Protective Effect of Galangin Methylation Modification Based on Cell Imaging on Inflammatory Lung Injury and Its Molecular Mechanism.

Background: Recently, inflammation has become a major threat to human health. Studies have confirmed that some Chinese traditional medicine ingredients may effectively interfere with the expression of inflammatory mediators through epigenetic modification, showing a great potential of the application. Objective: To investigate the role of the PPAR/DNMT3A pathway in the reversal of galangin-mediated inflammatory lung injury, promote the development of new anti-inflammatory drugs, reduce the side effects of chemical synthetic drugs on the body, and prove the effectiveness and safety of galangin in inhibiting inflammatory response and injury. Methods: 120 rats were randomly divided into 6 groups: (Group 1) LPS group; (Group 2) LPS + galangin group; (Group 3) LPS + galangin + GW9662 group; (Group 4) LPS + galangin + DNMT3A siRNA group; (Group 5) LPS + galangin + siRNA negative group; (Group 6) control group. The model of inflammatory lung injury was established by intrathecal instillation of LPS in the first five groups and NS in the control group. SD survival rate was recorded every 24 hours after modeling, lasting for 168 hours. The lung tissues were taken 168 hours after the establishment of the model. The pathological morphology of lung tissue was observed after the staining under the light microscope, and the lung dry/wet weight ratio was calculated after drying. After NS was perfused into lung tissue, the lavage fluid was collected and the levels of IL-6 and TNF-a were measured by ELISA. The contents of PPAR, DNMT3A, phosphorylated p65, and ERK in monocytes were detected by the WB method, and the binding contents of p65 and AP-1 in the promoter regions of IL-6 and TNF-a genes were detected by the Chip-qPCR method. Results: Intraperitoneal injection of galangin could inhibit the synthesis of alveolar inflammatory factors (TFs) in the SD model of lung injury induced by LPS, reduce the degree of pathological injury of lung tissue, and improve the survival rate of the SD model. GW9662 can completely reverse the protective effect, while DNMT3A interference can only partially block its protective effect. In addition, galangin could significantly inhibit the LPS-induced expression of p65 and AP-1 in alveolar monocytes and their binding content in the promoter region of inflammatory genes by activating PPAR/DNMT3A pathway. GW9662 could completely reverse the inhibitory effect of galangin. DNMT3A interference could restore the binding content of transcription factors at the promoter of the inflammatory gene but had no significant effect on its synthesis. Conclusion: Galangin can interfere with the binding of transcription factors to inflammatory gene promoters through the methylation modification induced by PPAR/DNMT3A pathway, so as to inhibit the synthesis of inflammatory molecules and reverse inflammatory lung injury.

Inhibitory effect of alpinetin on IL-6 expression by promoting cytosine methylation in CpG islands in the IL-6 promoter region.

BACKGROUND: Alpinetin is a flavonoid which exerts antibacterial and anti-inflammatory functions. In order to prove that the induced methylation is an important mechanism for alpinetin in regulating the expression of inflammatory factor Interleukin-6 (IL-6), we detected the dinucleotide methylation status of CpG islands in the IL-6 promoter region and IL-6 level after treatment of RAW246.7 murine macrophages with alpinetin. METHODS: After RAW246.7 murine macrophages were treated with alpinetin, alpinetin + GW9662 (the peroxisome proliferator-activated receptor (PPAR) antagonist), and alpinetin + DNA methyltransferase 3 alpha (DNMT3A) siRNA for 96 hr, CpG islands were analyzed using time-of-flight mass spectrophotometry (TOF-MS) and bisulfite sequencing polymerase chain reaction (BSP). Dinucleotide methylation status of the CpG islands in the IL-6 promoter region was analyzed by methylation-specific Polymerase Chain Reaction (PCR). IL-6 level was detected using the enzyme-linked immunosorbent assay (ELISA) method. Pearson's correlation analysis was conducted to test for potential correlation between the methylation status of CpG islands in the IL-6 promoter region and IL-6 level in RAW 246.7 cells. RESULTS: Alpinetin promoted dinucleotide methylation status of two CpG islands in the IL-6 promoter region stretching 500-2500 bp upstream of the transcriptional start site (TSS) (p < .05). This promoting effect was more significant for the CpG island stretching 500-1500 bp long. The methylation ratio of dinucleotide at this position was significantly inversely correlated with the level of IL-6 (p < .05). PPAR antagonist GW9662 and interference of DNMT3A could reverse both the alpinetin-induced methylation and inhibitory effects on IL-6 expression. CONCLUSION: Alpinetin could induce dinucleotide methylation status of CpG islands in the IL-6 promoter region by activating methyltransferase, thus inhibiting IL-6 expression in murine macrophages.

Construction, expression and characterization of a fusion protein HBscFv-IFNγ in Komagatella (Pichia) pastoris X33.

HBscFv-IFNγ, a fusion protein constructed by fusing γ-interferon (IFNγ) with an antibody fragment HBscFv for the purpose of targeted delivery of the cytokine IFNγ, was designed in order to enhance its therapeutic efficacy through increasing its hepatoma localization. HBscFv and IFNγ were connected into HBscFv-IFNγ by the linker (Gly4Ser)3, and then the multicopy recombinant plasmids pPICZαA/(HBscFv-IFNγ)1,2,4 were constructed and transformed into Komagatella (Pichia) pastoris X33. The engineering strain X4, which had much higher copy number and could secretively express HBscFv-IFNγ, was screened from transformed X33 by qPCR. Results from SDS-PAGE, Western blotting and ELISA indicated that HBscFv-IFNγ displayed an excellent immunoreaction against HBsAg. The culture supernatant of X4 was purified by 14F7 affinity chromatography to obtain the fusion protein HBscFv-IFNγ in a purity of 95-98%. The HBscFv-IFNγ was able to bind 27.9% HBsAg in the serum of HBV transgenic mice, showing that the antibody of HBscFv-IFNγ has high binding affinity against HBsAg. The expressing of the recombinant HBscFv-IFNγ in P. pastoris provides a promising and inexpensive diagnostic reagent for preventing HBV infection.

Analysis of carotenogenic genes promoters and WRKY transcription factors in response to salt stress in Dunaliella bardawil.

The unicellular alga Dunaliella bardawil is a highly salt-tolerant organism, capable of accumulating glycerol, glycine betaine and ß-carotene under salt stress, and has been considered as an excellent model organism to investigate the molecular mechanisms of salt stress responses. In this study, several carotenogenic genes (DbCRTISO, DbZISO, DbLycE and DbChyB), DbBADH genes involved in glycine betaine synthesis and genes encoding probable WRKY transcription factors from D. bardawil were isolated, and promoters of DbCRTISO and DbChyB were cloned. The promoters of DbPSY, DbLycB, DbGGPS, DbCRTISO and DbChyB contained the salt-regulated element (SRE), GT1GMSCAM4, while the DbGGPS promoter has another SRE, DRECRTCOREAT. All promoters of the carotenogenic genes had light-regulated elements and W-box cis-acting elements. Most WRKY transcription factors can bind to the W-box, and play roles in abiotic stress. qRT-PCR analysis showed that salt stress up-regulated both carotenogenic genes and WRKY transcription factors. In contrast, the transcription levels of DbBADH showed minor changes. In D. bardawil, it appears that carotenoid over-accumulation allows for the long-term adaptation to salt stress, while the rapid modulation of glycine betaine biosynthesis provides an initial response.

Characterization and expression of AMP-forming Acetyl-CoA Synthetase from Dunaliella tertiolecta and its response to nitrogen starvation stress.

AMP-forming acetyl-CoA synthetase (ACS) catalyzes the formation of acetyl-CoA. Here, a cDNA of ACS from Dunaliella tertiolecta (DtACS) was isolated using RACEs. The full-length DtACS cDNA (GenBank: KT692941) is 2,464 bp with a putative ORF of 2,184 bp, which encodes 727 amino acids with a predicted molecular weight of 79.72 kDa. DtACS has a close relationship with Chlamydomonas reinhardtii and Volvox carteri f. nagariensis. ACSs existing in Bacteria, Archaea and Eukaryota share ten conserved motifs (A1-A10) and three signature motifs (I-III) of the acyl-adenylate/thioester forming enzyme superfamily. DtACS was expressed in E. coli BL21 as Trx-His-tagged fusion protein (~100 kDa) and the enzymatic activity was detected. The recombinant DtACS was purified by HisTrap(TM) HP affinity chromatography to obtain a specific activity of 52.873 U/mg with a yield of 56.26%, which approached the specific activity of ACS isolated from other eukaryotes. Kinetic analysis indicated that the Km of DtACS was 3.59 mM for potassium acetate, and the purified DtACS exhibited a temperature optimum of 37 °C and a pH optimum of 8.0. In addition, the expression levels of DtACS were increased after nitrogen starvation cultivation, indicating that ACS activity may be related to the lipid accumulation under nitrogen deficient condition.

Characterization and Functional Identification of a Gene Encoding Geranylgeranyl Diphosphate Synthase from Dunaliella bardawil.

Geranylgeranyl diphosphate synthase (GGPS) catalyzes the biosynthesis of geranylgeranyl diphosphate, a key precursor for carotenoid biosynthesis. In this study, a full-length cDNA encoding GGPS from Dunaliella bardawil (DbGGPS) was isolated by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of DbGGPS was 1814 bp, containing a 1074 bp ORF encoding 357 amino acids with a calculated mass of 38.88 kDa. Analysis of DbGGPS genomic DNA revealed that it contained 10 exons and 9 introns. It was predicted that DbGGPS possessed a 48 amino acid transit peptide at its N terminus. Bioinformatic analysis revealed that DbGGPS was a member of a group of polyprenyltransferases with five conserved domains and two highly conserved aspartate-rich motifs. Using heterologous expression, carotenoid complementation assay, and gene deletion analysis, it was shown that the coding region of DbGGPS encodes a functional GGPS. This provides new gene sources for carotenoid genetic engineering.