ABSTRACT
Objective:To investigate the neuroprotective effect of Danggui Shaoyaosan （DSS） in a rat model of amyloid-<italic>β</italic>-peptide<sub>1-42</sub> （A<italic>β</italic><sub>1-42</sub>）-induced Alzheimer's disease （AD） as well as its regulatory effect on NOD-like receptor protein 3 （NLRP3）/cysteinyl aspartate-specific protease-1 （Caspase-1） signaling pathway. Method:The AD animal model was established via intracerebral injection of A<italic>β</italic><sub>1-42</sub> and treated with different concentrations of DSS after the division of rats into the sham operation group， model group， as well as the high-， medium-， and low-dose DSS groups. Morris water maze test was conducted to determine the learning and memory abilities of rats. The morphology and function of neurons were detected by hematoxylin-eosin （HE） staining and Golgi staining， followed by immunofluorescence co-localization of NLRP3 inflammasome activation. The mRNA expression levels of interleukin （IL）-1<italic>β</italic> and IL-18 were measured by Real-time polymerase chain reaction （Real-time PCR）， and the protein expression levels of NLRP3， Caspase-1， and IL-1<italic>β </italic>were assayed by Western blot. Result:Compared with the sham operation group， the model group exhibited significantly decreased learning and memory abilities （<italic>P</italic><0.01）， impaired neuronal morphology and function， up-regulated IL-1<italic>β</italic> and IL-18 mRNA expression， enhanced NLRP3 inflammasome activation， and elevated NLRP3， Caspase-1， and IL-1<italic>β</italic> protein expression （<italic>P</italic><0.01）. Compared with the model group， DSS at both medium and high doses remarkably improved the learning and memory abilities of AD rats （<italic>P</italic><0.05， <italic>P</italic><0.01）， restored neuronal morphology and function， down-regulated the mRNA expression levels of inflammatory factors IL-1<italic>β</italic> and IL-18， reduced the activation of NLRP3 inflammasomes， and lowered the protein expression levels of NLRP3， Caspase-1， and IL-1<italic>β</italic> （<italic>P</italic><0.01）. Conclusion:DSS inhibits inflammasome activation and neuroinflammatory response possibly by regulating the NLRP3/Caspase-1 signaling pathway， thus exerting the neuroprotective effect.
ABSTRACT
The objective of this research was to clone 1-deoxy-D-xylulose 5-phosphate reductoisomerase gene (MoDXR) and its promoter sequence from Morinda officinalis and carry out bioinformatic analysis, cis-acting elements analysis, and prokaryotic expression. On the basis of the MoDXR gene sequence obtained from the M. officinalis transcriptome and with NCBI-ORFfinder analysis, a pair of specific primers were designed, and used for RT-PCR amplification. The promoter region sequence at the 5′ end of MoDXR gene was isolated by the genome walking technique. Localization of MoDXR was carried out by subcellular analysis. The prokaryotic expression plasmid pET-28a-MoDXR was constructed and transfected into Escherichia coli BL21(DE3) chemically-competent cells; the recombiant plasmid expressed fusion protein after the induction by IPTG. The full-length cDNA of MoDXR was 2 015 bp,and open reading frame (ORF) size was 1 425 bp, and it encoded 474 amino acid residues and had a molecular mass of 51.27 kD. Sequence comparison with BlastP to the NCBI database revealed that MoDXR had high sequence similarity with many other DXRs, such as Coffea arabica DXR (CaDXR) and Rauvolfia verticillata DXR (RvDXR). A phylogenetic tree revealed that MoDXR had its closest relationship with DXR from Coffea arabica and Gardenia jasminoides. The subcellular localization revealed that MoDXR protein was located on the chloroplast. Plantcare analysis indicated that the promoter region sequence of MoDXR was 1 493 bp, covering multiple light, stress, and hormone-responsive cis-regulatory elements; protein electrophoresis showed that the expressed protein was the anticipated size. This research lays the foundation for further purification and structural and functional characterization of the MoDXR protein.
ABSTRACT
In order to explore MYB transcription factors related to developmental processes and secondary metabolism in Morinda officinalis, we analyzed MoMYB expression based on transcriptome data from three tissues (root, stem and leaf). We used this analysis to provide a theoretical foundation for regulating the metabolism of M. officinalis. RNA-seq data along with the five databases including PFAM and plantTFDB and others were used to screen and classify MoMYB, including GO functional annotation and classification, subcellular localization, signal peptide prediction, conserved motif discovery, and comparative phylogenetic analysis. RT-qPCR was carried out to detect tissue-specific expression differences of MoMYB genes. According to transcriptome data, 109 MoMYB sequences were identified and divided into four classes, containing 51 sequences related to R2R3-MYB. Subcellular localization analysis indicated that a majority of sequences were located in nucleus. Blast2GO analysis showed that 109 MoMYB sequences were classified into three major functional ontologies including molecular function (112), biological processes (76) and cellular components (239). The R2-MYB conserved motif of 51 R2R3-MYB sequences possessed three significantly conserved tryptophan residues, whereas a phenylalanine replaced the first tryptophan in R3-MYB. The results of multiple sequence alignment and phylogenetic analysis revealed that the R2R3-MYB was distributed in all subgroups, apart from the S10, S19 and S21 subgroups. RT-qPCR indicated that several R2R3-MYB genes were differentially expressed among the three tissues, and this finding was consistent with transcriptome data. The 109 MoMYB sequences were annotated and divided into different classes, which lays the foundation for further study on MYB transcriptional factors in M. officinalis.