A comparative metabolomics study between grain-sized moxibustion and suspended moxibustion on rats with gastric ulcers.

Grain-sized moxibustion (GS-Moxi) and suspended moxibustion (S-Moxi) represent the two typical local heat therapies in Traditional Chinese Medicine (TCM) and have been extensively used in treating gastric ulcers (GU) in China. However, the difference in biological response between the two moxibustion therapies in treating GU remains unclear. Here we investigated the therapeutic effect and potential mechanistic difference underlying the two moxibustion methods. Ethanol-induced GU model was established and was treated with GS-Moxi or S-Moxi at ST36 and ST21 for 5 days separately. And then, gastric histopathological examination, immunohistochemical staining for repair factors (EGFR, VEGF, Ki67), and 1H NMR-based metabolomics analysis of plasma and stomach of rats were conducted. We found GS-Moxi and S-Moxi effectively alleviated gastric damage and significantly increased the expression of related repair factors. However, S-Moxi corrected aberrant energy metabolism and lipids metabolism in GU rats but had little effect on neurotransmitter-related metabolism, while GS-Moxi regulated energy metabolism and neurotransmitter-related metabolism in GU rats but had no effect on lipids metabolism. We further proposed that the main target of S-Moxi may be liver and vasculature, whereas GS-Moxi specially targeted the stomach via regulating nervous system. This study strongly verified the outstanding gastroprotective effects of moxibustion and enriched our understanding of the varied biological responses triggered by different moxibustion methods.

[Nuclear magnetic resonance-based metabolomics revealed metabolite profiles participate in intervention effect of refined moxibustion in gastric ulcer model rats].

OBJECTIVE: To investigate the effect of refined moxibustion on expression of gastric mucosal epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF), and changes of metabolite profiles in gastric ulcer (GU) rats, so as to analyze its mechanism underlying improvement of GU. METHODS: Male SD rats were randomized into control, model, acupoint moxibustion groups (n=6 per group). The GU model was induced by cold-restraint stress. The ignited refined moxa was applied to bilateral "Liangmen" (ST21) and "Zusanli" (ST36) for 3 cones/acupoint, once daily for 7 days. Then, we employed 1H NMR-based metabolomics approach to analyze the metabolic profiles of serum and stomach tissue samples. The conventional histopathological changes of the gastric mucosa were observed by H.E. stain and the expressions of EGFR and VEGF in the gastric mucosa were detected by immunohistochemistry. RESULTS: Compared to the control group, the expression levels of EGFR and VEGF were significantly increased in the model group (P<0.01, P<0.05), and further notably up-regulated in the acupoint moxibustion group (P<0.001, P<0.01). Results of H.E. staining showed damage of the folds of gastric mucosa, disordered arrangement of the glands, infiltration of inflammatory cells and unclear structure of gastric mucosa in the model group, which was relatively milder in the acupoint moxibustion group. 1H-NMR technical analysis showed that in comparison with the control group, 11 and 11 metabolites in the stomach extract and plasma were increased, 10 in the gastric tissue and 3 in the plasma were decreased in the GU model group; while in comparison with the model group, 17 differently expressed metabolites in the gastric extract and 10 metabolites in the plasma restored to their levels of control group after the acupoint moxibustion intervention. These metabolites participate in 12 metabolic pathways including glycine, serine and threonine metabolism, glutathione metabolism, glycine metabolism, alanine, aspartic acid and glutamic acid metabolism, purine metabolism, glyoxylic acid and digarboxylic acid metabolism, biosynthesis of aminoacyl-tRNA, amino sugar and nucleotide sugar metabolism, cysteine and methionine metabolism, citrate cycle, pyruvate metabolism, and the mutual conversion of pentose and glucuronate，suggesting their involvement in moxibustion-induced improvement of GU. CONCLUSION: Refined moxibustion at ST21 and ST36 can up-regulate the expression of EGFR and VEGF in the gastric mucosa and lessen gastric mucosal injury, which may be related to its effects in reducing GU-induced metabolic disorders, including sugar, purine, amino acid, and phospholipid metabolism and antioxidant defense system.

Predicting the potential toxicity of 26 components in Cassiae semen using in silico and in vitro approaches.

Cassiae semen are dried and ripe seeds of Cassia obtusifolia L. or Cassia tora L. (Fabaceae) and have been made into roasted tea or used as a traditional medicine in Asian countries. However, it was reported to result in liver and renal toxicity. The components of Cassiae semen that induce hepatotoxicity or nephrotoxicity remain unknown. In the present study, we evaluate the potential toxicity of 26 newly isolated compounds from Cassiae semen using quantitative structure-activity relationship (QSAR) methods and co-culture of hepatic and renal cell approaches, and we aim to illustrate the relationship between the structural characteristics and cytotoxicity by general linear models (GLMs). Both the QSAR models and co-culture of hepatic and renal cell systems predicted that 6 compounds were potentially hepatotoxic, 10 compounds were potentially nephrotoxic, and specific anthraquinones and anthraquinone-glucosides were potential toxicants in Cassiae semen. Specific groups such as -OH and -OCH3 at the R1, R2, R3, and R7 positions influenced the cytotoxicity.

[Endogenous metabolites involving effects of moxibustion on injured gastric mucosal tissue in rats with stomach heat or cold syndrome based on nuclear magnetic resonance spectroscopy].

OBJECTIVE: To investigate the profile of metabolites of gastric mucosa involving the effectiveness of moxibustion in the treatment of syndromes of stomach heat (SH) and stomach cold (SC) by 1H-nuclear magnetic resonance (1H-NMR) spectroscopy in rats, so as to reveal its mechanisms underlying improvement of gastric disorders. METHODS: Male SD rats were randomly divided into control, SH-model, SC-model, SH-moxibustion and SC-moxibustion groups (n＝6 rats/group). The SH-model and SC-model were established by gavage of pepper liquid plus ethanol, and ice water plus NaOH, respectively. Moxibustion was applied to bilateral "Zusanli" (ST36) and "Liangmen"(ST21) for 20 min, once daily for 7days. Histopathological changes of the gastric tissue were observed by H．E. staining. Differential metabolites in the gastric mucosal tissue were detected and the relevant metabolic pathways analyzed by using 1H-NMR, pattern recognition method，and online MetPA (http: //www.metaboanalyst.ca). RESULTS: Compared with the control group, the body mass was decreased significantly from the 4th to 14th day after modeling (P<0.05，P<0.01). After the treatment, the body mass was obviously increased from the 10th day on in both SH-EA and SC-EA groups relevant to the SH and SC model group, respectively (P<0.05，P<0.01). H．E. staining showed severe damage of the columnar epithelial structure of the gastric mucosa and inflammatory cell infiltration in the SH group, and inflammatory cell infiltration in the SC model group, which were relatively milder in both moxibustion groups. 1H-NMR analysis displayed a total of 16 potential biomarkers in the injured gastric mucosa of SH syndrome and 14 biomarkers for the SC syndrome after mode-ling, and 13 metabolites related to SH moxibustion and 8 metabolites related to SC moxibustion after moxibustion interventions, respectively. After moxibustion, among the 13 differential metabolites of the SH syndrome, the effectively up-regulated candidates were isoleucine, creatinine, choline and lactate (P<0.05), and the down-regulated ones were choline phosphate, glycine, alanine, urine pyrimidine, tyrosine, phenylalanine, hypoxanthine, adenosine and nicotinamide (P<0.05). Among the 8 metabolites related to the SC syndrome, creatinine, ethanolamine, choline, adenosine and nicotinamide were markedly increased (P<0.05), and glycine, creatine phosphate and tyrosine remarkably decreased in their levels after moxibustion (P<0.05). MetPA showed that moxibustion could regulate 10 metabolic pathways for SH syndrome and 7 metabolic pathways for SC syndrome. Metabolites and metabolic pathways are mainly involved in functions of amino acid metabolism, energy metabolism and inflammatory response. CONCLUSION: The idea of "moxibustion could be used for heat syndrome" has metabolic substance basis, and its efficacy in repairing the injured gastric mucosa involves regulation of amino acid metabolism, energy balance and inflammation response, and moxibustion for SH and SC syndromes has both generality and specificity in regulating metabolic activities.

Effect of euphorbia factor L1 on oxidative stress, apoptosis, and autophagy in human gastric epithelial cells.

BACKGROUND: Euphorbia factor L1 (EFL1), a lathyrane-type diterpenoid from the medicinal herb Euphorbia lathyris L. (Euphorbiaceae), has been reported for many decades to induce gastric irritation, but the underlying mechanisms remain unclear. PURPOSE: The objective of this study was to investigate EFL1-induced cytotoxicity and the potential mechanisms of action on the human normal gastric epithelial cell GES-1. METHODS: GES-1 cells were treated with EFL1 (12.5-200 µM) for different time intervals, and cell survival, LDH release, intracellular reactive oxygen species (ROS), malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity were detected. Mitochondrial membrane potential (MMP) assay, DAPI staining, DNA fragment assay, and annexin V-FITC/PI staining were performed. The interaction between EFL1 and Bcl-2, cytochrome c, caspase-9, caspase-3, PI3K, AKT, and mTOR proteins was simulated by molecular docking. The mRNA and protein expression of apoptosis and autophagy factors were detected by RT-qPCR and Western blotting. RESULTS: EFL1 decreased survival, increased LDH leakage, and induced abnormal production of ROS, MDA and SOD in GES-1 cells. Mitochondria-mediated apoptosis was characterized by decreased MMP, condensed nuclei, fragmented DNA, and increased apoptosis rate. EFL1 interacted with proteins via hydrogen bonding. The mRNA, total or phosphorylated protein expression of Bcl-2, mitochondrial cytochrome c, PI3K, AKT, mTOR and p62 were downregulated; in contrast, those of cytoplasmic cytochrome c, cleaved caspase-9, cleaved caspase-3, LC3-ll and Beclin-1 were upregulated. CONCLUSION: These findings indicated that EFL1 decreased the survival of GES-1 cells through EFL1-induced oxidative stress, activation of the mitochondria-mediated apoptosis as well as autophagy via inhibition of the PI3K/AKT/mTOR pathway.

Anthraquinones in the aqueous extract of Cassiae semen cause liver injury in rats through lipid metabolism disorder.

BACKGROUND: Cassiae semen has been used as the tea or medicine component to treat hyperlipidemia or for hepatoprotection. However, Cassiae semen was reported to be a potentially hepatotoxic herb, and the underlying hepatotoxicity mechanisms or specific hepatotoxic components of Cassiae semen are unknown. PURPOSE: In this study, we aimed to explore the potential hepatotoxicity mechanisms and the hepatotoxic components of Cassiae semen. METHODS: Both young adult male and female SD rats were orally administrated with the aqueous extract of the seeds of Senna obtusifolia (L.) H.S.Irwin & Barneby at doses of 4.73, 15.75, 47.30 g/kg for 28 days, and the body weight, liver coefficient, bile acids, histopathology, serum levels of TC, TG, LDL, HDL, ALP, ALT, AST, and LDH were examined. Lipidomic analysis of rat serum was performed by LC-MS to investigate the specifically changed lipids caused by the aqueous extract treatment. The components absorbed in plasma were detected by UHPLC-Q-Exactive-MS. MTT assay was used to evaluate the cytotoxicity of these components absorbed in plasma. RESULTS: The serum levels of ALP, AST, ALT, LDH were increased on day 7 with some of which gradually dropped to normal level on day 28. In high dose of the aqueous extract treated group, the histopathological changes were observed based on the cytoplasmic vacuolation in the liver and the increase of bile acids, indicating the hepatotoxicity of the aqueous extract. The changes of TC, TG, LDL, HDL indicated the disorder of lipid metabolism. By comparing the difference in lipids between high dose group and control group, the results showed that the alterations were primarily focused on glycerophospholipid metabolism in both male and female rats. In addition, the glycerolipid metabolism in female rats also changed. Further analyses found that PC (18:2/20:4) and LysoPC 18:0 were significantly increased. Among these phytochemicals detected in plasma, nine components in the aqueous extract were considered to have the highest concentrations, particularly some types of anthraquinones (AQs) existing in Cassiae semen (AQs-in-CS), such as obtusifolin, aurantio-obtusin, and obtusin. The MTT assay showed that emodin, obtusifolin, rhein, aurantio-obtusin, and obtusin inhibited cell viability. Considering plasma concentrations and cytotoxicity of these components, our study indicates that the AQs-in-CS (obtusifolin, aurantio-obtusin and obtusin), emodin and rhein are the potential hepatotoxic phytochemicals in the aqueous extract.

[Toxicokinetics of bakuchiol, hepatic and renal toxicity in rats after single oral administration of Psoraleae Fructus and combination with Glycyrrhizae Radix et Rhizoma].

To study the toxicokinetics of bakuchiol, hepatic and renal toxicity in rats after single oral administration of Psoraleae Fructus and combined with Glycyrrhizae Radix et Rhizoma, in order to provide scientific evidences for clinical safe medication use. A total of 35 SD rats were randomly divided into seven groups: vehicle (distilled water) control group, Glycyrrhizae Radix et Rhizoma group, positive control (aristolochic acid A) group, Psoraleae Fructus (40 g x kg(-1)) group( both male and female rats), Psoraleae Fructus and Glycyrrhizae Radix et Rhizoma (40 +20) g x kg(-1) group (both male and female rats). HPLC-UV method was used to determine the concentration of bakuchiol in rat plasma at different time points after single oral administration. Plasma alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), plasma creatinine (Cr), N-acetyl-ß-D-glucosaminidase (NAG) and kidney injury molecule 1 (Kim-1) were measured after administration for 24 h. The main toxicokinetics parameters of bakuchiol in rats exert significantly gender difference. When Psoraleae Fructus combination with Glycyrrhizae Radix et Rhizoma, the total area under the plasma concentration-time curve( AUC), C(max), and plasma clearance (CL) of bakuchiol were increased, respectively; CL, half-life (t½) were decreased, and T(max) were prolonged. The biochemical indicators (including ALT, AST, BUN, Cr and KIM-1 level) in different dose of Psoraleae Fructus groups, were found no statistically significant difference when compared with vehicle control group. The level of NAG in both Psoraleae Fructus and compatibility with Glycyrrhizae Radix et Rhizoma groups were significant increased (P < 0.05). There are obvious effects on toxicokinetics of bakuchiol in rats when Psoraleae Fructus combined with Glycyrrhizae Radix et Rhizoma. Renal toxicity induced by Psoraleae Fructus at high dose was observed after single oral administration and no liver damage in rats was found.