In women with preeclampsia (PE), endothelial cell (EC) dysfunction can lead to altered secretion of paracrine factors that induce peripheral vasoconstriction and proteinuria. This study examined the hypothesis that PE sera may directly or indirectly, through human umbilical vein ECs (HUVECs), stimulate phospholipase C-gamma1-1,4,5-trisphosphate (PLC-gamma1-IP3) signaling, thereby increasing protein kinase C-alpha (PKC-alpha) activity, collagen I expression and intracellular Ca2+ concentrations ([Ca2+]i) in human umbilical artery smooth muscle cells (HUASMCs). HUASMCs and HUVECs were cocultured with normal or PE sera before PLC-gamma1 silencing. Increased PLC-gamma1 and IP3 receptor (IP3R) phosphorylation was observed in cocultured HUASMCs stimulated with PE sera (P<0.05). In addition, PE serum significantly increased HUASMC viability and reduced their apoptosis (P<0.05); these effects were abrogated with PLC-gamma1 silencing. Compared with normal sera, PE sera increased [Ca2+]i in cocultured HUASMCs (P<0.05), which was inhibited by PLC-gamma1 and IP3R silencing. Finally, PE sera-induced PKC-alpha activity and collagen I expression was inhibited by PLC-gamma1 small interfering RNA (siRNA) (P<0.05). These results suggest that vasoactive substances in the PE serum may induce deposition in the extracellular matrix through the activation of PLC-gamma1, which may in turn result in thickening and hardening of the placental vascular wall, placental blood supply shortage, fetal hypoxia-ischemia and intrauterine growth retardation or intrauterine fetal death. PE sera increased [Ca2+]i and induced PKC-alpha activation and collagen I expression in cocultured HUASMCs via the PLC-gamma1 pathway.
Adult ; Apoptosis ; Calcium/*metabolism ; Cell Line ; Cell Survival ; Cells, Cultured ; Coculture Techniques ; Collagen Type I/analysis/*metabolism ; Female ; Human Umbilical Vein Endothelial Cells ; Humans ; Muscle, Smooth, Vascular/*cytology/metabolism ; Phospholipase C gamma/genetics/*metabolism ; Pre-Eclampsia/*blood/*metabolism/pathology ; Pregnancy ; Protein Kinase C-alpha/metabolism ; RNA Interference ; *Signal Transduction ; Young Adult

Objective To investigate the mechanism that mediates the inhibitory effect of Xinfeng Capsule(XFC)on interleukin(IL)-1β-induced impairment of chondrocytes.Methods XFC-medicated serum was collected from SD rats with XFC gavage,and its optimal concentration for chondrocyte treatment was determined using Cell Counting Kit-8 assay and flow cytometry.Dual luciferase reporter analysis was performed to analyze the targeting relationship between miR-502-5p and TRAF2.In cultured human chondrocytes induced with IL-1β,the effects of transfection with miR-502-5p inhibitor and XFC-medicated serum,alone or in combination,on expression levels of IL-1β,tumor necrosis factor-α(TNF-α),IL-4,and IL-10 were examined with ELISA,and the changes in the expressions of collagen type Ⅱ alpha 1(COL2A1),matrix metalloproteinase 13(MMP13),adisintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTS5),and miR-502-5p/TRAF2/NF-κB axis gene expression were detected using RT-qPCR,Western blotting,and immunofluorescence assay.Results In cultured human chondrocytes,treatment with IL-1β significantly decreased the cell viability,increased cell apoptosis rate,lowered miR-502-5p,IL-4,IL-10,and COL2A1 expressions,and enhanced IL-1β,TNF-α,ADAMTS5,MMP13,TRAF2,and NF-κB p65 expressions(P<0.05),and these changes were significantly improved by treatment with XFC-medicated serum at the optimal concentration of 20％(P<0.05).Transfection of the chondrocytes with miR-502-5p inhibitor resulted in elevated expressions of IL-1β,TNF-α,ADAMTS5,MMP13,TRAF2,and NF-κB p65 and lowered expressions of miR-502-5p,IL-4,IL-10,and COL2A1,and XFC-medicated serum obviously reversed the effects of miR-502-5p inhibitor.Conclusion XFC can inhibit IL-1β-induced inflammatory response and ECM degradation in cultured human chondrocytes possibly by regulating the miR-502-5p/TRAF2/NF-κB axis.

Objective To investigate the mechanism that mediates the inhibitory effect of Xinfeng Capsule(XFC)on interleukin(IL)-1β-induced impairment of chondrocytes.Methods XFC-medicated serum was collected from SD rats with XFC gavage,and its optimal concentration for chondrocyte treatment was determined using Cell Counting Kit-8 assay and flow cytometry.Dual luciferase reporter analysis was performed to analyze the targeting relationship between miR-502-5p and TRAF2.In cultured human chondrocytes induced with IL-1β,the effects of transfection with miR-502-5p inhibitor and XFC-medicated serum,alone or in combination,on expression levels of IL-1β,tumor necrosis factor-α(TNF-α),IL-4,and IL-10 were examined with ELISA,and the changes in the expressions of collagen type Ⅱ alpha 1(COL2A1),matrix metalloproteinase 13(MMP13),adisintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTS5),and miR-502-5p/TRAF2/NF-κB axis gene expression were detected using RT-qPCR,Western blotting,and immunofluorescence assay.Results In cultured human chondrocytes,treatment with IL-1β significantly decreased the cell viability,increased cell apoptosis rate,lowered miR-502-5p,IL-4,IL-10,and COL2A1 expressions,and enhanced IL-1β,TNF-α,ADAMTS5,MMP13,TRAF2,and NF-κB p65 expressions(P<0.05),and these changes were significantly improved by treatment with XFC-medicated serum at the optimal concentration of 20％(P<0.05).Transfection of the chondrocytes with miR-502-5p inhibitor resulted in elevated expressions of IL-1β,TNF-α,ADAMTS5,MMP13,TRAF2,and NF-κB p65 and lowered expressions of miR-502-5p,IL-4,IL-10,and COL2A1,and XFC-medicated serum obviously reversed the effects of miR-502-5p inhibitor.Conclusion XFC can inhibit IL-1β-induced inflammatory response and ECM degradation in cultured human chondrocytes possibly by regulating the miR-502-5p/TRAF2/NF-κB axis.

Ascosphaera apis spores containing a dark-colored pigment infect honeybee larvae, resulting in a large-scale collapse of the bee colony due to chalkbrood disease. However, little is known about the pigment or whether it plays a role in bee infection caused by A. apis. In this study, the pigment was isolated by alkali extraction, acid hydrolysis, and repeated precipitation. Ultraviolet (UV) analysis revealed that the pigment had a color value of 273, a maximum absorption peak at 195 nm, and a high alkaline solubility (7.67%) and acid precipitability. Further chemical structure analysis of the pigment, including elemental composition, Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, mass spectrometry, and nuclear magnetic resonance (NMR), proved that it was a eumelanin with a typical indole structure. The molecular formula of melanin is C₁₀H₆O₄N₂, and its molecular weight is 409 Da. Melanin has hydroxyl, carboxyl, amino, and phenolic groups that can potentially chelate to metal ions. Antioxidant function analyses showed that A. apis melanin had a high scavenging activity against superoxide, hydroxyl, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, and a high reducing ability to Fe³⁺. Indirect immunofluorescence assay (IFA), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) analyses showed that A. apis melanin was located on the spore wall. The spore wall localization, antioxidant activity, and metal ion chelating properties of fungal melanin have been suggested to contribute to spore pathogenicity. However, further infection experiments showed that melanin-deficient spores did not reduce the mortality of bee larvae, indicating that melanin does not increase the virulence of A. apis spores. This study is the first report on melanin produced by A. apis, providing an important background reference for further study on its role in A. apis.
Animals ; Antioxidants/pharmacology* ; Larva ; Melanins ; Molecular Structure ; Onygenales

ObjectiveThis study aims to explore the effects of Huangqin Qingre Chubi capsules-containing serum on the expression of CircRNA_0001543/nuclear factor-kappa B （NF-κB） in co-cultured peripheral blood mononuclear cells （PBMCs） and human fibroblast-like synoviocytes （FLSs） from patients with ankylosing spondylitis （AS）. MethodsVenous blood was collected from patients with AS to isolate PBMCs. FLSs were co-cultured with AS patients' PBMCs， and FLSs were harvested after co-culture for subsequent experiments. The normal control group consisted of normal FLSs， while the model group comprised co-cultured AS PBMCs and FLSs to simulate AS pathology. The Huangqin Qingre Chubi capsules group involved adding Huangqin Qingre Chubi capsules-containing serum to the co-cultured cells（6.48 g·kg^-1）. To investigate the effect of HQC-containing serum on the viability of co-cultured cells， and the experiment was divided into the following groups based on the dilution concentration： blank group， 10% HQC group， 20% HQC group， and 30% HQC group.To study the influence of the optimal concentration of HQC-containing serum on cytokine and pathway indicators in each group， the experiment was divided into three groups： normal group， model group， and optimal concentration HQC-containing serum group.For the validation of the transfection efficiency of the CircRNA_0001543 interference plasmid， the experiment was divided into the following groups： blank group， si-NC group （with transfection reagent）， si-circ_0001543-1 group （with transfection reagent and interference plasmid No. 1 targeting circ_0001543）， si-circ_0001543-2 group （with transfection reagent and interference plasmid No. 2 targeting circ_0001543）， and si-circ_0001543-3 group （with transfection reagent and interference plasmid No. 3 targeting circ_0001543）.For the validation of the transfection efficiency of the CircRNA_0001543 overexpression plasmid， the experiment was divided into the following groups： blank group， OE-NC group （with transfection reagent）， and OE-circ_0001543 group （with transfection reagent and overexpression plasmid targeting circ_0001543）.To study the effects of CircRNA_0001543 interference/overexpression on cytokine and pathway indicators in each group， the experiment was divided into the following groups： si-NC group， si-CircRNA_0001543 group， OE-NC group， and OE-CircRNA_0001543 group. Enzyme-linked immunosorbent assay （ELISA） was used to detect levels of interleukin-1β （IL-1β）， IL-10， IL-37， and tumor necrosis factor-α （TNF-α）. Real-time quantitative polymerase chain reaction （Real-time PCR） was utilized to measure the expression of CircRNA_0001543， IκBα， and NF-κB p65. ResultsAfter 48 hours， 30% Huangqin Qingre Chubi Capsules-containing serum significantly inhibited the proliferation of co-cultured PBMCs and FLSs， which was determined to be the optimal experimental drug-containing serum concentration. Compared with those in the normal group， the expressions of NF-κB p65 mRNA， IκBα mRNA， IL-1β， and TNF-α in the model group were significantly increased （P<0.01）， while the expressions of CircRNA_0001543 mRNA， IL-10， and IL-37 were significantly decreased （P<0.01）. Compared with those in the model group， the expressions of NF-κB p65 mRNA， IκBα mRNA， IL-1β， and TNF-α in the Huangqin Qingre Chubi Capsules-containing serum group were significantly decreased （P<0.05）， and the expressions of CircRNA_0001543 mRNA， IL-10， and IL-37 were significantly increased （P<0.05）， with the most prominent changes in the 30% drug-containing serum group （P<0.01）. Compared with that in the si-NC group， the expression of CircRNA_0001543 was significantly reduced in the si-CircRNA_0001543 group （P<0.01）. Compared with that in the OE-NC group， the expression of CircRNA_0001543 was significantly increased in the OE-CircRNA_0001543 group （P<0.01）， indicating that the si-CircRNA_0001543 and OE-CircRNA_0001543 plasmids were successfully transfected. Based on the optimal drug-containing serum of Huangqin Qingre Chubi Capsules， si-CircRNA_0001543 transfection led to significantly increased expressions of NF-κB p65 mRNA， IκBα mRNA， IL-1β， and TNF-α and decreased the expressions of IL-10 and IL-37 （P<0.01）. In contrast， OE-CircRNA_0001543 transfection significantly decreased the expressions of NF-κB p65 mRNA， IκBα mRNA， IL-1β， and TNF-α （P<0.01） and increased the expressions of IL-10 and IL-37 （P<0.01）. ConclusionHuangqin Qingre Chubi capsules-containing serum can improve immune inflammation in AS by increasing the expression of CircRNA_0001543， regulating the NF-κB pathway， suppressing pro-inflammatory cytokines， and enhancing anti-inflammatory cytokine expression.