Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.
Real-Time Polymerase Chain Reaction/methods* ; Paeonia/genetics* ; Actins/genetics* ; Reproducibility of Results ; Transcriptome ; Glyceraldehyde-3-Phosphate Dehydrogenases/genetics* ; Reference Standards ; Gene Expression Profiling/methods*

Paeoniflorin, a representative pinane monoterpene glycoside, is the main active component and quality index of Paeoniae Radix Alba and Paeoniae Radix Rubra.The possible biosynthesis of paeoniflorin is as follows: GPP is derived from mevalonate(MVA) and/or 2-C-methyl-D-erythritol 4-phosphate(MEP) pathway(s) followed by the catalysis with terpene synthase, cytochrome P450(CYP450), UDP-glucuronosyltransferase(UGT), and acyltransferase(AT), respectively.This study aims to explore the genes rela-ted to the biosynthesis of paeoniflorin.To be specific, the cDNA libraries for flowers, leaves, and roots of Paeonia lactiflora were established and sequenced.A total of 30 609 open reading frames(ORFs) were yielded.Through functional annotation and expression analysis of all CYP450 genes in the transcriptome, 11 CYP450 genes belonging to CYP71 A and CYP71 D subfamilies and showing expression trend consistent with monoterpene synthase PlPIN that may be involved in paeoniflorin biosynthesis were screened out.Subsequently, 7 UGT genes and 9 AT genes demonstrating the expression trend consistent with PlPIN which were possibly involved in paeoniflorin biosynthesis were further screened by functional annotation analysis, full-length sequence analysis, expression analysis, and phylogeny analysis.This study provided a systematic screening method with smaller number of candidate genes, thus reducing the workload of functional gene verification.The result laid a foundation for analyzing the biosynthesis pathway of paeoniflorin and the formation mechanism.
Bridged-Ring Compounds ; Gene Expression Profiling ; Glucosides/metabolism* ; Monoterpenes/metabolism* ; Paeonia/genetics*

This study investigated the protective effects of Moutan Cortex polysaccharides components(MCPC) on the renal tissues of diabetic nephropathy(DN) rats and explored their regulation effect on inflammatory response and oxidative stress. The DN rat model was induced by high-glucose and high-fat diet combined with streptozotocin(STZ), and then the rats were randomly divided into control group, model group, positive group and MCPC high(120 mg·kg~(-1)·d~(-1)), low(60 mg·kg~(-1)·d~(-1)) dose groups. After 12 weeks treatment, blood was taken from the orbit of the rats, and then they were sacrificed before the kidney tissues were collected. The serum and tissues were detected for related biochemical indicators and pathological changes of the kidney. Immunohistochemical methods were used to determine the expression of FN and ColⅣ in the kidney tissue of DN rats. Compared with the model group, blood glucose, serum creatinine, blood urea nitrogen and 24 h urine protein in the MCPC high-dose group were significantly reduced(P<0.01). The results of HE, PAS, Masson staining showed that glomerular basement membrane thickening, Bowman's capsule narrowing and inflammatory cell infiltration in DN rats were improved in the MCPC high-dose group; the activity of T-SOD and GSH-Px in serum significantly increased(P<0.001), and the expression level of FN significantly decreased(P<0.001). The high-dose MCPC treatment could effectively inhibit the abnormal expression of Col Ⅳ(P<0.001) and significantly reduce the levels of AGEs and RAGE in serum(P<0.001), the content of VCAM-1 and IL-1β in serum(P<0.001), and the levels of IL-1β mRNA in kidney tissue(P<0.001), but failed to effectively reduce VCAM-1 mRNA levels in kidney tissues. The high-dose MCPC could significantly improve pathological injury of renal tissue and related renal indicators in DN rats, and achieve renal protection in DN rats mainly by regulating oxidative stress and inflammatory factors.
Animals ; Diabetes Mellitus, Experimental/genetics* ; Diabetic Nephropathies/genetics* ; Drugs, Chinese Herbal ; Kidney ; Paeonia ; Polysaccharides/pharmacology* ; Rats

Cerebral ischemia is one of the most common diseases in China, and the drug pair of Chuanxiong Rhizoma and Paeoniae Radix Rubra can intervene in cerebral ischemia to reduce the inflammatory response of cerebral ischemia and apoptosis. To reveal the intervention mechanism of Chuanxiong Rhizoma-Paeoniae Radix Rubra drug pair on cerebral ischemia systematically, computer network pharmacology technology was used in this paper to predict the target and signaling pathway of the drug pair on the intervention of cerebral ischemia, and then the molecular docking technology was used to further analyze the mechanism of the intervention. The target results were then verified by the rat cerebral ischemia model. The target network results showed that the active compounds of Chuanxiong Rhizoma-Paeoniae Radix Rubra for cerebral ischemic disease contained 30 compounds, 38 targets and 9 pathways. The main compounds included phenolic acids in Chuanxiong Rhizoma and monoterpene glycosides in Paeoniae Radix Rubra. The key targets involved mitogen-activated protein kinase 1(MAPK1), steroid receptor coactivator(SRC), epidermal growth factor receptor(EGFR), mitogen-activated protein kinase 14(MAPK14), caspase-3(CASP3), caspase-7(CASP7), estrogen receptor 1(ESR1), and mitogen-activated protein kinase 8(MAPK8), etc. The target gene functions were biased towards protein kinase activity, protein autophosphorylation, peptidyl-serine phosphorylation and protein serine/threonine kinase activity, etc. The important KEGG pathways involved Ras signaling pathway, ErbB signaling pathway and VEGF signaling pathway. Molecular docking results showed that catechin, oxypaeoniflorin, albiflorin, paeoniflorin and benzoylpaeoniflorin had strong binding ability with MAPK1, SRC, EGFR, MAPK14 and CASP7. MCAO rat experimental results showed that Chuanxiong Rhizoma-Paeoniae Radix Rubra significantly improved the cerebral ischemia injury and interstitial edema, and significantly reduced the activation of caspase-7 and the phosphorylation of ERK1/2. The Chuanxiong Rhizoma-Paeoniae Radix Rubra drug pair alleviated cerebral ischemia injury through a network model of multi-phenotype intervention by promoting cell proliferation and differentiation, reducing inflammatory factor expression, protecting nerve cells from death and figh-ting against neuronal cell apoptosis, with its action signaling pathway most related to Ras signaling pathway, ErbB signaling pathway and VEGF signaling pathway. This study provides the basis for clinical intervention of Chuanxiong Rhizoma-Paeoniae Radix Rubra drug pair on cerebral ischemia, and also provides ideas for the modernization of drug pairs.
Animals ; Brain Ischemia/genetics* ; Cerebral Infarction ; Drugs, Chinese Herbal ; Molecular Docking Simulation ; Paeonia ; Rats ; Rhizome

The traditionally used oriental herbal medicine Moutan Cortex Radicis [MCR; Paeonia Suffruticosa Andrews (Paeoniaceae)] exerts anti-inflammatory, anti-spasmodic, and analgesic effects. In the present study, we investigated the therapeutic effects of differently fractioned MCR extracts in a 6-hydroxydopamine (OHDA)-induced Parkinson's disease model and neuro-blastoma B65 cells. Ethanol-extracted MCR was fractionated by n-hexane, butanol, and distilled water. Adult Sprague-Dawley rats were treated first with 20 μg of 6-OHDA, followed by three MCR extract fractions (100 or 200 mg·kg) for 14 consecutive days. In the behavioral rotation experiment, the MCR extract-treated groups showed significantly decreased number of net turns compared with the 6-OHDA control group. The three fractions also significantly inhibited the reduction in tyrosine hydroxylase-positive cells in the substantia nigra pars compacta following 6-OHDA neurotoxicity. Western blotting analysis revealed significantly reduced tyrosine hydroxylase expression in the substantia nigra pars compacta in the 6-OHDA-treated group, which was significantly inhibited by the n-hexane or distilled water fractions of MCR. B65 cells were exposed to the extract fractions for 24 h prior to addition of 6-OHDA for 30 min; treatment with n-hexane or distilled water fractions of MCR reduced apoptotic cell death induced by 6-OHDA neurotoxicity and inhibited nitric oxide production and neuronal nitric oxide synthase expression. These results showed that n-hexane- and distilled water-fractioned MCR extracts inhibited 6-OHDA-induced neurotoxicity by suppressing nitric oxide production and neuronal nitric oxide synthase activity, suggesting that MCR extracts could serve as a novel candidate treatment for the patients with Parkinson's disease.
Animals ; Anti-Inflammatory Agents ; pharmacology ; therapeutic use ; Antiparkinson Agents ; pharmacology ; therapeutic use ; Cell Death ; drug effects ; Cell Line ; Disease Models, Animal ; Drugs, Chinese Herbal ; chemistry ; Neurons ; pathology ; Nitric Oxide ; analysis ; Nitric Oxide Synthase Type I ; biosynthesis ; Oxidopamine ; toxicity ; Paeonia ; chemistry ; Parkinsonian Disorders ; chemically induced ; drug therapy ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Plants, Medicinal ; Rats ; Rats, Sprague-Dawley ; Substantia Nigra ; drug effects ; enzymology ; Tyrosine 3-Monooxygenase ; genetics ; metabolism

The present study was designed to investigate the therapeutic effcts of Moutan Cortex (CM, root bark of Paeonia suffruticosa Andr) and Paeoniae Radix Rubra (PR, root of Paeonia veitchii Lynch) on metabolic disorders, focusing on the infuence of CM and PR on the obesity-related gut microbiota homeostasis. The diet-induced obese (DIO) mouse model was used to test the therapeutic effects of CM and PR. The mice were orally administered with CM and PR for 6 weeks, and oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed to evaluate the insulin sensitivity of the mice. Sterol-regulatory element binding proteins (SREBPs) and their target genes were measured by quantitative RT-PCR. High-throughput 16S ribosomal RNA (16S rRNA) gene sequencing technology was used to determine the composition of gut microbiota, and the metabolites in serum were analyzed by GC-MS. Our results indicated that CM and PR combination alleviated obese and insulin resistance in the DIO mice, leading to increased glucose uptake and gene expression in muscle and liver, and down-regulated SREBPs and their target genes in liver. Interesting, neither the CM-PR extracts, nor the major components of CM and PR did not affect SREBPs activity in cultured cells. Meanwhile, CM and PR significantly modulated the gut microbiota of the high-fat diet (HFD) treated mice, similar to metformin, and CM-PR reversed the overall microbiota composition similar to the normal chow diet (NCD) treated mice. In conclusion, our results provide novel mechanisms of action for the effects of CM and PR in treating DIO-induced dysregulation of sugar and lipid metabolism.
Animals ; Blood Glucose ; metabolism ; Diet, High-Fat ; adverse effects ; Drugs, Chinese Herbal ; administration & dosage ; Gastrointestinal Microbiome ; drug effects ; Homeostasis ; drug effects ; Humans ; Insulin ; metabolism ; Male ; Metabolic Diseases ; drug therapy ; genetics ; metabolism ; microbiology ; Mice ; Mice, Inbred C57BL ; Paeonia ; chemistry ; Sterol Regulatory Element Binding Proteins ; genetics ; metabolism

Paeonia lactiflora root (baishao in Chinese) is a commonly used herb in traditional Chinese medicines (TCM). Two isomers, paeoniflorin (PF) and albiflorin (AF), are isolated from P. lactiflora. The present study aimed to investigate the protective effects of PF and AF on myelosuppression induced by chemotherapy in mice and to explore the underlying mechanisms. The mouse myelosuppression model was established by intraperitoneal (i.p.) injection of cyclophosphamide (CP, 200 mg·kg(-1)). The blood cell counts were performed. The thymus index and spleen index were also determined and bone morrow histological examination was performed. The levels of tumor necrosis factor-α (TNF-α) in serum and colony-stimulating factor (G-CSF) in plasma were measured by Enzyme-Linked Immunosorbent Assays (ELISA) and the serum levels of interleukin-3 (IL-3), granulocyte-macrophagecolony-stimulatingfactor (GM-CSF), and interleukin-6 (IL-6) were measured by radioimmunoassay (RIA). The levels of mRNA expression protein of IL-3, GM-CSF and G-CSF in spleen and bone marrow cells were determined respectively. PF and AF significantly increased the white blood cell (WBC) counts and reversed the atrophy of thymus. They also increased the serum levels of GM-CSF and IL-3 and the plasma level of G-CSF and reduced the level of TNF-α in serum. PF enhanced the mRNA level of IL-3 and AF enhanced the mRNA levels of GM-CSF and G-CSF in the spleen. PF and AF both increased the protein levels of GM-CSF and G-CSF in bone marrow cells. In conclusion, our results demonstrated that PF and AF promoted the recovery of bone marrow hemopoietic function in the mouse myelosuppression model.
Animals ; Antineoplastic Agents ; adverse effects ; Bridged-Ring Compounds ; administration & dosage ; Cyclophosphamide ; adverse effects ; Drugs, Chinese Herbal ; administration & dosage ; Glucosides ; administration & dosage ; Granulocyte Colony-Stimulating Factor ; genetics ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; metabolism ; Hematologic Diseases ; etiology ; genetics ; metabolism ; prevention & control ; Humans ; Interleukin-3 ; genetics ; metabolism ; Interleukin-6 ; metabolism ; Male ; Mice ; Monoterpenes ; administration & dosage ; Paeonia ; chemistry ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVETo study the effect of different composition structures of total paeony glycoside (TPG) component and total phenolic acid of Ligusticum chuanxiong ( TLPA) on sodium dithionite (Na2S2O4) -induced human umbilical vein endothelial cells (HUVEC) hypoxic injury. The baseline geometric proportion was used to design different components structure. And then the best structure of components by cell injury model were optimized.

METHODA HUVEC hypoxic injury model was established by being induced of Na2S2O4. Cell viability was measured by MTI colorimetric method, intracellular superoxide dismutase (SOD) activity, malondialdehyde (MDA), lactate dehydrogenase( LDH) levels, nitric oxide (NO) contents were measured by kits. At last, Western blot analysis was used to detect the expression of two proteins, Bcl-2 and Bax.

RESULTCompared with the model group, TPG component, TLPA component at different composition structures can significantly increase SOD activity and decrease MDA, LDH, NO levels (P < 0.01, P < 0.05). Paeoniae Radix Rubra and Chuanxiong Rhizoma components can downregulate the expression of Bax protein and upregulate the expression of Bcl-2 protein. The ratio of Bcl-2 and Bax was significantly increased (P < 0 01, P < 0 05), it means that cell apoptosis was inhibited. The results indicate that among all the component composition structures, TPG and TLPA component at the proportion of 8: 2 had the best protection on hypoxic injury of endothelial cells.

CONCLUSIONTPG component and TLPA component can resist HUVEC hypoxia injury, the protective effect was the most evident under the structure of 8: 2, which may be due to the inhibition of intracellular lipid peroxidation and cell apoptosis.

Apoptosis ; drug effects ; Cell Line ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Glycosides ; analysis ; pharmacology ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Hydroxybenzoates ; analysis ; pharmacology ; Hypoxia ; drug therapy ; genetics ; metabolism ; physiopathology ; Malondialdehyde ; metabolism ; Oxygen ; metabolism ; Paeonia ; chemistry ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Rhizome ; chemistry

OBJECTIVETo investigate the effect of Moutan Cortex on mesangial proliferation and basement membrane thickening induced by advanced glycation end products (AGEs).

METHODThe glomerular mesangial cells (MC) injury model was established by inducing by AGEs. The cell were divided into 6 groups: the blank group ( BSA, 200 mg L-1) , the model group (AGEs, 200 mg L-1), the positive control group (AG, 10 mmol L L-1), and drug administration groups, namely the Moutan Cortex-treated high-dose group (2 x 10(-4) g mL(- 1)), the Moutan Cortex-treated medium-dose group (1 x 10(-4) g mL-1 ), and the Moutan Cortex-treated low-dose group (0. 5 x 10(-4) g . mL(-1)). The MTT method was performed to observe the effect of Moutan Cortex on the proliferation of MC. The content of fibronectin (FN) and collagen secretion 1V (Col IV) in cell supernatant were detected by ELISA kits. The western blot analysis was carried out to observe the FN expression. The Real-time PCR analysis was applied to examine the Col IV mRNA expression.

RESULTAGEs significantly increased AGEs-induced MC proliferation and FN and Col 1V secretion. The western blot analysis showed that MC could down-regulate the FN expression of MC secretion. According to the results of the real-time PCR assay, MC could down-regulate AGEs-induced MC secretion Col IV mRNA expression.

CONCLUSIONMC had a certain protective effect on MC cultured under AGEs conditions. MC could remarkably inhibit the composition and secretion of Col IV and FN in matrix and the basement membrane thickening, and provide an experimental basis for the treatment of diabetic nephropathy.

Animals ; Basement Membrane ; drug effects ; metabolism ; Cell Line ; Cell Proliferation ; drug effects ; Collagen Type IV ; genetics ; secretion ; Drugs, Chinese Herbal ; pharmacology ; Fibronectins ; biosynthesis ; Gene Expression Regulation ; drug effects ; Glycation End Products, Advanced ; adverse effects ; Mesangial Cells ; cytology ; drug effects ; metabolism ; secretion ; Paeonia

To explore a new method to identify Moutan Cortex to guarantee its safe use, internal transcribed spacer 2 (ITS2) sequence was used to identify Moutan Cortex and its adulterants. DNA was extracted and target fragments were amplified. Sequences were analyzed and assembled by CodonCode Aligner V3.7.1. Genetic distances were computed and phylogenetic tree was constructed based on kimura 2-parameter (K2P) model by MEGA 5.0. The length of the 20 ITS2 sequences of Moutan Cortex from nine different places is 227 bp, and no variation site was detected. The maximum inter-specificK2P distance of Moutan Cortex is 0, the minimum intra-specific K2P distance is 0.041, the average intra-specific K2P distance is 0.222. According to NJ analysis, Moutan Cortex from different places can get together as one branch with bootstrap support values 99%, which indicates Moutan Cortex can be easily distinguished from its adulterants. Using ITS2 sequence can accurately identify Moutan Cortex and its adulterants, it is an effective supplementary to traditional identification methods.
Base Sequence ; China ; DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drug Contamination ; prevention & control ; Drugs, Chinese Herbal ; chemistry ; classification ; Molecular Sequence Data ; Paeonia ; classification ; genetics ; Phylogeny ; Quality Control