Anti-rheumatoid arthritis potential of Rhododendron molle G. Don leaf extract in adjuvant induced arthritis rats.

AIM OF THE STUDY: The aim of this study was to test the anti-rheumatic arthritis effects of Rhododendron molle G. Don leaf extract in arthritis rats and inflammatory RAW 264.7 cells. Preliminary analysis and comparison of potential medicinal components of three polar extracts by HPLC and UHPLC-Q-TOF-MS. MATERIALS AND METHODS: SD rats were subcutaneously injected with complete Freund's adjuvant (CFA) to induce inflammation on the right hind paw. RAW 264.7 cells were induced by lipopolysaccharide (LPS) to established cell inflammatory model. The volume of rat hind paw was measured with a volume meter to detect swelling, and the weight of rats was measured with an electronic balance. The severity of arthritis in rats was evaluated by arthritis score. The pathological sections of rat hind paw joints were observed by hematoxylin-eosin staining, and the contents of IL-6 and IL-1ß in serum were detected. qRT-PCR was used to detect the expression of IL-1ß, IL-6, TNF-α and COX-2 genes in RAW 264.7 cells. The release of nitric oxide was measured by Griess reaction. The expression levels of IL-6 and IL-1ß were detected by Western-Blot. RESULTS: and discussion: The chloroform extract from R. molle leaves (CERL), Ethyl acetate extract from R. molle leaves (EERL), n-butanol extract from R. molle leaves (BERL) could significantly inhibit hind paws swelling and reduce arthritis index in arthritis rats. And it showed dose dependence. Compared with tripterygium glycosides (TG) tablets, an effective drug of RA treatment, CERL have better anti-RA effect after administration. In addition, the three kinds of the polar extracts of Rhododendron molle leaves (PERL) had lower toxicity, with the LD50 279.87, 239.65, 500.08 (mg/kg) respectively, while TG group's LD50 was 96.00 (mg/kg). In vitro experiments showed that the three PERLs can significantly inhibit the level of pro-inflammatory factors and inflammatory mediator, such as TNF-α, IL-1ß, IL-6, COX-2 and NO, which were consistent with their anti-RA ability. Among the three kinds of PERLs, CERL showed the best inhibitory activity. CONCLUSION: The R. molle leaf is a potential medicinal part for the treatment of RA. This study explored the anti-RA and anti-inflammatory activities of CERL, EERL, BERL, which laid a foundation for further promoting the clinical application of R. molle.

Analysis of the genetic diversity and population structure of Monochasma savatieri Franch. ex Maxim using novel EST-SSR markers.

BACKGROUND: Monochasma savatieri Franch. ex Maxim is a medicinally valuable herb. However, the collection and protection of the wild germplasm resources of M. savatieri are still insufficient, and their genetic diversity and population structure have been poorly studied. RESULTS: We collected and examined 46 M. savatieri individuals from Fujian, Hunan, Jiangxi, and Zhejiang provinces for genetic diversity and population structure, using 33 newly developed expressed sequence tag-simple sequence repeat (EST-SSR) markers. Applying these markers, we detected a total of 208 alleles, with an average of 6.303 alleles per locus. The polymorphic information content varied from 0.138 to 0.884 (average: 0.668), indicating a high level of polymorphism. At the population level, there was a low degree of genetic diversity among populations (I = 0.535, He = 0.342), with Zhejiang individuals showing the highest genetic diversity among the four populations (Fst = 0.497), which indicated little gene flow within the M. savatieri populations (Nm = 0.253). Mantel test analysis revealed a significant positive correlation between geographical and genetic distance among populations (R2 = 0.3304, p < 0.05), and structure and principal coordinate analyses supported classification of populations into three clusters, which was consistent with the findings of cluster analysis. CONCLUSIONS: As a rare medicinal plants, the protection of M. savatieri does not look optimistic, and accordingly, protective efforts should be beefed up on the natural wild populations. This study provided novel tools and insights for designing effective collection and conservation strategies for M. savatieri.

Identification of Phosphorus Stress Related Proteins in the Seedlings of Dongxiang Wild Rice (Oryza Rufipogon Griff.) Using Label-Free Quantitative Proteomic Analysis.

Phosphorus (P) deficiency tolerance in rice is a complex character controlled by polygenes. Through proteomics analysis, we could find more low P tolerance related proteins in unique P-deficiency tolerance germplasm Dongxiang wild rice (Oryza Rufipogon, DXWR), which will provide the basis for the research of its regulation mechanism. In this study, a proteomic approach as well as joint analysis with transcriptome data were conducted to identify potential unique low P response genes in DXWR during seedlings. The results showed that 3589 significant differential accumulation proteins were identified between the low P and the normal P treated root samples of DXWR. The degree of change was more than 1.5 times, including 60 up-regulated and 15 downregulated proteins, 24 of which also detected expression changes of more than 1.5-fold in the transcriptome data. Through quantitative trait locus (QTLs) matching analysis, seven genes corresponding to the significantly different expression proteins identified in this study were found to be uncharacterized and distributed in the QTLs interval related to low P tolerance, two of which (LOC_Os12g09620 and LOC_Os03g40670) were detected at both transcriptome and proteome levels. Based on the comprehensive analysis, it was found that DXWR could increase the expression of purple acid phosphatases (PAPs), membrane location of P transporters (PTs), rhizosphere area, and alternative splicing, and it could decrease reactive oxygen species (ROS) activity to deal with low P stress. This study would provide some useful insights in cloning the P-deficiency tolerance genes from wild rice, as well as elucidating the molecular mechanism of low P resistance in DXWR.

Cytological Observation and Transcriptome Comparative Analysis of Self-Pollination and Cross-Pollination in Dendrobium Officinale.

Dendrobium officinale is a rare and traditional medicinal plant with high pharmacological and nutritional value. The self-incompatibility mechanism of D. officinale reproductive isolation was formed in the long-term evolution process, but intraspecific hybridization of different germplasm resources leads to a large gap in the yield, quality, and medicinal value of D. officinale. To investigate the biological mechanism of self-incompatibility in D. officinale, cytological observation and the transcriptome analysis was carried out on the samples of self-pollination and cross-pollination in D. officinale. Results for self-pollination showed that the pollen tubes could grow in the style at 2 h, but most of pollen tubes stopped growing at 4 h, while a large number of cross-pollinated pollen tubes grew along the placental space to the base of ovary, indicating that the self-incompatibility of D. officinale may be gametophyte self-incompatibility. A total of 63.41 G basesum of D. officinale style samples from non-pollinated, self-pollination, and cross-pollination by RNA-seq were obtained, and a total of 1944, 1758, and 475 differentially expressed genes (DEGs) in the comparison of CK (non-pollinated) vs. HF (cross-pollination sample), CK vs. SF (self-pollination sample) and SF vs. HF were identified, respectively. Forty-one candidate genes related to self-incompatibility were found by function annotation of DEGs, including 6 Ca2+ signal genes, 4 armed repeat containing (ARC) related genes, 11 S-locus receptor kinase (SRK) related genes, 2 Exo70 family genes, 9 ubiquitin related genes, 1 fatty acid related gene, 6 amino acid-related genes, 1 pollen-specific leucine-rich repeat extensin-like protein (LRX) related gene and 1 lectin receptor-like kinases (RLKs) related gene, showed that self-incompatibility mechanism of D. officinale involves the interaction of multiple genes and pathways. The results can provide a basis for the study of the self-incompatibility mechanism of D. officinale, and provide ideas for the preservation and utilization of high-quality resources of D. officinale.

Variability of Volatile Compounds in the Medicinal Plant Dendrobium officinale from Different Regions.

Dendrobium officinale Kimura et Migo, a rare and traditional medicinal plant, contains many nutrients such as polysaccharides, alkaloids, amino acids and so on. Different growth environment and intraspecific hybridization of different germplasm resources lead to large differences in the yield, quality and medicinal value of D. officinale. Here, the volatile compounds of D. officinale from four producing regions (Zhejiang, Fujian, Yunnan and Jiangxi) were analyzed to provide a certain reference value for the selection of a specific medicinal component in D. officinale breeding. Fresh stems of D. officinale germplasm resources were collected, and the chemical constituents were determined by gas chromatography-mass spectrometry. A total of 101 volatile compounds were identified, of which esters and alcohols accounted for 23 and 22. Hexacosane is the highest relative content of all volatile components. The highest content of hexacosane was observed in YA1 from Yunnan was 34.41%, and the lowest (23.41%) in JA1 from Jiangxi. Moreover, 5-10 unique substances were determined in different regions. A total of 17 medicinal components were detected, and three unique medicinal components were detected only in YA1, revealing that YA1 can provide raw materials for the application of specific medicinal substances extraction. A total of four toxic components were detected, but no toxic components were detected in JA1 from Jiangxi, suggested that the germplasm resources from Jiangxi could be exploited efficiently for breeding superior D. officinale specimens. The results provide a theoretical basis for the collection, protection and utilization of D. officinale germplasm resources in different regions.

The Anti-Inflammatory Properties of Rhododendron molle Leaf Extract in LPS-Induced RAW264.7.

Rhododendron molle G.Don is a well-known traditional medicine which has been used to treat rheumatic inflammation. In this study, an inflammatory model of lipopolysaccharide (LPS)-stimulated RAW264.7 cells was established to analyze the anti-inflammatory effect and potential mechanism of the methanol extract of R. molle leaves (RLE). The production of NO and the expression of tumor necrosis factor by LPS were detected by Griess reaction and enzyme linked immunosorbent assay (ELISA). The mRNA expression of TNF-α, IL-1ß, IL-6, COX-2 and iNOS was measured by qRT-PCR assay. Griess and qRT-PCR showed that the RLE could significantly concentration-dependently inhibit NO production and the expression of many pro-inflammatory cytokines and inflammatory-related enzymes. Scanning electron microscope (SEM) analysis indicated that RLE could inhibit LPS-stimulated RAW264.7 macrophages activation. The protein level of TNF-α and IL-1ß were decreased over 50 % at 100 µg/ml of RLE, as detected by ELISA. These results indicated that RLE had strong anti-inflammatory and immunomodulatory activity.

Transcriptome analysis of phosphorus stress responsiveness in the seedlings of Dongxiang wild rice (Oryza rufipogon Griff.).

BACKGROUND: Low phosphorus availability is a major factor restricting rice growth. Dongxiang wild rice (Oryza rufipogon Griff.) has many useful genes lacking in cultivated rice, including stress resistance to phosphorus deficiency, cold, salt and drought, which is considered to be a precious germplasm resource for rice breeding. However, the molecular mechanism of regulation of phosphorus deficiency tolerance is not clear. RESULTS: In this study, cDNA libraries were constructed from the leaf and root tissues of phosphorus stressed and untreated Dongxiang wild rice seedlings, and transcriptome sequencing was performed with the goal of elucidating the molecular mechanisms involved in phosphorus stress response. The results indicated that 1184 transcripts were differentially expressed in the leaves (323 up-regulated and 861 down-regulated) and 986 transcripts were differentially expressed in the roots (756 up-regulated and 230 down-regulated). 43 genes were up-regulated both in leaves and roots, 38 genes were up-regulated in roots but down-regulated in leaves, and only 2 genes were down-regulated in roots but up-regulated in leaves. Among these differentially expressed genes, the detection of many transcription factors and functional genes demonstrated that multiple regulatory pathways were involved in phosphorus deficiency tolerance. Meanwhile, the differentially expressed genes were also annotated with gene ontology terms and key pathways via functional classification and Kyoto Encyclopedia of Gene and Genomes pathway mapping, respectively. A set of the most important candidate genes was then identified by combining the differentially expressed genes found in the present study with previously identified phosphorus deficiency tolerance quantitative trait loci. CONCLUSION: The present work provides abundant genomic information for functional dissection of the phosphorus deficiency resistance of Dongxiang wild rice, which will be help to understand the biological regulatory mechanisms of phosphorus deficiency tolerance in Dongxiang wild rice.

Transcriptome analysis of phosphorus stress responsiveness in the seedlings of Dongxiang wild rice (Oryza rufipogon Griff.)

Abstract Background: Low phosphorus availability is a major factor restricting rice growth. Dongxiang wild rice (Oryza rufipogon Griff.) has many useful genes lacking in cultivated rice, including stress resistance to phosphorus deficiency, cold, salt and drought, which is considered to be a precious germplasm resource for rice breeding. However, the molecular mechanism of regulation of phosphorus deficiency tolerance is not clear. Results: In this study, cDNA libraries were constructed from the leaf and root tissues of phosphorus stressed and untreated Dongxiang wild rice seedlings, and transcriptome sequencing was performed with the goal of elucidating the molecular mechanisms involved in phosphorus stress response. The results indicated that 1184 transcripts were differentially expressed in the leaves (323 up-regulated and 861 down-regulated) and 986 transcripts were differentially expressed in the roots (756 up-regulated and 230 down-regulated). 43 genes were up-regulated both in leaves and roots, 38 genes were up-regulated in roots but down-regulated in leaves, and only 2 genes were down-regulated in roots but up-regulated in leaves. Among these differentially expressed genes, the detection of many transcription factors and functional genes demonstrated that multiple regulatory pathways were involved in phosphorus deficiency tolerance. Meanwhile, the differentially expressed genes were also annotated with gene ontology terms and key pathways via functional classification and Kyoto Encyclopedia of Gene and Genomes pathway mapping, respectively. A set of the most important candidate genes was then identified by combining the differentially expressed genes found in the present study with previously identified phosphorus deficiency tolerance quantitative trait loci. Conclusion: The present work provides abundant genomic information for functional dissection of the phosphorus deficiency resistance of Dongxiang wild rice, which will be help to understand the biological regulatory mechanisms of phosphorus deficiency tolerance in Dongxiang wild rice.

Investigation of the adsorption characteristics of natural organic matter from typical Chinese surface waters onto alumina using quartz crystal microbalance with dissipation.

The adsorption of natural organic matter (NOM) from eight typical Chinese surface waters onto alumina was investigated using quartz crystal microbalance with dissipation monitoring (QCM-D). The adsorbed masses of NOM varied between 25ngcm(-2) and 64ngcm(-2), and these showed significant correlation with geographical location, and NOM character and concentrations. Adsorbed mass correlated with DOC concentration (slope k=0.0676, R(2)=0.61) and hydrophobic acid (HoA) and weakly hydrophobic acid (WHoA) (k=0.0342 and 0.0183; R(2)=0.49 and 0.52 for HoA and WHoA, respectively) constituents present in the water samples. The process of adsorbed layer formation was investigated from changes in the ΔD/Δf ratio and viscosity of adsorbed layer with injected time. The adsorbed layer viscosity increased exponentially with injected time (R(2)>0.99) for most samples. Samples with low DOC concentration (k=-1091.8, R(2)=0.55) and low content of HoA and WHoA (k=-524.33 and -322.76; R(2)=0.41 and 0.64 for HoA and WHoA, respectively), the slope of logarithm viscosity value is steeper, the property of adsorbed layer and NOM is more inconsistent. The QCM-D technique provides a method to view the process of complexation between NOM and coagulant, and can provide useful information to establish a quantitative calculation model of the coagulation process.