Arbutin Alleviates LPS Induced Sepsis Pneumonia in Mice.

The aim of this study was to investigate the effects of arbutin (AR) on lipopolysaccharide (LPS)-induced sepsis pneumonia. LPS-induced mice and A549 cells were used to establish septic pneumonia model. AR significantly decreased lung wet-to-dry weight (W/D) ratio, lung myeloperoxidase (MPO) activity and ameliorated lung histopathological changes. In addition, AR increased super oxide dismutase (SOD) activity, decreased malondialdehyde (MDA) content and levels of cytokines including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-ß) and interleukin-6 (IL-6) in bronchoalveolar lavage fluid (BALF) in mice. Furthermore, the results demonstrated that AR inhibited the JAK2/STAT3/NF-κB pathway in LPS-induced A549 cells which was further confirmed by siRNA JAK2 experiment. The experimental results indicated that the protective mechanism of AR on sepsis pneumonia might be attributed partly to the inhibition of cytokine production and JAK2/STAT3/NF-κB pathway.

De novo Sequencing and Transcriptome Analysis of Pinellia ternata Identify the Candidate Genes Involved in the Biosynthesis of Benzoic Acid and Ephedrine.

BACKGROUND: The medicinal herb, Pinellia ternata, is purported to be an anti-emetic with analgesic and sedative effects. Alkaloids are the main biologically active compounds in P. ternata, especially ephedrine that is a phenylpropylamino alkaloid specifically produced by Ephedra and Catha edulis. However, how ephedrine is synthesized in plants is uncertain. Only the phenylalanine ammonia lyase (PAL) and relevant genes in this pathway have been characterized. Genomic information of P. ternata is also unavailable. RESULTS: We analyzed the transcriptome of the tuber of P. ternata with the Illumina HiSeq™ 2000 sequencing platform. 66,813,052 high-quality reads were generated, and these reads were assembled de novo into 89,068 unigenes. Most known genes involved in benzoic acid biosynthesis were identified in the unigene dataset of P. ternata, and the expression patterns of some ephedrine biosynthesis-related genes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Also, 14,468 simple sequence repeats (SSRs) were identified from 12,000 unigenes. Twenty primer pairs for SSRs were randomly selected for the validation of their amplification effect. CONCLUSION: RNA-seq data was used for the first time to provide a comprehensive gene information on P. ternata at the transcriptional level. These data will advance molecular genetics in this valuable medicinal plant.

Siwu decoction attenuates oxonate-induced hyperuricemia and kidney inflammation in mice.

The aim of the study was to investigate the effects of Siwu decoction on hyperuricemia, kidney inflammation, and dysfunction in hyperuricemic mice. Siwu decoction at 363.8, 727.5, and 1 455 mg·kg(-1) was orally administered to potassium oxonate-induced hyperuricemic mice for 7 days. Serum urate, creatinine, and blood urea nitrogen levels and hepatic xanthine oxidase (XOD) activity were measured. The protein levels of hepatic XOD and renal urate transporter 1 (URAT1), glucose transporter 9 (GLUT9), organic anion transporters 1 (OAT1), ATP-binding cassette subfamily G member 2 (ABCG2), organic cation transporter 1 (OCT1), OCT2, organic cation/carnitine transporter 1 (OCTN1), OCNT2, Nod-like receptor family, pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein (ASC), Caspase-1, and interleukin-1ß (IL-1ß) were determined by Western blotting. Renal histopathology change was obtained following hematoxylin-eosin staining. Our results indicated that Siwu decoction significantly reduced serum urate, creatinine and blood urea nitrogen levels and increased fractional excretion of uric acid in hyperuricemic mice. It effectively reduced hepatic XOD activity and protein levels in this animal model. Furthermore, Siwu decoction down-regulated URAT1 and GLUT9 protein levels, and up-regulated the protein levels of OAT1, ABCG2, OCT1, OCT2, OCTN1, and OCTN2 in the kidney of the hyperuricemic mice. Additionally, Siwu decoction remarkably reduced renal protein levels of NLRP3, ASC, Caspase-1, and IL-1ß in the hyperuricemic mice. These results suggested that Siwu decoction exhibited anti-hyperuricemic and anti-inflammatory effects by inhibiting hepatic XOD activity, regulating renal organic ion transporter expression, and suppressing renal NLRP3 inflammasome activation, providing the evidence for its use in the treatment of hyperuricemia and associated kidney inflammation.

Candidate Genes Involved in the Biosynthesis of Triterpenoid Saponins in Platycodon grandiflorum Identified by Transcriptome Analysis.

BACKGROUND: Platycodon grandiflorum is the only species in the genus Platycodon of the family Campanulaceae, which has been traditionally used as a medicinal plant for its lung-heat-clearing, antitussive, and expectorant properties in China, Japanese, and Korean. Oleanane-type triterpenoid saponins were the main chemical components of P. grandiflorum and platycodin D was the abundant and main bioactive component, but little is known about their biosynthesis in plants. Hence, P. grandiflorum is an ideal medicinal plant for studying the biosynthesis of Oleanane-type saponins. In addition, the genomic information of this important herbal plant is unavailable. PRINCIPAL FINDINGS: A total of 58,580,566 clean reads were obtained, which were assembled into 34,053 unigenes, with an average length of 936 bp and N50 of 1,661 bp by analyzing the transcriptome data of P. grandiflorum. Among these 34,053 unigenes, 22,409 unigenes (65.80%) were annotated based on the information available from public databases, including Nr, NCBI, Swiss-Prot, KOG, and KEGG. Furthermore, 21 candidate cytochrome P450 genes and 17 candidate UDP-glycosyltransferase genes most likely involved in triterpenoid saponins biosynthesis pathway were discovered from the transcriptome sequencing of P. grandiflorum. In addition, 10,626 SSRs were identified based on the transcriptome data, which would provide abundant candidates of molecular markers for genetic diversity and genetic map for this medicinal plant. CONCLUSION: The genomic data obtained from P. grandiflorum, especially the identification of putative genes involved in triterpenoid saponins biosynthesis pathway, will facilitate our understanding of the biosynthesis of triterpenoid saponins at molecular level.

Venenum Bufonis induces rat neuroinflammation by activiating NF-κB pathway and attenuation of BDNF.

ETHNOPHARMACOLOGICAL RELEVANCE: Venenum Bufonis (VB), also called toad venom, has been widely used in clinic as a cardiotonic, anohyne and antineoplastic agents both in China and other Asian countries. However, its neurotoxicity and cardiotoxicity limit its wide clinical application. Compared with extensive attention attracted with cardiotoxicity, the toxic effect of VB on Central Nervous System (CNS) is much less studied. AIM OF THE RESEARCH: This study was performed to examine the neurotoxicity caused by VB on Sprague Dawley (SD) rats, then to clarify the mechanism in vivo by investigating its action on the neuroinflammation which possibly attributed to the activation of nuclear factor κB (NF-κB) pathway and the attenuation of brain-derived neurotrophic factor (BDNF). MATERIALS AND METHODS: Rats administrated with 0.5% carboxymethyl cellulose sodium salt (CMC-Na) aqueous solution and VB (100mg/kg, 200mg/kg and 400mg/kg) were sacrificed at 2h, 4h, 6h, 8h, 24h and 48h. The brain level of neurotransmitters and their corresponding receptors, pro-inflammatory cytokines, BDNF/TrkB and NF-κB pathway-related proteins were examined, respectively. RESULTS: VB administration induced severe neurologic damage and neuroinflammation, as indicated by the disordered 5-hydroxytryptamine (5-HT), dopamine (DA) and their corresponding receptors, together with the over production of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). VB also notably promoted the expression of p-NF-κBp65, p-IκBα, p-IKKα and p-IKKß and down-regulated the expression of BDNF and TrkB. CONCLUSION: This study demonstrates that VB triggers neurotoxicity which probably is induced by neuroinflammation via activating of NF-κB pathway and attenuating the expression of BDNF.

TXNIP/TRX/NF-κB and MAPK/NF-κB pathways involved in the cardiotoxicity induced by Venenum Bufonis in rats.

Venenum Bufonis (VB) is a widely used traditional medicine with serious cardiotoxic effects. The inflammatory response has been studied to clarify the mechanism of the cardiotoxicity induced by VB for the first time. In the present study, Sprague Dawley (SD) rats, were administered VB (100, 200, and 400 mg/kg) intragastrically, experienced disturbed ECGs (lowered heart rate and elevated ST-segment), increased levels of serum indicators (creatine kinase (CK), creatine kinase isoenzyme-MB (CK-MB), alanine aminotransferase (ALT), aspartate aminotransferase (AST)) and serum interleukin (IL-6, IL-1ß, TNF-α) at 2 h, 4 h, 6 h, 8 h, 24 h, and 48 h, which reflected that an inflammatory response, together with cardiotoxicity, were involved in VB-treated rats. In addition, the elevated serum level of MDA and the down-regulated SOD, CAT, GSH, and GPx levels indicated the appearance of oxidative stress in the VB-treated group. Furthermore, based on the enhanced expression levels of TXNIP, p-NF-κBp65, p-IκBα, p-IKKα, p-IKKß, p-ERK, p-JNK, and p-P38 and the obvious myocardial degeneration, it is proposed that VB-induced cardiotoxicity may promote an inflammatory response through the TXNIP/TRX/NF-κB and MAPK/NF-κB pathways. The observed inflammatory mechanism induced by VB may provide a theoretical reference for the toxic effects and clinical application of VB.

Transcriptomic comparison of the self-pollinated and cross-pollinated flowers of Erigeron breviscapus to analyze candidate self-incompatibility-associated genes.

BACKGROUND: Self-incompatibility (SI) is a widespread and important mating system that promotes outcrossing in plants. Erigeron breviscapus, a medicinal herb used widely in traditional Chinese medicine, is a self-incompatible species of Asteraceae. However, the genetic characteristics of SI responses in E. breviscapus remain largely unknown. To understand the possible mechanisms of E. breviscapus in response to SI, we performed a comparative transcriptomic analysis with capitulum of E. breviscapus after self- and cross-pollination, which may provide valuable information for analyzing the candidate SI-associated genes of E. breviscapus. METHODS: Using a high-throughput next-generation sequencing (Illumina) approach, the transcriptionexpression profiling of the different genes of E. breviscapus were obtained, some results were verified by quantitative real time PCR (qRT-PCR). RESULTS: After assembly, 63,485 gene models were obtained (average gene size 882 bp; N50 = 1485 bp), among which 38,540 unigenes (60.70% of total gene models) were annotated by comparisons with four public databases (Nr, Swiss-Prot, KEGG and COG): 38,338 unigenes (60.38% of total gene models) showed high homology with sequences in the Nr database. Differentially expressed genes were identified among the three cDNA libraries (non-, self- and cross-pollinated capitulum of E. breviscapus), and approximately 230 genes might be associated with SI responses. Several these genes were upregulated in self-pollinated capitulum but downregulated in cross-pollinated capitulum, such as SRLK (SRK-like) and its downstream signal factor, MLPK. qRT-PCR confirmed that the expression patterns of EbSRLK1 and EbSRLK3 genes were not closely related to SI of E. breviscapus. CONCLUSIONS: This work represents the first large-scale analysis of gene expression in the self-pollinated and cross-pollinated flowers of E. breviscapus. A larger number of notable genes potentially involved in SI responses showed differential expression, including genes playing crucial roles in cell-cell communication, signal transduction and the pollination process. We thus hypothesized that those genes showing differential expression and encoding critical regulators of SI responses, such as MLPK, ARC1, CaM, Exo70A1, MAP, SF21 and Nod, might affect SI responses in E. breviscapus. Taken together, our study provides a pool of SI-related genes in E. breviscapus and offers a valuable resource for elucidating the mechanisms of SI in Asteraceae.

[Effect of Xianxiong decoction on acute lung injury mice induced by lipopolysaccharide].

OBJECTIVE: To investigate the effect of Xianxiong decoction on the mice with acute lung injury induced by lipopolysaccharide. METHOD: Eighty female ICR mice were randomly divided into 8 groups: model group, Xianxiong decoction group, Daxianxiong decoction group, Xianxiong decoction group without Kansui Radix group, Xianxiong decoction group without Glycyrrhizae Radix et Rhizoma group, Glycyrrhizae Radix et Rhizoma and Kansui Radix group, normal group and control group. Animals of each group, except normal group, were undertaken intraperitoneal injection and intranasal inhalation of lipopolysaccharide (LPS) on day 1, 2, 3 to establish acute lung injury (ALI) model. 30 min after modeling, 0.2 mL corresponding drugs were administrated to each mice, dexam ethasone and normal saline were given to the mice of control group and normal group respectively. White blood cell in blood, neutrophil percentage of blood and bronchoalveolar lavage fluid (BALF) supernatant, the ratio of wet and dry lung tissue ( W/D), histopathological changes of lung tissue were estimated. Sixty ICR mice were randomly divided into normal, model, control, high, middle and low dose Xianxiong decoction groups and were modeled in the same way. ELISA was applied to detect the level of NF-kappaB, TNF-alpha and IL-6 in BALF, PCR for NF-kappaB and TNF-alpha mRNA in lung tissue, and Western blot for NF-kappaB and TNF-alpha. Half of 20 ICR mice were administrated with Xianxiong decoction of its maximum tolerant normal saline. RESULT: Compared with model group, the number of WBC in blood of Xianxiong decoction group mice decreased (P < 0.01), percentage of neutrophils in both blood and BALF decreased as well (P < 0.01, P < 0.05); it also significantly reduced the ratio of W/D (P < 0.01); and found the alveolar wall, the number of inflammatory cells infiltrating improved, compared with model group. Xianxiong decoction reduced the level of NF-kappaB, TNF-alpha and IL-6 in BALF (P < 0.01, P < 0.01, P < 0.05); its high and low dose groups only found TNF-alpha level declined. Five mice died 24 h after administration of Xianxiong decoction which indicated its toxicity when other influential factors were considered. CONCLUSION: Xianxiong decoction is effective on the ALI mice induced by LPS, but it is of toxicity at 3 g x mL(-1).

Simiao pill ameliorates renal glomerular injury via increasing Sirt1 expression and suppressing NF-κB/NLRP3 inflammasome activation in high fructose-fed rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Simiao pill is one of the most frequently prescriptions in traditional Chinese medicine to treat hyperuricemia and gout. This study was to investigate the protective effects of Simiao pill on renal glomerular injury in a rat model of high fructose intake. MATERIALS AND METHODS: Sprague-Dawley male rats were given 10% fructose in drinking water and standard laboratory chow for 4 weeks to induce hyperuricemia and metabolic syndrome. Then fructose-fed animals were randomly divided into four groups receiving water, Simiao pill (78.87 and 157.74 mg/kg) and allopurinol (5mg/kg) daily for next 6 weeks, respectively. Serum levels of uric acid, creatinine, triglyceride, total cholesterol, low density lipoprotein, blood urea nitrogen, insulin, as well as urinary albumin were measured. Oral glucose tolerance test (OGTT) was carried out. Kidney pathological changes were detected using periodic-acid schiff-stained (PAS) staining and transmission electron microscopy (TEM) analysis. Glomerular protein levels of nephrin, podocin, CD2-associated protein (CD2AP), interleukin (IL)-1ß, sirtuin 1 (Sirt1), nuclear factor kappaB (NF-κB) and pyrin domain containing 3 (NLRP3) inflammasome were measured by Western blot. RESULTS: Simiao pill effectively restored high fructose-induced hyperuricemia and metabolic syndrome in rats. Simiao pill significantly increased protein levels of nephrin, podocin and CD2AP in renal glomeruli, improved renal inflammatory cell infiltration into interstitium and glomerular injury in high fructose-fed rats with reduction of urine albumin levels. Furthermore, Simiao pill up-regulated Sirt1 protein levels and suppressed NF-κB/NLRP3 inflammasome activation to reduce IL-1ß in renal glomeruli of high fructose-fed rats. CONCLUSIONS: The renal protective effects of Simiao pill may be associated with up-regulation of Sirt1 expression and suppression of NF-κB/NLRP3 inflammasome activation to reduce renal glomerular injury in high fructose-fed rats with metabolic syndrome.

Transcriptome analysis of Panax vietnamensis var. fuscidicus discovers putative ocotillol-type ginsenosides biosynthesis genes and genetic markers.

BACKGROUND: P. vietnamensis var. fuscidiscus, called "Yesanqi" in Chinese, is a new variety of P. vietnamensis, which was first found in Jinping County, the southern part of Yunnan Province, China. Compared with other Panax plants, this species contains higher content of ocotillol-type saponin, majonoside R2. Despite the pharmacological importance of ocotillol-type saponins, little is known about their biosynthesis in plants. Hence, P. vietnamensis var. fuscidiscus is a suitable medicinal herbal plant species to study biosynthesis of ocotillol-type saponins. In addition, the available genomic information of this important herbal plant is lacking. RESULTS: To investigate the P. vietnamensis var. fuscidiscus transcriptome, Illumina HiSeq™ 2000 sequencing platform was employed. We produced 114,703,210 clean reads, assembled into 126,758 unigenes, with an average length of 1,304 bp and N50 of 2,108 bp. Among these 126,758 unigenes, 85,214 unigenes (67.23%) were annotated based on the information available from the public databases. The transcripts encoding the known enzymes involved in triterpenoid saponins biosynthesis were identified in our Illumina dataset. A full-length cDNA of three Squalene epoxidase (SE) genes were obtained using reverse transcription PCR (RT-PCR) and the expression patterns of ten unigenes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Furthermore, 15 candidate cytochrome P450 genes and 17 candidate UDP-glycosyltransferase genes most likely to involve in triterpenoid saponins biosynthesis pathway were discovered from transcriptome sequencing of P. vietnamensis var. fuscidiscus. We further analyzed the data and found 21,320 simple sequence repeats (SSRs), 30 primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism in 13 P. vietnamensis var. fuscidiscus accessions. Meanwhile, five major triterpene saponins in roots of P. vietnamensis var. fuscidicus were determined using high performance liquid chromatography (HPLC) and evaporative light scattering detector (ELSD). CONCLUSIONS: The genomic resources generated from P. vietnamensis var. fuscidiscus provide new insights into the identification of putative genes involved in triterpenoid saponins biosynthesis pathway. This will facilitate our understanding of the biosynthesis of triterpenoid saponins at molecular level. The SSR markers identified and developed in this study show genetic diversity for this important crop and will contribute to marker-assisted breeding for P. vietnamensis var. fuscidiscus.

Ginkgo biloba extracts attenuate lipopolysaccharide-induced inflammatory responses in acute lung injury by inhibiting the COX-2 and NF-κB pathways.

In the present study, we analyzed the role of Ginkgo biloba extract in lipopolysaccharide(LPS)-induced acute lung injury (ALI). ALI was induced in mice by intratracheal instillation of LPS. G. biloba extract (12 and 24 mg·kg(-1)) and dexamethasone (2 mg·kg(-1)), as a positive control, were given by i.p. injection. The cells in the bronchoalveolar lavage fluid (BALF) were counted. The degree of animal lung edema was evaluated by measuring the wet/dry weight ratio. The superoxidase dismutase (SOD) and myeloperoxidase (MPO) activities were assayed by SOD and MPO kits, respectively. The levels of inflammatory mediators, tumor necrosis factor-a, interleukin-1b, and interleukin-6, were assayed by enzyme-linked immunosorbent assay. Pathological changes of lung tissues were observed by H&E staining. The levels of NF-κB p65 and COX-2 expression were detected by Western blotting. Compared to the LPS group, the treatment with the G. biloba extract at 12 and 24 mg·kg(-1) markedly attenuated the inflammatory cell numbers in the BALF, decreased NF-κB p65 and COX-2 expression, and improved SOD activity, and inhibited MPO activity. The histological changes of the lungs were also significantly improved. The results indicated that G. biloba extract has a protective effect on LPS-induced acute lung injury in mice. The protective mechanism of G. biloba extract may be partly attributed to the inhibition of NF-κB p65 and COX-2 activation.

Tectorigenin inhibits the inflammation of LPS-induced acute lung injury in mice.

AIM: In a previous study, the anti-inflammatory effects of tectorigenin were disclosed. In this study, the anti-inflammatory effects of tectorigenin on acute lung injury using a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model were investigated METHOD: The cell-count in the bronchoalveolar lavage fluid (BALF) was measured. The animal lung edema degree was evaluated by the wet/dry weight (W/D) ratio. The superoxidase dismutase (SOD) activity and myeloperoxidase (MPO) activity was assayed using SOD and MPO kits, respectively. The levels of inflammatory mediators, including tumor necrosis factor-α (TNF-α), IL-1ß, and IL-6 were assayed using an enzyme-linked immunosorbent assay method. Pathological changes of lung tissues were observed through HE staining. The inflammatory signal pathway related protein nuclear factor NF-κB p65 mRNA expression was measured by real-time PCR, and the protein level of NF-κB p65 was measured using Western blotting analysis. RESULTS: The data showed that treatment with the tectorigenin markedly attenuated the inflammatory cell numbers in the BALF, decreased nuclear factor NF-κB p65 mRNA level and protein level in the lungs, and improved SOD activity and inhibited MPO activity. Histological studies showed that tectorigenin substantially inhibited LPS-induced neutrophils in lung tissue compared with the model group. CONCLUSION: The results indicated that tectorigenin had a protective effect on LPS-induced ALI in mice.

Ma Huang Tang ameliorates asthma though modulation of Th1/Th2 cytokines and inhibition of Th17 cells in ovalbumin-sensitized mice.

AIM: Ma Huang Tang (Ephedra decoction, MHT) is a famous classical formula from Shang Han Lun by Zhang Zhongjing in the Han Dynasty. The anti-asthmatic effects of MHT and the possible mechanisms were tested. METHOD: An asthma model was established by ovalbumin (OVA)-induction in mice. A total of forty-eight mice were randomly assigned to six experimental groups: control, model, dexamethasone (2 mg·kg(-1)) and MHT (5, 10, and 20 mg·kg(-1)). Airway resistance (Raw) was measured by the forced oscillation technique, histological studies were evaluated by hematoxylin and eosin (HE) staining, Th1/Th2 and Th17 cytokines were evaluated by enzyme-linked immunosorbent assay (ELISA), and Th17 cells were evaluated by flow cytometry (FCM). RESULTS: This study demonstrated that MHT inhibited OVA-induced increases in Raw and eosinophil count; interleukin (IL)-4 and IL-17 levels were recovered in bronchoalveolar lavage fluid, increased IFN-γ level in bronchoalveolar lavage fluid. Histological studies demonstrated that MHT substantially inhibited OVA-induced eosinophilia in lung tissue. Flow cytometry studies demonstrated that MHT substantially inhibited Th17 cells. CONCLUSION: These findings suggest that MHT may effectively ameliorate the progression of asthma, and could be further investigated for potential use as a therapy for patients with allergic asthma.

Toxicity of daphnane-type diterpenoids from Genkwa Flos and their pharmacokinetic profile in rat.

Daphnane-type diterpenoids (DDs) are the main types of plant diterpene orthoesters known and have remarkable biological activities. However, the in vivo toxicity and pharmacokinetic profile of DDs remains unkonwn. The aim of this study was to investigate the toxicity and pharmacokinetic profile of DDs from Genkwa Flos (Thymelaeaceae). The toxicity of diterpenoids was evaluated after oral administration of total diterpenoids extract from Genkwa Flos to rats, and the blood concentration of diterpenoids was analyzed by ultra performance liquid chromatography tandem triple-quadrupole mass spectrometry (UPLC-TQ-MS). The diterpenoids were confirmed to be the toxic components of Genkwa Flos. The pharmacokinetic profile of these diterpenoids was quite different due to their different structures. Although the contents of yuanhuafine and yuanhuapine were low in the extract, the blood concentrations were extremely high. In contrary, the contents of genkwanine F and Wikstroemia factor M1 in the extract were much higher, but they could not be detected in the blood. This result implied that yuanhuafine and yuanhuapine but not genkwanine F and Wikstroemia factor M1 were the potentail toxic components of Genkwa Flos in vivo. This paper shows for the first time the toxicity of diterpenoids from Genkwa Flos was correlated with their blood concentration and when DDs were used for medicinal purposes, their contents in herb as well as their blood concentrations should be considered.