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Carthami Flos, as a traditional blood-activating and stasis-resolving drug, possesses anti-tumor, anti-inflammatory, and immunomodulatory pharmacological activities. Flavonoid glycosides are the main bioactive components in Carthamus tinctorius. Glycosyltransferase deserves to be studied in depth as a downstream modification enzyme in the biosynthesis of active glycoside compounds. This study reported a flavonoid glycosyltransferase CtUGT49 from C. tinctorius based on the transcriptome data, followed by bioinformatic analysis and the investigation of enzymatic properties. The open reading frame(ORF) of the gene was 1 416 bp, encoding 471 amino acid residues with the molecular weight of about 52 kDa. Phylogenetic analysis showed that CtUGT49 belonged to the UGT73 family. According to in vitro enzymatic results, CtUGT49 could catalyze naringenin chalcone to the prunin and choerospondin, and catalyze phloretin to phlorizin and trilobatin, exhibiting good substrate versatility. After the recombinant protein CtUGT49 was obtained by hetero-logous expression and purification, the enzymatic properties of CtUGT49 catalyzing the formation of prunin from naringenin chalcone were investigated. The results showed that the optimal pH value for CtUGT49 catalysis was 7.0, the optimal temperature was 37 ℃, and the highest substrate conversion rate was achieved after 8 h of reaction. The results of enzymatic kinetic parameters showed that the K_m value was 209.90 μmol·L~(-1) and k_(cat) was 48.36 s~(-1) calculated with the method of Michaelis-Menten plot. The discovery of the novel glycosyltransferase CtUGT49 is important for enriching the library of glycosylation tool enzymes and provides a basis for analyzing the glycosylation process of flavonoid glycosides in C. tinctorius.
Assuntos
Carthamus tinctorius/química , Filogenia , Flavonoides/análise , Glicosídeos/análise , Glicosiltransferases/genética , Anti-Inflamatórios , ChalconasRESUMO
Objective:To observe the effect of Tongxie Yaofang on the expressions of colon serotonin transporter (SERT), liver 5-hydroxytryptamine<sub>2A</sub> receptor (5-HT<sub>2A</sub>R) protein, serum 5-HT and inflammatory factors in ulcerative colitis (UC) model rats of liver stagnation and spleen deficiency, in order to explore the basis of syndrome of liver stagnation and spleen deficiency and the intervention mechanism of Tongxie Yaofang. Method:Fifty male SD rats were randomly divided into blank control group, model group, high, medium and low-dose Tongxie Yaofang group (10,5,2.5 g·kg<sup>-1</sup>), and salazosulacil group (0.3 g·kg<sup>-1</sup>). The ulcerative colitis model of liver depression and spleen deficiency was established by 2,4,6-trinitrobenzene sulfonic acid (TNBS)/ethanol solution enema + restraint stress + diet loss. After successful modeling, the samples were collected after 21 days of drug intervention. Htoxylin eosin (HE) staining and oil red staining were used to observe the pathological changes of colon and liver in each group. Serum interleukin-6 (IL-6), IL-9, 5-HT and superoxide dismutase (SOD) were detected by enzyme linked immunosorbent assay (ELISA). Protein expressions of SERT in the colons and 5-HT<sub>2A</sub>R in liver of rats were detected by Western blot. Result:Compared with the normal group, obvious ulcers were formed in the colon and lipid droplets in the liver increased in the model group, serum levels of IL-6, IL-9 and 5-HT in the model group increased, while the level of SOD decreased (<italic>P</italic><0.05). The protein expression of SERT in colon decreased, whereas the protein expression of 5-HT<sub>2A</sub>R in liver increased (<italic>P</italic><0.05). Compare with model group, the pathological damage of colon was improved, and the formation of lipid droplets in liver was reduced in high, medium-dose Tongxie Yaofang groups and sulfasalazine group. The serum levels of IL-6, IL-9 and 5-HT decreased, while the level of SOD increased in Tongxie Yaofang group and sulfasalazine group (<italic>P</italic><0.05). The protein expression of SERT in colon increased in high,low-dose Tongxie Yaofang groups and sulfasalazine group, and the protein expression of 5-HT<sub>2A</sub>R in liver decreased in medium, low dose Tongxie Yaofang groups and sulfasalazine group (<italic>P</italic><0.05). Conclusion:Tongxie Yaofang may reduce the content of 5-HT, and regulate the intestinal motility and sensory system by up-regulating the expression of SERT in the colon, inhibit the expressions of IL-6,IL-9 and other inflammatory factors, and play an anti-inflammatory role, reduce the content of 5-HT and the expression of 5-HT<sub>2A</sub>R in the liver, increase the level of SOD, regulate emotion and lipid metabolism in the liver, and then exert the intervention effect on ulcerative colitis with liver depression and spleen deficiency on the whole.
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Mecicinal plants boast abundant natural compounds with significant pharmacological activity, and such compounds, featuring diversified and complex structures, can be used for research and development of drugs. At present, these natural compounds are directly extracted from herbs which, however, suffer from damaged wild resources and shortage of planting resources attributing to the increasing demand. Moreover, the low content in medicinal plants and complex structures are another challenge to the research and development of drugs. Heterologous synthesis with synthetic biology methods is a solution that has attracted wide attention. Synthetic bio-logy for the production of natural active compounds in Chinese medicinal plants involves the exploration of key enzymes in compound bio-synthetic pathways from plants, analysis of enzyme functions and mechanisms, and reconstruction and optimization of biosynthetic pathways in microorganisms for efficient synthesis of compounds. This study briefed the development process of synthetic biology and the biosynthetic pathways of terpenoids, alkaloids, and flavonoids, and summarized the related strategies of synthetic biology such as the reconstruction and optimization of metabolic pathways, regulation of fermentation process, and strain improvement, and the latest applications of heterogeneous synthetic biology in the production of natural compounds from Chinese medicinals. This study is expected to serve as a reference for the efficient production of terpenoids, alkaloids, flavonoids, and other active compounds from Chinese medicinal plants with strategies of synthetic biology.
Assuntos
Alcaloides , Vias Biossintéticas , China , Plantas Medicinais , Biologia SintéticaRESUMO
Objective:To observe the effect of serum of kidney Yang deficiency rats on the expression of β-catenin,osteoprotegerin(OPG) and nuclear transcription factor-κB receptor activator ligand (RANKL) in the co-culture system and regulatory of icariin on it, and to explore the possible mechanism of inducing osteoporosis.Method:The 16 male SD rats were randomly divided into blank group and model group, 8 rats in each group. 10 mL·kg-1 adenine was administrated to stomach to establish kidney yang deficiency model. Serum was separated and extracted after the model was established successfully. Isolation and culture of osteoblast(OB) and osteoclast(OC) in vitro, OB was observed and identified by alkaline phosphatase(ALP),alizarin red and Giemsa staining, OC was identified by tartrate resistant acid phosphatase(TRAP) staining, OB-OC co-culture system was established in transwell cell, icariin group(100 μmol·L-1), blank group, icariin(100 μmol·L-1) + serum group, serum group and Dickkopf1(DKK-1) drug(100 μg·L-1) were set up in group , 2 days after intervention of co-culture system, OC was counted, ALP and TRAP in supernatant were detected by microplate enzyme labeling, and the expression of OPG,β-catenin and RANKL in each group was detected by Western blot.Result:Compared with blank group, the ALP activity,β-catenin and OPG protein expression in serum group were significant reduction (P<0.05), while the OC quantity, TRAP activity and RANKL protein expression were marked increase (P<0.05). Compared with serum group, ALP activity of icariin group decreased significantly (P<0.01), Compared with icariin group, ALP activity and OPG protein expression decreased (P<0.05), trap activity and RANKL expression increased (P <0.05) in icariin + serum group.Conclusion:The serum of kidney Yang deficiency rats can induce the occurrence of osteoporosis, and the mechanism of action may be through inhibition of ALP activity, down regulating the expression of β-catenin and OPG protein, increasing the activity of TRAP and the expression of RANKL protein.
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<p><b>OBJECTIVE</b>To study the value of 3D visualization technique in breast-preserving surgery for breast cancer with immediate breast reconstruction using laparoscopically harvested pedicled latissimus dorsi muscle flap.</p><p><b>METHODS</b>From January, 2015 to May, 2016, 30 patients with breast cancer underwent breast-preserving surgery with immediate breast reconstruction using pedicled latissimus dorsi muscle flap. The CT data of the arterial phase and venous phase were collected preoperatively and imported into the self-developed medical image 3D visualization system for image segmentation and 3D reconstruction. The 3D models were imported into the simulation surgery platform for virtual surgery to prepare for subsequent surgeries. The cosmetic outcomes of the patients were evaluated 6 months after the surgery. Another 18 patients with breast cancer who underwent laparoscopic latissimus dorsi muscle breast reconstruction without using 3D visualization technique from January to December, 2014 served as the control group. The data of the operative time, intraoperative blood loss and postoperative appearance of the breasts were analyzed.</p><p><b>RESULTS</b>The reconstructed 3D model clearly displayed the anatomical structures of the breast, armpit, latissimus dorsi muscle and vessels and their anatomical relationship in all the 30 cases. Immediate breast reconstruction was performed successfully in all the cases with median operation time of 226 min (range, 210 to 420 min), a median blood loss of 95 mL (range, 73 to 132 mL). Evaluation of the appearance of the breast showed excellent results in 22 cases, good appearance in 6 cases and acceptable appearance in 2 cases. In the control group, the median operation time was 283 min (range, 256 to 313 min) and the median blood loss was 107 mL (range, 79 to 147 mL) with excellent appearance of the breasts in 10 cases, good appearance in 4 cases and acceptable appearance in 4 cases.</p><p><b>CONCLUSION</b>3D reconstruction technique can clearly display the morphology of the latissimus dorsi and the thoracic dorsal artery, allows calculation of the volume of the breast and the latissimus dorsi, and helps in defining the scope of resection of the latissimus dorsi to avoid injuries of the pedicled vessels. This technique also helps to shorten the operation time, reduce intraoperative bleeding, and improve the appearance of the reconstructed breast using pedicled latissimus dorsi muscle flap.</p>
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<p><b>OBJECTIVE</b>To study the clinical value of digital 3D technique combined with nanocarbon-aided navigation in endoscopic sentinel lymph node biopsy for breast cancer.</p><p><b>METHODS</b>Thirty-nine female patients with stage I/II breast cancer admitted in our hospital between September 2014 and September 2015 were recruited. CT lymphography data of the patients were segmented to reconstruct digital 3D models, which were imported into FreeForm Modeling Surgical System Platform for visual simulation surgery before operation. Endoscopic sentinel lymph node biopsy and endoscopic axillary lymph node dissection were then carried out, and the accuracy and clinical value of digital 3D technique in endoscopic sentinel lymph node biopsy were analyzed.</p><p><b>RESULTS</b>s The 3D models faithfully represented the surgical anatomy of the patients and clearly displayed the 3D relationship among the sentinel lymph nodes, axillary lymph nodes, axillary vein, pectoralis major, pectoralis minor muscle and latissimus dorsi. In the biopsy, the detection rate of sentinel lymph nodes was 100% in the patients with a coincidence rate of 87.18% (34/39), a sensitivity of 91.67% (11/12), and a false negative rate of 8.33% (1/12). Complications such as limb pain, swelling, wound infection, and subcutaneouseroma were not found in these patients 6 months after the operation.</p><p><b>CONCLUSION</b>Endoscopic sentinel lymph node biopsy assisted by digital 3D technique and nanocarbon-aided navigation allows a high detection rate of sentinel lymph nodes with a high sensitivity and a low false negative rate and can serve as a new method for sentinel lymph node biopsy for breast cancer.</p>