The role of PHF8 and TLR4 in osteogenic differentiation of periodontal ligament cells in inflammatory environment.

BACKGROUND: Histone methylation is considered to play an important role in the occurrence and development of periodontitis. Plant homeodomain finger protein 8 (PHF8), a histone demethylase, has been shown to regulate inflammation and osteogenic differentiation of bone marrow stromal cells (BMSCs). This study aimed to detect the functions of PHF8 and TLR4 in osteogenic differentiation in an inflammatory environment induced by Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) METHODS: A periodontitis mouse model was established, and the mice were treated with TAK-242. Immunohistochemical staining was used to detect the expression of PHF8 in periodontal tissue. Periodontal ligament cells (PDLCs) were treated with mineralization induction medium supplemented with Pg-LPS and/or TAK-242, and a Cell Counting Kit-8 (CCK-8) assay was used to detect the proliferation of PDLCs. Real-time PCR and western blotting were used to detect the mRNA and protein expression levels, respectively, of PHF8, toll-like receptor 4 (TLR4) and the other osteogenic markers alkaline phosphatase (ALP), osteocalcin (OCN), Special AT-rich sequence-binding protein 2 (Satb2) and Runt-related transcription factor 2 (Runx2) RESULTS: Periodontitis reduced PHF8 expression in periodontal tissue, and TAK-242 partially reversed this downregulation. An in vitro experiment revealed that the mRNA and protein expression levels of PHF8 were significantly upregulated during the osteogenic differentiation of PDLCs. Alizarin red staining showed that the mineralized nodules of PDLCs in osteogenic induction group were more than those in control group. Real-time PCR and western blot results indicated that Pg-LPS inhibited PHF8 expression and upregulated TLR4 expression in PDLCs. TAK-242 inhibited TLR4 and partially reversed the inhibition of PHF8 expression and osteogenic differentiation induced by Pg-LPS in PDLCs CONCLUSION: PHF8 and TLR4 play important roles in periodontitis. Pg-LPS inhibits the expression of PHF8 via upregulation of TLR4 and might further inhibit the osteogenic differentiation of PDLCs. However, the specific mechanisms involved remain to be explored.

Effects of calcium treatment on malate metabolism and γ-aminobutyric acid (GABA) pathway in postharvest apple fruit.

Calcium treatment effects on malate metabolism and the GABA pathway in 'Cripps Pink' apple fruit during storage were investigated. Postharvest apple fruit treated with 1% and 4% calcium chloride solutions were stored at 25 ± 1 °C. The 4% calcium treatment suppressed declines in titratable acidity and malate content and increased succinate and oxalate concentrations. Calcium treatment also reduced the respiration rate and decreased ethylene production peak during storage. Moreover, 4% calcium treatment significantly enhanced cyNAD-MDH and PEPC activities and upregulated MdMDH1, MdMDH2, MdPEPC1 and MdPEPC2 expression while inhibiting cyNADP-ME and PEPCK activities and downregulating MdME1, MdME4 and MdPEPCK2 expression. Surprisingly, calcium treatment changed the content of some free amino acids (GABA, proline, alanine, aspartic acid and glutamate), two of which (glutamate and GABA) are primary metabolites of the GABA pathway. Furthermore, calcium application enhanced GABA pathway activity by increasing MdGAD1, MdGAD2, MdGABA-T1/2 and MdSSADH transcript levels.

Exogenous γ-Aminobutyric Acid Treatment That Contributes to Regulation of Malate Metabolism and Ethylene Synthesis in Apple Fruit during Storage.

Organic acid is an important indicator of fruit quality, and malate is the predominant organic acid in apple fruit. However, the regulation of malate metabolism in postharvest fruit is rarely reported. Here, we found that, compared with a control treatment, a 10 mM γ-aminobutyric acid (GABA) treatment remarkably delayed the loss of tiftratable acidity and malate and increased the succinate and oxalate contents in "Cripps Pink" fruit stored in polyethylene bags at room temperature. The higher malate levels in GABA-treated fruit were accompanied by higher activities of cytosolic nicotinamide adenine dinucleotide-dependent malate dehydrogenase (cyNAD-MDH) and phosphoenolpyruvate carboxylase (PEPC) but lower cytosolic NAD phosphate-dependent malic enzyme (cyNADP-ME) and phosphoenolpyruvate carboxykinase (PEPCK) activities than those seen in control fruit. Notably, ethylene production was significantly reduced by GABA treatment, paralleling the downregulation of MdACS, MdACO, and MdERF expression. Meanwhile, GABA treatment also enhanced the activity of the GABA shunt and promoted the accumulation of GABA. This study provides new insights into the regulation of malate metabolism and reports for the first time the possible interplay between GABA and ethylene signaling pathways in apple fruit during postharvest storage.

Role of Brassinosteroids in Persimmon ( Diospyros kaki L.) Fruit Ripening.

Brassinosteroids (BRs) are phytohormones that regulate numerous processes including fruit ripening. In this study, persimmon ( Diospyros kaki L.) fruits were treated with 24-epibrassinolide (EBR) or brassinazole (Brz, a BR biosynthesis inhibitor) and then stored at ambient temperature. The results show that endogenous BR contents gradually increased during persimmon fruit ripening. EBR treatment significantly increased both the content of water-soluble pectin and the activities of polygalacturonase, pectate lyase, and endo-1,4-beta-glucanase but significantly reduced the content of acid-soluble pectin and cellulose, resulting in rapid fruit softening. The EBR treatment also promoted ethylene production and respiration rate. In contrast, Brz treatment delayed persimmon fruit ripening. qRT-PCR analysis showed that DkPG1, DkPL1, DkPE2, DkEGase1, DkACO2, DkACS1, and DkACS2 were up-regulated (especially a 38-fold increase in DkEGase1) in the fruit of the EBR-treated group. These results suggest that BRs are involved in persimmon fruit ripening by influencing cell-wall-degrading enzymes and ethylene biosynthesis.