The effect of Huanglian Jiedu Decoction on the intestinal flora of type 2 diabetes mellitus(T2DM) was investigated using 16S rRNA sequencing technology. Sixty rats were randomly divided into a normal group(10 rats) and a modeling group(50 rats). After one week of adaptive feeding, a high-fat diet + streptozotocin was given for modeling, and fasting blood glucose >16.7 mmol·L~(-1) was considered a sign of successful modeling. The modeling group was randomly divided into the model group, high-, medium-, and low-dose groups of Huanglian Jiedu Decoction, and metformin group. After seven days of intragastric treatment, the feces, colon, and pancreatic tissue of each group of rats were collected, and the pathological changes of the colon and pancreatic tissue of each group were observed by hematoxylin-eosin staining. The changes in the intestinal flora structure of each group were observed by the 16S rRNA sequencing method. The results showed that compared with the model group, the high-, medium-, and low-dose of Huanglian Jiedu Decoction reduced fasting blood glucose levels to different degrees and showed no significant changes in body weight. The number of islet cells increased, and intestinal mucosal damage attenuated. Alpha diversity analysis revealed that Huanglian Jiedu Decoction reduced the abundance and diversity of intestinal flora in rats with T2DM; at the phylum level, low-and mediam-dose of Huanglian Jiedu Decoction reduced the abundance of Bacteroidota, Proteobacteria, and Desulfobacterota and increased the abundance of Firmicute and Bacteroidota/Firmicutes, while the high-dose of Huanglian Jiedu Decoction increased the relative abundance of Proteobacteria and Bacteroidota/Firmicutes ratio, and decreaseal the relative; abundance of Firmicute; at the genus level, Huanglian Jiedu Decoction increased the relative abundance of Allobaculum, Blautia, and Lactobacillus; LEfse analysis revealed that the biomarker of low-and medium-dose groups of Huanglian Jiedu Decoction was Lactobacillus, and the structure of the intestinal flora of the low-dose group of Huanglian Jiedu Decoction was highly similar to that of the metformin group. PICRUSt2 function prediction revealed that Huanglian Jiedu Decoction mainly affected carbohydrate and amino acid metabolic pathways. It suggested that Huanglian Jiedu Decoction could reduce fasting blood glucose and increase the number of islet cells in rats with T2DM, and its mechanism of action may be related to increasing the abundance of short-chain fatty acid-producing strains and Lactobacillus and affecting carbohydrate and amino acid metabolic pathways.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Diabetes Mellitus, Type 2/metabolism* ; Gastrointestinal Microbiome/drug effects* ; Rats ; Male ; Rats, Sprague-Dawley ; Humans ; Bacteria/drug effects* ; Blood Glucose/metabolism*

Sucrose synthase plays a crucial role in the plant sugar metabolism pathway by catalyzing the production of uridine diphosphate (UDP)-glucose, which serves as a bioactive glycosyl donor for various metabolic processes. In this study, a sucrose synthase gene named CtSus was cloned from Cistanche tubulosa, a traditional Chinese medicine known for its rich content of diverse glycosides, based on transcriptome analysis results. The open reading frame of CtSus was 2 418 bp long encoding 805 amino acids. Sequence analysis revealed conserved domains associated with the plant sucrose synthase family at both the N-terminal and C-terminal regions of CtSus protein. Phylogenetic analysis demonstrated that CtSus shares a close evolutionary relationship with sucrose synthases from other plants within the same order. Functional identification of CtSus was performed through whole-cell catalysis coupling with UGT71BD1, a characterized glycosyltransferase enzyme. The results showed that the introduction of CtSus significantly enhanced the conversion rate of glycosylation catalyzed by UGT71BD1. The soluble expression of CtSus in Escherichia coli was further achieved using the pCold^TM TF expression vector. In vitro enzymatic assay indicated the activity of CtSus to catalyze the formation of UDP-glucose in the presence of sucrose and UDP. Real-time quantitative-polymerase chain reaction results showed that the expression patterns of CtSus gene in different parts of C. tubulosa and the suspension cell cultures of C. tubulosa under drought stress correlated with the accumulation patterns observed for phenylethanol glycosides, respectively. Furthermore, key amino acids and their interactions between enzyme and substrate were explored based on protein structure prediction and molecular docking results. Overall, our findings identify a sucrose synthase CtSus responsible for the supply of the active glycosyl donor UDP-glucose during the biosynthesis of glycoside products in C. tubulosa and have also provided a gene element that can be utilized in engineering strain construction for glycoside products production.

Objective To investigate the correlation between qi stagnation and phlegm stasis syndrome in the young and middle-aged population and the detection rate of thyroid nodules.Methods The clinical data of those who participated in the questionnaire survey and took thyroid ultrasonography at Dongguan Hospital of Guangzhou University of Chinese Medicine from June 1 to December 1,2023 were collected.The clinical information covered age,gender,family history,body mass index(BMI),related symptoms,and ultrasonographic findings.And then the related data were statistically analyzed.Results(1)The clinical data of 196 cases were collected,of which 65 cases(33.16%)suffered from thyroid nodules,50 cases(25.51%)were differentiated as qi stagnation and phlegm stasis syndrome,53 cases(27.04%)had qi depression constitution of traditional Chinese medicine(TCM),55 cases(28.06%)had blood stasis constitution,and 48 cases(24.49%)had phlegm-dampness constitution.(2)The results of univariate analysis showed that the relevant factors for thyroid nodules included female,family history,qi stagnation and phlegm stasis syndrome,qi depression constitution,blood stasis constitution,phlegm-dampness constitution,dizziness and headache,neck stiffness,swallowing discomfort,lump on the surface of the body,dysmenorrhea and amenorrhea,tightness in the chest,distending pain in hypochondrium,depressed in spirits,emotional vulnerability,distending pain in breast,gloomy complexion,darkish lips,dark circles around the eyes,heaviness of the body,eyelid edema,and profuse sputum,and the differences were all statistically significant(P＜0.05 or P＜0.01).(3)Multivariate Logistic regression analysis was performed on the basis of univariate analysis,and the results showed that qi stagnation and phlegm stasis syndrome(OR:4.03,95%CI:1.85-8.77),phlegm-dampness constitution(OR:4.68,95%CI:2.06-10.63),and lump on the surface of the body(OR:2.97,95%CI:1.11-7.95)were the influencing factors for thyroid nodules.(4)A prediction model for detecting thyroid nodules was constructed:logit(P)=-1.607+1.39×qi stagnation and phlegm stasis syndrome(0 expressing absence,1 expressing presence)+1.54×phlegm-dampness constitution(0 expressing absence,1 expressing presence)+1.09×lump on the surface of the body(0 expressing absence,1 expressing presence).The model was evaluated by using the receiver operating characteristic(ROC)curve,and the area under the curve(AUC)was 0.75(95%CI:0.67-0.83,P＜0.001).Conclusion In the young and middle-aged population,qi stagnation and phlegm stasis are the risk factors for the detectable rate of thyroid nodules.The early identification,risk prediction and timely intervention for the population with qi stagnation and phlegm stasis will be helpful for the prevention and treatment of thyroid nodules.

As a biocatalyst, enzyme has the advantages of high catalytic efficiency, strong reaction selectivity, specific target products, mild reaction conditions, and environmental friendliness, and serves as an important tool for the synthesis of complex organic molecules. With the continuous development of gene sequencing technology, molecular biology, genetic manipulation, and other technologies, the diversity of enzymes increases steadily and the reactions that can be catalyzed are also gradually diversified. In the process of enzyme-catalyzed synthesis, the majority of common enzymatic reactions can be achieved by single enzyme catalysis, while many complex reactions often require the participation of two or more enzymes. Therefore, the combination of multiple enzymes together to construct the multi-enzyme cascade reactions has become a research hotspot in the field of biochemistry. Nowadays, the biosynthetic pathways of more natural products with complex structures have been clarified, and secondary metabolic enzymes with novel catalytic activities have been identified, discovered, and combined in enzymatic synthesis of natural/unnatural molecules with diverse structures. This study summarized a series of examples of multi-enzyme-catalyzed cascades and highlighted the application of cascade catalysis methods in the synthesis of carbohydrates, nucleosides, flavonoids, terpenes, alkaloids, and chiral molecules. Furthermore, the existing problems and solutions of multi-enzyme-catalyzed cascade method were discussed, and the future development direction was prospected.
Biological Products/chemistry* ; Catalysis ; Alkaloids ; Biocatalysis

This study aimed to investigate the effective substances and mechanism of Yishen Guluo Mixture in the treatment of chronic glomerulonephritis(CGN) based on metabolomics and serum pharmacochemistry. The rat model of CGN was induced by cationic bovine serum albumin(C-BSA). After intragastric administration of Yishen Guluo Mixture, the biochemical indexes related to renal function(24-hour urinary protein, serum urea nitrogen, and creatinine) were determined, and the efficacy evaluations such as histopathological observation were carried out. The serum biomarkers of Yishen Guluo Mixture in the treatment of CGN were screened out by ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry(UPLC-Q-TOF-MS) combined with multivariate statistical analysis, and the metabolic pathways were analyzed. According to the mass spectrum ion fragment information and metabolic pathway, the components absorbed into the blood(prototypes and metabolites) from Yishen Guluo Mixture were identified and analyzed by using PeakView 1.2 and MetabolitePilot 2.0.4. By integrating metabolomics and serum pharmacochemistry data, a mathematical model of correlation analysis between serum biomarkers and components absorbed into blood was constructed to screen out the potential effective substances of Yishen Guluo Mixture in the treatment of CGN. Yishen Guluo mixture significantly decreased the levels of 24-hour urinary protein, serum urea nitrogen, and creatinine in rats with CGN, and improved the pathological damage of the kidney tissue. Twenty serum biomarkers of Yishen Guluo Mixture in the treatment of CGN, such as arachidonic acid and lysophosphatidylcholine, were screened out, involving arachidonic acid metabolism, glycerol phosphatide metabolism, and other pathways. Based on the serum pharmacochemistry, 8 prototype components and 20 metabolites in the serum-containing Yishen Guluo Mixture were identified. According to the metabolomics and correlation analysis of serum pharmacochemistry, 12 compounds such as genistein absorbed into the blood from Yishen Guluo Mixture were selected as the potential effective substances for the treatment of CGN. Based on metabolomics and serum pharmacochemistry, the effective substances and mechanism of Yishen Guluo Mixture in the treatment of CGN are analyzed and explained in this study, which provides a new idea for the development of innovative traditional Chinese medicine for the treatment of CGN.
Animals ; Rats ; Arachidonic Acid ; Biomarkers/blood* ; Blood Proteins ; Chromatography, High Pressure Liquid ; Creatinine ; Drugs, Chinese Herbal/therapeutic use* ; Glomerulonephritis/metabolism* ; Metabolomics ; Urea ; Chronic Disease ; Disease Models, Animal ; Complex Mixtures/therapeutic use*

In this study, we investigated the anti-osteoporotic activity and mechanism of action of extract of Panax quiquefolium L. based on zebrafish model combined with metabolomics technology. A zebrafish model of prednisolone-induced osteoporosis was used to compare the anti-osteoporotic activity of Panax quiquefolium L., and the expression of osteoblast-associated genes and osteoclast-associated genes in zebrafish was detected by quantitative real-time PCR (qRT-PCR), using bone fluorescence area and fluorescence density as evaluation indexes. Metabolomics based on ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) was used to explore the change patterns of biomarkers and the metabolic pathways affected. The results showed that the 50% ethanol extracts of Panax quiquefolium L. from Jilin, Canada, Wenden and the United States can significantly improve the bone fluorescence area of zebrafish compared with model group. Furthermore, four sources 50% ethanol extracts of Panax quiquefolium L. except United States also can significantly improve the bone fluorescence density of zebrafish. In addition, PCR showed that extract of Panax quiquefolium L. can significantly up-regulated the expression of vitamin D receptor b (vdrb), collagen type I α2 (col1a2) and cysteine-rich acidic secreted protein (sparc) genes, and down-regulated the expression of matrix metalloproteinase 9 (mmp9), anti-tartrase acid phosphatase (trap) and cathepsin K (ctsk) genes. Metabolomic analysis identified 24 key differential metabolites. Furthermore, pathway analysis showed that Panax quiquefolium L. could regulate the levels of 10 key biomarkers by participating in purine metabolism, tricarboxylic acid cycle and pentose phosphate metabolism and improve the osteoporosis status of zebrafish. This study preliminically revealed the anti-osteoporosis mechanism of 50% ethanol extract from Panax quiquefolium L. through multi-component, multi-target and multi-pathway and also provides theoretical basis for clinical development and utilization of anti-osteoporosis products of Panax quiquefolium L. This experiment was approved by the Experimental Animal Welfare Ethics Committee of the Institute of Biology, Shandong Academy of Sciences (approval number: SWS20181002).

Dihydroflavonol 4-reductase (DFR) plays an essential role in the biosynthesis of anthocyanin and regulation of plant flower color. Based on the transcriptome data of Cistanche tubulosa (Schenk) Wight, a full-length cDNA sequence of CtDFR gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR). CtDFR contains an open reading frame (ORF) of 1 263 bp which encodes 420 amino acids with a predicted molecular weight of 47.5 kDa. The sequence analysis showed that CtDFR contains a nicotinamide adenine dinucleotide phosphate (NADPH) binding domain and a specific substrate binding domain. The expression analysis indicated that CtDFR was highly expressed in red and purple flowers, and the relative expression levels were 4.04 and 19.37 times higher than those of white flowers, respectively. The recombinant CtDFR protein was expressed in E.coli BL21 (DE3) using vector pET-28a-CtDFR and was purified. In vitro enzyme activity analysis, CtDFR could reduce three types of dihydroflavonols including dihydrokaempferol, dihydroquercetin, and dihydromyricetin to leucopelargonidin, leucocyanidin and leucodelphinidin. Subcellular localization analysis showed that CtDFR was mainly localized in the cytoplasm. These results demonstrate that CtDFR plays an important role in regulation of flower color in C. tubulosa and make a valuable contribution for the further investigation on the regulation mechanism of C. tubulosa (Schenk) Wight flower color.

Adult ; Aged ; China/epidemiology* ; Cohort Studies ; Female ; Humans ; Male ; Metabolic Syndrome/etiology* ; Middle Aged ; Parks, Recreational/supply & distribution* ; Prevalence ; Residence Characteristics/statistics & numerical data* ; Rural Population/statistics & numerical data* ; Young Adult

The present study investigated the anti-oxidative and anti-apoptotic effects and molecular mechanisms of catalpol on the H_2O_2-induced pancreatic β-cells(INS-1 cells).The oxidative damage model of INS-1 cells was induced and optimized by the stimulation of H_2O_2 of different concentrations for different time.CCK-8 assay was used to detect cell viability after catalpol intervention(1, 5, 10, 20, 40, 80, and 160 μmol·L~(-1)) for 24 h.Intracellular reactive oxygen species(ROS), superoxide dismutase(SOD), and lipid peroxide malondialdehyde(MDA) were measured by DCFH-DA fluorescent probe, WST-1, and TBA respectively.Moreover, the apo-ptotic effect was detected by AO-EB and Annexin V-FITC/PI staining.In addition, the protein expression levels were detected by Wes-tern blot, and intracellular insulin concentration was measured by ELISA.The results showed that the oxidative damage model of INS-1 cells was stably induced by 50 μmol·L~(-1) H_2O_2 treatment for 2 h, and catalpol at 1-80 μmol·L~(-1) did not affect cell viability of INS-1 cells.Compared with the conditions in the model group, 1, 5, and 10 μmol·L~(-1) catalpol intervention for 2 h could protect INS-1 cells from oxidative damage(P<0.001), reduce ROS and MDA, increase SOD, and inhibit excessive cell apoptosis.Moreover, 1, 5, and 10 μmol·L~(-1) catalpol could also up-regulate the phosphorylation of nuclear transcription factor NF-E2 related factors, negatively regulate Kelch-like ECH-associated protein 1(Keap1), phosphorylation of extracellular signal-regulated kinase(ERK), and heme oxyge-nase 1(HO-1), and promote the protein expression of pancreatic-duodenal homeobox factor-1(PDX-1) and glucose transporter 2(GLUT2).In addition, 1, 5, and 10 μmol·L~(-1) catalpol increased insulin secretion of INS-1 cells under oxidative damage in the high-glucose culture medium, indicating function recovery of pancreatic β cells.PDX-1 is a key nuclear transcription factor of pancreatic β cell function that directly regulates GLUT2 and insulin synthesis, and affects glucose homeostasis.In conclusion, catalpol can reduce the oxidative damage and apoptosis of INS-1 cells, activate antioxidant pathway, protect the function of pancreatic β cells, and improve insulin synthesis and secretion.
Apoptosis ; Glucose/metabolism* ; Insulin/metabolism* ; Insulin-Secreting Cells/metabolism* ; Iridoid Glucosides ; Kelch-Like ECH-Associated Protein 1/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Oxidative Stress ; Reactive Oxygen Species/metabolism* ; Superoxide Dismutase/metabolism*