IiAGL6 participates in the regulation of stamen development and pollen formation in Isatis indigotica.

The AGL6 (AGMOUSE LIKE 6) gene is a member of the SEP subfamily and functions as an E-class floral homeotic gene in the development of floral organs. In this study, we cloned IiAGL6, the orthologous gene of AGL6 in Isatis indigotica. The constitutive expression of IiAGL6 in Arabidopsis thaliana resulted in a late-flowering phenotype and the development of curly leaves during the vegetative growth period. Abnormal changes in floral organ development were observed during the reproductive stage. In woad plants, suppression of IiAGL6 using TRV-VIGS (tobacco rattle virus-mediated virus-induced gene silencing) decreased the number of stamens and led to the formation of aberrant anthers. Similar changes in stamen development were also observed in miRNA-AGL6 transgenic Arabidopsis plants. Yeast two-hybrid and BiFC tests showed that IiAGL6 can interact with other MADS-box proteins in woad; thus, playing a key role in defining the identities of floral organs, particularly during stamen formation. These findings might provide novel insights and help investigate the biological roles of MADS transcription factors in I. indigotica.

BzATP reverses ferroptosis-induced gut microbiota disorders in collagen-induced arthritis mice.

Recent studies suggested that altered gut microbiota may be related to the pathogenesis of rheumatoid arthritis (RA), albeit the exact mechanisms are unknown. In this study, we aimed to discover the particular mechanism of RA treatment by microbiota by investigating the effects of ferroptosis on gut microbiota and its metabolites in collagen-induced arthritis (CIA) mice. Mice were divided into five groups: control, CIA, erastin, BzATP, and BzATP + erastin group. We performed 16S rDNA sequencing and metabolomics analysis on mouse feces and found that erastin and BzATP altered the microbiota and metabolites. The findings demonstrated that the microbiota was significantly disturbed at the phylum (Proteobacteria, Firmicutes, and Bacteroidota) and genus level (Lachnospiraceae_NK4A136, Lactobacillus, and Bifidobacterium) in the CIA group, and erastin exacerbated this disturbance. Unexpectedly, BzATP treatment could repair the disruptive effects of erastin. Additionally, there were significant variations in metabolites between each group. Erastin worsened metabolite abnormalities in CIA mice, while BzATP mitigated them, consistent with the microbiota results. These findings provide novel perspectives and insights into the therapy of RA.

SMSCs-derived sEV overexpressing miR-433-3p inhibits angiogenesis induced by sEV released from synoviocytes under triggering of ferroptosis.

BACKGROUND: Ferroptosis is characterized by accumulation of lipid peroxides that leads to oxidative stress. In progressive rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) suffered from oxidative stress induced by generation of excess reactive oxygen species (ROS) and survived from elevated lipid oxidation. However the phenomenon of abnormal synovial fibroblasts proliferation under ferroptotic stress remain to be explained and the effects of this event on disease progression of RA need to be investigated. METHODS: FLS from RA patients (RA-FLS) were stimulated with LPS as an inflammatory model in vitro, and simultaneously treated with ferroptosis inducer Erastin/RSL3 or inhibitor ferrostatin-1. Besides, small extracellular vesicles (sEV) from the supernatant of RA-FLS culture under Erastin/RSL3 management were isolated. The degree of ferroptosis in cells were evaluated by Lipid-ROS detection via flowcytometry and ferroptosis marker protein expression determined by western bloting. The expression of core component of ESCRT-III CHMP4A and CHMP5 was determined by western bloting, and knockdown of CHMP4A was further performed to detect the influence of ESCRT-III complex on ferroptosis as well as LPS/Erastin induced sEV (LPS/Erastin-sEV) releasing. Moreover, miR-433-3p level in the isolated sEV was evaluated by RT-qPCR and interaction of miR-433-3p with FOXO1/VEGF axis were evaluated. MiR-433-3p was overexpressed in synovial mesenchymal stem cells (SMSCs) via miR-433-3p mimics transfection. RA-FLS was co-cultured with human dermal microvascular endothelial cells (HDMECs). LPS/Erastin-sEV or sEV derived from miR-433-3p-overexpressing SMSCs (miR-433-3p-SMSCs-sEV) were added to the co-culture system, and supernatants from co-culture without sEV were given to HDMECs. Angiogenic activity of HDMECs were identified by transwell test and endothelial tube formation analysis. Erastin-sEV and miR-433-3p-SMSCs-sEV were also administrated in collagen-induced arthritis (CIA) mouse model respectively, and progression of arthritis were evaluated. RESULTS: Ferroptosis of RA-FLS was triggered by LPS/Erastin and accompanied with increased expression of ESCRT-III core components as well as elevated release of sEV from RA-FLS. HDMECs' migration and tube formation in vitro was significantly induced/suppressed by supernatants from co-culture under management of Erastin-sEV/miR-433-3p-SMSCs-sEV due to varied VEGF expression regulated by miR-433-3p targeting FOXO1. MiR-433-3p-SMSCs-sEV could inhibit the Erastin-sEV promoted VEGF expression and mitigated arthritis severity. CONCLUSION: Erastin-sEV could aggravate synovial angiogenesis and promote arthritis progression. Administration of miR-433-3p-SMSCs-sEV may be a potential novel therapeutic method as significant antagonism to Erastin-sEV for RA treatment.

Potential predictive and therapeutic applications of small extracellular vesicles-derived circPARD3B in osteoarthritis.

Background: Heterogeneous phenotypes that display distinct common characteristics of osteoarthritis (OA) are not well defined and will be helpful in identifying more customized therapeutic options for OA. Circular RNAs (circRNAs) have attracted more and more attention due to their role in the progression of OA. Investigating the role of circRNAs in the pathogenesis of OA will contribute to the phenotyping of OA and to individualized treatment. Methods: Small extracellular vesicles (sEV) were isolated from serum samples from patients with OA of different stages and sEV-derived circPARD3B was determined using RT-qPCR analysis. CircPARD3B expression in a stimulated coculture that included OA fibroblast-like synoviocytes (OA-FLS) as well as human dermal microvascular endothelial cells (HDMECs), plus the effects of circPARD3B on the expression of vascular endothelial growth factor (VEGF) long with angiogenic activity, were evaluated in vitro. Based on bioinformatics analysis and luciferase reporter assay (LRA), MiR-326 and sirtuin 1 (SIRT1) were found to be interactive partners of circPARD3B. Mesenchymal stem cells (SMSCs) overexpressing circPARD3B were constructed and SMSCs-derived sEV with overexpressed circPARD3B (OE-circPARD3B-SMSCs-sEV) were obtained to explore the effect of the intervention of circPARD3B combined with SMSCs-sEV-based therapy in vitro and in a OA model induced by collagenase in vivo. Results: Serum sEV-linked circPARD3B was indentified to be significantly decreased in the inflammatory phenotype of OA. Overexpression of circPARD3B was found to inhibit the expression of VEGF, as well as the angiogenesis induced by VEGF in a IL-1ß stimulated the co-culture of OA-FLS as well as HDMECs. CircPARD3B is directly bound to miR-326. SIRT1 was considered a novel miR-326 target gene. OE-circPARD3B-SMSCs-sEV significantly reduced VEGF expression in coculture of OA-FLS and HDMECs. Injection of OE-circPARD3B-SMSCs-sEV could also reduce synovial VEGF; additionally, it could further ameliorate OA in the mouse model of OA in vivo. Conclusion: Serum sEV circPARD3B is a potential biomarker that enables the identification of the inflammatory phenotype of patients with OA. Correspondingly, intracellular transfer of circPARD3B through OE-circPARD3B-SMSCs-sEV could postpone disease progression through a functional module regulated angiogenesis of circPARD3B-miR-326-SIRT1, providing a novel therapeutic strategy for OA.

Arsenic trioxide modulates the composition and metabolic function of the gut microbiota in a mouse model of rheumatoid arthritis.

The mechanism of rheumatoid arthritis (RA) has been widely investigated, and studies on the use of arsenic trioxide (ATO) in the treatment of RA have been reported in recent years. However, the exact mechanism of action of ATO in RA remains unclear. This study explores alterations in the gut microbiota and metabolism during ATO treatment in a mouse model of RA and provides an integrative analysis of the biomechanism. The purpose of this study was to verify whether ATO can alleviate RA by altering the gut microbiota. In this study, the mice were randomly divided into four different groups: the normal control (NC) group, the collagen-induced arthritis (CIA) group, the ATO 1.0 mg/kg/day group, and the ATO 2.0 mg/kg/day group. Fecal samples were collected. Through 16S rDNA gene sequencing and metabolomic analysis, the effect of ATO on the composition and metabolites of gut microbiota in CIA mice was investigated. The results showed that compared with NC mice, CIA mice showed differences at both the phylum level (Firmicutes and Bacteroidetes) and the genus level (Muribaculaceae_unclassified and Alistipes). Meanwhile, many metabolites were significantly changed between the two groups, including benzoic acid and (s)-2-acetolactate. However, these alterations were partially reversed in ATO-treated CIA mice. These results indicated that ATO treatment modulated gut microbiota disorder and improved fecal metabolite abnormalities. In conclusion, this study provided important evidence for alterations of the gut microbiota and metabolites and the role of these alterations in a potential novel mechanism of ATO treatment in RA.

Functional identification of the different regions in B-class floral homeotic MADS-box proteins IiAP3 and IiPI from Isatis indigotica.

APETALA3 (AP3) and PISTILLATA (PI) are B-class MADS-box floral homeotic genes of Arabidopsis and are involved in specifying the identity of petals and stamens. In the present work, IiAP3 and IiPI, the respective orthologous genes of AP3 and PI, were cloned from Isatis indigotica. By expressing in ap3-6 and pi-1 homozygous mutant and in wild-type Arabidopsis under the control of AP3 promoter or CaMV 35S promoter, we demonstrated that IiAP3 and IiPI were functionally equivalent to AP3 and PI of Arabidopsis. Referring to previous reports and the research results in the present work, expression patterns of AP3 and PI homologs are not the same in different angiosperms possessing diverse floral structures. It suggests that the alterations in expression may contribute to the changing morphology of flowers. To further determine the relationship between IiAP3 and IiPI, the coding sequences of the different structural regions in these two proteins were swapped with each other, and the data collected from transgenic Arabidopsis plants of the chimeric constructs suggested that MADS domain was irreplaceable for the function of IiAP3, K domain of IiAP3 was involved in specifying the identity of stamens, K domain of IiPI was mainly related to the formation of petals, and C-terminal region of IiPI was involved in characterization of stamens. In addition, a complete KC region of these two proteins was more effective in phenotypic complementation of the mutants.

SEPALLATA--like genes of Isatis indigotica can affect the architecture of the inflorescences and the development of the floral organs.

BACKGROUND: The architecture of inflorescence and the development of floral organs can influence the yield of seeds and have a significant impact on plant propagation. E-class floral homeotic MADS-box genes exhibit important roles in regulation of floral transition and differentiation of floral organs. Woad (Isatis indigotica) possesses unique inflorescence, floral organs and fruit. However, very little research has been carried out to determine the function of MADS-box genes in this medicinal cruciferous plant species. RESULTS: SEPALLATA orthologs in I. indigotica were cloned by degenerate PCR. The sequence possessing the highest identity with SEP2 and SEP4 of Arabidopsis were named as IiSEP2 and IiSEP4, respectively. Constitutive expression of IiSEP2 in Columbia (Col-0) ecotype of Arabidopsis led to early flowering, and the number of the flowers and the lateral branches was reduced, indicating an alteration in architecture of the inflorescences. Moreover, the number of the floral organs was declined, the sepals were turned into carpelloid tissues bearing stigmatic papillae and ovules, and secondary flower could be produced in apetalous terminal flowers. In 35S::IiSEP4-GFP transgenic Arabidopsis plants in Landsberg erecta (Ler) genetic background, the number of the floral organs was decreased, sepals were converted into curly carpelloid structures, accompanied by generation of ovules. Simultaneously, the size of petals, stamens and siliques was diminished. In 35S::IiSEP4-GFP transgenic plants of apetalous ap1 cal double mutant in Ler genetic background, the cauliflower phenotype was attenuated significantly, and the petal formation could be rescued. Occasionally, chimeric organs composed of petaloid and sepaloid tissues, or petaloid and stamineous tissues, were produced in IiSEP4 transgenic plants of apl cal double mutant. It suggested that overexpression of IiSEP4 could restore the capacity in petal differentiation. Silencing of IiSEP4 by Virus-Induced Gene Silencing (VIGS) can delay the flowering time, and reduce the number and size of the floral organs in woad flowers. CONCLUSION: All the results showed that SEPALLATA-like genes could influence the architecture of the inflorescence and the determinacy of the floral meristems, and was also related to development of the floral organs.

Therapeutic Potential of Exosomal circRNA Derived from Synovial Mesenchymal Cells via Targeting circEDIL3/miR-485-3p/PIAS3/STAT3/VEGF Functional Module in Rheumatoid Arthritis.

BACKGROUND: Synovial inflammation and its associated activation of angiogenesis play critical roles in rheumatoid arthritis (RA). Exosomes, as carriers of genetic information including circular RNAs (circRNAs), have been explored as delivery vehicles for therapeutic molecules. However, the effects of synovial mesenchymal stem cells (SMSCs)-derived exosomal circRNAs and their mechanisms of action in RA progression remain unclear. METHODS: SMSCs-derived exosomes (SMSCs-Exos) were administered to a co-culture of RA fibroblast-like synoviocytes (RA-FLS) and human dermal microvascular endothelial cells (HDMECs) in vitro as well as to a collagen-induced arthritis (CIA) mouse model in vivo. Their effects on VEGF expression and angiogenic activity in vitro and the therapeutic efficacy in vivo were evaluated. Exosomes from circEDIL3-overexpressing SMSCs (Ad-circEDIL3-SMSCs-Exos) were used to further determine the role of circEDIL3 in SMSCs-Exo-based therapy. RESULTS: Both SMSCs-Exos and Ad-circEDIL3-SMSCs-Exos significantly downregulated the expression of VEGF induced by the IL-6/sIL-6R complex in the supernatants of RA-FLS and HDMECs co-culture as well as in the cell lysate of co-cultured RA-FLS, and the extent of reduction was more pronounced in the latter. Subsequent experiments showed that angiogenic activity was significantly downregulated by SMSCs-Exos and Ad-circEDIL3-SMSCs-Exos due to reduced VEGF expression. CircEDIL3 functioned as a sponge for miR-485-3p, which targeted PIAS3. PIAS3 is known to suppress STAT3 activity and reduce downstream VEGF. Injection of SMSCs-Exos or Ad-circEDIL3-SMSCs-Exos reduced synovial VEGF and consequently ameliorated arthritis severity in the CIA mouse model. CONCLUSION: The intracellular transfer of circEDIL3 by SMSCs-Exos may be a potential novel therapeutic strategy for RA.

Angiogenesis is Inhibited by Arsenic Trioxide Through Downregulation of the CircHIPK3/miR-149-5p/FOXO1/VEGF Functional Module in Rheumatoid Arthritis.

Angiogenesis is a crucial event in the pathogenesis of rheumatoid arthritis (RA). Arsenic trioxide (ATO, As2O3) has been reported to inhibit synovial angiogenesis via the vascular endothelial growth factor (VEGF)-centered functional module. However, the exact mechanisms of ATO on VEGF modulation remain unclear. Circular RNAs (circRNAs) are emerging as important regulators in RA, and the detailed mechanisms remain largely unknown. Here, we reported a circRNA (circHIPK3), the expression of which was significantly increased in RA fibroblast-like synoviocytes (RA-FLS) after TNF-α induction. Moreover, VEGF content in the supernatants of a RA-FLS and human dermal microvascular endothelial cell (HDMEC) co-culture as well as in RA-FLS co-cultured was significantly elevated in accordance with circHIPK3 levels. This increased VEGF expression may significantly upregulate endothelial tube formation and transwell migration, as well as microvessel sprouting in the ex vivo aortic ring assay. CircHIPK3 was further illustrated to be a sponge for the forkhead box transcription factor O1 (FOXO1)-targeting miR-149-5p, leading to the changing expression of the downstream VEGF. These networked factors mainly form a functional module regulating angiogenesis in RA-FLS, and the expression of this functional module could be significantly downregulated by ATO with a consistently reduced vascularity in vitro. In the collagen-induced arthritis (CIA) mice model, an intra-articular injection of the adeno-associated virus-si-circHIPK3 or ATO was demonstrated to alleviate the synovial VEGF expression and arthritis severity respectively. Thus, we elucidate a previously unknown mechanism between circRNAs and RA, and ATO has a significant protective effect on RA-FLS and CIA synovium via its inhibition of the angiogenic functional module of circHIPK3/miR-149-5p/FOXO1/VEGF, suggesting great potential for the combination therapy of ATO with circHIPK3 silencing.

Signals in systemic acquired resistance of plants against microbial pathogens.

After a local infection by the microbial pathogens, plants will produce strong resistance in distal tissues to cope with the subsequent biotic attacks. This type of the resistance in the whole plant is termed as systemic acquired resistance (SAR). The priming of SAR can confer the robust defense responses and the broad-spectrum disease resistances in plants. In general, SAR is activated by the signal substances generated at the local sites of infection, and these small signaling molecules can be rapidly transported to the systemic tissues through the phloem. In the last two decades, numerous endogenous metabolites were proved to be the potential elicitors of SAR, including methyl salicylate (MeSA), azelaic acid (AzA), glycerol-3-phosphate (G3P), free radicals (NO and ROS), pipecolic acid (Pip), N-hydroxy-pipecolic acid (NHP), dehydroabietinal (DA), monoterpenes (α-pinene and ß-pinene) and NAD(P). In the meantime, the proteins associated with the transport of these signaling molecules were also identified, such as DIR1 (DEFECTIVE IN INDUCED RESISTANCE 1) and AZI1 (AZELAIC ACID INDUCED 1). This review summarizes the recent findings related to synthesis, transport and interaction of the different signal substances in SAR.

Metabolomics analysis identifies metabolites associated with systemic acquired resistance in Arabidopsis.

BACKGROUND: Systemic acquired resistance (SAR) is a type of plant defense response that provides a long-lasting resistance to broad-spectrum pathogens in uninfected distal tissues following an initial localized infection. However, little information is available at present on the biological basis of SAR at the molecular level, especially in uninfected distal leaves. METHODS: In the present work, we used two SAR-inducing pathogens, avirulent Pseudomonas syringae pv. maculicola ES4326 harboring avrRpm1 (Psm avrRpm1) and virulent P. syringae pv. maculicola ES4326 (Psm ES4326), to induce SAR in Arabidopsis ecotype Col-0. A metabolomics approach based on ultra-high-performance liquid chromatography (UPLC) coupled with mass spectrometry (MS) was used to identify SAR-related metabolites in infected local leaves, and in uninfected distal leaves. RESULTS: Differentially accumulated metabolites were distinguished by statistical analyses. The results showed that both the primary metabolism and the secondary metabolism were significantly altered in infected local leaves and in uninfected distal leaves, including phenolic compounds, amino acids, nucleotides, organic acids, and many other metabolites. CONCLUSIONS: The content of amino acids and phenolic compounds increased in uninfected distal leaves, suggesting their contribution to the establishment of SAR. In addition, 2'-hydroxy-4, 4', 6'-trimethoxychalcone, phenylalanine, and p-coumaric acid were identified as potential components which may play important roles both in basic resistance and in SAR. This work provides a reference for understanding of the metabolic mechanism associated with SAR in plants, which will be useful for further investigation of the molecular basis of the systemic immunity.

Tobacco NtabSPL6-2 can enhance local and systemic resistances of Arabidopsis thaliana to bacterial and fungal pathogens.

NtabSPL6-2 of Nicotiana tabacum was introduced into Arabidopsis by Agrobacterium-mediated floral-dip method. Compared to wild-type Col-0 plants, the arrangement of cauline leaves in NtabSPL6-2 transgenic plants was converted into opposite from simple and alternate, and the margin of rosette leaves was serrated. NtabSPL6-2 transgenic plants possessed a significantly greater fresh weight. Subcellular localization by fusion with GFP confirmed that the encoded product of NtabSPL6-2 existed in the nucleus. The leaves of NtabSPL6-2 transgenic plants exhibited an enhanced capacity to restrain the bacterial reproduction after infection by Pseudomonas syringae, accompanied by higher expression of the pathogenesis-related gene PR1 in the infiltrated leaves, indicating NtabSPL6-2 could improve the defense response of Arabidopsis to P. syringae at the local sites. Similarly, it was confirmed that NtabSPL6-2 could enhance the systemic acquired resistance of Arabidopsis in response to P. syringae. In addition, the area of necrotic plaque appearing on the transgenic leaves inoculated with Botrytis cinerea was smaller and accompanied by an upregulation of PR1 and PR5, indicating NtabSPL6-2 transgenic leaves were less susceptible to the fungal pathogen. Moreover, there was less accumulation of reactive oxygen species (H2O2 and O2-) and malondialdehyde in the local infected sites of transgenic plants, whereas the wild-type Col-0 plants were more oxidatively injured after infestation by B. cinerea.

Cloning of a SEPALLATA4-like gene (IiSEP4) in Isatis indigotica Fortune and characterization of its function in Arabidopsis thaliana.

E-class MADS-box genes, SEPALLATA (SEP), participate in various aspects of plant development together with B-, C- and D-class MADS-box genes. IiSEP4, a homologous gene of SEP4, was cloned from Isatis indigotica. IiSEP4 was highly expressed in sepals, and its mRNA was mildly detected in leaves, inflorescences, flowers, stamens and young silicles. Constitutive expression of IiSEP4 in Arabidopsis thaliana caused early flowering, accompanied by the reduction of flowers and floral organs. Moreover, the sepals in some flowers were transformed into carpelloid structures with stigmatic papillae, and obviously accompanied by ovule formation. Yeast two-hybrid assays demonstrated that IiSEP4 interacts with other woad MADS proteins to determine the identity of floral organs. These findings reveal the important roles of IiSEP4 in floral development of I. indigotica. The results of this study can lay a foundation for further study on biological functions of MADS transcriptional factors in I. indigotica.