Serum metabolomics analysis of biomarkers and metabolic pathways in patients with colorectal cancer associated with spleen-deficiency and qi-stagnation syndrome or damp-heat syndrome: a prospective cohort study.

Objective: To profile the serum metabolites and metabolic pathways in colorectal cancer (CRC) patients associated with spleen-deficiency and qi-stagnation syndrome (SDQSS) or damp-heat syndrome (DHS). Methods: From May 2020 to January 2021, CRC patients diagnosed with traditional Chinese medicine (TCM) syndromes of SDQSS or DHS were enrolled. The clinicopathological data of the SDQSS and DHS groups were compared. The serum samples were analyzed by liquid chromatography-mass spectrometry (LC-MS). The variable importance in the projection >1, fold change ≥3 or ≤0.333, and P value ≤0.05 were used to identify differential metabolites between the two groups. Furthermore, areas under the receiver operating characteristic (ROC) curve > 0.9 were applied to select biomarkers with good predictive performance. The enrichment metabolic pathways were searched through the database of Kyoto Encyclopedia of Genes and Genomes. Results: 60 CRC patients were included (30 SDQSS and 30 DHS). The level of alanine aminotransferase was marginally significantly higher in the DHS group than the SDQSS group (P = 0.051). The other baseline clinicopathological characteristics were all comparable between the two groups. 23 differential serum metabolites were identified, among which 16 were significantly up-regulated and 7 were significantly down-regulated in the SDQSS group compared with the DHS group. ROC curve analysis showed that (S)-3-methyl-2-oxopentanoic acid, neocembrene, 1-aminocyclopropanecarboxylic acid, 3-methyl-3-hydroxypentanedioate, and nicotine were symbolic differential metabolites with higher predictive power. The top five enrichment signalling pathways were valine, leucine and isoleucine biosynthesis; lysosome; nicotine addiction; fructose and mannose metabolism; and pertussis. Conclusion: Our study identifies the differential metabolites and characteristic metabolic pathways among CRC patients with SDQSS or DHS, offering the possibility of accurate and objective syndrome differentiation and TCM treatment for CRC patients.

Clinicopathological features and expression of regulatory mechanism of the Wnt signaling pathway in colorectal sessile serrated adenomas/polyps with different syndrome types.

BACKGROUND: Colorectal cancer (CRC) is the third most common cancer worldwide, with the fourth highest mortality among all cancers. Reportedly, in addition to adenomas, serrated polyps, which account for 15%-30% of CRCs, can also develop into CRCs through the serrated pathway. Sessile serrated adenomas/polyps (SSAs/Ps), a type of serrated polyps, are easily misdiagnosed during endoscopy. AIM: To observe the difference in the Wnt signaling pathway expression in SSAs/Ps patients with different syndrome types. METHODS: From January 2021 to December 2021, patients with SSAs/Ps were recruited from the Endoscopy Room of Shanghai Traditional Chinese Medicine-Integrated Hospital, affiliated with Shanghai University of Traditional Chinese Medicine. Thirty cases each of large intestine damp-heat (Da-Chang-Shi-Re, DCSR) syndrome and spleen-stomach weakness (Pi-Wei-Xu-Ruo) syndrome were reported. Baseline comparison of the general data, typical tongue coating, colonoscopy findings, and hematoxylin and eosin findings was performed in each group. The expression of the Wnt pathway-related proteins, namely ß-catenin, adenomatous polyposis coli, and mutated in colorectal cancer, were analyzed using immunohistochemistry. RESULTS: Significant differences were observed with respect to the SSAs/Ps size between the two groups of patients with different syndrome types (P = 0.001). The other aspects did not differ between the two groups. The Wnt signaling pathway was activated in patients with SSAs/Ps belonging to both groups, which was manifested as ß-catenin protein translocation into the nucleus. However, SSAs/Ps patients with DCSR syndrome had more nucleation, higher ß-catenin expression, and negative regulatory factor (adenomatous polyposis coli and mutated in colorectal cancer) expression (P < 0.0001) than SSA/P patients with Pi-Wei-Xu-Ruo syndrome. In addition, the SSA/P size was linearly correlated with the related protein expression. CONCLUSION: Patients with DCSR syndrome had a more obvious Wnt signaling pathway activation and a higher risk of carcinogenesis. A high-quality colonoscopic diagnosis was essential. The thorough assessment of clinical diseases can be improved by combining the diseases of Western medicine with the syndromes of traditional Chinese medicine.

HMGB1/PTEN/PI3K axis participates in the peripheral immune cell differentiation in two representative TCM syndromes of chronic hepatitis B patients.

Liver depression and spleen deficiency syndrome (LDSDS) and spleen-gastric damp-heat syndrome (SGDHS) are two major traditional Chinese medicine syndromes observed in chronic hepatitis B (CHB). Both syndromes exhibit significant differences in the pathogenesis and prognosis, and are closely related to the immune system. However, the underlying mechanisms are largely unknown. This study aimed to explore the immunoregulatory mechanisms of the two syndromes and promote the differentiation precision between the two syndromes. Thirty-six patients with CHB (18 LDSDS patients and 18 SGDHS patients) and 14 healthy controls were recruited into this study and blood was collected from all the subjects for testing. We studied the contents of T lymphocytes by flow cytometry and the expression levels of HMGB1/PTEN/PI3K axis proteins by enzyme-linked immunosorbent assay (Elisa). Protein-protein interaction (PPI) networks among HMGB1/PTEN/PI3K axis were constructed for functional enrichment. The correlations between T lymphocytes and proteins were analyzed by constructing multiple regression equations. The results revealed that the CD8+ T cells level in the two syndromes were lower than that in healthy controls, and the levels of Th17, Treg cells, and HMGB1, PI3K, PDK1, Akt were higher than those of the healthy controls (p < 0.05). Moreover, the levels of CD4+ T, Th17 cells, and HMGB1, PTEN, PI3K in LDSDS were higher than SGDHS (p < 0.05). PPI network indicated that HMGB1/PTEN/PI3K axis participated in T cell activation and liver pathology. Our results revealed that HMGB1/PTEN/PI3K axis may play an important role in regulating the formation of peripheral immune differences between the two syndromes. CD4+ T and Th17 are two representative immune cells that may serve as potential biological markers for LDSDS and SGDHS in CHB.

Metabolomic Analysis of Urine in Rat Model with Spleen-stomach Damp-heat Syndrome / 中国实验方剂学杂志

ObjectiveTaking the rat model of spleen-stomach damp-heat syndrome（SSDHS） as the research object， this study aimed to investigate the potential biomarkers of SSDHS and the related metabolic pathways based on urine metabolomics， and tried to reveal the essence of SSDHS at the level of endogenous small molecular metabolites. MethodSixteen SD rats were randomly divided into normal and model groups. The normal group was fed normal chow and the model group was fed with 200 g·L-1 honey water daily， and lard and Chinese Baijiu alternately on alternate days for 17 days. The SSDHS model rats were exposed to external dampness-heat environment with temperature at 30-34 ℃， relative humidity of 95% for 2 h at the same time every day from the 10th day for 7 d. Then， the model was evaluated by observing the general conditions of the rats， measuring the contents of motilin（MTL） and gastrin（GT） in plasma by enzyme-linked immunosorbent assay（ELISA）， and examining the histopathology of gastronitestinal tissues. In additon， the urine metabolomics analysis was performed by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， and the detection conditions was as follows：ACQUITY™ UPLC BEH C18 column（2.1 mm×100 mm， 1.7 μm）， mobile phase of 0.1% formic acid aqueous solution（A）-0.1% formic acid acetonitrile solution（B） for gradient elution （0-3 min， 1%-18%B； 3-8 min， 18%-40%B； 8-10 min， 40%-100%B）， the flow rate of 0.4 mL·min-1， electrospray ionization（ESI） in positive and negative ion modes， scanning range of m/z 50-1 000. The univariate and multivariate statistical analysis were constructed for screening inter-group differential ions， the element composition was calculated according to the precise relative molecular weight， and ion information was matched with databases such as Human Metabolome Database（HMDB） to identify biomarkers. Kyoto Encyclopedia of Genes and Genomes（KEGG） database was used to obtain the biological information of metabolites， and their associated metabolic pathways were analyzed by MetaboAnalyst 5.0. ResultCompared with the normal group， the rectal temperature of the model group increased significantly（P<0.01）， the levels of plasma MTL and GT decreased significantly（P<0.05， P<0.01）， and pathological changes such as bleeding， congestion and inflammatory infiltration in the gastric and colonic tissues. A total of 25 differential metabolites such as L-histidine， citric acid and isocitric acid were found to be the potential biomarker of SSDHS by urine metabolomics， 13 of which were phase Ⅱ metabolites of endogenous substances（glucuronic acid conjugates， sulfuric acid conjugates and acetyl conjugates）， involving the metabolic pathways of histidine metabolism， tricarboxylic acid cycle， glyoxylate and dicarboxylate metabolism. ConclusionSSDHS primarily causes disorders of histidine metabolism， tricarboxylic acid cycle， glyoxylate and dicarboxylate metabolism， as well as the imbalance of the activation/inactivation of endogenous metabolites， which may involve the immune response， material and energy metabolism， inflammatory response and intestinal flora， and may provide a basis for the establishment and application of SSDHS model.

A Retrospective Clinical Study on the Correlation between the Theory of Five Movements and Six Climates， and Kidney Deficiency with Governor Vessel Cold Syndrome and Kidney Deficiency Damp-Heat Syndrome in Ankylosing Spondylitis / 中医杂志

ObjectiveTo explore the relationship between the kidney deficiency and governor vessel cold syndrome and the kidney deficiency damp-heat syndrome in ankylosing spondylitis （AS） patients and the five evolutive phases and six climatic factors of their birth and onset year based on the theory of five movements and six climates （FMSC）. MethodsTotally 1791 patients with AS who were admitted to China-Japan Friendship Hospital from September 2010 to September 2020 and met the diagnostic and inclusion criteria were selected in this study. The clinical data were classified into two types of syndromes， kidney deficiency and governor vessel cold syndrome and the kidney deficiency damp-heat syndrome based on the diagnostic criteria of traditional Chinese medicine syndromes. The date of birth and the year of disease onset were converted into FMSC symbols according to the perpetual almanac （《万年历》）， and the two could be converted into the terrestrial branch， year evolutive phase， host evolutive phase， guest evolutive phase， host climatic qi， guest climatic qi， celestial manager qi， guest climatic qi adding to fixed host qi， combined analysis of five evolutive phases and six climatic factors， solar terms， and season of the date of birth， as well as the terrestrial branch， year evolutive phase， and celestial manager qi of the year of disease onset. Univariate analyses were performed using the two independent samples t-test or the Mann Whitney U-test， the Pearson （Pearson） χ2 test， or one-way logistic regression analyses， and variables for which statistical significance existed in the one-way analyses were included in the multivariate logistic regression analyses. General conditions， clinical manifestations， physical signs， laboratory indicators ［including C reactive protein （CRP）， erythrocyte sedimentation rate （ESR）， and humans leukocyte antigen B27 （HLA-B27）］， measurement （including occipital wall distance， jaw peduncle distance， finger-to-ground distance， thoracic range of motion， and Schober experiment）， and distribution of FMSC of birth and disease onset between AS patients with kidney deficiency and governor vessel cold syndrome and with the kidney deficiency damp-heat syndrome were compared， and the association between FMSC and AS patients with kidney deficiency and governor vessel cold syndrome and the kidney deficiency damp-heat syndrome was studied. ResultsThe differences in ESR， CRP， chest mobility， occurrence of achilles tendon enthesitis， and peripheral arthritis between the two groups of patients were statistically significant （P＜0.05）. Single factor analysis found that taking kidney deficiency and governor vessel cold syndrome as control the following FMSC factors increases the risk of developing kidney deficiency damp-heat syndrome： excess of water in year evolutive phase at birth， excess of wood in host evolutive phase at birth， excess of wood in guest evolutive phase at birth， excess of wood in year evolutive phase of onset， deficiency of metal in year evolutive phase at birth （OR = 2.000， P = 0.004）， excess of metal in host evolutive phase at birth （OR = 1.745， P = 0.024） or excess of wood （OR = 1.781， P = 0.023）， deficiency of fire in guest evolutive phase at birth （OR = 1.689， P = 0.049） or deficiency of wood （OR = 1.901， P = 0.018） or excess of metal （OR = 2.163， P = 0.004）， excess of water in year evolutive phase at the disease onset （OR = 1.880 ， P = 0.013） or deficiency of wood （OR = 1.707， P = 0.022）. Multivariate logistic regression analysis found that the risk of developing kidney deficiency damp-heat syndrome in AS was increased by deficiency of metal in year evolutive phase at birth， excess of metal in host evolutive phase at birth， higher level of ESR， greater the chest mobility， incidence of concomitant Achilles tendon enthesitis and peripheral arthritis. ConclusionThe year evolutive phase and host evolutive phase at birth play a significant role in the development of kidney deficiency and governor vessel cold syndrome AS. Risk of developing kidney deficiency damp-heat syndrome can be increased by excess of water or deficiency of metal in year evolutive phase at birth， and excess of wood or excess of metal in host evolutive phase at birth and the kidney deficiency damp-heat syndrome in ankylosing spondylitis.

Distinct common signatures of gut microbiota associated with damp-heat syndrome in patients with different chronic liver diseases.

Background: Chronic hepatitis B (CHB) and non-alcoholic fatty liver disease (NAFLD) are prevalent in China. According to traditional Chinese medicine (TCM) theory, damp-heat (DH) syndrome is common in chronic liver disease. However, the biological characteristics related to quantitative diagnosis remain to be determined. This study aimed to identify the consistent alterations in the gut microbiota associated with DH syndrome in patients with CHB or NAFLD. Methods: A total of 405 individuals were recruited, of which 146 were participants who met the consistent TCM diagnosis by three senior TCM physicians and were typical syndromes. All participants were required to provide fresh stool and serum samples. The gut microbiota was assessed by fecal 16S rRNA gene sequencing, and the serum metabolite profiles of participants were quantified by an ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) system. DH syndrome-related bacteria taxa were identified based on the 146 individuals with typical syndromes and validated in all 405 volunteers. Results: The results showed that CHB and NAFLD patients with typical TCM DH syndrome had consistently elevated serum total bile acid (TBA) levels. Significant alterations in microbial community were observed according to TCM syndromes identification. A total of 870 microbial operational taxonomic units and 21 serum metabolites showed the same variation trends in both the CHB and NAFLD DH syndrome groups. The functional analysis predicts consistent dysregulation of bile acid metabolism. Five genera (Agathobacter, Dorea, Lachnospiraceae_NC2004_group, Subdoligranulum, and unclassified_c__Clostridia) significantly decreased in abundance in patients with DH syndrome. We utilize these five genera combined with TBA to construct a random forest classifier model to predict TCM diagnosis. The diagnostic receiver-operator characteristic (ROC) areas for DH syndrome were 0.818 and 0.791 in internal tenfold cross-validation and the test set based on all 405 individuals, respectively. Conclusion: There are common signatures of gut microbiota associated with DH syndrome in patients with different chronic liver diseases. Serum TBA combined with DH-related genera provides a good diagnostic potential for DH syndrome in chronic liver disease.

A Mouse Model of Damp-Heat Syndrome in Traditional Chinese Medicine and Its Impact on Pancreatic Tumor Growth.

Background: Damp-heat syndrome is one of the most important syndrome types in the traditional Chinese medicine (TCM) syndrome differentiation and treatment system, as well as the core pathogenesis of pancreatic cancer (PC) which remains a challenge to medical researchers due to its insidious onset and poor prognosis. Great attention has been given to the impact of damp-heat syndrome on tumorigenesis and progression, but less attention has been given to damp-heat modeling per se. Studying PC in a proper damp-heat syndrome animal model can recapitulate the actual pathological process and contribute to treatment strategy improvement. Methods: Here, an optimized damp-heat syndrome mouse model was established based on our prior experience. The Fibonacci method was applied to determine the maximum tolerated dosage of alcohol for mice. Damp-heat syndrome modeling with the old and new methods was performed in parallel of comparative study about general appearance, food intake, water consumption and survival. Major organs, including the liver, kidneys, lungs, pancreas, spleen, intestines and testes, were collected for histological evaluation. Complete blood counts and biochemical tests were conducted to characterize changes in blood circulation. PC cells were subcutaneously inoculated into mice with damp-heat syndrome to explore the impact of damp-heat syndrome on PC growth. Hematoxylin-eosin staining, Masson staining and immunohistochemistry were performed for pathological evaluation. A chemokine microarray was applied to screen the cytokines mediating the proliferation-promoting effects of damp-heat syndrome, and quantitative polymerase chain reaction and Western blotting were conducted for results validation. Results: The new modeling method has the advantages of mouse-friendly features, easily accessible materials, simple operation, and good stability. More importantly, a set of systematic indicators was proposed for model evaluation. The new modeling method verified the pancreatic tumor-promoting role of damp-heat syndrome. Damp-heat syndrome induced the proliferation of cancer-associated fibroblasts and promoted desmoplasia. In addition, circulating and tumor-located chemokine levels were altered by damp-heat syndrome, characterized by tumor promotion and immune suppression. Conclusions: This study established a stable and reproducible murine model of damp-heat syndrome in TCM with systematic evaluation methods. Cancer associated fibroblast-mediated desmoplasia and chemokine production contribute to the tumor-promoting effect of damp-heat syndrome on PC.

[Effect of Gegen Qinlian Decoction on cardiac diastolic function of diabetic mice with damp-heat syndrome].

This study was designed to explore the effect and mechanism of Gegen Qinlian Decoction(GQD) on cardiac function of diabetic mice with damp-heat syndrome. The db/db diabetic mice were exposed to the damp-heat environment test chamber for inducing the damp-heat syndrome. Forty-eight six-week-old db/db mice were randomly divided into six groups, namely the db/db diabetic model group, db/db diabetic mouse with damp-heat syndrome(db/db-dh) group, db/db diabetic mouse with damp-heat syndrome treated with low-dose GQD(db/db-dh+GQD-L) group, db/db-dh+GQD-M(medium-dose) group, db/db-dh+GQD-H(high-dose) group, and db/db-dh+lipro(liprostatin-1, the inhibitor of ferroptosis) group, with eight six-week-old db/m mice classified into the control group. The results showed that mice presented with the damp-heat syndrome after exposure to the "high-fat diet" and "damp-heat environment", manifested as the elevated fasting blood glucose, reduced food intake, low urine output, diarrhea, listlessness, loose and coarse hair, and dark yellow and lusterless fur. However, the intragastric administration of the high-dose GQD for 10 weeks ameliorated the above-mentioned symptoms, inhibited myocardial hypertrophy and fibrosis, and improved the cardiac diastolic function of db/db-dh mice. qPCR suggested that GQD regulated the expression of ferroptosis-related genes, weakened the lipid peroxidation in the myocardium, and up-regulated glutathione peroxidase 4(GPX4) expression in comparison with those in the db/db-dh group. At the same time, the ferroptosis inhibitor liprostatin-1 significantly improved the cardiac function and reversed the cardiac remodeling of db/db-dh mice. It can be concluded that the damp-heat syndrome may aggravate myocardial ferroptosis and accelerate cardiac remodeling of db/db mice, thus leading to diastolic dysfunction. GQD is able to improve cardiac remodeling and diastolic function in diabetic mice with damp-heat syndrome, which may be related to its inhibition of myocardial ferroptosis.

Effect of Jiedu Huayu Tongfu prescription on intestinal flora in patients with hepatitis B cirrhosis with liver-gallbladder damp-heat syndrome / 临床肝胆病杂志

Objective To investigate the regulatory effect of Jiedu Huayu Tongfu prescription on intestinal homeostasis in patients with hepatitis B cirrhosis with liver-gallbladder damp-heat syndrome, as well as its effect on endotoxin, inflammatory factors, and cellular immune function. Methods A total of 72 patients who attended The First Affiliated Hospital of Henan University of Chinese Medicine from June 2019 to January 2021 and met the diagnostic and inclusion criteria were enrolled as subjects and then randomly divided into observation group and control group, with 36 patients in each group. In the treatment group, 2 patients were lost to follow-up, 2 patients were excluded, and 32 patients completed the study; in the control group, 2 patients were lost to follow-up, 1 patient was excluded, and 33 patients completed the study. In addition to the basic treatment including antiviral therapy and liver-protecting treatment, the patients in the observation group were given Jiedu Huayu Tongfu granules, and those in the control group were given oral administration of Bifidobacterium tetravaccine tablets; the course of treatment was 4 weeks for both groups. The 16S rDNA sequencing technique was used for sequencing of fecal flora, and the two groups were measured in terms of the changes in liver function [alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), and albumin (Alb)], endotoxin (ET), levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and T lymphocyte subsets (CD3 + T, CD4 + T, CD8 + T, and CD4 + /CD8 + ) after treatment. For normally distributed continuous data with homogeneity of variance, the paired t -test was used for comparison within each group, and the independent samples t -test was used for comparison between two groups; the Wilcoxon rank-sum test was used for comparison of non-normally distributed continuous data. The chi-square test was used for comparison of categorical data. Results The observation group had a significantly higher overall response rate than the control group (87.5% vs 60.6%, χ 2 =-2.299, P =0.022). After treatment, both groups had significant reductions in ALT, AST, and TBil and a significant increase in Alb (all P 0.05). Conclusion Jiedu Huayu Tongfu prescription, in combination with etiological and basic treatments, can alleviate clinical symptoms, reduce liver injury, and improve cellular immune function in patients with hepatitis B cirrhosis with liver-gallbladder damp-heat syndrome. Jiedu Huayu Tongfu prescription can improve the imbalance of intestinal flora by increasing the abundance of the probiotic bacteria such as Firmicutes, Lactobacillus, and Bifidobacterium and the pathogenic bacteria such as Bacteroidetes and Cercozoa, and its effect in further improving liver and immune function may be associated with the regulation of intestinal microecology.

Effect of Modified Da Chaihutang on FXR/FGF15/FGFR4 Signaling Pathway in Mice with Cholesterol Gallstone Differentiated into Damp-Heat Syndrome / 中国实验方剂学杂志

ObjectiveTo observe the effect of modified Da Chaihutang on cholesterol gallstone （CS） in mice due to damp-heat based on the farnesoid X receptor （FXR）/fibroblast growth factor 15 （FGF15）/fibroblast growth factor receptor 4 （FGFR4） pathway and explore the molecular biological mechanisms of CS differentiated into damp-heat syndrome from the perspective of correspondence between prescription and syndrome. MethodForty-eight six-week-old mice were randomly divided into the blank group， model group， modified Da Chaihutang （23.4 g·kg-1） group， and ursodeoxycholic acid （0.12 g·kg-1） group， with 12 mice in each group. The ones in the latter three groups were exposed to "internal dampness + external dampness + high-cholesterol diet" for 12 weeks for inducing CS due to damp-heat. Mice in the modified Da Chaihutang group and ursodeoxycholic acid group were gavaged with the corresponding drugs， while those in the model and blank groups with the same amount of normal saline for a total of four weeks. Before and after modeling， mice in each group were subjected to open field tests for determining their activities and mental states. Such general conditions as body mass， food intake， fur， and urine and stool of mice in each group were observed and recorded weekly for judging the damp-heat syndrome. After the intervention， the sampled liver and gallbladder tissues of mice in each group were stained with hematoxylin-eosin （HE） staining， and the serum γ-glutamyltransferase （GGT）， alkaline phosphatase （ALP）， and total bilirubin （TBIL） were determined. The total cholesterol （TC） and total bile acid （TBA） contents in bile were measured by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expression levels of FXR， FGF15， FGFR4， and cholesterol 7α-hydroxylase gene （CYP7A1） were assayed by real-time fluorescence quantitative polynucleotide chain reaction （Real-time PCR） and Western blot. ResultCompared with the blank group， the model group exhibited enlarged gallbladder， brown turbid bile with flocculent precipitation visible to the naked eye， obvious damp-heat syndrome， lipoid degeneration in the liver tissue， rough and thickened gallbladder wall， elevated ALP， GGT， and TBIL in serum （P<0.01） and TC in bile （P<0.01）， reduced TBA （P<0.01）， up-regulated FXR， FGF15， and FGFR4 mRNA and protein expression in ileum （P<0.05， P<0.01）， and down-regulated CYP7A1 mRNA and protein expression （P<0.01）. Compared with the model group， the two medication groups displayed improved bile turbidity， and the bile in the modified Da Chaihutang group became clearer. After intervention， the damp-heat syndrome of mice in the modified Da Chaihutang group was significantly alleviated. The liver and gallbladder lesions of mice in the two medication groups were significantly relieved， manifested as reduced serum ALP， GGT， and TBIL （P<0.01）. The reduction in ALP and TBIL of the modified Da Chaihutang group was more significant （P<0.01）. The TC contents in the bile of mice from the two medication groups were significantly lowered， whereas the TBA contents were elevated （P<0.01）， with more significant changes present in the modified Da Chaihutang group （P<0.01）. The mRNA and protein expression levels of FXR， FGF15， and FGFR4 in the modified Da Chaihutang group were down-regulated （P<0.05， P<0.01）， while the mRNA and protein expression levels of CYP7A1 rose （P<0.05）， except that the elevation in FGF15 and FGFR4 protein expression and reduction in CYP7A1 protein expression were not significant. The mRNA and protein expression levels of FXR， FGF15， and FGFR4 in the ursodeoxycholic acid group all decreased， among which the reduction in FXR was remarkable （P<0.05）， and the mRNA and protein expression levels of CYP7A1 were significantly up-regulated （P<0.05）. ConclusionModified Da Chaihutang significantly improves the stone， liver function， bile composition， abnormal cholesterol-bile acid metabolism， and damp-heat syndrome in the model mice of CS differentiated into damp-heat syndrome， which may be related to its regulation of key factors FXR， FGF15， FGFR4， and CYP7A1 mRNA and protein expression in the cholesterol-bile acid metabolism pathway.

Gender differences in Damp-Heat Syndrome: A review.

Gender differences have important biological significance for medical research. In this study, a bias towards males was identified in animal experiments of Damp-Heat Syndrome in traditional Chinese medicine, as was first proposed by a data mining method. Combined with the correlation between Damp-Heat Syndrome in traditional Chinese medicine and Gender differences, it was considered that Gender-related factors have a significant influence on the development of Damp-Heat Syndrome in traditional Chinese medicine. However, most traditional Chinese medicine studies ignore the key significance of Gender-related factors. This study emphasises that the development of modern traditional Chinese medicine research needs to pay full attention to the biological significance of Gender-related factors and to apply this concept to the research on the Gender equivalence strategy in basic research and the practice of personalised medical diagnosis and clinical treatment.

Integrated analyses of miRNA and mRNA profiles in leukocytes and serums in traditional Chinese medicine (TCM)-defined Pi-qi-deficiency syndrome and Pi-wei damp-heat syndrome resulting from chronic atrophic gastritis.

BACKGROUND: To investigate the microRNA (miRNA)-gene interactions underlying leukocyte functions and characteristics, especially the potential serum biomarkers, implicated in the traditional Chinese medicine (TCM)-defined Pi-qi-deficiency syndrome (PQDS) and Pi-wei damp-heat syndrome (PDHS) resulting from chronic atrophic gastritis (CAG). METHODS: Using RNA/miRNA-sequencing approach, compared with healthy control population, we identified the PDHS- or PQDS-specific miRNAs and genes in leukocytes or serums, especially the Zheng (syndrome)-specific miRNA-gene interactions, and further decoded their functions and pathways. RESULTS: Despite being the TCM-defined Zhengs resulting from the same disease of CAG, the Zheng-specific genes and miRNAs were not same. The PDHS-specific leukocyte genes were mainly involved in defense and immune responses, including NOD-like receptor signaling and several synapses-related pathways. The expression upregulation of PDHS-specific genes enriched in the neutrophil degranulation pathway, indicated the enhanced leukocyte degranulation activation. The PQDS-specific genes in leukocytes were implicated in inflammatory response, extracellular matrix (ECM) organization and collagen catabolism. They could be enriched in MAPK and IL17 signaling and helper T cell differentiation pathways, especially the pathways associated with cell-to-cell adhesion/junction and communication such as cell adhesion molecules, ECM organization and ECM-receptor interaction, probably contributing to the characteristics and functions of leukocytes. Also, the experimentally-supported miRNA-gene interactions, concerned with COL4A2, COL26A1, SPP1 and PROCR, were implicated in the regulation of pathways related to cell-to-cell adhesion/junction and communication, suggesting the potential roles of the PQDS-specific miRNA-gene interactions for the characteristic and functional changes of leukocytes. Interestingly, the PQDS-specific miRNAs in the serums and the corresponding leukocytes, seemed to have the common roles in contributing to the characteristics and functions of leukocytes. Importantly, the hsa-miR-122-5p could be a potential biomarker, capable of being contained and carried in plasma exosomes and much higher expression in both the leukocytes and corresponding serums in the CAG patients with PQDS rather than PDHS. CONCLUSIONS: These results may provide new insights into the characteristic and functional changes of leukocytes in the two Zhengs, PDHS and PQDS, especially the miRNA-mediated gene regulation underlying leukocyte characteristics and functions, with potential leukocyte and serum biomarkers for future application in integrative medicine. Trial registration ClinicalTrials.gov, NCT02915393. Registered on September 17, 2016.

Efficacy and Mechanism of Qingre Lishi Prescription in Treating Children with Acute Bacterial Lower Urinary Tract Infection of Bladder Damp-heat Syndrome / 中国实验方剂学杂志

Objective:To observe the efficacy of Qingre Lishi prescription in treating children with acute bacterial lower urinary tract infection of bladder damp-heat syndrome， and to explore its mechanism of action. Method:Eighty children with acute bacterial lower urinary tract infection of late bladder damp-heat syndrome who were admitted to the Affiliated Hospital of Changchun University of Chinese Medicine were divided into control group and observation group， 40 cases in each group. Patients in control group were given Bazhengsan for oral treatment on basis of basic treatment， while patients in observation group were given Qingre Lishi prescription for oral administration plus external washing treatment. After two weeks of treatment， the clinical and etiological effect， traditional Chinese medicine （TCM） syndrome scores， antipyretic time and urinary negative time， adverse reactions， and urine pathogens （<italic>Escherichia coli， Enterococcus faecalis， Strange proteus， Klebsiella pneumoniae</italic>）， serum inflammatory factor indicators ［tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， interleukin-6 （IL-6）， interleukin-8 （IL-8）， calcium lowering PCT， white blood cell count （WBC） and serum C-reactive protein （CRP）］， immune function indicators ［T cell subsets （CD3⁺， CD4⁺， CD8⁺） and complement （C3， C4）］ were comapred between two groups. Result:The clinical efficacy of observation group was 92.50% （37/40）， which was significantly higher than 65.00% （26/40） in control group （<italic>χ²</italic>=9.038， <italic>P</italic><0.01）， the etiological efficacy of observation group was 87.50% （35/40）， which was significantly higher than 60.00% （24/40） in control group （<italic>χ²</italic>=7.813， <italic>P</italic><0.01）. After treatment， the scores of TCM syndromes of the two groups were significantly reduced （<italic>P</italic><0.05）. The scores of fever， frequent urination， urgent urination， painful urination， difficulty urinating and abdominal pain in two groups were significantly lower than those before treatment （<italic>P</italic><0.05）， and the TCM syndrome scores in observation group were lower than those in control group （<italic>P</italic><0.05）， the antipyretic time and urinary bacteria turning negative time of observation group were significantly lower than those in control group （<italic>P</italic><0.05）， the <italic>Escherichia coli， Enterococcus faecalis， Proteus mirabilis， Klebsiella pneumoniae</italic> pathogenic bacteria detected in both groups were both significantly lower than those before treatment （<italic>P</italic><0.05）. After treatment， the levels of inflammatory factors such as TNF-<italic>α</italic>， IL-6， IL-8， PCT， WBC and CRP in two groups were significantly lower than those before treatment （<italic>P</italic><0.05）， the immune function of the two groups was significantly improved， and the levels of CD3⁺， CD4⁺， C3， and C4 in observation group were higher than those in control group（<italic>P</italic><0.05）， and the CD8⁺ level was lower than that in control group （<italic>P</italic><0.05）. The incidence of adverse reactions had no significant difference between two groups. Conclusion:Qingre Lishi prescription has good clinical effect in treating children with acute bacterial lower urinary tract infection with bladder damp-heat syndrome. It can improve TCM syndromes and clinical symptoms. Its mechanism is related to inhibiting pathogenic bacteria， reducing inflammation， and improving immune function， and it has good security.

Berberine Attenuates Arterial Plaque Formation in Atherosclerotic Rats with Damp-Heat Syndrome via Regulating Autophagy.

PURPOSE: Berberine (BBR) is an effective component of Huanglian and has shown to attenuate atherosclerosis (AS); however, the detailed mechanism of BBR-mediated protective actions against AS remains elusive. This study was undertaken to examine the effects of BBR on aortic atherosclerotic plaque stability and the expression of autophagy-related proteins in AS rats with damp-heat syndrome or yang deficiency. METHODS: Thirty SD rats were randomly divided into (1) control (CON); (2) damp-heat syndrome atherosclerosis (AS + DH); (3) yang deficiency syndrome atherosclerosis (AS + YX); (4) damp-heat syndrome atherosclerosis + BBR (AS + DH + BBR); (5) yang deficiency syndrome, atherosclerosis + BBR (AS + YX + BBR); and (6) damp-heat syndrome, atherosclerosis + BBR + 3-methyladenine (AS + DH + BBR + 3-MA) (n = 5/group) groups. Pathological morphology, macrophage plaque infiltration, inflammation, and LC3-II and P62 expression were assessed. RESULTS: Compared with the CON group, the AS + DH and AS + YX groups had an increased plaque area in the aortic tissue with substantial foam cell and macrophage infiltration, and increased levels of IL-1ß and TNF-α (P < 0.01). After four weeks of BBR intervention, the plaque area in the AS + DH + BBR group was reduced with decreased foam cells and macrophage infiltration, and decreased levels of TNF-α and IL-1ß, whereas LC3-II protein expression was increased and P62 protein expression was decreased in the AS + DH + BBR group when compared to AS + DH group. In addition, the AS + DH + BBR + 3-MA group exhibited a significantly enlarged plaque, substantial foam cell and macrophage infiltration, increased levels of IL-1ß and TNF-α, and decreased LC3-II and P62 (P < 0.01) expression when compared to the AS + DH + BBR group. CONCLUSION: Our results indicated that the BBR could inhibit arterial plaque formation and alleviate the inflammatory response in the aortic tissues in the AS rats with damp-heat syndrome possibly via promoting autophagy. The molecular mechanisms of BBR-mediated protective effects in this animal model still require further investigation.

[Differences in intestinal absorption of Gegen Qinlian Decoction between normal rats and rats with large intestinal damp-heat syndrome].

The study aimed to compare the difference in intestinal absorption of the components of Gegen Qinlian Decoction between normal rats and those with large intestinal damp-heat syndrome in the pathological state, in order to explore the rational application of Gegen Qinlian Decoction in the treatment of large intestinal damp-heat syndrome. Puerarin, daidzin, liquiritin, scutellarin, baicalin, wogonoside, coptisine, jatrorrhizine, berberine and palmatine were used as the detection indexes in the in vitro everted gut sacs absorption experiment. The cumulative absorption amount(Q/µg) and the absorption rate(K_a) of each component in each intestine segment were calculated and compared. It was found that the absorption of each component in different intestinal segments were linear absorption, with R~2 greater than 0.9, which conformed to the zero-order absorption rate. There were differences between normal rats and model rats in the absorption of the components in Gegen Qinlian Decoction with the same concentration. Intestinal absorption of most components of Gegen Qinlian Decoction in the model of large intestinal damp-heat syndrome increased to some extent. The components of Gegen Qinlian Decoction with the concentration of 200 g·L~(-1) had the highest absorption in the jejunum of the model rats, and the absorption in the ileum, duodenum and colon successively decreased except daidzin and baicalin. In terms of the absorption rate constant, the absorption in the duodenum and jejunum were significantly increased(P<0.01) compared with normal rats, and the absorption in the ileum was significantly decreased(P<0.01) compared with normal rats. In addition, the absorption of puerarin, daidzin, glycyrrhizin, coptisine and berberine increased selectivity in the colon. Therefore, pathological model animals were recommended in the study of the components relating to absorption effect, in order to really lay a research foundation for the symptomatic treatment of large intestinal damp-heat syndrome.

Screening and bioinformatic analysis of serum microRNA expression profiles in patients with Pi-deficiency syndrome / 第二军医大学学报

ObjectiveTo explore the pathogenesis and syndrome differentiation of Pi-deficiency syndrome (PDS) from microRNA (miRNA) levels through screening and bioinformatic analysis of serum miRNA expression in PDS patients. MethodsFour hyperlipemia patients with PDS, 4 hyperlipemia patients with Pi-Wei damp-heat syndrome (PWDS) and 5 healthy volunteers were recruited. Their serum RNA was used in miRNA quantitative PCR array experiment. Serum miRNA expression profiles in PDS patients were screened to perform bioinformatic analysis. ResultsNine candidate miRNAs (6 upregulated and 3 downregulated) were screened from PDS patients. These miRNAs were able to clearly distinguish among PDS patients, PWDS patients and healthy volunteers. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed 83 target genes controlled by 6 up-regulated miRNAs were significantly enriched in 7 pathways, which were mainly involved in cytokine-cytokine receptor interaction, pathogens of infectious diseases, immune/inflammatory-related signaling pathway and pancreatic cancer; and 365 target genes controlled by 3 down-regulated miRNAs were significantly enriched in 5 pathways, which were mainly involved in signaling pathways of neurotrophin and phosphatidylinositol, RNA transport, and metabolisms of inositol phosphate and amino acid. ConclusionOur findings provide potential miRNA biomarkers for clinical syndrome differentiation of PDS patients, as well as information for understanding and studying the pathogenesis of PDS patients.

Evaluation of Different Effect of Pith-nodecayed and Pith-decayed Products of Scutellariae Radix on Large Intestine Damp-heat Syndrome Based on Pharmacological Indexes / 中国实验方剂学杂志

Objective::To compare the pharmacodynamic effects of pith-nodecayed and pith-decayed products of Scutellariae Radix on rats with large intestine damp-heat syndrome, and to demonstrate the scientificness of dividing Scutellariae Radix into pith-nodecayed and pith-decayed products as medicines by modern pharmacological test. Method::Rats were randomly divided into blank group, model group, low-and high-dose group of pith-nodecayed products (0.9, 3.6 g·kg-1), low-and high-dose group of pith-decayed products (0.9, 3.6 g·kg-1), Scutellariae Radix group (0.9 g·kg-1), compound berberine tablets group (positive drug group, 0.045 g·kg-1), and 8 rats in each group. Taking model rats with large intestine damp-heat syndrome, the body temperature, thymus index, spleen index, pathological sections of colon and ileum, inflammatory factors and Secretory immunoglobulin (SIg) A content were selected as indexes to evaluate the therapeutic effect of pith-nodecayed and pith-decayed products on large intestine damp-heat syndrome, and make comprehensive evaluation of the difference in efficacy between them. Partial least squares-discriminant analysis (PLS-DA) was employed to analyze the pharmacological indexes of these two products against large intestine damp-heat syndrome. Result::Pith-nodecayed and pith-decayed products of Scutellariae Radix with different doses could reduce the body temperature, thymus index, spleen index, contents of interleukin (IL)-2, IL-6, IL-1β in serum and SIgA content in intestinal mucosa, and most of them had significant differences (P<0.05, P<0.01). Compared with the isodose group of pith-decayed products, the effect of corresponding dose group of pith-nodecayed products was better, and most of them had significant differences (P<0.05, P<0.01). PLS-DA results indicated that there were significant differences in the pharmacological effects of pith-nodecayed and pith-decayed products, and they were clustered on one side, respectively. Conclusion::Both of pith-nodecayed and pith-decayed products of Scutellariae Radix have therapeutic effect on large intestine damp-heat syndrome with distinctly different strength of action, and pith-nodecayed products is superior to pith-decayed products, which verify the scientific nature of pith-nodecayed products was specializedly used to treat bowel disease in ancient times.

Advances Research on Mechanism of Traditional Chinese MedicineAgainst Damp-heat Syndrome / 中国实验方剂学杂志

By summarizing the existing researches on the prevention and treatment of damp-heat syndrome with traditional Chinese medicine（TCM）， this paper digitized the TCM syndromes of damp-heat syndrome， enriched the TCM theories， and provided reference for the prevention and treatment of damp-heat syndrome and provided innovative ideas for the research of damp-heat syndrome. Damp-heat syndrome can cause pathological changes all over the body. It is easy to induce a variety of acute febrile diseases， including ulcerative colitis， chronic gastritis， chronic hepatitis B， chronic kidney disease， irritable bowel syndrome， pneumonia， cough and asthma， which seriously affects the health of the body. Damp-heat syndrome can be divided into syndrome of damp-heat in upper Jiao， syndrome of damp-heat in middle Jiao and syndrome of damp-heat in lower Jiao .At the same time it can be divided into damp-heat obstructing the lung， large intestine damp-heat， damp-heat containing the spleen， liver-gallbladder damp-heat， and bladder damp-heat syndrome ， nephritic damp-heat syndrome by simulating clinical pathogeny according to basic theories of TCM. Heat-clearing and dampness-expelling is the treatment principle of damp-heat syndrome. TCM achieves the purpose of treating damp-heat syndrome by virtue of its anti-inflammation， anti-oxidation， regulation of intestinal flora， lowering blood glucose， lowering blood lipid and enhancing immunity. Based on the two aspects of TCM and prescription， the application， mainly in the syndrome differentiation of triple energizer differentiation， visceral syndrome differentiation is complementary， pharmacological action and mechanism of TCM in prevention and treatment of damp heat syndrome are reviewed in detail， which is a concrete analysis and discussion， in order to provide relevant experience for data integration and system construction of damp heat syndrome， and provide new ideas and help for follow-up clinical drug research and development and optimization innovation of damp heat syndrome. At present， the material basis and relevant prevention and treatment mechanism of dampness-heat syndrome are still not clear in China， so it is necessary to further study and explore its potential biological diagnostic markers， strengthen the research and development of new Chinese medicine preparation against dampness-heat， and elaborate the mechanism of action of Chinese medicine on damp-heat syndrome in multiple ways and directions.

Clinical Efficacy of Changyanqing Mixture on Chronic Recurrent Ulcerative Colitis with Damp-heat Ayndrome of Large Intestine / 中国实验方剂学杂志

Objective::To observe the clinical efficacy of Changyanqing mixture on chronic recurrent ulcerative colitis (UC) with damp-heat syndrome of large intestine and the effect on the recurrence of disease, in order to discuss the mechanism of action in terms of the neuro-endocrine-immune inflammation network. Method::One hundred and twelve patients were randomly divided into control group (55 cases) and observation group (57 cases) by random number table. Patients in control group got mesalazine slow release tablets, 0.1 g/time, 4 times/days, and those the score of Mayo≥7 were added with prednisone acetate tablets, 0.75 mg·kg-1·d-1, and bifidobacterium viable powder with warm water after dinner, 1 pack/day, 2 times/days. In addition to the therapy of control group, patients in observation group were also given Changyanqing mixture in the morning and evening, 1 pack/day. A course of treatment was 6 weeks, and patients got further consultation once a week. During the remission stage, patients in both groups got mesalazine slow release tablets, 0.5 g/time, 3 times/days, and patients in observation group were added with Changyanqing mixture until the score of damp-heat syndrome of large intestine reduced by more than 90%. The number of patients entering the remission stage of 6 weeks and the time of remission stage were recorded. Before and after treatment, colonoscopy was detected, and Geboes index, Baron, damp-heat syndrome of large intestine and Mayo were scored. And levels of peripheral blood interleukin-6 (IL-6), IL-8, IL-10, IL-17, vasoactive intestinal peptide (VIP), motilin (MTL) and neuropeptide (NPY) were detected, and relapse at the 24-week follow-up was recorded. Result::After the 6-week treatment, the clinical efficacy in observation group was 100%, which was higher than 89.09%in control group (P<0.05). And the healing rate of mucosa was 96.4%, which was higher than 81.82%in control group (P<0.05). And the response rate in two groups was 100%. At the 6th month after the treatment, the clinical remission rate in observation group was 91.23%, which was higher than 76.36%in control group (χ2=4.581, P<0.05). And the average remission time was shorter than that in control group (P<0.01). After treatment, scores of colonic mucosa, Geboes index, colonic mucosa and Mayo were all lower than those in control group (P<0.01). And levels of IL-6, IL-8, IL-17, VIP and MTL were lower than those in control group (P<0.01), while levels of IL-10 and NPY were higher than those in control group (P<0.01). The relapse rate in observation group was 17.54%, which was lower than 38.18%in control group (χ2=5.955, P<0.05). And the mean recurrence time was longer than that in control group (P<0.01). Conclusion::In addition to the routine western medicine therapy, Changyanqing mixture can alleviate the condition of patients by shortening the course of the disease, reducing the recurrence rate, delaying the recurrence time, and regulating the nerve-endocrine-immune inflammation network.

Network pharmacology based investigation into the effect and mechanism of Modified Sijunzi Decoction against the subtypes of chronic atrophic gastritis.

Chronic atrophic gastritis (CAG) is a common digestive disease without specific treatment. According to syndrome differentiation, traditional Chinese medicine (TCM) classified it into different syndromes and has achieved significant therapeutic effects. In this study, immune repertoire sequencing techniques combined with symptom scores, electronic gastroscopy as well as pathologic changes were used to evaluate the effect and the underlying mechanism of Modified Sijunzi Decoction (MSD) in treating CAG. The results showed that MSD could relieve CAG symptoms, improve pathologic changes in CAG with fatigue and tiredness symptom, but with no help in CAG with reversal heat symptom. Moreover, MSD could regulate immune disorders in CAG with fatigue and tiredness symptom, and 7 TCR biomarkers were explored in CAG patients with immune disorders. All these results indicated that MSD is effective in treating CAG patients with fatigue and tiredness symptom by tonifying the spleen qi, suggesting that CAG treatment based on syndrome differentiation is reasonable.