Metabonomics and 16S rRNA gene sequencing to study the therapeutic mechanism of Danggui Sini decoction on collagen-induced rheumatoid arthritis rats with Cold Bi syndrome.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by persistent joint inflammation. The development of rheumatoid arthritis is directly correlated with the disturbance of gut microbiome and its metabolites. RA can be effectively treated with the Danggui Sini decoction (DSD), a Traditional Chinese medicine (TCM) prescription from the Treatise on Febrile Diseases. Further research is needed to clarify the precise mechanism of DSD in the treatment of RA. In this study, 1H NMR metabonomics and 16 S rRNA gene sequencing techniques were used to clarify the intervention of DSD on CIA-induced RA. The results of 1H NMR metabolomics of feces revealed that five metabolites (alanine, glucose, taurine, betaine, and xylose) were disturbed, which could be regarded as potential biomarkers of RA. The intestinal microbiome of RA rats had changed, according to the results of 16 S rRNA gene sequencing; eight microbes (g_norank_f_Eubacterium_coprostanoligenes_group, g_Ruminococcus_torques_group, g_Dubosiell, g_Lactobacillus, g_norank_f_Desulfovibrionaceae, g_Bacteroides, g_Oscillibacter, and g_Romboutsia) occurred significantly at the genus level, and DSD significantly impacted six of them (g_Dubosiell, g_Lactobacillus, g_norank_f_Eubacterium_coprostanoligenes_group, g_Ruminococcus_torques_grou, g_Bacteroides, and g_Romboutsia). Three of them (g_norank_f_Eubacterium_ coprostanoligenes_group, g_Romboutsia, and g_Lactobacillus) were regarded as key microbiomes for DSD to treat RA, and three common metabolic pathways (taurine and hypotaurine metabolism; alanine, aspartate, and glutamate metabolism; primary bile acid biosynthesis) were discovered based on the 1H NMR metabonomics and PICRUST2 prediction of 16 S rRNA gene sequencing. Six SCFAs in feces (acetic acid, butyric acid, propionic acid, caproic acid, isobutyric acid, and valeric acid) increased significantly in RA, according to the outcomes of targeting SCFAs, while five SCFAs (acetic acid, butyric acid, propionic acid, caproic acid, and valeric acid) had decreased significantly due to DSD treatment. In conclusion, our study indicated that DSD could regulate RA's metabolic disorder by affecting intestinal microbiome and its metabolites. It also establishes a framework for future research into exploiting gut microbes therapeutic to treat RA.

Combining 1H-NMR-based metabonomics and network pharmacology to dissect the mechanism of antidepression effect of Milletia speciosa Champ on mouse with chronic unpredictable mild stress-induced depression.

OBJECTIVES: Milletia speciosa Champ (MS), a traditional Chinese medicine, has the abilities of antistress, antifatigue, anti-oxidation and so on. In our previous study, MS was found to antidepression while the underlying mechanism of which needs further elucidation. METHODS: Here, a proton nuclear magnetic resonance (1H-NMR)-based metabonomics combined network pharmacology research approach was performed to investigate the antidepressive mechanism of MS act on mouse with chronic unpredictable mild stress-induced depression. KEY FINDINGS: Results showed that MS could alleviate the ethology of depression (including sucrose preference degree, crossing lattice numbers and stand-up times) and disordered biochemical parameters (5-hydroxytryptamine, norepinephrine and brain-derived neurotrophic factor). Metabonomics study and network pharmacology analysis showed that MS might improve depression through synergistically regulating five targets including Maoa, Maob, Ache, Ido1 and Comt, and three metabolic pathways such as tryptophan metabolism, synthesis of neurotransmitter and phospholipid metabolism. CONCLUSIONS: This study for the first time preliminary clarified the potential antidepressive mechanism of MS and provided theoretical basis for developing MS into novel effective antidepressant.

Serum metabolomics reveals the intervention mechanism and compatible regularity of Chaihu Shu Gan San on chronic unpredictable mild stress-induced depression rat model.

OBJECTIVES: To provide a comprehensive study of the intervention mechanism and compatible regularity of Chaihu Shu Gan San (CSGS) in a chronic unpredictable mild stress (CUMS)-induced depression model. METHODS: Ethological study and ELISA assay were applied to measure the phenotypes of depression after CUMS stimulate and assess the antidepressant activity of fluoxetine, CSGS and its compatibilities. The serum metabolic profile changes were revealed by untargeted Q/TOF MS-based metabolomics followed by multivariate statistical analysis. KEY FINDINGS: CSGS exhibits an significant intervention effect on CUMS-induced depression. After the multivariate statistical analysis, 17 potential serum biomarkers were identified and 16 of them could be regulated by CSGS. The intervention of CSGS on CUMS-induced depression involved five key pathways. Moreover, each functional unit (monarch, minister, assistant and guide medicine) in CSGS regulates different metabolites and metabolic pathways to achieve different effects on antidepressant; however, their intervention efficacies are inferior to the holistic formula, which may be due to the synergism of bioactive ingredients in the seven herbs of CSGS. CONCLUSIONS: CSGS produced an obvious antidepressant activity. The comprehensive and holistic metabolomics approach could be a powerful tool to study the intervention mechanism and the compatibility rule of traditional Chinese medicine.

Dynamic urinary metabolomics analysis based on UHPLC-Q-TOF/MS to investigate the potential biomarkers of blood stasis syndrome and the effects of Danggui Sini decoction.

Blood stasis syndrome (BSS) is one of the common syndromes in traditional Chinese medicine (TCM). It involves abnormal blood circulation, which can progress to produce many severe diseases. Danggui Sini decoction (DSD) is a classical TCM prescription frequently used to treat BSS by decreasing blood stasis and improving blood circulation. However, understanding of the therapeutic mechanism of DSD during the development of BSS is still limited, as the development of BSS is a slow dynamic process. Therefore, a dynamic urinary metabolomics analysis based on ultra-high-performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry (UHPLC-Q-TOF/MS) combined with multivariate statistical analysis was used to explore the distinctive metabolic patterns of BSS development and the efficacy of DSD. The dynamic changes of endogenous metabolites over time revealed the progression of BSS and allowed the overall efficacy of DSD in rats with BSS to be evaluated. The effects of the DSD compatibilities were also explored. A total of 21 metabolites were identified during the development of BSS. They are involved in the metabolic pathways of tryptophan metabolism, phenylalanine metabolism, riboflavin metabolism, nicotinate and nicotinamide metabolism, pentose and glucuronate interconversions, histidine metabolism, steroid hormone biosynthesis, and starch and sucrose metabolism. A receiver operating characteristic (ROC) curve analysis showed that 10 metabolites with an area under the curve (AUC) value >0.9, which can be used as potential biomarkers for the diagnosis of BSS. In conclusion, a dynamic urinary metabolomics approach was applied to identify potential biomarkers of the development of BSS and to clarify the therapeutic mechanism of DSD in BSS. The results could provide a theoretical basis for further research on the therapeutic mechanism of DSD.

Comparative analysis of the compatibility effects of Danggui-Sini Decoction on a blood stasis syndrome rat model using untargeted metabolomics.

Danggui-Sini Decoction (DSD) is one of the most widely used traditional Chinese medicine formulae (TCMF) for treating various diseases caused by cold coagulation and blood stasis due to its effect of nourishing blood to warm meridians in clinical use. However, studies of the mechanism of how it dispels blood stasis and its compatible regularity are challenging because of the complex pathophysiology of blood stasis syndrome (BSS) and the complexity of DSD, with multiple active ingredients acting on different targets. Observing variations of endogenous metabolites in rats with BSS after administering DSD may further our understanding of the mechanism of BSS and the compatible regularity of DSD. In this study, to understand the pathogenesis of BSS and assess the compatibility effects of DSD, an ultra-performance liquid chromatography quadrupole-time of flight mass spectrometry-based untargeted metabolomics approach was used. Serum metabolic profiles in rats with BSS that was induced by an ice water bath associated with subcutaneous injection of epinephrine hydrochloride were compared with the intervention groups which were administered with DSD or its compatibility. Using pattern recognition analysis, a clear separation between the BSS model and control group was observed; DSD and its compatibility intervention groups were clustered closer toward the control than the model group, which corroborates results of hemorheology studies. In addition, 20 metabolites were considered as potential biomarkers associated with the development of BSS. Nine metabolites were regulated by DSD in intervening blood stasis, they were considered to be correlated with the effect of nourishing blood to warm meridians. Additionally, the results suggested that the intervention effect of DSD on BSS may involve regulating four pathways, namely, arachidonic acid metabolism, glycerophospholipid metabolism, bile acid biosynthesis, and pyruvate metabolism. Moreover, each functional unit (monarch, minister, and assistant) in DSD regulates different metabolites and metabolic pathways to achieve different effects on dispelling blood stasis; however, their intervention efficacies are inferior to the holistic formula, which may be due to the synergism of the bioactive ingredients in seven herbs of DSD. This study demonstrated that metabolomics is a powerful tool for evaluating the efficacy and compatibility effects of traditional Chinese medicine (TCM).

Investigation of the hepatoprotective effect of Corydalis saxicola Bunting on carbon tetrachloride-induced liver fibrosis in rats by 1H-NMR-based metabonomics and network pharmacology approaches.

Liver fibrosis is a common consequence of chronic liver diseases resulting from multiple etiologies. Furthermore, prolonged unresolved liver fibrosis may gradually progress to cirrhosis, and eventually evolve into hepatocellular carcinoma (HCC). Corydalis saxicola Bunting (CS), a type of traditional Chinese folk medicine, has been reported to have hepatoprotective effects on the liver. However, the exact mechanism of how it cures liver fibrosis requires further elucidation. In this work, an integrated approach combining proton nuclear magnetic resonance (1H-NMR)-based metabonomics and network pharmacology was adopted to elucidate the anti-fibrosis mechanism of CS. Metabonomic study of serum biochemical changes by carbon tetrachloride (CCl4)-induced liver fibrosis in rats after CS treatment were performed using 1H-NMR analysis. Metabolic profiling by means of partial least squares-discriminate analysis (PLS-DA) indicated that the metabolic perturbation caused by CCl4 was reduced after CS treatment. As a result, lipids, leucine, alanine, acetate, O-acetyl-glycoprotein and creatine were significantly restored after CS treatment, which regulated valine, leucine and isoleucine metabolism; arginine and proline metabolism; lipid metabolism and pyruvate metabolism. Additionally, 157 potential targets of CS and 265 targets of liver fibrosis were identified by means of network pharmacology. Subsequently, 5 target proteins, which are the intersection of potential CS targets and liver fibrosis targets, indicated that CS has potential anti-fibrosis effects through regulating alanine aminotransferase (ALT) activity, the farnesoid X receptor (FXR), cyclooxygenase-2 (COX-2), matrix metalloproteinase-1 (MMP-1) and angiotensinogen. Chelerythrine and sanguinarine were the potential active compounds in CS for treating liver fibrosis through regulating ALT activity. This study is the first report to study the anti-fibrosis effects of CS on the basis of combining a metabonomics and network pharmacology approaches, and it may be a potentially powerful tool to study the efficacy and mechanisms of traditional Chinese folk medicines.

Metabolomics analysis of Danggui Sini decoction on treatment of collagen-induced arthritis in rats.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by persistent joint inflammation leading to bone and cartilage damage and even disability. However, the pathogenesis of RA is multi-factorial and to a large degree, remains unknown. Danggui Sini decoction (DSD), a traditional Chinese medicine (TCM) formula, has been widely used as a remedy for rheumatoid arthritis (RA) in recent years. In our study, 1H-nuclear magnetic resonance (1H NMR) based metabolomics analysis of 7 potential biomarkers, including taurine (1), urea (2), betaine (3), pyruvate (4), hippurate (5), succinate (6) and acetone (7) was performed to investigate the progression of RA and assess the efficacy of DSD in collagen-induced arthritis (CIA) rats. According to pathway analysis using identified metabolites and correlation construction, taurine and hypotaurine metabolism, gut microbiota metabolism, pyruvate metabolism, glycolysis/gluconeogenesis, the citrate cycle (TCA cycle) and lipid metabolism were recognized as being the most influenced metabolic pathways associated with RA. As a result, deviations of metabolites 1, 3, 4, 5, 6 and 7 in CIA rats were improved by DSD, which suggested that DSD mediated the abnormal metabolic pathways synergistically. In summary, the efficacy and its underlying therapeutic mechanisms of DSD on RA were systematically investigated and expect to provide a new insight in relevant studies of other TCM formulas.

Urinary metabonomics study of the hepatoprotective effects of total alkaloids from Corydalis saxicola Bunting on carbon tetrachloride-induced chronic hepatotoxicity in rats using 1H NMR analysis.

Chronic liver injury has been shown to cause liver fibrosis due to the sustained pathophysiological wound healing response of the liver, and eventually progresses to cirrhosis. The total alkaloids of Corydalis saxicola Bunting (TACS), a collection of important bioactive ingredients derived from the traditional Chinese folk medicine Corydalis saxicola Bunting (CS), have been reported to have protective effects on the liver. However, the underlying molecular mechanisms need further elucidation. In this study, the urinary metabonomics and the biochemical changes in rats with carbon tetrachloride (CCl4)-induced chronic liver injury due to treatment TACS or administration of the positive control drug-bifendate were studied via proton nuclear magnetic resonance (1H NMR) analysis. Partial least squares-discriminate analysis (PLS-DA) suggested that metabolic perturbation caused by CCl4 damage was recovered with TACS and bifendate treatment. A total of seven metabolites including 2-oxoglutarate, citrate, dimethylamine, taurine, phenylacetylglycine, creatinine and hippurate were considered as potential biomarkers involved in the development of CCl4-induced chronic liver injury. According to pathway analysis using identified metabolites and correlation network construction, the tricarboxylic acid (TCA) cycle, gut microbiota metabolism and taurine and hypotaurine metabolism were recognized as the most affected metabolic pathways associated with CCl4 chronic hepatotoxicity. Notably, the changes in 2-oxoglutarate, citrate, taurine and hippurate during the process of CCl4-induced chronic liver injury were significantly restored by TACS treatment, which suggested that TACS synergistically mediated the regulation of multiple metabolic pathways including the TCA cycle, gut microbiota metabolism and taurine and hypotaurine metabolism. This study could bring valuable insight to evaluating the efficacy of TACS intervention therapy, help deepen the understanding of the hepatoprotective mechanisms of TACS and enable optimal diagnosis of chronic liver injury.

Serum metabonomics study of the hepatoprotective effect of Corydalis saxicola Bunting on carbon tetrachloride-induced acute hepatotoxicity in rats by (1)H NMR analysis.

Corydalis saxicola Bunting (CS), a traditional Chinese folk medicine, has been effectively used for treating liver disease in Zhuang nationality in South China. However, the exact hepatoprotective mechanism of CS was still looking forward to further elucidation by far. In present work, metabonomic study of biochemical changes in the serum of carbon tetrachloride (CCl4)-induced acute liver injury rats after CS treatment were performed using proton nuclear magnetic resonance ((1)H-NMR) analysis. Metabolic profiling by means of principal components analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) indicated that the metabolic perturbation caused by CCl4 was reduced by CS treatment. A total of 9 metabolites including isoleucine (1), lactate (2), alanine (3), glutamine (4), acetone (5), succinate (6), phosphocholine (7), d-glucose (8) and glycerol (9) were considered as potential biomarkers involved in the development of CCl4-induced acute liver injury. According to pathway analysis by metabolites identified and correlation network construction by Pearson's correlation coefficency matrix, alanine, aspartate and glutamate metabolism and glycerolipid metabolism were recognized as the most influenced metabolic pathways associated with CCl4 injury. As a result, notably, deviations of metabolites 1, 3, 4, 7 and 9 in the process of CCl4-induced acute liver injury were improved by CS treatment, which suggested that CS mediated synergistically abnormalities of the metabolic pathways, composed of alanine, aspartate and glutamate metabolism and glycerolipid metabolism. In this study, it was the first report to investigate the hepatoprotective effect of the CS based on metabonomics strategy, which may be a potentially powerful tool to interpret the action mechanism of traditional Chinese folk medicines.

Induction of apoptosis by Uncaria tomentosa through reactive oxygen species production, cytochrome c release, and caspases activation in human leukemia cells.

Uncaria tomentosa (Wild.) DC., found in the Amazon rain forest in South-America and known commonly as cat's claw, has been used in traditional medicine to prevent and treat inflammation and cancer. Recently, it has been found to possess potent anti-inflammation activities. In this study, we extracted cat's claw using four different solvents of different polarities and compared their relative influence on proliferation in human premyelocytic leukemia HL-60 cell lines. Cat's claw n-hexane extracts (CC-H), ethyl acetate extracts (CC-EA) and n-butanol extracts (CC-B) had a greater anti-cancer effect on HL-60 cells than those extracted with methanol (CC-M). Furthermore, CC-EA induced DNA fragmentation in HL-60 cells in a clearly more a concentration- and time-dependent manner than the other extracts. CC-EA-induced cell death was characterized by cell body shrinkage and chromatin condensation. Further investigating the molecular mechanism behind CC-EA-induced apoptosis, sells treated with CC-EA underwent a rapid loss of mitochondrial transmembrane (DeltaPsi(m)) potential, stimulation of phosphatidylserine flip-flop, release of mitochondrial cytochrome c into cytosol, induction of caspase-3 activity in a time-dependent manner, and induced the cleavage of DNA fragmentation factor (DFF-45) and PARP poly-(ADP-ribose) polymerase (PARP). CC-EA promoted the up-regulation of Fas before the processing and activation of procaspase-8 and cleavage of Bid. In addition, the apoptosis induced by CC-EA was accompanied by up-regulation of Bax, down-regulation of Bcl-X(L) and cleavage of Mcl-1, suggesting that CC-EA may have some compounds that have anti-cancer activities and that further studies using cat's claw extracts need to be pursued. Taken together, the results of our studies show clearly that CC-EA's induction of apoptosis in HL-60 cells may make it very important in the development of medicine that can trigger chemopreventive actions in the body.

Effect of radix paeoniae rubra on expression of p38 MAPK/iNOS/HO-1 in rats with lipopolysaccharide-induced acute lung injury / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the effect of radix paeoniae rubra (RPR) on expression of p38 mitogen activated protein kinase (MAPK)/iNOS/HO-1 in rats with lipopolysaccharide-induced acute lung injury and explore the molecular mechanism.</p><p><b>METHODS</b>Forty healthy male Wistar rats, weighing 200-250 g, aged 6-8 weeks (mean equal to 7 weeks), provided by the Experimental Center, Medical College, Wuhan University, Wuhan, China, were employed in this study. Under anesthesia with 7% chloraldurat (5 ml/kg body weight) through intraperitoneal injection, the trachea of the rat was exposed and an arterial puncture needle pricked into the trachea via cricothyroid membrane. Then they were randomly divided into five groups: 8 rats receiving 1 ml normal saline through the puncture needle (Group A), 8 receiving 1 ml lipopolysaccharide (LPS, 2.5 mg/kg, Group B), 8 receiving LPS and RPR (30 mg/kg, pumped through the femoral vein for 2 hours, Group C), 8 receiving RPR 2 hours before dripping LPS (Group D), and 8 receiving hemin (75 micromol/L through intraperitoneal injection) 18 hours before dripping LPS (Group E). After 6 hours of LPS dripping, blood samples were obtained through the carotid artery to perform blood gas analysis, then all the rats were exsanguinated to death and specimens of lung tissues were obtained. The pathomorphological changes of the lung tissues were observed. The expression of p38 MAPK/iNOS/HO-1, the neutrophil ratio, protein content in alveolar irrigating solution and malonaldehyde (MDA) content in the lung tissues were also detected.</p><p><b>RESULTS</b>Compared with Group A, the expression of p38 MAPK, iNOS and HO-1 markedly increased in Groups B, C, D, and E (P < 0.01). But in Groups C, D and E the expression of p38 MAPK and iNOS were significantly lower than that of Group B, while expression of HO-1 was obviously higher than that of Group B (P < 0.05). The protein content, the ratio of neutrophils in bronchoalveolar lavage fluid (BALF), the content of MDA and the activities of serum NO in Group B were significantly higher than those of Group A (P < 0.01). There was a significant decrease in the level of arterial bicarbonate and partial pressure of oxygen in Group B (P < 0.01). Compared with Group B, these indexes of lung injury were significantly lower while the levels of arterial bicarbonate and partial pressure of oxygen increased significantly in Groups C, D and E (P < 0.05 or P < 0.01). Under light microscope, the pathological changes induced by LPS were significantly attenuated by RPR and hemin.</p><p><b>CONCLUSIONS</b>The high expression of MAPK plays an important role in lipopolysaccharide-induced acute lung injury. Protective effect of RPR on lipopolysaccharide-induced acute lung injury may be related to the inhibition of the abnormal high expression of p38 MAPK/iNOS/HO-1.</p>