An integrated network analysis, RNA-seq and in vivo validation approaches to explore the protective mechanism of Mongolian medicine formulae Ruda-6 against indomethacin-induced gastric ulcer in rats.

Gastric ulcer (GU) is one of the most prevalent digestive diseases that seriously affects people's health. Previous studies have demonstrated the anti-GU effect of Ruda-6 (RD-6), a classic formulae of traditional Mongolian medicine. However, the underlying mechanism of RD-6 against GU remains elusive. Thus, we conducted an integrative approach of network analysis, RNA-seq, and in vivo validation experiment to elucidate the therapeutic mechanisms of RD-6 in preventing GU. A network analysis was performed to predict the potential targets of RD-6. Rats were pretreated with RD-6 at different doses for 21 days, followed by GU induction with indomethacin injection. The ulcer index and inhibition rates were calculated, and the levels of inflammatory related factors were determined by ELISA. The gastroprotective mechanism of RD-6 against ulceration was verified by RNA-seq and the key pathway was detected by in vivo validation. As the network analysis predicted, RD-6 exerts anti-GU effects by regulating 75 targets and 160 signaling pathways. Animal experiment results suggested that pretreatment with RD-6 significantly ameliorated the gastric mucosal injury and inflammatory response, as evidenced by a reduced ulcer index, decreased interleukin (IL)-1ß, IL-6, and IL-17 levels, and increased prostaglandin E2 (PGE2) levels in the GU model rats induced by indomethacin. RNA-seq data identified four potential hub genes that were primarily involved in the IL-17 signaling pathway. Furthermore, in vivo validation experiment showed that RD-6 inhibited the IL-17 signaling pathway by down-regulating the expression of IL17RA, proto-oncogene C-Fos (FOS), IL1B and prostaglandin-endoperoxide synthase 2 (PTGS2). Taken together, the present study provides evidence that RD-6 could effectively protect against indomethacin-induced GU, which might be attributed to suppressed inflammation. The IL-17 signaling pathway may be one of the crucial mechanisms that mediates the effect of RD-6.

Mongolian medicine formulae Ruda-6 alleviates indomethacin-induced gastric ulcer by regulating gut microbiome and serum metabolomics in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Ruda-6 (RD-6), a typical traditional Mongolian medicine formulae consisting of 6 herbs, has been traditionally used in treating gastric disorders. Even though it has been shown to protect against gastric ulcers (GU) in animal models, the gut microbiome and serum metabololite-related mechanisms that prevent GU are not well understood. AIM OF THE STUDY: This study was conducted to evaluate the gastroprotective mechanism of RD-6 associated with the alteration of the gut microbiome and serum metabolic profiles in GU rats. MATERIALS AND METHODS: RD-6 (0.27, 1.35 and 2.7 g/kg) or ranitidine (40 mg/kg) were orally administered in rats for three weeks before the induction of gastric ulcer using indomethacin (30 mg/kg, single oral dose). The gastric ulcer index, ulcer area, H&E staining, and the levels of TNF-α, iNOS, MPO and MDA were quantified to evaluate the ulcer inhibitory effects of RD-6. Then, 16S rRNA gene sequencing combined with LC-MS metabolic profiling was performed to investigate the effect of RD-6 on the gut microbiota and serum metabolites in rats. Moreover, a spearman analysis was used to calculate the correlation coefficient between the different microbiota and the metabolites. RESULTS: RD-6 inhibited the gastric lesion damage caused by indomethacin in rats, decreased the ulcer index by 50.29% (p < 0.05), reduced the levels of TNF-α, iNOS, MDA and MPO in gastric tissue. Additionally, RD-6 reshaped the diversity and microbial composition, and reversed the reduced bacteria including [Eubacterium]_xylanophilum group, Sellimonas, Desulfovibrio, and UCG-009, and the increased bacteria Aquamicrobium caused by indomethacin induction. Furthermore, RD-6 regulated the levels of metabolites including amino acids and organic acids, and these affected metabolites were involved in taurine and hypotaurine metabolism and tryptophan metabolism. Spearman analysis revealed that the perturbed gut microbiota were closely related to the changes in differential serum metabolites. CONCLUSION: In view of the 16S rRNA gene sequencing and LC-MS metabolic results, the present study suggests the mechanism of RD-6 ameliorating GU via modulating intestinal microbiota and their metabolites.

Effects of the Expressions and Variants of the CAST Gene on the Fatty Acid Composition of the Longissimus Thoracis Muscle of Grazing Sonid Sheep.

Fatty acid (FA) composition has an important impact on the nutrition and flavor of meat, and on consumer health, and is receiving more attention in the sheep industry. This study aimed to evaluate the relationship between the expression levels of the CAST gene and the FA composition in the longissimus thoracis (LL) muscle, to identify novel variants of CAST, and to perform association analysis with the FA composition in grazing Sonid lambs. The correlation results showed that high expression levels of CAST are correlated with better FA compositions and classes in LL. For association studies, the results showed that c.1210C>T and c.1437G>A in LD-M, and c.2097C>T mutations are associated with some compositions and classes of FA in the LL of grazing Sonid sheep. Two missense c.646G>C (G216R) and c.1210C>T (R404C) mutations were predicted to influence the Calpain_inhib domains of CAST. Thus, the correlation results and associated mutations are expected to be genetic selection markers for the FA composition and meat quality of grazing Sonid lamb muscle and provide new insights into sheep meat quality traits influenced by the ovine CAST gene.

RNA-Seq Reveals Protective Mechanisms of Mongolian Medicine Molor-Dabos-4 on Acute Indomethacin-Induced Gastric Ulcers in Rats.

This study aimed to apply transcriptomics to determine how Molor-Dabos-4 (MD-4) protects healthy rats against indomethacin (IND)-induced gastric ulcers and to identify the mechanism behind this protective effect. Rats were pretreated with MD-4 (0.3, 1.5, or 3 g/kg per day) for 21 days before inducing gastric ulcers by oral administration with indomethacin (30 mg/kg). Unulcerated and untreated healthy rats were used as controls. Effects of the treatment were assessed based on the ulcer index, histological and pathological examinations, and indicators of inflammation, which were determined by enzyme-linked immunosorbent assay. Transcriptomic analysis was performed for identifying potential pharmacological mechanisms. Eventually, after identifying potential target genes, the latter were validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). After pretreatment with MD-4, gastric ulcers, along with other histopathological features, were reduced. MD-4 significantly (p < 0.05) increased the superoxide dismutase (SOD) levels in ulcers and reduced pepsin, TNF-α, and IL-6 levels. RNA-seq analysis identified a number of target genes on which MD-4 could potentially act. Many of these genes were involved in pathways that were linked to anti-inflammatory and antioxidant responses, and other protective mechanisms for the gastric mucosa. qRT-PCR showed that altered expression of the selected genes, such as Srm, Ryr-1, Eno3, Prkag3, and Eef1a2, was consistent with the transcriptome results. MD-4 exerts protective effects against IND-induced gastric ulcers by reducing inflammatory cytokines and pepsin and increasing the expression of SOD levels. Downregulation of Srm, Ryr-1, Eno3, Prkag3, and Eef1a2 genes involved in regulating arginine and proline metabolism, calcium signaling pathway, HIF-1 signaling pathway, oxytocin signaling pathway, and legionellosis are possibly involved in MD-4-mediated protection against gastric ulcers.

Metabolomics investigation on antiobesity effects of Corydalis bungeana on high-fat high-sugar diet-induced obese rats.

Objective: Corydalis bungeana (CB) is a well-used medicinal herb in Mongolian folk medicine and has been traditionally applied as an antiobesity agent. However, the evidence-based pharmacological effects of CB and its specific metabolic alterations in the obese model are not entirely understood. This study aimed to utilize untargeted metabolomic techniques to identify biomarkers and gain mechanistic insight into the serum metabolite alterations associated with weight loss and lipid metabolism in obese rats. Methods: A high-fat high-sugar (HFHS) diet was used to induce obese models in rats. CB extract was orally gavaged at 0.18, 0.9 and 1.8 g/kg doses for six weeks, and feed intake, body weight, fat pad weight, and blood indexes were measured. Blood serum metabolites were evaluated by gas chromatography/quadrupole time-of-flight tandem mass spectrometry (GC-TOF/MS). Results: The results showed that compared with the obese group, the administration of CB extract caused significant decreases in body weight (P < 0.05), feed intake, Lee's index, and perirenal, mesenteric, epididymal fat weight. CB extract also reduced blood triglyceride and total cholesterol levels (P < 0.05) of obese rats. Metabolomic findings showed that nine differential metabolites, including pyruvic acid, D-glucuronic acid, malic acid, dimethylglycine, oxoglutaric acid, pantothenic acid, sorbitol acid, fumaric acid and glucose 6-phosphate were identified under CB treatment and altered metabolic pathways such as TCA cycle, pantothenate and CoA biosynthesis, and glycolysis/gluconeogenesis. Conclusion: This study demonstrated weight loss and lipid lowering effects of CB on HFHS diet-induced obese rats and identified nine metabolites as potential biomarkers for evaluating the favorable therapeutic mechanism of CB via regulation of lipid and glucose metabolism.

[Anti-obesity and lipid-lowering mechanism of Corydalis Bungeanae Herba: based on intestinal microflora and metabolomics].

This study aims to explore anti-obesity and lipid-lowering mechanism of Corydalis Bungeanae Herba(CB) based on intestinal microflora and metabolomics. Specifically, high-fat high-sugar diet(HFHS, 10 weeks) was used to induce obesity in rats. Then the model rats were randomized into the model group, low-dose(0.18 g·kg~(-1)), medium-dose(0.9 g·kg~(-1)), and high-dose(1.8 g·kg~(-1)) CBH groups, and orlistat group(0.03 g·kg~(-1)), 12 in each group. Rats which received normal diet were used as control. The body weight and feed intake of rats were recorded every week. After 6 weeks of administration, rats were killed and gastric emptying and small intestinal propulsion were examined. Enzyme-linked immunosorbent assay(ELISA) was employed to analyze serum indexes, and liver and perirenal fat were collected for haematoxilin-eosin(HE) staining. Rat feces and serum were gathered for 16 S rDNA sequencing and metabolomics analysis and Spearman's correlation analysis was performed to explore the correlation between differential microflora and differential metabolites. The result showed that CBH extract decreased body weight, feed intake, and serum cholecystokinin(CCK), triglyceride(TG), and total cholesterol(TC), delayed gastric emptying, and reduced fat accumulation in liver and perirenal adiposity as compared with rats in the model group. In addition, Lachnospiraceae and Sutterellaceaecan significantly decreased in the model group, but CBH extract up-regulated their abundance. Moreover, the abundance of Prevotellaceae was significantly raised by HFHS, but CBH decreased it. Glutaric acid, glyceric acid, hippuric acid, malic acid, glyceric acid, oxoglutaric acid, fumaric acid/succinic acid, oxoglutaric acid/isocitric acid, D-glucuronic acid, cholic acid were the main deferentially expressed metabolites and significantly correlated with Sutterellaceae and Prevotellaceae. These key metabolites and microbiota mainly involved in tricarboxylic acid(TCA) cycle, glucose metabolism, amino acid metabolism, and energy metabolism. This study proved that CBH can efficiently improve body weight and blood lipids, reduce adipocyte volume, and positively regulate the intestinal microflora and serum metabolites, thereby achieving the anti-obesity and lipid-owering effect.

Hepatoprotective Effects of Ixeris chinensis on Nonalcoholic Fatty Liver Disease Induced by High-Fat Diet in Mice: An Integrated Gut Microbiota and Metabolomic Analysis.

Ixeris chinensis (Thunb.) Nakai (IC) is a folk medicinal herb used in Mongolian medical clinics for the treatment of hepatitis and fatty liver diseases even though its pharmacological mechanism has not been well characterized. This study investigated the hepatoprotective mechanism of IC on mice with nonalcoholic fatty liver disease (NAFLD) by integrating gut microbiota and metabolomic analysis. A high-fat diet (HFD) was used to develop nonalcoholic fatty liver disease, after which the mice were treated with oral IC (0.5, 1.5 and 3.0 g/kg) for 10 weeks. HFD induced NAFLD and the therapeutic effects were characterized by pathological and histological evaluations, and the serum indicators were analyzed by ELISA. The gut microbial and metabolite profiles were studied by 16S rRNA sequencing and untargeted metabolomic analysis, respectively. The results showed that the administration of IC resulted in significant decreases in body weight; liver index; serum biomarkers such as ALT, TG, and LDL-C; and the liver inflammatory factors IL-1ß, IL-6, and TNF-α. The 16S rRNA sequencing results showed that administration of IC extract altered both the composition and abundance of the gut microbiota. Untargeted metabolomic analysis of liver samples detected a total of 212 metabolites, of which 128 were differentially expressed between the HFD and IC group. IC was found to significantly alter the levels of metabolites such as L-glutamic acid, pyridoxal, ornithine, L-aspartic acid, D-proline, and N4-acetylaminobutanal, which are involved in the regulation of glutamine and glutamate, Vitamin B6 metabolism, and arginine and proline metabolic pathways. Correlation analysis indicated that the effects of the IC extract on metabolites were associated with alterations in the abundance of Akkermansiaceae, Lachnospiraceae, and Muribaculaceae. Our study revealed that IC has a potential hepatoprotective effect in NAFLD and that its function might be linked to improvements in the composition of gut microbiota and their metabolites.

Metabolomics investigation on antiobesity effects of Corydalis bungeana on high-fat high-sugar diet-induced obese rats / 中草药·英文版

Objective: Corydalis bungeana (CB) is a well-used medicinal herb in Mongolian folk medicine and has been traditionally applied as an antiobesity agent. However, the evidence-based pharmacological effects of CB and its specific metabolic alterations in the obese model are not entirely understood. This study aimed to utilize untargeted metabolomic techniques to identify biomarkers and gain mechanistic insight into the serum metabolite alterations associated with weight loss and lipid metabolism in obese rats. Methods: A high-fat high-sugar (HFHS) diet was used to induce obese models in rats. CB extract was orally gavaged at 0.18, 0.9 and 1.8 g/kg doses for six weeks, and feed intake, body weight, fat pad weight, and blood indexes were measured. Blood serum metabolites were evaluated by gas chromatography/quadrupole time-of-flight tandem mass spectrometry (GC-TOF/MS). Results: The results showed that compared with the obese group, the administration of CB extract caused significant decreases in body weight (P < 0.05), feed intake, Lee's index, and perirenal, mesenteric, epididymal fat weight. CB extract also reduced blood triglyceride and total cholesterol levels (P < 0.05) of obese rats. Metabolomic findings showed that nine differential metabolites, including pyruvic acid, D-glucuronic acid, malic acid, dimethylglycine, oxoglutaric acid, pantothenic acid, sorbitol acid, fumaric acid and glucose 6-phosphate were identified under CB treatment and altered metabolic pathways such as TCA cycle, pantothenate and CoA biosynthesis, and glycolysis/gluconeogenesis. Conclusion: This study demonstrated weight loss and lipid lowering effects of CB on HFHS diet-induced obese rats and identified nine metabolites as potential biomarkers for evaluating the favorable therapeutic mechanism of CB via regulation of lipid and glucose metabolism.

Evaluation of the skill ，adherence and effectiveness of medication in asthma patients from a pharmacist perspective / 中国药房

OBJECTIVE To analyze th e evaluation methods of the skill ，adherence and effectiveness of medication in bronchial asthma （hereinafter referred to as “asthma”）patients from a pharmacist perspective ，and to provide reference for pharmacists to carry out clinical intervention researches and management of asthma patients. METHODS Referring to diagnosis and treatment guidelines ，evidence-based medicine literature and clinical practice experience ，the evaluation methods of asthma patients ’ medication with clinical value were expounded from three aspects including medication skill （inhalation technology ），adherence and effectiveness. RESULTS Inhalation technique rating form was an important evaluation method of medication skills in asthma patients. Medication adherence could be evaluated by Morisky Medication Adherence Scale or the Medication Adherence Report Scale for Asthma. Effectiveness evaluation methods contained direct evaluation indexes （such as clinical manifestations ，situation of acute exacerbation and relieving medication ，examination indexes such as pulmonary function indexes and fractional concentration of exhaled nitric oxide ）and indirect evaluation tools （i.e. various scales ，including Asthma Control Test ，Children-Asthma Control Test，the Test for Respiratory and Asthma Control in Kids ，asthma related quality of life scales ，etc.）CONCLUSIONS The combination of the evaluation methods of medication skill ， adherence and effectiveness contributes to assessing the pharmacotherapy effect of asthma patients. Pharmacists should apply and perfect these evaluation methods in practice ，so as to conduct better pharmaceutical intervention on asthma patients.

Improvement Effects of Safflower Yellow on Lung Function of Chronic Obstructive Pulmonary Disease Model Rats / 中国药房

OBJECTIVE：To study the improvement effects of saf flower yellow on lung function in chronic obstructive pulmonary disease （COPD）model rats. METHODS ：SD rats were randomly divided into control group ，model group ，positive control group （dexamethasone，0.09 mg/kg），safflower yellow low-dose ，medium-dose and high-dose groups （5，10，20 mg/kg）， with 10 rats in control group and 11 rats in other groups. Except for control group ，other groups were given lipopolysaccharide combined with fumigation to induce COPD model. After modeling ，control group and model group were given constant volume of normal saline intragastrically ，and administration groups were given relevant medicine intragastrically ，once a day ，for consecutive 12 weeks. After last medication ，the levels of TNF-α，IL-6 and IL- 8 were detected by ELISA. The levels of blood gas indexes （PaO2，SaO2 and PaCO 2）in whole blood were detected by blood gas analyzer. The levels of lung function indexes （FVC，FEV1， FEV1/FVC，PEF and MMEF ）were detected by lung function analyzer. The expression of TLR 4，NF-κB and I κB-α protein were detected by Western blot. HE staining was used to observe the pathological changes of lung tissue. RESULTS ：Compared with control group ，the serum levels of TNF-α，IL-6 and IL- 8，the level of PaCO 2 in whole blood as well as the protein expression of TLR4 and NF-κB in lung tissue were increased significantly in model group（P＜0.01）；the levels of PaO 2 and SaO 2 in whole blood，the levels of lung function as FVC ，FEV1，FEV1/ FVC，PEF and MMEF as well as the protein expression of IκBα in lung tissue were decreased significantly（P＜0.01）； there were obvious degeneration and necrosis in the epithelial cells of lung tissue ，and obvious inflammatory infiltration in the interstitial cells. Compared with model group ，the levels o f inflammatory factors in serum ，blood gas indexes in whole blood and lung function indexes as well as the expression of related protein in lung tissue （except for IκBα in low-dose group）were reversed significantly in safflower yellow groups （P＜0.05 or P＜ 0.01）；the necrosis ，exfoliation and inflammatory infiltration of epithelial cells in lung tissue were improved in varying degrees. CONCLUSIONS：Safflower yellow can significantly improve the lung function of COPD model rats ，and its mechanism may be related to inhibiting the expression of inflammatory factors and regulating the expression of TLR 4/NF-κB pathway-related proteins.

Epithelial-mesenchymal transition and metastasis of colon cancer cells induced by the FAK pathway in cancer-associated fibroblasts.

OBJECTIVE: The role and mechanism of tetrathiomolybdate (TM) in cancer-associated fibroblasts (CAFs) in colon cancer using three-dimensional (3D) culture were investigated, and the associations between the focal adhesion kinase (FAK) pathway and epithelial-mesenchymal transition (EMT) in CAFs were explored. METHODS: A 3D co-culture model of colon cancer LOVO cells with CAFs and normal fibroblasts (NFs) was established using Matrigel as a scaffold material. The differential expression of LOXL2 (lysyl oxidase-like 2) in the supernatant of CAFs and NFs was determined using ELISA, and expression levels of EMT-related proteins and FAK signaling pathway-related proteins were determined using western blot. RESULTS: LOXL2 levels secreted by CAFs were higher compared with that secreted by NFs. In the CAF + LOVO group, compared with the LOVO group, E-cadherin expression decreased significantly, while N-cadherin and F-PAK expression increased significantly. TM results were opposite compared with the above results. CONCLUSIONS: CAFs stimulate EMT in human colon cancer LOVO cells by secreting LOXL2 to activate the FAK signaling pathway, thereby promoting tumor metastasis. TM inhibited the occurrence of EMT in the CAF-induced colon cancer LOVO cell line, thereby reducing the invasion and metastasis of colon cancer cells.

Effects of flavonoids from Allium mongolicum Regel on growth performance and growth-related hormones in meat sheep.

This study was conducted to investigate the effects of different doses of flavonoids from Allium mongolicum Regel on the production performance and neuroendocrine hormones in meat sheep and to determine the optimum dosage of Allium mongolicum Regel flavonoids to add to the basal diet of dry lot-feeding meat sheep. Sixty meat sheep (initial body weight = 39.9 ± 3.2 kg; 6-month-old) were randomly assigned to 4 groups (15 sheep per group). The sheep in the control group were fed a basal diet, and the 3 experimental groups were fed the basal diet supplemented with flavonoids at 11, 22 and 33 mg/kg. Blood samples were collected via the jugular vein at d 0, 15, 30, 45, and 60 to determine the neuroendocrine hormone levels. The fasting weight of the sheep was measured during the experimental period, and feed offered and refusals were recorded daily. The basal diet supplemented with flavonoids from 11 to 33 mg/kg significantly increased the daily weight gain and average daily feed intake (P < 0.05) and significantly decreased the feed conversion ratio (P < 0.05), but there were no differences among the supplementation groups (P > 0.05). Starting on d 30, the growth hormone (GH) and insulin-like growth factor-1 (IGF-1) levels in the sera of the sheep in the supplementation groups increased significantly (P < 0.05), and the increases occurred in a time-dependent manner. Compared with control group, after d 30, the serum corticosterone (CORT) levels were reduced in the sheep that consumed the basal diet supplemented with 22 mg/kg flavonoids (P < 0.05), but among the other experimental groups, there was a non-significant effect (P > 0.05). The serum adrenocorticotropic hormone (ACTH) levels were increased by the supplementation of flavonoids, but compared with the control group, the effect was not significant. The basal diet supplemented with flavonoids at levels from 11 to 33 mg/kg had a significant effect on the production performance and neuroendocrine hormone levels of meat sheep, and the effect occurred in a time-dependent manner. The effect was especially obvious after 30 d of feeding.

Effect of tumor suppressor gene cyclin-dependent kinase inhibitor 2A wild-type and A148T mutant on the cell cycle of human ovarian cancer cells.

Single-base substitution may affect the function of genes. This study identified a single-base substitution of G for A in codon 148 of cyclin-dependent kinase inhibitor 2A (CDKN2A/p16) by sequencing human ovarian cancer cell line UACC-1598. As a tumor suppressor gene, the expression of CDKN2A/p16 should be strictly controlled. In order to control CDKN2A/p16 gene expression, an inducible pTUNE vector system was selected. Using recombinant DNA technology, a CDKN2A/p16-A148T and CDKN2A/p16-wild-type gene expression system was successfully constructed to investigate whether this single-base substitution affects the function of CDKN2A/p16. For the wild-type and the mutant, expression of CDKN2A/p16-green fluorescent protein fusion protein increased markedly following isopropyl-ß-D-thiogalactoside induction, and was accompanied by significant G1 arrest in the transfected human ovarian cancer SKOV3 cell line. The inducible vectors used in this study, CDKN2A/p16-wild-type and CDKN2A/p16-A148T open reading frame, may be useful for further investigation into whether this somatic mutation could alter the function of CDKN2A/p16 as a tumor suppressor gene. In summary, CDKN2A/p16-A148T was identified in ovarian cancer cells, and this single-base substitution did not affect the ability of CDKN2A/p16 to arrest the cell cycle.