BACKGROUND AND PURPOSE:While pain and itch are both commonly associated with chronic dermatitis (CD), the molecular mechanisms underlying these debilitating symptoms is not well understood. This study aims to identify novel, endogenous compounds that mediate CD-associated pain and itch. EXPERIMENTAL APPROACH:Lesional skin of CD model mice was examined using unbiased metabolomic analysis to identify candidate pain or itch inducing compounds in CD. Sphingosine-1-phosphate (S1P) concentration in CD model skin was analysed using UPLC/MS/MS. Behaviour, calcium imaging and immunofluorescence staining were used to determine the pain and itch effects and mechanisms of the identified CD-related compounds. KEY RESULTS:In the lesional skin of CD model mice, 136 compounds were significantly changed. These compounds are predominately associated with the sphingolipids metabolism pathway. S1P is significantly increased in the lesional skin . The TRPV4 channel was critical for S1P induced itch and pain. Sphingosine kinase 2 (SPHK2), the key enzyme controlling S1P synthesis, was significantly increased in lesional skin. ABC294640, a SPHK2 inhibitor, significantly decreased S1P concentration in lesional CD model skin, as well as in model associated epidermal hyperplasia and chronic pain and itch. In CD patients, SPHK2 expression and S1P concentration were significantly elevated compared to healthy control skin. CONCLUSION AND IMPLICATIONS:Our results indicate that, in CD, increased S1P induces chronic pain and itch partly through TRPV4. Inhibition of S1P synthesis or the S1P/S1P receptor-TRPV4 pathway are promising treatment strategies for CD-associated pain and itch.

Rationale: Noxious stimuli are often perceived as itchy in patients with chronic dermatitis (CD); however, itch and pain mechanisms of CD are not known.Methods: TRPV1 involvement in CD was analyzed using a SADBE induced CD-like mouse model, and several loss-and gain-of-function mouse models.Trigeminal TRPV1 channel and MrgprA3 + neuron functions were analyzed by calcium imaging and whole-cell patch-clamp recordings.Lesional CD-like skin from mice were analyzed by unbiased metabolomic analysis.20-HETE availability in human and mouse skin were determined by LC/MS and ELISA.And finally, HET0016, a selective 20-HETE synthase inhibitor, was used to evaluate if blocking skin TRPV1 activation alleviates CD-associated chronic itch or pain.Results: While normally a pain inducing chemical, capsaicin induced both itch and pain in mice with CD condition.DREADD silencing of MrgprA3 + primary sensory neurons in these mice selectively decreased capsaicin induced scratching, but not pain-related wiping behavior.In the mice with CD condition, MrgprA3 + neurons showed elevated ERK phosphorylation.Further experiments showed that MrgprA3 + neurons from MrgprA3;Braf mice, which have constitutively active BRAF in MrgprA3 + neurons, were significantly more excitable and responded more strongly to capsaicin.Importantly, capsaicin induced both itch and pain in MrgprA3;Braf mice in an MrgprA3 + neuron dependent manner.Finally, the arachidonic acid metabolite 20-HETE, which can activate TRPV1, was significantly elevated in the lesional skin of mice and patients with CD.Treatment with the selective 20-HETE synthase inhibitor HET0016 alleviated itch in mice with CD condition. Conclusion:Our results demonstrate that 20-HETE activates TRPV1 channels on sensitized MrgprA3 + neurons, and induces allokinesis in lesional CD skin.Blockade of 20-HETE synthesis or silencing of TRPV1-MrgprA3 + neuron signaling offers promising therapeutic strategies for alleviating CD-associated chronic itch.

(CG) is widely used as a traditional Chinese medicine for wound treatment. In this study, we aimed to determine the effects of CG extract (CGE) on diabetic wound healing and the commensal wound microbiome.

Background Calvatia gigantea (CG) is widely used as a traditional Chinese medicine for wound treatment. In this study, we aimed to determine the effects of CG extract (CGE) on diabetic wound healing and the commensal wound microbiome.Method A wound model was established using leptin receptor-deficient db/db mice, with untreated mice as the control group and CGE-treated mice as the treatment group. The wound healing rate, inflammation and histology were analyzed. Additionally, wound microbiome was evaluated via 16S ribosomal RNA (rRNA) gene sequencing.Results CGE significantly accelerated the healing of diabetic ulcer wounds, facilitated re-epithelialization, and downregulated the transcription levels of the inflammatory cytokines, interleukin-1 beta and tumor necrosis factor-alpha. Furthermore, CGE treatment positively affected the wound microbiome, promoting diversity of the microbial community and enrichment of Escherichia-Shigella bacteria in the CGE-treated group.Conclusions Overall, CGE enhanced diabetic wound healing by modulating the wound microbiome and facilitating macrophage polarization during inflammation. These findings suggest modulation of the commensal wound microbiome using medicinal plants as a potential therapeutic strategy for diabetic wounds. Graphical Abstract

Angelica sinensis is a medicinal plant widely used to treat multiple diseases in Asia and Europe, which contains numerous active components with therapeutic value. The interaction between root and rhizosphere microorganisms is crucial for the growth and quality formation of medicinal plants. But the micro-plant-metabolite regulation patterns for A. sinensis remain largely undetermined. Here, we collected roots and rhizosphere soils from A. sinensis in seedling stage (M) and picking stage (G), respectively cultivated for one year and two years, generated metabolite for roots, microbiota data for rhizospheres, and conducted a comprehensive analysis. Changes in metabolic and microbial communities of A.sinensis over growth were distinct. The composition of rhizosphere microbes in G was dominated by proteobacteria, which had a strong correlation with the synthesis of organic acids, while in M was dominated by Actinobacteria, which had a strong correlation with the synthesis of phthalide and other organoheterocyclic compounds, flavonoids, amines, and fatty acid. Additionally, co-occurrence network analysis identified that Arthrobacter was found to be strongly correlated with the accumulation of senkyunolide A and n-butylidenephthalide. JGI 0001001.H03 was found to be strongly correlated with the accumulation of chlorogenic acid. Based on rhizosphere microorganisms, this study investigated the correlation between root metabolism and rhizosphere microbiota of A. sinensis at different growth stages in traditional geoherb region, which could provide references for exploring the quality formation mechanism of A. sinensis in the future.

The roots of Angelica sinensis (Oliv.) Diels are well known for their efficacy in promoting blood circulation. Although many studies have indicated that phthalides are the main chemical components responsible for the pharmacological properties of A. sinensis, the phthalide biosynthetic pathway and enzymes that transform different phthalides are still poorly understood. We identified 108 potential candidate isoforms for phthalide accumulation using transcriptome and metabolite profile analyses. Then, six enzymes, including phospho-2-dehydro-3-deoxyheptonate aldolase 2, shikimate dehydrogenase, primary amine oxidase, polyphenol oxidase, tyrosine decarboxylase, and shikimate O-hydroxycinnamoyl transferase, were identified and proven to be involved in phthalide accumulation by heterologously expressing these proteins in Escherichia coli. We proposed a possible mechanism underlying phthalide transformation and biosynthetic pathways in A. sinensis based on our findings. The results of our study can provide valuable information for understanding the mechanisms underlying phthalide accumulation and transformation and enable further development of quality control during the cultivation of A. sinensis.

目的 基于网络药理学方法和斑马鱼模型探讨瓜蒌薤白半夏汤干预慢性阻塞性肺疾病(chronic obstructive pulmoriary disease,COPD)的潜在作用机制.方法 运用中药系统药理学数据库与分析平台(TCMSP)、中医药百科全书数据库(ETCM)、中医药综合数据库(TCMID)以及国内外相关文献检索瓜蒌薤白半夏汤的主要活性成分,通过TCMSP和SwissTargetPrediction数据库预测靶点;通过GeneCards数据库、在线人类孟德尔遗传数据库(OMIM)和TCMSP数据库获取COPD相关靶点,并采用交集法筛选出与瓜蒌薤白半夏汤活性成分作用的共同靶点.运用Cytoscape 3.7.1软件构建"中药-活性成分-靶点"网络,通过String数据库构建蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络;通过DAVID数据库对共有基因进行基因本体(gene ontology,GO)功能富集分析及京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析;借助斑马鱼炎症模型对筛选的靶点进行实验验证.结果 瓜蒌薤白半夏汤共筛选出成分955个,潜在活性成分106个,核心活性成分如芹菜素、茴香酸、美迪紫檀素、金圣草黄素、香叶木素等74个,对应靶点1134个;PPI网络分析获得瓜蒌薤白半夏汤干预COPD的关键作用靶点408个,包括碳酸酐酶9(carbonic anhydrase 9,CA9)、前列腺素内过氧化物合酶1 (prostaglandin-endoperoxide synthase 1,PTGS1)、胰岛素(insulin,INS)、白细胞介素-6(interleukin-6,IL-6)、丝氨酸/苏氨酸蛋白激酶1(serine/threonine protein kinase 1,AKT1)、肿瘤坏死因子(tumor necrosis factor,TNF)、丝裂原活化蛋白激酶1(mitogen-activated protein kinase 1,MAPK1)、MAPK3、血管内皮生长因子A(vascular endothelial growth factorA,VEGFA)、表皮生长因子受体(epidermal growth factor receptor,EGFR)等;GO功能及KEGG通路富集分析得出与瓜蒌半夏薤白汤干预COPD作用相关的生物过程1096个,如炎症反应、细胞溶质、酶结合等;相关信号通路125条,如癌症通路、TNF信号通路、前列腺癌、缺氧诱导因子1信号通路、神经活性配体-受体相互作用等.斑马鱼炎症模型实验结果显示,瓜蒌薤白半夏汤显著降低IL-6、B淋巴细胞2(B-cell lymphoma 2,BCL2)和EGFR mRNA表达水平(P＜0.05),降低半胱氨酸蛋白酶8(Caspases 8,CASP8)mRNA表达水平.结论 瓜蒌薤白半夏汤可能通过作用于EGFR、IL-6、BCL2、CASP8等靶点,调节多条信号通路,从而发挥对COPD的治疗作用.

Plant-microorganism interaction in the rhizosphere is thought to play an important role in the formation of soil fertility, transformation and absorption of nutrients, growth and development of medicinal plants, and accumulation of medicinal ingredients. Yet, the role that they play in the phthalides accumulation of Angelica sinensis (Oliv.) Diels remains unclear. In the present study, we report a correlative analysis between rhizosphere microorganisms and phthalides accumulation in A. sinensis from Gansu, China where was the major production areas. Meanwhile, Bacillus was explored the potential functions in the plant growth and phthalide accumulation. Results revealed that the common bacterial species detected in six samples comprised 1150 OTUs which were involved in 368 genera, and predominant taxa include Actinobacteria, Acidobacteria, and Proteobacteria. The average contents of the six phthalides were 4.0329 mg/g. The correlation analysis indicated that 20 high abundance strains showed positive or negative correlations with phthalides accumulation. Flavobacterium, Nitrospira, Gaiella, Bradyrhizobium, Mycobacterium, Bacillus, RB41, Blastococcus, Nocardioides, and Solirubrobacter may be the key strains that affect phthalides accumulation on the genus level. By the plant-bacterial co-culture and fermentation, Bacillus which were isolated from rhizosphere soils can promote the plant growth, biomass accumulation and increased the contents of the butylidenephthalide (36∼415%) while the ligustilide (12∼67%) was decreased. Altogether, there is an interaction between rhizosphere microorganisms and phthalides accumulation in A. sinensis, Bacillus could promote butylidenephthalide accumulation while inhibiting ligustilide accumulation.

The root of Angelica sinensis is known throughout Asia for its traditional efficacy in invigorating and promoting blood circulation. The wild germplasm resources of A. sinensis was in short supply, and most of the commercial medicinal materials come from cultivation. To obtain the differences in the transcriptional levels of wild and cultivated of A. sinensis, the full-length transcriptome of A. sinensis was analyzed using PacBio SMRT three-generation high-throughput sequencing technology. Using the high-throughput sequencing platform Illumina HiSeq X Ten PE150, a root transcriptome dataset of wild and cultivated A.sinensis was obtained. The transcriptome sequencing analyses obtained 16.5 Gb database in wild and cultivated A.sinensis, after assembly steps, we obtain 113 906 transcript sequences(insfroms) with an average length of 1 466 nt. BLAST analysis indicated that 109 113(accounting for 95.79% of the total insfroms), 93 276(81.89%),60 638(53.24%),48 928(42.95%),42 876(37.64%)isofroms were successfully annotated in the NR, Swiss-port, GO, KO and KOG databases, respectively. The annotation information can be classified into three categories of biological processes, cellular components and molecular functions of GO classification, involving 128 KEGG standard metabolic pathways. Analysis of 25 463 differential insfroms, 15 090 higher expression in wild A. sinensis, and 10 373 higher in cultivated A. sinensis. In the enrichment analysis of GO and KEGG, differential insfroms were concentrated on the pathway of plant-pathogen interaction, MAPK signaling pathway-plant and plant hormone signal transduction. In this study, high-throughput sequencing was used to obtain the full-length transcription information of A. sinensis, and the overall characteristics of A. sinensis genetic information were clarified. By comparing the differential expression of wild and cultivated A. sinensis at the genetic level, it provides basic information for further screening and breeding of A. sinensis germplasm resources, resistance research and secondary metabolic pathway analysis.