[Effect of moxibustion at "Mingmen" (GV 4) and "Guanyuan" (CV 4) on immune function in healthy rats based on intestinal flora].

OBJECTIVE: To observe the effects of moxibustion at "Mingmen" (GV 4) and "Guanyuan" (CV 4) on immune function and intestinal flora in healthy rats, thereby investigating the underlying mechanism of moxibustion on immune function. METHODS: Twenty 8-week-old SD rats were randomly divided into a young blank group and a young moxibustion group, with 10 rats in each group. Similarly, twenty 8-month-old SD rats were randomly divided into a middle-aged blank group and a middle-aged moxibustion group, with 10 rats in each group. The rats in the two moxibustion groups received moxibustion at "Mingmen" (GV 4) and "Guanyuan" (CV 4), 15 min per session, once daily, five times a week, for a total of four months. The rats in the two blank groups were fed under normal conditions. After the intervention, thymus and spleen indexes were calculated; the morphology of thymus and spleen tissues was observed using HE staining; the flow cytometry was used to detect the expression of CD and CD T lymphocytes and the CD/CD ratio was calculated; ELISA was used to measure the serum levels of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-17 (IL-17); 16S rRNA high-throughput sequencing was used to analyze the intestinal flora. Additionally, the correlation between the relative abundance of intestinal flora and serum levels of TNF-α, IFN-γ, IL-6, IL-10 and IL-17 was analyzed. RESULTS: Compared with the young blank group, the young moxibustion group exhibited an increase in the cortical area of thymus tissue with tighter lymphocyte arrangement; compared with the middle-aged blank group, the middle-aged moxibustion group showed an increase in thymus index (P<0.05) and an increase in the cortical area of thymus tissue. There were no significant differences in spleen index between the 2 moxibustion groups and the 2 blank groups (P>0.05). There were no significant differences in the expression of CD, CD, and CD/CD ratio between the 2 moxibustion groups and the corresponding blank groups (P>0.05). Compared with the young blank group, the young moxibustion group had elevated IL-6 level (P<0.05); compared with the middle-aged blank group, the middle-aged moxibustion group had decreased IL-10 and IL-17 levels (P<0.05). Compared with the young blank group, the young moxibustion group exhibited increased Sobs index, Ace index, and Chao index (P<0.01, P<0.05), as well as increased relative abundance of Spirochaetota, Treponema, Turicibacter, Rikenellaceae_RC9_gut_group (P<0.05), and decreased relative abundance of Dubosiella (P<0.05). Compared with the middle-aged blank group, the middle-aged moxibustion group had increased relative abundance of Spirochaetota, Treponema, norank_f_Peptococcaceae (P<0.05), and decreased relative abundance of Proteobacteria, Allobaculum, and Faecalibaculum (P<0.05). Correlation analysis revealed that relative abundance of Eubacterium_xylanophilum_group and unclassified _f_Lachnospiraceae was negatively correlated with serum TNF-α level (r=-0.39, P=0.03; r=-0.24, P=0.04), while relative abundance of norank_f_norank_o_Clostridia_UCG-014 and Lactobacillus was positively correlated with serum TNF-α level (r=0.37, P=0.04; r=0.43, P=0.02). The relative abundance of Roseburia and Monoglobus was negatively correlated with serum IFN-γ level (r=-0.40, P=0.02; r=-0.44, P=0.01), while relative abundance of Lactobacillus was positively correlated with serum IL-10 level (r=0.43, P=0.02). CONCLUSION: Moxibustion could improve immune function in healthy rats, and its mechanism may be related to the regulation of relative abundance of intestinal flora.

Tea Polyphenols Reduce Inflammation of Orbital Fibroblasts in Graves' Ophthalmopathy via the NF-κB/NLRP3 Pathway.

OBJECTIVE: This study aimed to explore the effects of tea polyphenols (TP) on inflammation of orbital fibroblasts in Graves' ophthalmopathy (GO) and to provide new ideas for GO treatment. METHODS: Primary orbital fibroblasts were extracted from orbital adipose/connective tissues of patients with and without GO. Real-time quantitative PCR (RT-qPCR) was used to detect the expression of interleukin (IL)-6, IL-1ß, and monocyte chemotactic protein (MCP)-1 in non-GO and GO orbital fibroblasts. The CCK-8 assay was used to determine the appropriate concentration of TP for subsequent experiments. RT-qPCR and enzyme-linked immunosorbent assay (ELISA) were performed to investigate the effects of TP on lipopolysaccharide (LPS)-induced production of inflammatory cytokines. Nuclear factor-κB (NF-κB) expression was measured using Western blotting analysis. NOD-like receptor 3 (NLRP3) expression was detected using both Western blotting analysis and immunofluorescence staining. RESULTS: The mRNA levels of IL-6, IL-1ß, and MCP-1 in GO orbital fibroblasts were significantly higher than those in non-GO cells. TP treatment significantly inhibited LPS-induced production of inflammatory factors, including IL-6, IL-1ß, and MCP-1. TP also inhibited the expression levels of NF-κB and NLRP3. Inflammation in the GO orbital fibroblasts was higher than that in non-GO cells. TP inhibited the production of inflammatory cytokines in GO orbital fibroblasts in vitro through the NF-κB/NLRP3 pathway. CONCLUSION: These findings suggest that TP may have a potential role in GO treatment.

Transcriptome Analysis of Rat Lungs Exposed to Moxa Smoke after Acute Toxicity Testing.

The increasing use of moxibustion has led to a debate concerning the safety of this treatment in human patients. Inhalation of cigarette smoke induces lung inflammation and granulomas, the proliferation of alveolar epithelial cells, and other toxic effects; therefore, it is important to assess the influence of inhaled moxa smoke on the lungs. In the present study, a novel poisoning cabinet was designed and used to assess the acute toxicity of moxa smoke in rats. We evaluated pathological changes in rat lung tissue and analyzed differentially expressed genes (DEGs) using RNA-seq and transcriptomic analyses. Our results show that the maximum tolerable dose of moxa smoke was 290.036 g/m³ and LC50 was 537.65 g/m³. Compared with that of the control group, the degree of inflammatory cell infiltration in the lung tissues of group A rats (all dead group) was increased, while that in group E rats (all live group) remained unchanged. GO and KEGG enrichment analyses showed that the DEGs implicated in cell components, binding, and cancer were significantly enriched in the experimental groups compared with the profile of the control group. The expressions of MAFF, HSPA1B, HSPA1A, AOC1, and MX2 determined using quantitative real-time PCR were similar to those determined using RNA-seq, confirming the reliability of RNA-seq data. Overall, our results provide a basis for future evaluations of moxibustion safety and the development of moxibustion-based technology.

Analysis of the Components in Moxa Smoke by GC-MS and Preliminary Discussion on Its Toxicity and Side Effects.

Moxibustion plays an important role in the prevention and treatment of diseases and the promotion of human health. In this study, the components in moxa smoke from Jiangxi Poai Biotechnology Co., Ltd., namely, Qing moxa sticks, were absorbed by five solvents (cyclohexane, ethyl acetate, n-butanol, anhydrous ethanol, and water) and identified by gas chromatography-mass spectrometry. The identification results of the smoke from the Qing moxa sticks that was absorbed in liquid are as follows: a total of 294 compounds were identified, including 139 in cyclohexane, 145 in ethyl acetate, 60 in n-butanol, 89 in anhydrous ethanol, and 77 in water, and of those, 112 toxic compounds were identified. Furthermore, Ingenuity Pathway Analysis software and the PubChem database were successfully applied to analyze the toxic compounds. There were 812 target proteins related to the toxic components, 25 molecular networks, and 54 biological pathways. The results showed that the toxic compounds of moxa smoke may have some side effects on the heart, liver, and kidney of humans. This study revealed that the components of moxa smoke are complex and diverse. Due to the findings of toxic compounds in moxa smoke, we recommend that moxibustion rooms should be equipped with ventilation equipment or enough artificial ventilation to ensure the health of patients and practitioners.

[Molecular mechanism analysis of Erzhi Wan in treatment of aging based on network pharmacology].

This present study aimed to explore the molecular mechanism of Erzhi Wan(a prescription of nourishing Yin and toni-fying liver and kidney) in treatment of aging by network pharmacology. The active constituents and target proteins of Erzhi Wan were searched from Traditional Chinese Medicine Systems Pharmacology Database(TCMSP) and PubChem databases respectively. Aging-related genes were searched from Gene and HAGR databases. Based on the Ingenuity Pathway Analysis(IPA), we analyzed the common molecular network, biological pathway and interaction sites between these two parts, and verified some of them by Western blot. Twelve active constituents of Erzhi Wan were screened by TCMSP databases, 69 protein targets were predicted through PubChem, and 148 aging-related genes were found in Gene and HAGR databases. IPA comparison showed that the molecular networks of these two were complex, with diversity of biological functions. The common pathways involved 292 pathways, mainly related to tumors. They acted on hypoxia inducible factor-1α gene(HIF1α), nuclear factor-E2 related factor(Nrf2/NFE2 L2), tumor necrosis factor(TNF) and other sites. Western blot results suggested that Erzhi Wan could down-regulate the expression of HIF1α, with statistical difference(P<0.05). It was concluded that, Erzhi Wan could intervene aging through improving pseudo-hypoxic microenvironment and inflammation. The molecular mechanism of Erzhi Wan in delaying aging was preliminarily revealed, which laid a foundation for further stu-dying the anti-aging mechanism of Erzhi Wan, and also provided a reference for the compatibility mechanism and extended application of Chinese medicine compounds.

Rapid screening and identification of lycodine-type alkaloids in Lycopodiaceae and Huperziaceae plants by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Lycodine-type alkaloids have gained significant interest owing to their unique skeletal characteristics and acetylcholinesterase activity. This study established a rapid and reliable method using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q/TOF-MS/MS) for comprehensive characterization of lycodine-type alkaloids for the first time. The lycodine-type alkaloids were detected successfully from Lycopodiastrum casuarinoides, Huperzia serrata and Phlegmarirus carinatus in seven plants of the Lycopodiaceae and Huperziaceae families, based on the established characteristic MS fragmentation of five known alkaloids. Furthermore, a total of 13 lycodine-type alkaloids were identified, of which three pairs of isomers were structurally characterized and differentiated. This study further improves mass analysis of lycodine-type alkaloids and demonstrates the superiority of UPLC with a high-resolution mass spectrometer for the rapid and sensitive structural elucidation of other trace active compounds. Copyright © 2016 John Wiley & Sons, Ltd.

Further C-15-acyl phragmalin derivatives from Chukrasia tabularis A. Juss.

Relying on characteristic double UV absorptions (210 and 270 nm), sixteen phragmalins with three types of enolic acyl substituents at C-15 were isolated directly from EtOH extracts of the seeds of Chukrasia tabularis A. Juss. Eight of these compounds possessed a C-15-acetyl phragmalin skeleton, and the basic carbon skeleton and absolute configuration of one of these was determined by NMR and X-ray diffraction analysis, while the structures of the other phragmalins were determined via NMR, HR-MS, and CD spectra. Additionally, all of the isolates were tested for inhibition against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 macrophages and cytotoxicity in SMMC-7721, MCF-7 and U2OS tumor cells.

Withanolides from Physalis minima and their inhibitory effects on nitric oxide production.

Six new withanolides (1-6), including two uncommon 1,10-seco withanolides (1 and 2), together with five known withanolides (7-11), were isolated from the whole plants of Physalis minima Linn.. The structures of new compounds were elucidated through spectroscopic methods, including (1)H, (13)C NMR, 2D-NMR, HRESIMS and circular dichroism (CD). Inhibitory effects of the isolates on nitric oxide (NO) production in lipopolysaccaride-activated RAW264.7 macrophages were evaluated. Compounds 2 and 5 showed strong inhibitory activities with IC50 values of 8.04 and 10.01 µM, respectively. Compounds 1, 9 and 10 exhibited moderate inhibitory activities with IC50 values from 25.54 to 43.58 µM.

Chemical characteristics combined with bioactivity for comprehensive evaluation of Panax ginseng C.A. Meyer in different ages and seasons based on HPLC-DAD and chemometric methods.

Panax ginseng C.A. Meyer has been known as a valuable traditional Chinese medicines for thousands years of history. Ginsenosides, the main active constituents, exhibit prominent immunoregulation effect. The present study first describes a holistic method based on chemical characteristic and lymphocyte proliferative capacity to evaluate systematically the quality of P. ginseng in thirty samples from different seasons during 2-6 years. The HPLC fingerprints were evaluated using principle component analysis (PCA) and hierarchical clustering analysis (HCA). The spectrum-efficacy model between HPLC fingerprints and T-lymphocyte proliferative activities was investigated by principal component regression (PCR) and partial least squares (PLS). The results indicated that the growth of the ginsenosides could be grouped into three periods and from August of the fifth year, P. ginseng appeared significant lymphocyte proliferative capacity. Close correlation existed between the spectrum-efficacy relationship and ginsenosides Rb1, Ro, Rc, Rb2 and Re were the main contributive components to the lymphocyte proliferative capacity. This comprehensive strategy, providing reliable and adequate scientific evidence, could be applied to other TCMs to ameliorate their quality control.

Chemical constituents from Trichilia connaroides and their nitric oxide production and α-glucosidase inhibitory activities.

Phytochemical investigation of the stem and bark of Trichilia connaroides led to the isolation of eight new nortriterpenoids (1-8), along with fifteen known compounds (9-23). Their structures were established based on extensive spectroscopic analysis. The absolute configuration of 2 was confirmed by X-ray crystallographic study. The nitric oxide production and α-glucosidase inhibitory effects for these isolates were evaluated: moderate to strong nitric oxide production inhibitory activities were found for 5, 6, and 11-15, with IC50 values ranging from 7.5 to 26.3 µM; marked α-glucosidase inhibitory effects were observed for 22 and 23, with IC50 values of 2.3 and 0.4 µM, respectively.

Bioactive benzofuran neolignans from Aristolochia fordiana.

Six new benzofuran neolignans (1-6) were isolated from the EtOAc-soluble fraction of the aerial part of Aristolochia fordiana, together with twelve known analogues (7-18). The structures of the isolated compounds were elucidated by spectroscopic methods. All isolates were evaluated for their inhibitory effects on nitric oxide production in lipopolysaccaride-activated RAW264.7 macrophages and for their cytotoxic activities on three human cancer cell lines. Compound 2 showed significant nitric oxide production inhibitory activity with an IC50 value of 10.00 µM, while compound 16 exhibited cytotoxic activity with an IC50 value of 11.9 µM against MG-63 and compound 18 of 9.15 µM against HepG2 cell lines, respectively.

Amomaxins A and B, two unprecedented rearranged labdane norditerpenoids with a nine-membered ring from Amomum maximum.

Amomaxins A (1) and B (2), featuring an unprecedented rearranged labdane norditerpene skeleton with a nine-membered ring, along with their biosynthetic related known compound isocoronarin D (3) were isolated from the roots of Amomum maximum. Their structures with absolute configurations were determined by spectroscopic data, CD experimentation, and single-crystal X-ray diffraction. Compound 2 showed an inhibitory effect on nitric oxide (NO) production in lipopolysaccharide-induced RAW264.7 macrophages.