Objective:To investigate improvement effect of astragalus polysaccharides(APS)on recurrent spontaneous abor-tion(RSA)in rats and its mechanism.Methods:Pregnant rats were randomly grouped into control group,RSA group,dexamethasone group,APS low,medium,high doses groups and LY294002 group.A RSA rat model was constructed,uterine organ index and embryo loss rate were calculated.HE staining was applied to observe histopathology changes of decidua.ELISA was applied to detect serum levels of estradiol(E2),progesterone(P),prolactin(PRL),cytokines such as IFN-γ,TNF-α,IL-4 and IL-10.Immunohisto-chemistry was applied to detect expression of decidua progesterone receptor(PR).Western blot was applied to detect expression of PI3K/AKT signaling pathway proteins of decidua.Results:Compared with control group,desquamated cells of rats in RSA group exhibit edema,disordered arrangement,nuclear pyknosis and disappearance,and decidua reduced interstitial blood vessels,uterine organ index,serum IL-4,IL-10,E2,P,PRL levels,decidua PR,p-PI3K,p-AKT expressions were obviously reduced,embryo loss rate,IFN-γ and TNF-α levels were obviously increased(P<0.05);compared with RSA group,morphology of decidual cells and interstitial blood vessels in dexamethasone group and APS low,medium and high doses groups were improved,uterine organ index,serum IL-4,IL-10,E2,P,PRL levels,decidua PR,p-PI3K,p-AKT expressions were obviously increased,embryo loss rate,serum IFN-γ and TNF-α levels were obviously reduced(P<0.05);LY294002 reversed improvement effect of high-dose APS on adverse pregnancy out-comes in RSA rats(P<0.05).Conclusion:APS can reduce miscarriage rate of RSA rats,regulate Th1/Th2 balance,whose mecha-nism may be related to activation of PI3K/AKT.

Objective:To investigate improvement effect of astragalus polysaccharides(APS)on recurrent spontaneous abor-tion(RSA)in rats and its mechanism.Methods:Pregnant rats were randomly grouped into control group,RSA group,dexamethasone group,APS low,medium,high doses groups and LY294002 group.A RSA rat model was constructed,uterine organ index and embryo loss rate were calculated.HE staining was applied to observe histopathology changes of decidua.ELISA was applied to detect serum levels of estradiol(E2),progesterone(P),prolactin(PRL),cytokines such as IFN-γ,TNF-α,IL-4 and IL-10.Immunohisto-chemistry was applied to detect expression of decidua progesterone receptor(PR).Western blot was applied to detect expression of PI3K/AKT signaling pathway proteins of decidua.Results:Compared with control group,desquamated cells of rats in RSA group exhibit edema,disordered arrangement,nuclear pyknosis and disappearance,and decidua reduced interstitial blood vessels,uterine organ index,serum IL-4,IL-10,E2,P,PRL levels,decidua PR,p-PI3K,p-AKT expressions were obviously reduced,embryo loss rate,IFN-γ and TNF-α levels were obviously increased(P<0.05);compared with RSA group,morphology of decidual cells and interstitial blood vessels in dexamethasone group and APS low,medium and high doses groups were improved,uterine organ index,serum IL-4,IL-10,E2,P,PRL levels,decidua PR,p-PI3K,p-AKT expressions were obviously increased,embryo loss rate,serum IFN-γ and TNF-α levels were obviously reduced(P<0.05);LY294002 reversed improvement effect of high-dose APS on adverse pregnancy out-comes in RSA rats(P<0.05).Conclusion:APS can reduce miscarriage rate of RSA rats,regulate Th1/Th2 balance,whose mecha-nism may be related to activation of PI3K/AKT.

Objective To evaluate the effect of different honey processing methods on the dissolution of Cynanchum salicifolia in vitro.Methods High performance liquid chromatography ① Carotene and β-Determination method of sitosterol content:chromatographic column:Dia monosil C18(250 mm×4.6 mm,5 μm).Mobile phase:methanol:0.05％phosphoric acid water=99∶1.Isocratic elution;Flow rate 1.0 mL·min-1;Detection wavelength 209 nm;Injection volume 10 μL.The column temperature is 30℃.②Determination method of glucose and fructose:the chromatographic column is Inspire NH2 sugar analysiscolumn(250 mm×4.6 mm,5 μm).The mobile phase was acetonitrile water(77∶23).The flow rate is 0.8 mL·min-1;The column temperature is 35℃;Injection volume 10 μL;Evaporative light scattering detector(ELSD):the drift tube temperature is 100℃,and the carrier gas flow rate is 2.8 L·min-1.Results ①The results showed that carotene and β-the determination of sitosterol showed a good linear relationship in the range of mass concentration(r＞0.9999).The average recoveries were 99.71％and 98.83％.The RSD of precision,stability and repeatability were all less than 2.0％.②The established method of glucose fructose showed a good linear relationship in the range of 0.05-0.50 mg·mL-1(r=0.9991).Conclusion The content determination method and in vitro dissolution test methodology of the study meet the requirements,which provides a scientific basis for the establishment of quality standards and modern research of honey roasted Paeonia salicifolia.

Objective To evaluate the effect of different honey processing methods on the dissolution of Cynanchum salicifolia in vitro.Methods High performance liquid chromatography ① Carotene and β-Determination method of sitosterol content:chromatographic column:Dia monosil C18(250 mm×4.6 mm,5 μm).Mobile phase:methanol:0.05％phosphoric acid water=99∶1.Isocratic elution;Flow rate 1.0 mL·min-1;Detection wavelength 209 nm;Injection volume 10 μL.The column temperature is 30℃.②Determination method of glucose and fructose:the chromatographic column is Inspire NH2 sugar analysiscolumn(250 mm×4.6 mm,5 μm).The mobile phase was acetonitrile water(77∶23).The flow rate is 0.8 mL·min-1;The column temperature is 35℃;Injection volume 10 μL;Evaporative light scattering detector(ELSD):the drift tube temperature is 100℃,and the carrier gas flow rate is 2.8 L·min-1.Results ①The results showed that carotene and β-the determination of sitosterol showed a good linear relationship in the range of mass concentration(r＞0.9999).The average recoveries were 99.71％and 98.83％.The RSD of precision,stability and repeatability were all less than 2.0％.②The established method of glucose fructose showed a good linear relationship in the range of 0.05-0.50 mg·mL-1(r=0.9991).Conclusion The content determination method and in vitro dissolution test methodology of the study meet the requirements,which provides a scientific basis for the establishment of quality standards and modern research of honey roasted Paeonia salicifolia.

l-Heptopyranoses are important components of bacterial polysaccharides and biological active secondary metabolites like septacidin (SEP), which represents a group of nucleoside antibiotics with antitumor, antifungal, and pain-relief activities. However, little is known about the formation mechanisms of those l-heptose moieties. In this study, we deciphered the biosynthetic pathway of the l,l-gluco-heptosamine moiety in SEPs by functional characterizing four genes and proposed that SepI initiates the process by oxidizing the 4'-hydroxyl of l-glycero-α-d-manno-heptose moiety of SEP-328 ( 2) to a keto group. Subsequently, SepJ (C5 epimerase) and SepA (C3 epimerase) shape the 4'-keto-l-heptopyranose moiety by sequential epimerization reactions. At the last step, an aminotransferase SepG installs the 4'-amino group of the l,l-gluco-heptosamine moiety to generate SEP-327 ( 3). An interesting phenomenon is that the SEP intermediates with 4'-keto-l-heptopyranose moieties exist as special bicyclic sugars with hemiacetal-hemiketal structures. Notably, l-pyranose is usually converted from d-pyranose by bifunctional C3/C5 epimerase. SepA is an unprecedented monofunctional l-pyranose C3 epimerase. Further in silico and experimental studies revealed that it represents an overlooked metal dependent-sugar epimerase family bearing vicinal oxygen chelate (VOC) architecture.

Natural cyclohexapeptide AFN A₁ fromStreptomyces alboflavus 313 has moderate antibacterial and antitumor activities. An artificial designed AFN A₁ homodimer, di-AFN A₁, is an antibiotic exhibiting 10 to 150 fold higher biological activities, compared with the monomer. Unfortunately, the yield of di-AFN A₁ is very low (0.09 ± 0.03 mg·L^-1) in the engineered strain Streptomyces alboflavus 313_hmtS (S. albo/313_hmtS), which is not friendly to be genetically engineered for titer improvement of di-AFN A₁ production. In this study, we constructed a biosynthetic gene cluster for di-AFN A₁ and increased its production through heterologous expression. During the collection of di-AFN A₁ biosynthetic genes, the afn genes were located at three sites of S. alboflavus 313 genome. The di-AFN A₁ biosynthetic gene cluster (BGC) was first assembled on one plasmid and introduced into the model strain Streptomyces lividans TK24, which produced di-AFN A₁ at a titer of 0.43 ± 0.01 mg·L^-1. To further increase the yield of di-AFN A₁, the di-AFN A₁ BGC was multiplied and split to mimic the natural afn biosynthetic genes, and the production of di-AFN A₁ increased to 0.62 ± 0.11 mg·L^-1 in S. lividans TK24 by the later strategy. Finally, different Streptomyces hosts were tested and the titer of di-AFN A₁ increased to 0.81 ± 0.17 mg·L^-1, about 8.0-fold higher than that in S. albo/313_hmtS. Successful heterologous expression of di-AFN A₁ with a remarkable increased titer will greatly facilitate the following synthetic biological study and drug development of this dimeric cyclohexapeptide.
Cloning, Molecular ; Streptomyces/metabolism* ; Multigene Family ; Anti-Bacterial Agents/metabolism* ; Plasmids/genetics*

OBJECTIVE To study the toxicity of the extract from Mi ao medicine Wikstroemia indica to zebrafish . METHODS Zebrafish embryo model was used as the object ，after exposure to W. indica extract （10，20，40 μg/mL），the number of spontaneous twitching within 1 min，heart rate within 10 s，the occurrence of malformation and death were detected and recorded . Zebrafish was used as the object ，after exposure to W. indica extract（10-100 μg/mL）for 24，48 and 72 h，and the median lethal concentration（LC50）of W. indica extract to zebrafish at different time points were calculated . After exposure to low ，medium and high concentration （27，37，51 μg/mL）of W. indica extract，the liver phenotype ，hepatocyte apoptosis and lipid deposition of zebrafish were observed ，and the activities of alanine aminotransferase （ALT），aspartate aminotransferase （AST）and lactate dehydrogenase（LDH）in liver tissue were detected . RESULTS Compared with blank group ，the number of spontaneous twitching ， malformation rate （except for 10 μg/mL group ）and mortality of embryos increased significantly in 10，20，40 μg/mL groups of W. indica extract，and the heart rate （except for 10，20 μg/mL group ）of embryos decreased significantly （P＜0.05）. LC50 of W. indica extract to zebrafish at 24，48 and 72 h were 39.850，28.300 and 21.490 μg/mL，respectively. After drug treatment ，the transparency of liver area of zebrafish in low ，medium and high concentration groups of W. indica extract reduced and their shape were enlarged；apoptosis and lipid deposition increased ；the activities of ALT ，AST and LDH in liver tissue were significantly increased （P＜0.05）. CONCLUSIONS Miao medicine W. indica extract had develop mental toxicity to zebrafish embryos and he patotoxicity to zebrafish .

Aim To investigate the protective effect of astragaloside IV (AS-Ⅳ) on human retinal pigment epithelium injury induced by methylglyoxal (MGO), and explore its molecular mechanism.Methods The injury of ARPE-19 cells was induced by MGO and the cell viability was measured by CCK-8 method.The morphology of cell nucleus was analyzed by Hoechst 33342 staining and the cell apoptosis was analyzed by flow cytometry to detect labbled Annexin V-FITC/PI.JC-1 staining and fluorescence probe DCFH-DA were employed to evaluate the change of mitochondrial membrane potential and reactive oxygen species (ROS).The levels of SOD, MDA, caspase-9 and caspase-3 were determined by respective kits.Western blot was used to analyse the expression of Bcl-2, Bax and PARP.Results AS-Ⅳ could significantly inhibit the decrease of cell viability induced by MGO, improve the morphology of cell nucleus, reduce the ARPE-19 cell apoptosis rate and the level of ROS and MDA, and increase the activity of SOD.Furthermore, AS-Ⅳ could enhance mitochondrial membrane potential, the ratio of Bcl-2/Bax and the expression of PARP, and inhibit the activation of caspase-9 and caspase-3.Conclusion AS-Ⅳ may protect ARPE-19 cells from the injury induced by MGO by increasing the antioxidant ability of ARPE-19 cells and inhibiting cell apoptosis.

OBJECTIVE:To establish a method for the content determination of berberine hydrochloride in the Jiangtang xiao-zhi tablets. METHODS:HPLC was conducted with the Symmetry C18 column. The mobile phase was acetonitrile-0.05 mol/L sodium dihydrogen phosphate(pH adjusted to 3.0 using phosphoric acid)(24∶76,V/V)at the flow rate of 1.0 ml/min. The detection wave-length was 345 nm,the column temperature was room temperature and injection volume was 10 μl. RESULTS:The linear range of berberine hydrochloride was 0.522-4.698 μg(r=0.999 9);RSDs of precision,stability and reproducibility tests were no more than 0.72%;the average recovery was 97.79%(RSD=2.09%,n=6). CONCLUSIONS:The method is simple,accurate,sensitive and reproducible,and can be used for the content determination of berberine hydrochloride.

Objective Recombinant human parathyroid hormone(1-34) [ rhPTH(1-34)] is the unique anabolic substance acting on the skeleton. The efficacy and safety of long-term administration of rhPTH(1-34) in Chinese postmenopausal women have not been evaluated. This study compared the clinical efficacy and safety of rhPTH(1-34) with elcatonin for treating postmenopausal women with osteoporosis in 11 urban areas of China. Methods A total of453 postmenopausal women with osteoporosis were enrolled in an 18-month, multi-center, randomized, controlled study. They were randomized to receive either rhPTH(1-34) 20 μg(200 U) daily for 18 months, or elcatonin 20 U weekly for 12 months. Lumbar spine ( L1-4) and femoral neck bone mineral density (BMD), fracture rate, back pain as well as biochemical markers of bone turnover ( serum bone-specific alkaline phosphatase was measured by radioimmunoassay; C-telopeptide/ creatinine ( CTX/ Cr) measured by quantitative sandwich enzyme-linked immunosorbent assay) at 6, 12, and 18 months. Adverse events were recorded. Results rhPTH(1-34) increased lumbar BMD more significantly than that did by elcatonin at 6 months( M6), 12 months (M12), and 18 months(M18; 4. 3% vs 1. 94% , 6. 8% vs 2. 72% , 9. 51% vs 2. 86% , P<0. 01). There was only a small but significant increase of femoral neck BMD at M18(2. 64% , P<0. 01) in rhPTH(1-34) groups. There were greater increases in bone turnover markers in the rhPTH(1-34) group than in the elcatonin group at M6, M12, and M18[serum bone-specific alkaline phosphatase(BSAP) 93. 67% vs -3. 56% , 117. 78% vs -4. 12% , 49. 24% vs-5. 81% , P<0. 01; urinary CTX/ Cr 250% vs -29. 5% , 330% vs -41. 4% , 273 % vs -10. 6% , P<0. 01]. rhPTH (1-34) showed similar effect of pain relief as elcatonin. The incidence of clinical fractures was 5. 36% (6 / 112) in elcatonin group and 3. 23% ( 11 / 341 ) in rhPTH ( 1-34 ) group ( P = 0. 303 ). Both treatments were well tolerated. Hypercaluria(9. 38% ) and hypercalcemia(7. 04% ) in rhPTH(1-34) group was transient and caused no clinical symptoms. Pruritus(8. 21% vs 2. 68, P=0. 044) and redness of injection site(4. 40% vs 0, P=0. 024) were more frequent in rhPTH(1-34). Nausea / vomiting(16. 07% vs 6. 16% , P = 0. 001) and hot flushes(7. 14% vs 0. 59% , P<0. 001) were more common in elcatonin group. Conclusion rhPTH(1-34) treatment was associated with greater increases in lumbar spine BMD and bone formation markers. It could increase femoral BMD after 18 months treatment. rhPTH(1-34) could ameliorate back pain effectively. The results of the present study indicate that rhPTH(1-34) is an effective, and safe agent in treating postmenopausal women with osteoporosis.