[Achyranthes bidentata polysaccharide inhibits the adipogenic differentiation of rat bone marrow mesenchymal stem cells by blocking the PPARγ/TRPV4 pathway].

Objective To investigate the effect of Achyranthes bidentata polysaccharides (ABPS) on adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and its mechanism. Methods Five SD rats were sacrificed, and the BMSCs were dissected and isolated. The BMSCs were adherently cultured to logarithmic growth phase after identification, and treated with different doses of ABPS for 48 hours. The cell survival rates were detected by MTT assay. The highest dose of ABPS without toxicity to BMSCs was selected for subsequent experiments. Cells were randomly divided into control group, ABPS group, rosiglitazone group and ABPS combined with rosiglitazone group. Cell survival rates were detected by MTT assay. Triglyceride (TG) levels in BMSCs were detected by spectrophotometry. Lipid droplet formation in BMSCs was observed by oil red O staining. The mRNA and protein expression of peroxisome proliferater-activated receptor γ (PPARγ), transient receptor potential vanilloid 4 (TRPV4) and CCAAT/enhancer binding protein α (C/EBPα) were detected by real time quantitative PCR and Western blot analysis. Results The dose of ABPS≤200 mg/L had no obvious toxic effect on the growth of BMSCs after 48 hours, and the cell survival rate of 400 mg/L ABPS group was lower. Compared with the control group, the ABPS group showed decreased levels in TG, decreased relative expression of PPARγ, TRPV4 and C/EBPα mRNA and protein, and the decreased number of cytoplasmic lipid droplets. In the rosiglitazone group, observation reported the decreased cell survival rate, increased TG level, increased relative expression levels of PPARγ, TRPV4 and C/EBPα mRNA and protein, along with the increased number of cytoplasmic lipid droplets. Compared with the ABPS group, the cell survival rate was decreased, TG level was increased, the relative expression levels of PPARγ, TRPV4 and C/EBPα mRNA and protein increased, and the number of cytoplasmic lipid droplets increased in the ABPS combined with rosiglitazone group. Compared with rosiglitazone group, the survival rate was increased, TG level was decreased, the relative expression levels of PPARγ, TRPV4 and C/EBPα mRNA and protein were decreased, and the number of cytoplasmic lipid droplets was decreased in the ABPS combined with rosiglitazone group. Conclusion ABPS can inhibit adipogenic differentiation of BMSCs, and the mechanism may be related to the regulation of PPARγ/TRPV4 pathway.

Network Pharmacology Analysis of the Effects of Achyranthis Bidentatae Radix Plus Semen Vaccariae on Migraine-induced Erectile Dysfunction.

BACKGROUND AND AIM: Achyranthis Bidentatae Radix plus Semen Vaccariae are traditional Chinese medicines, which have been widely applied in the treatment of migraine and Erectile Dysfunction (ED) for many years. This study verified the effect of Achyranthis Bidentatae Radix plus Semen Vaccariae in improving migraine-induced ED and explored its potential mechanism. METHODS: Key targets and signaling pathways of Achyranthis Bidentatae Radix plus Semen Vaccariae in migraine-induced erectile dysfunction treatment were predicted by network pharmacology. A rat model of migraine was established by nitroglycerin injection. Apomorphine was injected into rats to screen the migraine-induced erectile dysfunction model, Achyranthis Bidentatae Radix-Semen Vaccariae granule suspension administered, and erectile function evaluated. Hematoxylin and eosin staining was used to compare the histological structure of the penile tissue, while RT-qPCR and Western blotting were used to determine mRNA and protein levels, respectively. RESULTS: Screening allowed us to identify common targets for migraine and ED; the signaling pathway exhibiting the greatest change was the Myosin light chain kinase- Calcium (MLCK-CaM) signal pathway. From Western blotting and RT-qPCR, we found that the levels of MLCK mRNA and protein in rats from Group B rats were significantly higher (P <0.05) than those in Groups A and C. Furthermore, the mRNA and protein levels of CaM were significantly higher in Group B (P <0.05) than in Groups A and C. CONCLUSION: Data indicate that the regulatory effects of Achyranthis Bidentatae Radix plus Semen Vaccariae on migraine-induced ED in a rat model are mediated by the MLCK-CaM signaling pathway.

Integrated miRNA-mRNA analysis reveals the roles of miRNAs in the replanting benefit of Achyranthes bidentata roots.

The yield and quality of the medicinal plant Achyranthes bidentata can be increased when it is replanted into a field cultivated previously with the same crop, however, fundamental aspects of its biology (so-called "replanting benefit") still remain to be elucidated. miRNAs are sRNA molecules involved in the post-transcriptional regulation of gene expression in plant biological processes. Here, 267 conserved and 36 novel miRNAs were identified in A. bidentata roots. We compared the miRNA content of the roots (R1) from first-year planting with that of the roots (R2) of second-year replanting, and screened 21 differentially expressed (DE) miRNAs. Based on in silico functional analysis, integrated miRNA-mRNA datasets allowed the identification of 10 miRNA-target family modules, which might participate in the benefit. The expression profiles of the miRNA-target modules were potentially correlated with the presence of the replanting benefit. The indication was that the miRNA-responsive continuous monoculture could reprogram miRNA-mRNA expression patterns, which possibly promote the root growth and development, enhance its transport activity and strengthen its tolerance to various stresses, thereby improving A. bidentata productivity as observed in the replanting benefit. Our study provides basic data for further research on the molecular mechanisms of the benefit in A. bidentata.

Comparing and phylogenetic analysis chloroplast genome of three Achyranthes species.

In this study, the chloroplast genome sequencing of the Achyranthes longifolia, Achyranthes bidentata and Achyranthes aspera were performed by Next-generation sequencing technology. The results revealed that there were a length of 151,520 bp (A. longifolia), 151,284 bp (A. bidentata), 151,486 bp (A. aspera), respectively. These chloroplast genome have a highly conserved structure with a pair of inverted repeat (IR) regions (25,150 bp; 25,145 bp; 25,150 bp), a large single copy (LSC) regions (83,732 bp; 83,933 bp; 83,966 bp) and a small single copy (SSC) regions (17,252 bp; 17,263 bp; 17,254 bp) in A. bidentate, A. aspera and A. longifolia. There were 127 genes were annotated, which including 8 rRNA genes, 37 tRNA genes and 82 functional genes. The phylogenetic analysis strongly revealed that Achyranthes is monophyletic, and A. bidentata was the closest relationship with A. aspera and A. longifolia. A. bidentata and A. longifolia were clustered together, the three Achyranthes species had the same origin, then the gunes of Achyranthes is the closest relative to Alternanthera, and that forms a group with Alternanthera philoxeroides. The research laid a foundation and provided relevant basis for the identification of germplasm resources in the future.

The root transcriptome of Achyranthes bidentata and the identification of the genes involved in the replanting benefit.

KEY MESSAGE: The transcriptome profiling in replanting roots revealed that expression pattern changes of key genes promoted important metabolism pathways, antioxidant and pathogen defense systems, adjusted phytohormone signaling and inhibited lignin biosynthesis. The yield of the medicinal plant Achyranthes bidentata could be significantly increased when replanted into a field cultivated previously for the same crop, but the biological basis of this so-called "replanting benefit" is unknown. Here, the RNA-seq technique was used to identify candidate genes responsible for the benefit. The analysis of RNA-seq libraries prepared from mRNA extracted from the roots of first year planting (normal growth, NG) and second year replanting (consecutive monoculture, CM) yielded about 40.22 GB sequencing data. After de novo assembly, 87,256 unigenes were generated with an average length of 1060 bp. Among these unigenes, 55,604 were annotated with public databases, and 52,346 encoding sequences and 2881 transcription factors were identified. A contrast between the NG and CM libraries resulted in a set of 3899 differentially transcribed genes (DTGs). The DTGs related to the replanting benefit and their expression profiles were further analyzed by bioinformatics and qRT-PCR approaches. The major differences between the NG and CM transcriptomes included genes encoding products involved in glycolysis/gluconeogenesis, glutathione metabolism and antioxidant defense, in aspects of the plant/pathogen interaction, phytohormone signaling and phenylpropanoid biosynthesis. The indication was that replanting material enjoyed a stronger level of defense systems, a balance regulation of hormone signals and a suppression of lignin formation, thereby promoting root growth and development. The study provides considerable significant insights for a better understanding of the molecular mechanism of the replanting benefit and suggests their possible application in developing methods to reinforce the effects in medicinal plants.

Molecular identification of Achyranthis Bidentatae Radix by using DNA barcoding.

Achyranthis Bidentatae Radix has a long history in China as a commonly used herb that can be used to treat various diseases, including those related to the liver, muscles, bones, and kidneys. Recently, an increase in the number of adulterants has been reported, which affects the clinical safety of Achyranthis Bidentatae Radix. To identify adulterants of Achyranthis Bidentatae Radix, we collected samples from major regions and conducted an in-depth genetic comparison of the herb and its commonly used adulterants. We amplified and sequenced three genomic regions, internal transcribed spacer (ITS), psbA-trnH, and internal transcribed spacer 2 (ITS2), to confirm whether ITS2 is a suitable identifier for Achyranthis Bidentatae Radix. Results showed that the ITS2 sequence length of Achyranthis Bidentatae Radix was 199 bp, with no variation between samples. The inter-specific genetic distance of ITS2 between Achyranthis Bidentatae Radix and its adulterants was 0.390. Neighbor-joining trees showed that Achyranthis Bidentatae Radix and its adulterants are easily differentiated by monophyly. In conclusion, ITS2 regions accurately and effectively distinguished between Achyranthis Bidentatae Radix and its adulterants.

[Authentification of Radix Cyathule and its adulterants based on SCAR markers].

Chuanniuxi (Radix Cyathule) is one of the most important geo-herb in Sichuan province, which adulterants are Hongniuxi (Cyathula capitata) and Huainiuxi (Achyranthes bidentata). In this paper Chuanniuxi and its adulterants were identified by SCAR markers. Nineteen populations from Tianquan, Baoxin, Huili and Jinkouhe were collected and their RAPD fingerprints were established. Based on the RAPD patterns, two polymorphic bands F300 and F500 were selected, recycled, cloned and sequenced. According to the sequences two pairs of sequence characterized amplified regions (SCAR) primers were designed and used to amplify all materials to prove the efficiency of identification of the different populations. Chuanniuxi and Huiniuxi could be distinguished by the primer SC-320, Chuanniuxi and Hongniuxi could be distinguished by the primer SC-495. Combining the two SCAR markers, Chuanniuxi, Hongniuxi and huainiuxi could be identified effectively and quickly.

Discriminating between Achyranthis Bidentatae Radix and Cyathulae Radix in Chinese medicine preparations by nested PCR and DNA sequencing methods.

The widely used Achyranthis Bidentatae Radix (Niu Xi) in Chinese medicine preparation is easily misused and confused with Cyathulae Radix (Chuan Niu Xi). Taiwan authority has ruled that Achyranthis Bidentatae Radix and Cyathulae Radix should be individually entered in the given preparations. In this study, with the internal transcribed spacer (ITS) as a DNA marker, nested PCR and DNA sequencing were used to distinguish the two herbs in preparations. Authentic Achyranthis Bidentatae Radix and Cyathulae Radix were collected from the original country, mainland China, and twenty two samples of Chinese medicine preparations labeled with Niu Xi as the ingredient were purchased locally. The total DNA of all samples were extracted by organic solution and purified using a kit. For a standard, ITS regions of authentic Achyranthis Bidentatae Radix and Cyathulae Radix were amplified with PCR and analyzed by an auto-sequencer. The ITS regions of preparation samples of DNA were amplified by nested PCR and the data of sequences received from an auto-sequencer. We compared the ITS sequences of preparation samples with the sequences of a standard and Genbank database to check which Achyranthis Bidentatae Radix or Cyathulae Radix is in the preparation samples. The results showed that only one sample contained Achyranthis Bidentatae Radix and the others contained Cyathulae Radix. The Cyathulae Radix components in four preparation samples do not fit into Taiwan authority rules. And our method can be applied for the differentiation of Achyranthis Bidentatae Radix and Cyathulae Radix in Chinese medicine preparations.

[Study on the genetiey relationship between Chinese and Korean medicinal materials of niuxi with the method of RAPD].

OBJECTIVE: To study the hereditary relationship between Chinese and Korean medicinal materials of Niuxi achyranthis root. METHOD: Ten samples of four kinds of Niuxi was studied with the method of RAPD. RESULT: Expanded product showed total record of 100 spectrum bands, which proved that the hereditary gap between Korean self-produced A. japonica and A. bidentata is smallest. CONCLUSION: Korean self-produced A. japonica is near to A. Sidentata.