Transcriptome analysis of three medicinal plants of the genus Polygonatum: identification of genes involved in polysaccharide and steroidal saponins biosynthesis.

Polysaccharides and saponins are the main active components of Polygonati Rhizoma. Studying the molecular mechanism of their synthesis pathway is helpful in improving the content of active components at the molecular level. At present, transcriptome analysis of three Polygonatum species (Polygonatum sibiricum Red., Polygonatum cyrtonema Hua, Polygonatum kingianum Coll. et Hemsl.) has been reported, but no comparative study has been found on the transcriptome data of the three species. Transcriptome sequencing was performed on the rhizomes of three Polygonatum species based on high-throughput sequencing technology, and all transcripts were assembled. A total of 168,108 unigenes were generated after the removal of redundancy, of which 121,642 were annotated in seven databases. Through differential analysis and expression analysis of key enzyme genes in the synthesis pathway of three Polygonatum polysaccharides and steroidal saponins, 135 differentially expressed genes encoding 18 enzymes and 128 differentially expressed genes encoding 28 enzymes were identified, respectively. Numerous transcription factors are involved in the carbohydrate synthesis pathway. Quantitative real-time PCR was used to further verify the gene expression level. In this paper, we present a public transcriptome dataset of three medicinal plants of the genus Polygonatum, and analyze the key enzyme genes of polysaccharide and steroidal saponins synthesis pathway, which lays a foundation for improving the active component content of Polygonati Rhizoma by molecular means.

Myosin light chain kinase inhibitor ML7 improves vascular endothelial dysfunction and permeability via the mitogen-activated protein kinase pathway in a rabbit model of atherosclerosis.

Endothelial dysfunction (ED) and hyperpermeability are considered as the initiating steps in early atherosclerosis. Phosphorylation of myosin light chain (MLC) is key to cause vascular hyperpermeability via endothelial cell contraction. However, it is unclear whether MLC phosphorylation can also regulate the balance between contraction and relaxation of endothelial cells, thereby affecting endothelium-dependent diastolic function and leading to ED. The present study investigated relationships between ED and MLC phosphorylation and underlying mechanisms. Twenty-four male New Zealand white rabbits were randomly divided into three groups: control, AS, and ML7 (MLCK inhibitor) groups, and fed with normal diet, high-fat diet (HFD), and HFD plus oral ML7 (1 mg/kg daily) respectively. HFD-fed rabbits showed typical atheromatous lesions and endothelial hyperpermeability, and these lesions could be partly reversed following ML7 therapy. Western blotting revealed significant increased expression of myosin light chain kinase (MLCK) and phosphorylation of MLC, JNK, and ERK in the arterial wall of rabbits in the AS group compared with those of the control group (p < 0.05), whereas the ML7 group showed markedly decreased levels of these proteins compared with the AS group (p < 0.05). The endothelium-dependent relaxation rate was significantly reduced both in vitro and in vivo in AS group, and was improved using ML7 therapy. Taken together, these results indicate that MLCK expression and subsequent MLC phosphorylation increase vascular endothelial permeability and endothelium-dependent diastolic dysfunction by promoting endothelial cell contraction, which may be initiated by the activation of the MAP/ERK (MEK) and MAP/JNK (MEK) pathways.

MiRNA-155-5p Reduces Corneal Epithelial Permeability by Remodeling Epithelial Tight Junctions during Corneal Wound Healing.

PURPOSE: Corneal epithelial cells play a vital role in the function of the cornea by forming a physical barrier to protect the eye from invasion by external pathogenic agents. A recent study showed that miR-155 promotes cutaneous wound healing. However, its function in corneal epithelial wound healing is unknown. The present study examined whether miR-155-5p reduces corneal epithelial permeability by remodeling epithelial tight junctions during corneal wound healing. MATERIALS AND METHODS: Rat corneal wounds were produced by removing the central corneal epithelium with a blunt scalpel blade under a dissecting microscope. One eye of each rat was treated with topical miR-155-5p, and the other eye was treated with topical agomir negative control for 3 days before and after corneal epithelial wounding. Corneal epithelial permeability was assessed by the macromolecular osmosis method. Expression of zona occludens 1 (ZO-1), occludin, and myosin light chain kinase (MLCK) and phosphorylation of myosin light chain (MLC) were detected by Western blot. Human corneal epithelial (HCE) cells were cultured in the upper chamber of Transwell filters, and transepithelial electrical resistance (TER) was measured using a voltohmmeter. The distribution of ZO-1 and occludin in HCE cells treated with miR-155-5p was determined by immunofluorescence. RESULTS: miR-155-5p significantly promoted the repair of corneal epithelial injury and reduced the permeability of the corneal epithelium. It significantly decreased expression of MLCK and phosphorylation of MLC and increased expression of the tight junction proteins ZO-1 and occludin in corneal epithelial cells during corneal wound healing. miR-155-5p significantly increased TER, decreased MLCK expression and MLC phosphorylation, increased ZO-1 and occludin expression, and promoted anchoring of tight junction proteins in the cell membrane and remodeling in HEC cells. CONCLUSIONS: Our results suggest that miR-155-5p reduced corneal permeability and accelerated the recovery of corneal epithelial wounds by decreasing the expression of MLCK and phosphorylation of MLC and by remodeling tight junctions.