Objective To explore the mechanism of Huoxiang Zhengqi Dropping Pills in treating dampness encumbering the spleen and stomach type rats by metabolomics method based on gas chromatography-time of flight mass spectrometry(GC-TOF/MS).Methods Male SD rats were randomly divided into normal group,model group and Huoxiang Zhengqi Dropping Pills group(1 g·kg-1).The rat model of dampness encumbering the spleen and stomach was prepared by comprehensive physical modeling method combined with improper diet.Intragastric administration was performed once a day for 10 consecutive days.The macroscopic signs and behavioral indexes(body mass,body length,tail length,abdominal circumference index and spontaneous activities frequency)of rats were observed.GC-TOF/MS method was used to analyze the serum and urine metabolome of rats,identify potential biomarkers related to dampness encumbering the spleen and stomach,and analyze the metabolic pathway of Huoxiang Zhengqi Dropping Pills in rats with dampness encumbering the spleen and stomach.Results Compared with the normal group,the body mass,body length,tail length and spontaneous activities frequency of the rats in the model group were significantly reduced(P＜0.05),and the abdominal circumference index was significantly increased(P＜0.05).Compared with the model group,the body mass,body length and spontaneous activities frequency of the rats in the Huoxiang Zhengqi Dropping Pills group were significantly increased(P＜0.05),and the abdominal circumference index was significantly decreased(P＜0.05).A total of 38 and 44 potential biomarkers related to dampness encumbering the spleen and stomach were identified in rat serum and urine,respectively.Huoxiang Zhengqi Dropping Pills can effectively interfere with the serum and urine metabolic phenotypes of model rats,and can significantly reverse 17 and 13 potential biomarkers in serum and urine,involving a total of 7 metabolic pathways.Conclusion The intervention mechanism of Huoxiang Zhengqi Dropping Pills on rats with dampness encumbering the spleen and stomach syndrome may be related to the regulation of tryptophan metabolism,histidine metabolism,glutamate-glutamine metabolism,energy metabolism and intestinal flora.

OBJECTIVE To study the improvement effects of Cannabis sativa oil on the symptoms in dextran sodium sulfate （DSS）-induced ulcerative colitis （UC） model rats， and to investigate its effects on intestinal flora of rats. METHODS Forty SD rats were randomly divided into control group， model group， C. sativa oil group （1 g/kg） and sulfasalazine group （positive control， 300 mg/kg）， with 10 rats in each group. The rats in control group and model group were given 0.5% polysorbate 80 by gavage， and the rats in C. sativa oil group and sulfasalazine group were given corresponding drug solution by gavage once a day for 10 days. From the 4th day， rats in model group， C. sativa oil group and sulfasalazine group were given 4% DSS solution for 7 consecutive days to establish UC model. The body weight， disease activity index （DAI） score， colon length， colon weight， weight per unit length of colon， the pathological changes of colon tissue， and the contents of tumour necrosis factor-α （TNF-α）， interleukin-6 （IL-6） and IL-10 in serum of rats were determined. The changes of intestinal flora in rats were detected by high- throughput sequencing. RESULTS Compared with control group， the body weight and the length of colon were decreased significantly in model group， while DAI score， the weight of colon， weight per unit length of colon， serum contents of TNF-α and IL-6 were increased significantly， and the content of IL-10 was decreased significantly （P＜0.05）； epithelial layer of colon tissue fell off， inflammatory cells infiltrated and invaded the submucosa， and intestinal glands were disordered. Compared with model group， above indexes of C. sativa oil group and sulfasalazine group were reversed significantly （P＜0.05）， and related symptoms were improved significantly. The result of flora sequencing showed that ACE index and Chao1 index of model group were decreased significantly， compared with control group （P＜0.05）； while Chao1 index of C. sativa oil group was increased significantly， compared with model group （P＜0.05）. Compared with control group， 41 genera of bacteria in the model group changed； compared with model group， C. sativa oil could return 3 of the 41 genera to normal state， including Dubosilla， Porphyrobacter and Allobaculum. CONCLUSIONS C. sativa oil can improve the symptoms of UC model rats by regulating the diversity of intestinal flora， increasing beneficial bacteria and decreasing pathogenic bacteria.

Objective: To investigate the changes in coagulation of sepsis rats with protein-malnutrition or energy-malnutrition. Methods: 108 male Sprague-Dawley (SD) rats were divided into three groups by random number table, with 36 rats in each group. The rats in normal feeding group were given a free diet (27 g/d, containing 18% protein fodder), and the rats in protein-malnutrition group were given a low protein diet (27 g/d, containing 5% protein fodder). The rats in energy-malnutrition group were given a low energy diet (9 g/d, containing 18% protein fodder). After 4 weeks of continuous feeding, 8 rats from each group were sacrificed for malnutrition evaluation. The weights of body, thymus and spleen were measured. The percentages of spleen T lymphocyte subsets and M1 macrophage were determined by flow cytometry. Plasma interleukins (IL-6 and IL-10) levels were determined by enzyme-linked immunosorbent assay (ELISA). The remaining 28 rats in each group were collected for cecal ligation and puncture (CLP) to reproduce the sepsis model, 20 rats of which were used for Kaplan-Meier survival analysis, and the other 8 rats were sacrificed at 8 hours after CLP. The levels of plasma IL-6 and IL-10 were determined by ELISA, and the percentage of spleen M1 macrophages was determined by flow cytometry. The mRNA expressions of tissue factor (TF) and plasminogen activation inhibitor-1 (PAI-1) in liver tissue were determined by reverse transcription-polymerase chain reaction (RT-PCR). Pearson correlation method was used to analyze the correlation between the mRNA expressions of TF and PAI-1 and IL-6 in rats after CLP. Results: ① After 4 weeks of feeding, the rats in the normal feeding group and protein-malnutrition group gained weight, while those in the energy-malnutrition group lost 25% of their initial body weight. The weights of body, thymus and spleen in the protein-malnutrition group and the energy-malnutrition group were significantly lower than those in the normal feeding group. Compared with the normal feeding group and the protein-malnutrition group, the percentages of spleen CD3⁺T lymphocytes, CD4⁺T lymphocytes, M1 macrophages and plasma IL-6 levels were significantly increased in the energy-malnutrition group [CD3⁺T lymphocytes percentage: (52.1±3.7)% vs. (46.9±3.9)%, (44.5±2.2)%; CD4⁺T lymphocyte percentages: (35.0±3.6)% vs. (26.3±2.2)%, (25.1±2.3)%; M1 macrophage percentages: (8.7±2.0)% vs. (3.2±1.3)%, (4.2±1.1)%; IL-6 (ng/L): 129.4±16.2 vs. 48.1±10.0, 53.0±8.3, all P < 0.05]. ② Kaplan-Meier survival analysis at 7 days after CLP showed: all rats in the energy-malnutrition group died, and the 7-day cumulative survival rate was significantly lower than that in the normal feeding group and the protein-malnutrition group [0% (0/20) vs. 35% (7/20), 55% (11/20), both P < 0.05]. The mortality of the normal feeding group was 65%, which was consistent with moderate CLP mortality, indicating that the CLP model was successfully prepared. After CLP, the plasma IL-6 level in the protein-malnutrition group was significantly lower than that in the normal feeding group [IL-6 (ng/L): 154.6±34.7 vs. 233.4±41.2, P < 0.05]. Compared with the normal feeding group, the mRNA expressions of TF and PAI-1 in liver and plasma IL-6 levels in the energy-malnutrition group were significantly increased [TF mRNA (2^-ΔΔCT): 4.5±2.2 vs. 1.1±0.7, PAI-1 mRNA (2^-ΔΔCT): 3.3±1.8 vs. 1.3±0.9, IL-6 (ng/L): 382.7±118.2 vs. 233.4±41.2, all P < 0.05], the percentage of M1 macrophages in spleen was significantly lowered [(8.9±2.4)% vs. (15.2±5.4)%, P < 0.05]. There was no significant difference in plasma IL-10 level among all the groups. Correlation analysis showed that the mRNA expressions of TF and PAI-1 in the liver of rats after CLP were positively correlated with plasma IL-6 level (r₁ = 0.644, r₂ = 0.574, both P < 0.01). Conclusions Long-term sustained stress (starvation) leads to sustained chronic inflammatory state, and stimulated the release of related inflammatory factors and activation of the coagulation system after infection. And the inflammatory factors in sepsis rats without sustained stress protein malnutrition were significantly reduced.

OBJECTIVE: To investigate the changes in coagulation of sepsis rats with protein-malnutrition or energy-malnutrition. METHODS: 108 male Sprague-Dawley (SD) rats were divided into three groups by random number table, with 36 rats in each group. The rats in normal feeding group were given a free diet (27 g/d, containing 18% protein fodder), and the rats in protein-malnutrition group were given a low protein diet (27 g/d, containing 5% protein fodder). The rats in energy-malnutrition group were given a low energy diet (9 g/d, containing 18% protein fodder). After 4 weeks of continuous feeding, 8 rats from each group were sacrificed for malnutrition evaluation. The weights of body, thymus and spleen were measured. The percentages of spleen T lymphocyte subsets and M1 macrophage were determined by flow cytometry. Plasma interleukins (IL-6 and IL-10) levels were determined by enzyme-linked immunosorbent assay (ELISA). The remaining 28 rats in each group were collected for cecal ligation and puncture (CLP) to reproduce the sepsis model, 20 rats of which were used for Kaplan-Meier survival analysis, and the other 8 rats were sacrificed at 8 hours after CLP. The levels of plasma IL-6 and IL-10 were determined by ELISA, and the percentage of spleen M1 macrophages was determined by flow cytometry. The mRNA expressions of tissue factor (TF) and plasminogen activation inhibitor-1 (PAI-1) in liver tissue were determined by reverse transcription-polymerase chain reaction (RT-PCR). Pearson correlation method was used to analyze the correlation between the mRNA expressions of TF and PAI-1 and IL-6 in rats after CLP. RESULTS: (1) After 4 weeks of feeding, the rats in the normal feeding group and protein-malnutrition group gained weight, while those in the energy-malnutrition group lost 25% of their initial body weight. The weights of body, thymus and spleen in the protein-malnutrition group and the energy-malnutrition group were significantly lower than those in the normal feeding group. Compared with the normal feeding group and the protein-malnutrition group, the percentages of spleen CD3⁺T lymphocytes, CD4⁺T lymphocytes, M1 macrophages and plasma IL-6 levels were significantly increased in the energy-malnutrition group [CD3⁺T lymphocytes percentage: (52.1±3.7)% vs. (46.9±3.9)%, (44.5±2.2)%; CD4⁺T lymphocyte percentages: (35.0±3.6)% vs. (26.3±2.2)%, (25.1±2.3)%; M1 macrophage percentages: (8.7±2.0)% vs. (3.2±1.3)%, (4.2±1.1)%; IL-6 (ng/L): 129.4±16.2 vs. 48.1±10.0, 53.0±8.3, all P < 0.05]. (2) Kaplan-Meier survival analysis at 7 days after CLP showed: all rats in the energy-malnutrition group died, and the 7-day cumulative survival rate was significantly lower than that in the normal feeding group and the protein-malnutrition group [0% (0/20) vs. 35% (7/20), 55% (11/20), both P < 0.05]. The mortality of the normal feeding group was 65%, which was consistent with moderate CLP mortality, indicating that the CLP model was successfully prepared. After CLP, the plasma IL-6 level in the protein-malnutrition group was significantly lower than that in the normal feeding group [IL-6 (ng/L): 154.6±34.7 vs. 233.4±41.2, P < 0.05]. Compared with the normal feeding group, the mRNA expressions of TF and PAI-1 in liver and plasma IL-6 levels in the energy-malnutrition group were significantly increased [TF mRNA (2^-ΔΔCT): 4.5±2.2 vs. 1.1±0.7, PAI-1 mRNA (2^-ΔΔCT): 3.3±1.8 vs. 1.3±0.9, IL-6 (ng/L): 382.7±118.2 vs. 233.4±41.2, all P < 0.05], the percentage of M1 macrophages in spleen was significantly lowered [(8.9±2.4)% vs. (15.2±5.4)%, P < 0.05]. There was no significant difference in plasma IL-10 level among all the groups. Correlation analysis showed that the mRNA expressions of TF and PAI-1 in the liver of rats after CLP were positively correlated with plasma IL-6 level (r₁ = 0.644, r₂ = 0.574, both P < 0.01). CONCLUSIONS Long-term sustained stress (starvation) leads to sustained chronic inflammatory state, and stimulated the release of related inflammatory factors and activation of the coagulation system after infection. And the inflammatory factors in sepsis rats without sustained stress protein malnutrition were significantly reduced.
Animals ; Male ; Malnutrition ; Rats ; Rats, Sprague-Dawley ; Sepsis ; Spleen ; Tumor Necrosis Factor-alpha

OBJECTIVE:To elucidate the efficacy and mechanism of Hugan tablets in hepatoprotective effects from perspective of metabolic pathways. METHODS:36 male rats were randomly divided into normal group (0.5%sodium carboxymethyl cellu-lose),model group(0.5%sodium carboxymethyl cellulose)and Hugan tablets group(1.7 g/kg),12 in each group,intragastrically administrated once a day,for 9 d. After 1 h of last administration,rats in model group and Hugan tablets group were intraperitone-ally injected 50%CCl4 peanut oil solution 1 mL/kg to induce liver injury. After 24 h of modeling,malondialdehyde(MDA),super-oxide dismutase(SOD),glutathione peroxidase(GSH-Px)levels in liver tissue of rats were detected. Nuclear magnetic resonance spectroscopy(1H-NMR)metabolomics technique was adopted to establish the serum and liver metabolite profiles of rats,and the ef-fects of Hugan tablets on changes of metabolic profile and potential biomarkers in serum and liver of rats with CCl4-induced acute liver injury were analyzed. RESULTS:Compared with normal group,MDA level in liver tissue of rats in model group was signifi-cantly increased(P<0.05),SOD and GSH-Px levels were significantly reduced(P<0.05). Both body physiology and material me-tabolism of rats were obviously changed,and levels of 11 metabolic potential biomarkers in serum and 14 metabolic potential bio-markers in liver were significantly increased/decreased (P<0.05). Compared with model group,MDA level in liver tissue in Hugan tablets group was significantly reduced(P<0.05),SOD and GSH-Px levels were significantly increased(P<0.05). Serum and liver metabolism tended to be normal,6 metabolic potential biomarkers(isoleucine,leucine,3-hydroxybutyrate,acetone,ace-toacetate,choline) in serum and 8 metabolic potential biomarkers (3-hydroxybutyrate,alanine,glutamate,pyruvate,succinate, choline,lactate,glucose)in liver got significant callback(P<0.05). CONCLUSIONS:The hepatoprotective mechanism of Hugan tablets may be associated with antioxidative stress and regula-tion of lipid metabolism,glucose metabolism and amino acid metabolism.

To investigate the intervention effects of Morinda officinalis How. on 'Kidney-yang deficiency syndrome' induced by hydrocortisone in rats, the metabolic profiles of rat urine were characterized using proton nuclear magnetic resonance and principal component analysis (PCA) was applied to study the trajectory of urinary metabolic phenotype of rats with 'Kidney-yang deficiency syndrome' under administration of M. officinalis at different time points. Meanwhile, the intervention effects of M. officinalis on urinary metabolic potential biomarkers associated with 'Kidney-yang deficiency syndrome' were also discussed. The experimental results showed that in accordance to the increased time of administration, an obvious tendency was observed that clustering of the treatment group moved gradually closed to that of the control group. Eight potential biomarkers including citrate, succinate, alpha-ketoglutarate, lactate, betaine, sarcosine, alanine and taurine were definitely up- or down-regulated. In conclusion, the effectiveness of M. oficinalis on 'Kidney-yang deficiency syndrome' is proved using the established metabonomic method and the regulated metabolic pathways involve energy metabolism, transmethylation and transportation of amine. Meanwhile, the administration of M. officinalis can alleviate the kidney impairment induced by 'Kidney-yang deficiency syndrome'.

OBJECTIVETo investigate the metabolic profile of hydrocortisone-induced 'Kidney-yang deficiency syndrome'in rats and the intervention effects of Morinda officinalis.

METHODProton nuclear magnetic resonance (1H-NMR) technique was used to analyze the rat metabonome in serum. Orthogonal partial least squares discriminant analysis (OPLS-DA) were processed to analyze the metabonome difference between the control and hydrocortisone treated samples. Twelve potential biomarkers were selected, via the parameter of variable importance in the projection (VIP). Principal components analysis (PCA) was employed to process the data from the M. officinalis. treatment group and the intervention effects of M. officinalis, was investigated through the selected potential biomarkers.

RESULTAfter hydrocortisone treatment, the energy metabolism, amino acids metabolism and gut microflora environment were seriously disturbed and transmethylation was surpressed. M. officinalis could effectively alleviate the disturbance of energy and amino acids metabolism and enhance transmethylation, but could not modulate the gut microflora environment.

CONCLUSIONThe results obtained suggested that metabonomic studies could better reflect the whole status of metabolism in bio-systems, and could be treated as a potential powerful approach in pharmacological studies and investigation of the essence of 'syndrome' in traditional Chinese medicine.

Animals ; Biomarkers ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Kidney ; drug effects ; metabolism ; Male ; Metabolomics ; Morinda ; chemistry ; Rats ; Rats, Sprague-Dawley ; Yang Deficiency ; drug therapy ; metabolism