2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione isolated from Averrhoa carambola L. root inhibits high glucose-induced EMT in HK-2 cells through targeting the regulation of miR-21-5p/Smad7 signaling pathway.

OBJECTIVE: 2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione (DMDD) isolated from Averrhoa carambola L. root, has been proven therapeutic effects on diabetic kidney disease (DKD). This research aims to assess DMDD's effects on DKD and to investigate its underlying mechanisms, to establish DMDD as a novel pharmaceutical agent for DKD treatment. METHODS: The human renal tubular epithelial (HK-2) cells were induced by high glucose (HG) to mimic DKD and followed by DMDD treatment. The cytotoxicity of DMDD was assessed using the Cell Counting Kit-8 (CCK-8) assay. The migratory capacity of HK-2 cells was evaluated through transwell and scratch-wound assays. To investigate the effect of Smad7 and miR-21-5p, lentiviral transfection was employed in HK-2 cells. Additionally, the expression of proteins related to epithelial-mesenchymal transition (EMT) and TGFß1/Smad2/3 pathway was checked by QRT-PCR, Western blot, and immunofluorescence techniques. RESULTS: This study has shown that DMDD significantly suppresses cell migration and the expression of Vimentin, α-SMA, TGFß1, and p-Smad2/3 in HK-2 cells under HG conditions. Concurrently, DMDD enhances the protein expression of E-cadherin and Smad7. Intriguingly, the therapeutic effect of DMDD was abrogated upon Smad7 silencing. Further investigations revealed that DMDD effectively inhibits miR-21-5p expression, which is upregulated by HG. Downregulation of miR-21-5p inhibits the activation of the TGFß1/Smad2/3 pathway and EMT induced by HG. In contrast, overexpression of miR-21-5p negates DMDD's therapeutic benefits. CONCLUSION: DMDD mitigates EMT in HG-induced HK-2 cells by modulating the miR-21-5p/Smad7 pathway, thereby inhibiting renal fibrosis in DKD. These findings suggest that DMDD holds promise as a potential therapeutic agent for DKD.

First report of Watermelon silver mottle orthotospovirus infecting Siraitia grosvenorii in China.

Siraitia grosvenorii, known as "Luohanguo or monk fruit", is a perennial vine belonging to the family Cucurbitaceae. It is cultivated for its fruits, which are used as a Chinese traditional medicine to treat throat, lung and intestine ailments, or as raw material to extract sweet cucurbitane-glycosides as sugar substitute sweeteners (Chen et al., 2007). The production of S. grosvenorii is limited by viral diseases especially cucumber green mottle mosaic virus (CGMMV), papaya ringspot virus (PRSV), watermelon mosaic virus, and zucchini yellow mosaic virus (Liao et al., 2005; Xie et al., 2020). In 2022, virus-like disease consisting of leaf mottling, crinkling, and ringspot was observed on S. grosvenorii plants grown in an insect-proof greenhouse in Guilin City, Guangxi Province, China, with an incidence rate of ~17%. High-throughput sequencing (HTS) was applied to identify potential viruses in the diseased plants. Briefly, total RNA was extracted from a pool of 28 leaf samples (with or without symptoms) of S. grosvenorii using Trizol reagent according to manufacturer's instructions (Invitrogen, U.S.A.). The rRNA was depleted (Epicentre Ribo-zero™ rRNA Removal Kit, Epicentre, U.S.A.), before steps of cDNA library construction (NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®, NEB, U.S.A.), and sequencing (Hiseq 4000 platform, Illumina, U.S.A.). The subsequent bioinformatics analyses were performed according to Liu et al. (2021). HTS of the sample and raw reads processing resulted in 8.4 Gb clean data. The clean reads (150 bp) were de novo assembled into 87,414 contigs (≥200 bp), using CLC Genomics Workbench 21 (Qiagen, Germany). The contigs were annotated by local BLASTX, resulting in matches to CGMMV, PRSV, and watermelon silver mottle virus (WSMoV). Three contigs of 6,557 bp, 4,950 bp, and 3,594 bp were most identical to L (GenBank accession no. JX177647), M (MW051789), and S (KM242056) segments of WSMoV. The complete genome sequences corresponding to the contigs derived from the sample (designated as GL-1 variant of WSMoV, OQ401466-OQ401468) were obtained by reads mapping to segments of these isolates. The reads coverage was ≥99.75% in each RNA segment and the depth of the coverage was in a range of 74-285. To detect the presence of GL-1 in S. grosvenorii plants, three primer pairs D7280F/D7382R (5'-TGATAGCCTGATGAACACCA/5'-TGTCTCTAAACCTTCTACCGC, Tm = 55℃, product size 172 bp), D4512F/D4703R (5'-GCATTGAACTCGCTCACAC/5'-AGTAGACGACCCTGAAGACCT, Tm = 55℃, 192 bp), and D109F/D451R (5'-TTATGGCACAAGAGACAACAGAG/5'-GGGCGTTATGTTCAGTATATTGG, Tm = 56℃, 342 bp) were designed in the L, M, and S segments, respectively. Fresh symptomatic and asymptomatic leaf tissues (n=38) were collected from three fields and their extracted nucleic acids were individually tested with the primers designed by two-steps RT-PCR using TaKaRa RNA PCR kit Ver.3.0 (Takara, Japan). Expected amplicons were obtained in symptomatic samples (n=7) showing mottling, crinkling, and chlorosis. Other samples (n=31) with or without symptoms were negative to WSMoV infection. The amplicons were sequenced, and the sequences obtained shared >99% nt identities with the corresponding GL-1 sequences in GenBank. This is the first report of WSMoV on S. grosvenorii, which provides the basic information for virus disease management.

Metabolomic Analysis on the Mechanism of Nanoselenium Biofortification Improving the Siraitia grosvenorii Nutritional and Health Value.

Nanoselenium (nano-Se) foliar application is crucial for enhancing plant health. However, the mechanism by which nano-Se biofortification promotes the nutritional components of Siraitia grosvenorii remains unclear. In this study, nano-Se foliar application increased the carbohydrate and amino acid contents, including glucose (23.6%), fructose (39.7%), sucrose (60.6%), tryptophan (104.5%), glycine (85.9%), tyrosine (78.4%), phenylalanine (60.1%), glutamic acid (63.4%), and proline (52.5%). Nano-Se application enhanced apigenin (3.8 times), syringic acid (0.7 times), and 4-hydroxy-3,5-dimethoxycinnamic acid (1.4 times) of the phenylpropane biosynthesis pathways. Importantly, the SgCDS (31.1%), CYP-P450 (39.1%), and UGT (24.6%) were induced by nano-Se, which enhanced the mogroside V content (16.2%). Compared to the control, nano-Se treatment dramatically enhanced aromatic substances, including 2-butanone (51.9%), methylpropanal (146.3%), n-nonanal dimer (141.7%), pentanal (52.5%), and 2-pentanone (46.0%). In summary, nano-Se improves S. grosvenorii quality by increasing nutrients and volatile organic compounds and adjusting the phenylpropane pathway.