Methylation Characteristics of Perivisceral Fat Gene in Obese Rats with Phlegm-dampness Syndrome and the Effect of Wen Dan Decoction

Objective To observe the characteristics of gene methylation in obese rats with phlegm-dampness syndrome induced by the high-fat diet, and to study the effect of Wen Dan Decoction on gene methylation after the intervention. Methods Methylation sites of genes were detected by the MeDIP-seq method. Bioinformatics method was used to analyze the gene methylation characteristics of obesity with phlegmdampness syndrome and the effect of Wen Dan Decoction. Results (1) There were 3 242 methylation differential loci in dietinduced obesity with phlegm-dampness syndrome, of which 1 243 were down-regulated and 1 999 were up-regulated, involving 1 579 differential genes. GO analysis showed that "offactory receptor activity" and others were enriched. The possible signal pathways involved were "Olfactory transduction""Tuberculosis""Systemic lupus erythematosus" and "Ribosome".(2) After the intervention of Wen Dan Decoction in obesity with phlegmdampness syndrome, 4 046 different methylation loci were obtained, including 1 067 down-regulated loci and 2 979 up-regulated loci, involving 2 068 genes. GO analysis showed that "offactory receptor activity" and others were enriched. These genes involved seven signaling pathways, such as "Metabolic pathways".(3) Between diet-induced obesity with phlegm-dampness syndrome and Wen Dan Decoction intervening obesity with the phlegm-dampness syndrome, 582 common genes of methylation differential genes were obtained. After the intervention of Wen Dan Decoction, the number of GO enrichment items was more than that of obesity with phlegm-dampness syndrome, and even the same GO enrichment items involved more genes. Conclusions The phlegm-dampness syndrome of obesityinduced by diet had the characteristics of gene methylation changes, and the intervention of Wen Dan Decoction could also affect the status of gene methylation. The genes affected by Wen Dan Decoction were closely related to the methylation gene of phlegm-dampness syndrome of obesity-induced by diet but covered a wider range.

Proteomics analysis of liver proteins from rats with spleen-deficiency syndrome induced by chronic improper diet consumption and fatigue

Objective:To investigate a proteomics analysis of liver proteins from rats with spleen-deficiency syndrome (SDS) induced by chronic improper diet consumption and fatigue.Methods:This study used a liver proteomic profiling method to identify differentially expressed proteins and altered pathways involved in SDS rats.Specifically,we collected liver samples from a control group and a group with SDS induced by chronic improper diet consumption and fatigue for 4 weeks.The pooled liver proteins in each group were labeled with 8-plex isobaric tags for relative and absolute quantitation reagents.The labeled control and SDS group samples were pooled together and separated by high-pH reverse-phase liquid chromatography.The differentially expressed proteins from the liver proteomes were analyzed to identify potential biomarkers of SDS.Differentially expressed proteins were selected in conjunction with gene ontology and ingenuity pathway analysis.Results:We identified 2176 protein clusters with more than two peptides in the SDS group,with 141 proteins quantified as differentially expressed proteins.Of these,75 proteins were up-regulated,and 66 were down-regulated.Three activated signaling pathways,the thrombin,CXCR4 and synaptic long term depression signaling pathways,and a large multi-protein complex within the network were revealed in the liver proteomic analysis of SDS rats.Conclusions:This is the first report of a differential liver proteome under SDS conditions.The results suggest that the liver proteome partially reflects the pathological changes involved in SDS.The findings provide important information for comprehensively understanding the mechanisms of dysfunction or injury in the liver at the molecular level as a result of SDS.Furthermore,they provide a novel understanding of the connotation of SDS in the fleld of traditional Chinese Medicine.

Study on the Resolving Phlegm Effect of D-Limonene in Mice with Spleen Deficiency and Phlegm-Dampness Syndrome

Objective:According to Traditional Chinese Medicine theory,spleen deficiency and phlegm-dampness syndrome(SDPDS)are caused by abnormal water metabolism in the body because of spleen dysfunction.The purpose of this article was to evaluate the efficacy of D-limonene(DL)in resolving phlegm in mice with SDPDS from the perspective of regulating the level of aquaporin 3(AQP3).Methods:The model of SDPDS was induced in mice using the multifactor modeling method,which combines internal and external dampness.An artificial climate box was used to create a humid environment,whereas the irregular diet was caused by different feeding methods on odd-even days.The mice were divided into blank control,model group,DL low-dose,DL high-dose,and positive groups.The mice were modeled and treated for 7 day.Levels of gastrin and amylase(AMS)in the serum,mucus secretion in the trachea,and AQP3 in the tissue near the gastric cardia.Results:DL significantly reduced mucus secretion in the trachea(P<0.001).It also increased the level of AMS in the serum(P<0.01)and decreased the level of AQP3 in the gastric tissue(P<0.01).Conclusions:Mice with SDPDS exhibited disturbed water metabolism and significantly increased AQP3 levels.DL can restore the levels of AQP3 and plays an important role in resolving phlegm.This study may also help expand the efficacy of natural drugs containing DL and improve the utilization of natural drug resources.

Effect of Electro-acupuncture on Expression of IRS-1/PI3K/GLUT4 Pathway in Ovarian Granulosa Cells of Infertile Patients with Polycystic Ovary Syndrome-Insulin Resistance of Phlegm-Dampness Syndrome

Objective:To evaluate the effect of electro-acupuncture(EA)in infertile patients with phlegmdampness polycystic ovary syndrome-insulin resistance(PCOS-IR).Methods:Seventy-six PCOS-IR patients who underwnet in vitro fertilization and embryo transfer(IVF-ET)were equally assigned to two groups according to a random digital table:the EA group and the control group,with 38 cases in each group.Before undergoing IVF,the two groups were treated with EA or pseudo-acupuncture,respectively,for 3 menstrual cycles.The intervention was 25 min twice a week until the day of oocyte collection.The selected acupoints were Zhongwan(RN 12),Tianshu(ST 25),Daheng(SP 15),Daimai(GB 26),Qihai(CV 6),Guanyuan(CV 4),and bilateral points including Xuehai(SP 10),Fenglong(ST 40),Zusanli(ST 36),and Yinlingquan(SP 9).Evaluation of phlegm-dampness syndrome score and IR score were carried out before and after treatment.Additionally,the number of oocytes retrieved,transplantable embryo rate,high-quality embryo rate,clinical pregnancy rate and live birth rate were compared between the two groups.Real-time polymerase chain reaction analysis was used to monitor the m RNA expression of the insulin receptor substrate(IRS-1),phosphatidylinositiol 3-kinase(PI3 K)and glucose transport factor 4(GLUT4)in ovarian granulosa cells.Results:EA treatment reduced the phlegm-dampness syndrome score as well as the IR scores compared with the control group(P<0.05).No significant differences in the number of oocytes retrieved and clinical pregnancy rate between the two groups(P>0.05).Moreover,the transplantable embryo rate[49.0%(284/580)vs.41.9%(273/652)],high-quality embryo rate[36.6%(104/284)vs.27.8%(76/273)],and live birth rate[50%(19/38)vs.26.3%(10/38)]in the EA group were significantly higher than in the control group(P<0.05).Gene expression analyses revealed significantly elevated IRS-1,PI3 K and GLUT4 m RNA in ovarian granulosa cells of the EA group compared with the control group(P<0.05).Conclusions:EA may ameliorate the effects of phlegm-dampness syndrome and ovarian IR in PCOS-IR patients.Mechanistically,this effect might be through an upregulation of the IRS-1/PI3 K/GLUT4 signaling pathway,which may result in improved oocyte quality and embryonic development potential.(Registration No.Chi CTR1800015453)

Gut microbiota and Chinese medicine syndrome: altered fecal microbiotas in spleen(Pi)-deficient patients

OBJECTIVE:To investigate the fecal microbiota changes in patients with spleen(Pi)-deficiency(SPD),a common Chinese medicine syndrome with digestive and absorptive disturbances and to provide insight into the relationship between Chinese medicine syndrome and gut dysbiosis.METHODS:Fecal microbiotas from the stool samples of 53 SPD patients and 35 healthy subjects were analyzed via 16 S r RNA gene polymerase chain reaction(PCR)-denaturation gradient gel electrophoresis(DGGE).SPD-related marker genes from20 SPD patients and 20 healthy subjects were identified through gene sequencing,while some genes were quantified using quantitative PCR(q PCR).Discriminant analysis was conducted using SPSS software,and the canonical discriminant function formula for Pi-deficiency was established.RESULTS:Alterations in microbiota diversity and composition between the SPD and healthy groups were demonstrated via 16 S r RNA gene PCR-DGGE combined with multivariate statistical analysis.Fecal microbiota changes were also observed among different SPD-subtype patients.Eight SPD-related markers were found,and putative species corresponding to these markers were identified through gene sequencing,which may have potential associations with the common digestive dysfunctions in SPD patients.q PCR methods were established for two of these markers,which were significantly altered in the SPD patients.The canonical discriminant function formula was calculated for SPD,and the validity rates of these markers were over 85%.CONCLUSION:Fecal microbiotas are altered in patients with SPD,which may provide insight for further studies on clinically diagnosing and treating SPD.The results may also provide data to gain a better understanding of Traditional Chinese Medicine syndrome and gut dysbiosis.

Differentially Expressed Proteins in Rat Hippocampus after Chronic Immobilization Stress and Intervention Using Xiao Yao San Decoction

Objective To identify differentially expressed proteins in the hippocampus of rats after chronic immobilization stress(CIS)using a proteomics approach,and to study the effect of the Xiao Yao San(XYS)decoction on differentially expressed proteins.Methods Twenty-four Sprague Dawley rats were randomly assigned to one of four groups of equal body weight:control(non-stress),7-day stress,21-day stress and21-day stress+XYS treatment groups.Two-dimensional gel electrophoresis(2-DE)was used to detect differences in protein expression in rat hippocampus.One differentially expressed protein was measured and verified by western blotting.Results Seventeen proteins showed differential expression.Among these,eight could be identified:glial fibrillary acidic protein-2(GFAP-2),tubulin alpha-1c,cytoplasmic muscle actin2,14-3-3protein,β-2a tubulin,phosphatidylethanolamine binding protein,synucleinαsyn3,and a low molecular weight(18kD)protein.Six of these proteins exhibited increased expression,one showed decreased expression,and the other protein,which comprised five subtypes,were either increased or decreased.These proteins are known to be involved in immunity,signal transduction,cell cycle control,apoptosis,regulation of enzyme activity,cytoskeleton structure,and synaptic plasticity.GFAP-2was further analyzed,and its differential expression confirmed by western blotting.Conclusion Some proteins are differentially expressed in the hippocampus of rats under chronic stress.The biological functions of these differentially expressed proteins are varied.Finally,the XYS decoction can significantly up-or down-regulate these protein expression levels.