Research Of Molecular Markers And Effect In Anti-Inflammation Of Atheroclerosis On Cultivated Paeonia Of Paeonia Suffruticosa

In recent years, it has provide new methods and means in order to reveal the genetic diversity of medicinal plants and identify species of genuineness of Chinese herbal with the rapidly development of technology of molecular marker and DNA fingerprinting analysis, which was widely applied in crop germplasm identification. ITS region (including ITS-1, 5.8S and ITS-2) of ribosomal DNA has widely used in taxonomic studies because it has many fragments and easily amplification. It has highly variation of nucleotide sequence and conservation of length, which is easy to sort in relative group. While it's rich variation can also resolve questions of plant phylogeny in lower classification. Firstly, it is need to get high quality genomic DNA from medicinal herbs in these studies. We usually obtain purpose DNA from deal with fresh leaves. However, it is difficult to and (NO.KJ070304).extract DNA with tradition method from medicinal herbs, which containing large amount of amylose, protein and secondary metabolites serious interference Genome extraction.Several recent studies have provided evidence that the inflammatory chemokines (IL-8 NO, et al) plays an important role in the onset, development and evolution of atherosclerotic lesions. Paeonol (20-hydroxy-40-methoxyacetophenone), the main active compound of the radix of Paeonia suffruticosa, and the root cortex of Paeonia suffruticosa Andrews, has been reported has a widely pharmacological effect on anti-inflammatory, anti-spasmodic, antipyretic analgesics, and so on.In this study, we firstly establish a set of economic, fast and efficient method which can isolate the genomic DNA from Root Bark Chinese medicinal herb on the basis of the CTAB method. The extraction DNA improved by CTAB method was pure, integral, the value of A260/A280 was 1.8 to 2.0,the applicated bands of PCR is clearly and brightly ,which lay a good foundation for the following molecular biology experiments.Secondly, the coding region of the rDNA ITS gene in different groups of peonies were sequenced to study phylogenetic relationships of 19 Paeonia species. According to the characteristics and variation of ITS gene sequence of Paeonia from different area, the molecular markers of peonies was proposed by considering the discordance between various taxa. The rDNA ITS regions were compared with the GeneBank information (accession No. U27692) and analyzed and identified the polymorphism (SNP) sites in the different groups of peonies by Clustal X, MEGA 3.1. The length of rDNA ITS gene sequence of paeonia varieties is 652 bp, including ITS1 (267 bp), 5.8S (163 bp) and ITS1 (222 bp). The GC content is about 55.7% to 56.9%. Compared with the rDNA ITS gene sequences of paeonia (U27692), the specific 448 site in the samples tested which is C in database is lost. There is not only about 30 kinds of SNPs in non-coding region ITS1 and ITS2, but also some coding SNPs loci in the conservative region of 5.8S. Using ITS gene sequences, we were able to distinguish paeonia cultured in Chongqing from all other species of paeonia included in this study.Thirdly, the aim of present study is to explore the effects and mechanisms of paeonol on inflammation by lipopolysaccharides (LPS)-induced on human umbilical vein endothelial (HUVEC).The cells were treated with LPS and paeonol. IL-8 concentration was measured a commercially available sandwich ELISA. Moreover, IL-8 mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Extracellular NO and NOS were detected with an assay kit according to the manufacturer's protocol. Intracellular NO was monitored by flow cytometry and fluorescence microscope stained with DAF-FM diacetate. Annexin V and PI staining were used to analysis cell apoptosis and cell cycle, respectively. The results indicated that Paeonol could inhibit IL-8 productions induced by LPS in HUVECs. The expression of IL-8 mRNA was increased in a time-dependent manner after stimulation with LPS, whereas expression of IL-8 mRNA induced by LPS was significantly diminished by 50μg/ml paeonol. Paeonol increased the production of NO and NOS which was inhibited by LPS. In addition, our results also indicated that paeonol could control cell apoptosis and cell cycle arrest mediated by LPS.Obviously, rDNA ITS sequence can be used to identify the main peony cultivars of different regions. The inflammations on atherosclerosis induced by LPS can be influenced by paeonol. Our findings suggest a novel application for paeonol in the treatment of atherosclerosis of cardiovascular diseases.