexin, and vitexin were exceeding 80% at 100 µM. Taken together, the material foundation for the screen of GAs toxicity-attenuated ingredients were provided here. PRACTICAL APPLICATIONS: Ginkgo biloba extracts (EGBs) have been conducted to develop functional food which could increase blood circulation and enhance memory. Nevertheless, people in consumption of ginkgo products, often caused severe allergic reactions due to the potential allergens identified ginkgolic acids (GAs) of ginkgo products. We first find that the extracts of Celtis sinensis leaves can reduce GAs-induced damage on HepG2 liver cells. Then, the bioactive compounds in C. sinensis leaves were separated and purified based on anti-inflammatory activities against T cells. Quercetin, apigenin, and isovitexin showed well anti-inflammatory activities against Con A-activated T-lymphocytes and LPS activated RAW 264.7 macrophages. However, quercetin and apigenin are flavones O-glycosides which are rich in Ginkgo biloba. To solve the problems in Ginkgo biloba products caused by GAs, flavone C-glycoside (isovitexin) may be used for the further study in GAs toxicity-reduction.

Echinacoside ameliorates alcohol-induced oxidative stress and hepatic steatosis by affecting SREBP1c/FASN pathway via PPARα.

Alcoholic liver disease (ALD) is one of the most common health problems for drinkers, especially in men. Echinacoside (ECH), a natural phenylethanoid glycoside welcomed by the market, has been shown to have a variety of biological activities, such as neuroprotective, anti-fatigue, anti-diabetes and so on. Here, the protective effect and the underlying mechanism of ECH on ethanol-induced liver injuries were studied. In vitro, the HepG2 cells were treated with ECH prior to ethanol. In vivo, C57BL/6 J mice were fed a Lieber-DeCarli ethanol liquid diet and gave with or without 100 mg/kg ECH for 10 days. Our experiments showed that ECH significantly enhanced the levels of antioxidants and reduced the level of ROS, thus attenuating ethanol-induced oxidative stress. Besides, ECH attenuated lipid accumulation caused by ethanol, as evidenced by oil-red O staining, histological examination and the quantification of TG and TC. Finally, ECH increased the level of PPAR-α, and reduced the levels of SREBP-1c and FASN. When PPAR-α inhibitor was introduced in the system, the effects of ECH on SREBP-1c and FASN were reversed. Taken together, our study suggest that ECH can protect against ethanol-induced liver injuries via alleviating oxidative stress and hepatic steatosis by affecting SREBP-1c/FASN pathway via PPAR-α.