Apigenin suppresses mycoplasma-induced alveolar macrophages necroptosis via enhancing the methylation of TNF-α promoter by PPARγ-Uhrf1 axis.

BACKGROUND: Mycoplasma-associated pneumonia is characterized by severe lung inflammation and immunological dysfunction. However, current anti-mycoplasma agents used in clinical practice do not prevent dysfunction of alveolar macrophages caused by the high level of the cytokine tumor necrosis factor-α (TNF-α) after mycoplasma infection. Apigenin inhibits the production of TNF-α in variet inflammation associated disease. PURPOSE: This study aimed to investigate apigenin's effect on mycoplasma-induced alveolar immune cell injury and the mechanism by which it inhibits TNF-α transcription. METHODS: In this study, we performed a mouse model of Mycoplasma hyopneumoniae infection to evaluate the effect of apigenin on reducing mycoplasma-induced alveolar immune cell injury. Furthermore, we carried out transcriptome analysis, RNA interference assay, methylated DNA bisulfite sequencing assay, and chromatin immunoprecipitation assay to explore the mechanism of action for apigenin in reducing TNF-α. RESULTS: We discovered that M. hyopneumoniae infection-induced necroptosis in alveolar macrophages MH-S cells and primary mouse alveolar macrophages, which was activated by TNF-α autocrine. Apigenin inhibited M. hyopneumoniae-induced elevation of TNF-α and necroptosis in alveolar macrophages. Apigenin inhibited TNF-a mRNA production via increasing ubiquitin-like with PHD and RING finger domains 1 (Uhrf1)-dependent DNA methylation of the TNF-a promotor. Finally, we demonstrated that apigenin regulated Uhrf1 transcription via peroxisome proliferator activated receptor gamma (PPARγ) activation, which acts as a transcription factor binding to the Uhrf1 promoter and protected infected mice's lungs, and promoted alveolar macrophage survival. CONCLUTSION: This study identified a novel mechanism of action for apigenin in reducing alveolar macrophage necroptosis via the PPARγ/ Uhrf1/TNF-α pathway, which may have implications for the treatment of Mycoplasma pneumonia.

microRNA-125b Regulates Apoptosis by Targeting Bone Morphogenetic Protein Receptor 1B in Yak Granulosa Cells.

The intronic microRNA, miR-125b, plays a vital role in promyelocytic and hematopoietic stem cells, and in the development and apoptosis of cancer cells. In this study, we showed that miR-125b regulates granulosa cell (GC) apoptosis in the yak ovary. Bioinformatic analyses and luciferase reporter assays demonstrated that bone morphogenetic protein receptor type 1B (BMPR1B) is an miR-125b target. miR-125b overexpression induced apoptosis in yak GC, and affected the mRNA and protein expression of BMPR1B and the ratio of Bcl2/Bax. Silencing of miR-125b decreased the rate of yak GC apoptosis and increased the ratio of Bcl2/Bax. In addition, the effects of an miR-125b inhibitor were overturned by cotransfection with siRNA-BMPR1B2 (siRNA-299) in yak GC. Together, these results demonstrated that miR-125b regulates GC apoptosis in the yak ovary by targeting BMPR1B.

A quantitative method for measuring the antitumor potency of recombinant human endostatin in vivo.

Recombinant human endostatin (rhEndostatin) has been shown to inhibit tumor growth, but the variable antitumor activity of different rhEndostatin preparations has necessitated the development of an accurate, reproducible in vivo bioassay for evaluating the rhEndostatin activity. To assess the in vivo antitumor efficacy of rhEndostatin, H22 tumor-bearing mice received three doses of rhEndostatin and the potency of rhEndostatin preparations in inhibiting tumor growth was determined by ED(50)-potency assay and validated by dose-response parallel-line assay. There was a consistent and highly reproducible linear regression relationship between rhEndostatin dosage and tumor growth inhibition rate. The ED(50) values were determined from dose-response regression lines for seven rhEndostatin preparations with high reproducibility. On the basis of the current study, the potency of rhEndostatin preparations was assigned a value of 6.09 x 10(5) U/ampoule and a 95% confidence limit of 5.96 x 10(5)-6.22 x 10(5). We consider that this procedure can be served as a potential candidate pharmacopoeial method for potency measurement of different rhEndostatin preparations.

RNA interference remarkably suppresses bcl-2 gene expression in cancer cells in vitro and in vivo.

Bcl-2 is an anti-apoptotic protein. If the level of Bcl-2 protein can be reduced sufficiently in tumors using RNA interference (RNAi) to target the gene message, the apoptosis of tumor cells may be promoted. In this study, we synthesized 19 nucleotides (nts) small interference RNA (siRNA) constructs suppressing bcl-2 gene expression in human tumor cells (HeLaB2 and BGC-823 cell lines) in vitro. The bcl-2 gene expression levels were significantly reduced when these siRNA were transfected into experimental two tumor cells for 72 hours. The apoptosis process was also examined in the tumor cells. Here we synthesized siRNA from a DNA template under the control of the RNA polymerase III promoter in transfected tumor cells. Using this DNA vector-based approach, we found that the siRNA efficiently and specifically inhibited the synthesis of protein encoded by the bcl-2 gene in HeLaB2 and BGC-823 tumor cells. Tumor growth was inhibited by 66.5% with 2mg/kg pSilencer 3.1H1-bcl-2 in mouse liver tumor-bearing BALB/c mice. This approach may prove to be a valuable clinical technique for the analysis of specific gene functions and gene therapy of malignant tumors that utilize the bcl-2 gene via RNA interference.

The methods for determining the purity and in vitro or in vivo activity of recombinant human endostatin.

In order to establish the methods of high-performance liquid chromatography (HPLC) for determining the purity of recombinant human endostatin (rhEndostatin) and in vitro or in vivo activity of rhEndostatin, two columns were firstly used in HPLC analysis for determining the purity of rhEndostatin, including Waters Symmetry 300C4 (4.6 mm x 250 mm, 5 microm) and the Superdex75 HR 10/30. Cell lines, bovine capillary endothelial cells (BCEs) or human umbilical vein endothelial cells (HUVECs) expression human vascular endothelial growth factor (hVEGF) were used in method MTT or LDH as substrate, respectively. The bioactivity in vivo was assayed by the anti-tumor proliferation rate in H22 liver tumor-bearing mice. The results showed that the retention time of rhEndostatin sample was stable at 19.066 min or 11.506 min in reverse phase HPLC (RP-HPLC) or gel filtering HPLC (GF-HPLC). The stableness, repeat and recovery rates were over 99% in both methods and there was no statistical difference between these two methods (p > 0.05). In nonserum culture medium, rhEndostatin can sensitively and stably inhibit the proliferation of the HUVEC cells that were transfected with plasmid encoding hVEGF. LDH substrate methods is the most sensitive and stable method. The anti-tumor activity in H22 tumor-bearing mice was also highly repeatable and had an inhibition rate over 50% at 20 mg kg(-1) weight. As a conclusion, the RP-HPLC and GF-HPLC set up in this paper are highly repeatable, accurate and sensitive for detecting the purity of rhEndostatin. The bioactivity of rhEndostatin can be measured through detection the proliferation-inhibition on HUVECs transfectants with hVEGF in vitro or on H22 liver tumor in vivo.