DNA barcoding of Gentiana crassicaulis in Lijiang and evaluation of root processing methods / 药学学报

The key factors for producing the best quality Chinese herbal medicines are high-quality germplasm, suitable cultivation area and the proper processing methods for herbal raw materials. Gentiana crassicaulis in Gentiana (Sect. Cruciata), Gentianaceae is one of the original plants of the Chinese herb Qinjiao (Gentianae Macrophyllae Radix), and its type specimen was collected in Lijiang, Yunnan. There is a long planting history of the herb in this area. In this study a sampling plot was designated in these traditional planting areas. G. crassicaulis was planted and herbal raw materials were harvested from the plot. The raw materials were prepared locally and at a pharmaceutical factory in Shanghai using processing methods such as "sweating" or "no sweating", "slicing" or "no slicing" (whole root), and "stoving" or "no stoving" (air drying). The quality of all processed samples was evaluated. In addition, molecular markers were determined for identifying cultivated and wild samples from Lijiang, Yunnan. The results are as follows: ① Samples from the sampling plot and the field are taxonomically identified as Gentiana crassicaulis. ② A total of 270 sequences of trnC-GCA-petN, atpB-rbcL, psbN, ndhB-rps7 and ycf1 were obtained, and three genotypes were determined from the cultivated samples; the type III was shared by both cultivated and wild plants. Based on the molecular markers, a DNA barcoding method to identify cultivated and wild samples of G. crassicaulis from Lijiang, Yunnan was established. ③ Total content of loganic acid and gentiopicroside in all samples was ≥ 2.5%, and above the Chinese Pharmacopoeia (2020) limit. ④ In HPLC fingerprinting, 9 common peaks were assigned and similarity between all samples was > 0.999; and ⑤ In a PCA score plot all slice samples were clustered, while whole root samples were scattered. Therefore, our studies could provide basic data for optimizing the processing method, producing best quality Gentianae Macrophyllae Radix, and evaluating the quality of different ecotype varieties and the multiple origin of herbal medicines.

Exploring the clinical characters of Shugan Jieyu capsule through text mining / 中国中药杂志

In this paper,the potential climate factors affecting the Pairs polyphylla var. yunnanensis distribution in China at rational scales were selected from related literatures, using the sampling point geographic information from of P. polyphylla var. yunnanensis, combine the maximum entropy model (MaxEnt) with spatial analyst function of ArcGIS software, to study the climate suitability of P. polyphylla var. yunnanensis cultivating region in China and the leading climate factors. The results showed that, average rainfall in August, average rainfall in October, coefficient of variation of seasonal precipitation, the average temperature of the dry season, isothermal characteristic, average temperature in July were the leading climate factors affecting the potential distribution of P. polyphylla var. yunnanensis cultivating region in China, with their cumulative contribution rate reached 97.2% of all candidate climate factors. Existence probability of the region to be predicted of P. polyphylla var. yunnanensis through the constructed model, the climate unsuitable region, low, medium and high region of P. polyphylla var. yunnanensis in China were clarified and the threshold of climatic factors were gave and clarified the climate characteristics of the cultivating region in each climatic suitability division. The results of research can provide reference for production layout and introduction of P. polyphylla var. yunnanensis.

Characteristics and Impact Factors of Renal Threshold for Glucose Excretion in Patients with Type 2 Diabetes Mellitus

Sodium glucose co-transporter 2 (SGLT-2) inhibitors are newly developed but promising medicine for type 2 diabetes. However, patients with a different renal threshold for glucose excretion (RT(G)) may have a different reaction to this medicine. Therefore, the objective of this study was to investigate the characteristics of RT(G) and its impact factors in patients with type 2 diabetes mellitus (T2DM). The clinical and laboratory data of 36 healthy individuals and 168 in-hospital patients with T2DM were collected and analyzed, RTG was calculated using blood glucose (BG) measured by dynamic BG monitoring, urinary glucose excretion (UGE) and estimated glomerular filtration rate (eGFR). The characteristics of RT(G) were investigated. The risk factors for high RT(G) were analyzed using non-conditional logistic regression analysis. Our results found that RT(G) of the T2DM group was higher than that of the healthy individuals (P < 0.05); and 22.22% from the healthy individuals group but 58.33% from the T2DM group had high RT(G). Age, duration of diabetes, body mass index (BMI), and homeostasis model assessment insulin resistance index (HOMA-IR) were independently associated with high RT(G) (P < 0.05). Further stratified analysis revealed that RT(G) in T2DM patients increased with age, duration of diabetes, and BMI. In conclusion, RT(G) is increased in patients with T2DM, especially in those with longer diabetic duration, higher BMI, and those who are older. Therefore, these patients may be more sensitive to SGLT-2 inhibitors.

Downregulation of Aquaporin 4 Expression through Extracellular Signal-regulated Kinases1/2 Activation in Cultured Astrocytes Following Scratch-injury / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the role of extracellular signal-regulated kinase1/2 (ERK1/2) pathway in the regulation of aquaporin 4 (AQP4) expression in cultured astrocytes after scratch-injury.</p><p><b>METHODS</b>The scratch-injury model was produced in cultured astrocytes of rat by a 10-μL plastic pipette tip. The morphological changes of astrocytes and lactate dehydrogenase (LDH) leakages were observed to assess the degree of scratch-injury. AQP4 expression was detected by immunofluorescence staining and Western blot, and phosphorylated-ERK1/2 (p-ERK1/2) expression was determined by Western blot. To explore the effect of ERK1/2 pathway on AQP4 expression in scratch-injured astrocytes, 10 µmol/L U0126 (ERK1/2 inhibitor) was incubated in the medium at 30 min before the scratch-injury in some groups.</p><p><b>RESULTS</b>Increases in LDH leakage were observed at 1, 12, and 24 h after scratch-injury, and AQP4 expression was reduced simultaneously. Decrease in AQP4 expression was associated with a significant increase in ERK1/2 activation. Furthermore, pretreatment with U0126 blocked both ERK1/2 activation and decrease in AQP4 expression induced by scratch-injury.</p><p><b>CONCLUSION</b>These results indicate that ERK1/2 pathway down-regulates AQP4 expression in scratch-injured astrocytes, and ERK1/2 pathway might be a novel therapeutic target in reversing the effects of astrocytes that contribute to traumatic brain edema.</p>

miR-200c inhibits metastasis of breast cancer cells by targeting HMGB1 / 华中科技大学学报（医学）（英德文版）

miR-200c has been shown to regulate the epithelial-mesenchymal transition (EMT) by inhibiting ZEB1 and ZEB2 expression in breast cancer cells. This study further examined the role of miR-200c in the invasion and metastasis of breast cancer that goes beyond the regulation on ZEB1 and ZEB2 expression. In this study, the bioinformatics software (miRanda) was used to predict the target gene of miR-200c and Renilla luciferase assay to verify the result. The metastatic breast cancer cells MDA-MB-231 were cultured and transfected with the miR-200c mimic or inhibitor. The expressions of miR-200c and HMGB1 were detected by RT-PCR and Western blotting, respectively. Transwell assay and wound healing assay were employed to examine the invasive and migrating ability of transfected cells. Target prediction and Renilla luciferase analysis revealed that HMGB1 was a putative target gene of miR-200c. After transfection of MDA-MB-231 cells with the miR-200c mimic or inhibitor, the expression of miR-200c was significantly increased or decreased when compared with cells transfected with the miR-200c mimic NC or inhibitor NC. Moreover, the expression of HMGB1 was reversely correlated with that of miR-200c in transfected cells. Tranwell assay showed that the number of invasive cells was significantly reduced in miR-200c mimic group when compared with miR-200c inhibitor group. It was also found that the migrating ability of cells transfected with miR-200c mimics was much lower than that of cells transfected with miR-200c inhibitors. It was suggested that miR-200c can suppress the invasion and migration of breast cancer cells by regulating the expression of HMGB1. miR-200c and HMGB1 may become useful biomarkers for progression of breast cancer and targets of gene therapy.

miR-200c inhibits metastasis of breast cancer cells by targeting HMGB1 / 华中科技大学学报（医学）（英德文版）

miR-200c has been shown to regulate the epithelial-mesenchymal transition (EMT) by inhibiting ZEB1 and ZEB2 expression in breast cancer cells. This study further examined the role of miR-200c in the invasion and metastasis of breast cancer that goes beyond the regulation on ZEB1 and ZEB2 expression. In this study, the bioinformatics software (miRanda) was used to predict the target gene of miR-200c and Renilla luciferase assay to verify the result. The metastatic breast cancer cells MDA-MB-231 were cultured and transfected with the miR-200c mimic or inhibitor. The expressions of miR-200c and HMGB1 were detected by RT-PCR and Western blotting, respectively. Transwell assay and wound healing assay were employed to examine the invasive and migrating ability of transfected cells. Target prediction and Renilla luciferase analysis revealed that HMGB1 was a putative target gene of miR-200c. After transfection of MDA-MB-231 cells with the miR-200c mimic or inhibitor, the expression of miR-200c was significantly increased or decreased when compared with cells transfected with the miR-200c mimic NC or inhibitor NC. Moreover, the expression of HMGB1 was reversely correlated with that of miR-200c in transfected cells. Tranwell assay showed that the number of invasive cells was significantly reduced in miR-200c mimic group when compared with miR-200c inhibitor group. It was also found that the migrating ability of cells transfected with miR-200c mimics was much lower than that of cells transfected with miR-200c inhibitors. It was suggested that miR-200c can suppress the invasion and migration of breast cancer cells by regulating the expression of HMGB1. miR-200c and HMGB1 may become useful biomarkers for progression of breast cancer and targets of gene therapy.

Complete genomic analysis of a novel infectious bronchitis virus isolate / 病毒学报

The genome of CK/CH/SD09/005, an isolate of infectious bronchitis virus (IBV), was characterized to enable the further understanding of the epidemiology and evolution of IBV in China. Twenty-five pairs of primers were designed to amplify the full-length genome of CK/CH/SD09/005. The nucleotide sequence of CK/CH/SD09/005 was compared with reference IBV strains retrieved from GenBank. The phylogenic relationship between CK/CH/SD09/005 and the reference strains was analyzed based on S1 gene sequences. The complete genome of CK/CH/SD09/005 consisted of 27691 nucleotides (nt), excluding the 5' cap and 3' poly A tail. The whole-genome of CK/CH/SD09/005 shared 97 - 99% nucleotide sequence homology with the GX-NN09032 strain, which was the only complete genome that was closely related to CK/CH/SD09/005. When compared with all reference strains except GX-NN09032, CK/CH/SD09/005 showed the highest similarity to ck/CH/LDL/091022 and SDIB821/2012 (QX-like) in the replicase gene (Gene 1) and 3'UTR, with a sequence identity rate of 97% and 98%, respectively. However, CK/CH/SD09/005 exhibited lower levels of similarity with ck/CH/LDL/091022 and SDIB821/2012 in S-3a-3b-3c/ E-M-5a-5b-N with a sequence identity of 72% - 90%. CK/CH/SD09/005 showed the highest level of nucleotide identity with Korean strain 1011, and Chinese strains CK/CH/LXJ/02I, DK/CH/HN/ZZ2004 and YX10, in ORF 3c/E (97%), 5a (96%), 5b (99%) and N (96%), respectively. ORFs 3a, 3b and M of CK/CH/SD09/005 exhibited no more than 90% homology with the reference strains, excluding GX-NN09032. The phylogenic analysis based on the S1 gene revealed that CK/CH/SD09/005 and 39 published strains were classified into seven clades (genotypes). CK/CH/SD09/005 was distributed in clade IV with several isolates collected between 2007 and 2012. CK/CH/SD09/005 showed 66% - 69% and 72% - 81% nucleotide identities with the IBV strains of other six clades in the S1 and S2 subunits, respectively. More over, multiple substitutions were found throughout the entire S gene of CK/CH/SD09/005, while insertions and deletions were located within the S1 gene. These results indicated that CK/CH/SD09/005 is a novel variant that may be derived from the QX-like strains that are prevalent in China. Multiple genetic mechanisms, including recombinations, mutations, insertions and deletions, are likely to have contributed to the emergence of this IBV strain.

Biological characteristics of three Newcastle disease virus isolates and entire genome sequences analysis / 病毒学报

Three Newcastle disease virus (NDV) strains recovered from ND outbreaks in chickens and duck flocks in north china during 2009 to 2011 were completely sequenced and biologically characterized. All the strains were velogenic and had the velogenic motif 112R-R-Q-K-R-F117 which was consistent with the results of biological tests. Analysis of the variable region (nucleotide 47 to 420) of the F gene indicated that the three isolates belonged to genotype VII d. Cross hemagglutination inhibition test indicated that the antigen homology between three isolates and LaSota were 82.5%-89.4%, the homology between the two isolates from chicken was 90%. A cross-protection experiment in which specific-pathogen-free chickens vaccinated with LaSota were challenged by SDLY01 isolate showed that LaSota vaccine could provide complete protection against SDLY01, however virus discharge could be detected on fifth day. Challenge experiment in which Cherry Valley duck of 30 day old challenged with SD03 strain indicated that cherry valley duck had no disease in experiment period, but virus discharge could be detected from Larynx and cloaca until fifth day. Genome length of three NDV isolates was 15192bp and belonged to genotype VII d. Sequence analysis clarified that the whole genomic sequence of these three isolates shared high homology with NDV virus strains isolated from goose and duck over the same period, which elucidated that NDV isolated from goose, duck or chicken had close genetics and epidemiological relationship.

Effects of pravastatin on the proliferation and invasion of human hepatocarcinoma HepG2 cell line / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To observe the effects of pravastatin on the proliferation and invasion of human hepatocarcinoma HepG2 cell line.</p><p><b>METHODS</b>The effects of pravastatin on the proliferation, migration and invasion of HepG2 cells was observed by MTT assay, Boyden chamber assay and motility assay. p38 activity was measured, and the expression of p-p38, MKP-1, RhoC and MMP-2 was analyzed by Western blot.</p><p><b>RESULTS</b>Pravastatin inhibited the proliferation of HepG2 cells. The intracellular p38 activity and expressions of p-p38, RhoC and MMP-2 were decreased, while MKP-1 expression was elevated in pravastatin treated cells. In addition, pravastatin inhibited the invasion and motility.</p><p><b>CONCLUSION</b>Pravastatin can inhibit the proliferation and invasion of HepG2 cells.</p>

ShRNA against NSP4 gene inhibits the proliferation of bovine rotavirus in vitro / 病毒学报

Based on the NSP4 sequence of bovine rotavirus (BRV), the shRNA was designed and synthesized, and a shRNA recombinant lenti-virus vector RNAi-H1-89 was constructed. The recombinant RNAi-H1-89 Lenti-virus was packaged by transfecting the 293T cell with the recombinant vector RNAi-H1-89 and two helper plasmids using lipofectamine, and then used to infect MA104 cells. The MA104 cells were further infected with BRV strain G6 24h post-infection, with the LacZ shRNA recombinant lenti-virus as control. Thirty-six hours later, the CPE of the infected cells was observed under microscope, shRNA of NSP4 gene inhibited CPE in MA104 cell; the shRNA against NSP4 gene also inhibited NSP4 gene expression by RT-PCR, The virus titer in the cell culture supernatant was significant lower compared with the control group. The above results showed that RNAi-H1-89 against NSP4 gene could specifically silence NSP4 gene expression, and inhibit the proliferation of BRV.

Expression, purification of recombinant Luxi yellow cattle IFN-alpha fusion protein and its antiviral activities / 生物工程学报

Interferon a gene was cloned from genomic DNA of Chinese Luxi yellow cattle by PCR, and the PCR product was inserted into vector pET32a( + ) to make a recombinant plasmid pET32a( + )/BoIFN-alpha. The expression of BoIFN-alpha in Escherichia coli was induced by addition of IPTG. Sequence analysis showed that the Chinese Luxi yellow cattle IFN-alpha gene is composed of 498 nucleotides, encoding a mature polypeptide of 166 amino acids. Compared with other BoIFN-alpha subtypes, it shares the highest identity of 97.6% to the C-subtype. SDS-PAGE results showed that recombinant proteins were expressed in inclusion bodies in Escherichia coli with molecular weight of 40 kD and the recombinant proteins accounted for 26.7% of the whole proteins.The expressed product was purified by affinity chromatography with immobilized nickel chelating NTA (Ni-NTA) and its antiviral activities were tested on MDBK/VSV cell system. Its antiviral activities were 5 x 10(5) u/mg on MDBK/VSV cell system. The results showed that the expression plasmid was successfully constructed and BoIFN-alpha C2 protein was expressed in Escherichia coli. Moreover the purification had good effects on antiviral activities.