Screening of specific DNA barcode, identification of germplasm resources, and analysis of genetic diversity of Atractylodes chinenesis / 药学学报

italic>Atractylodes chinensis has important medicinal and economic values. In this study, the chloroplast genome sequences of four A. chinensis samples from different producing areas were sequenced using the Illumina platform. The specific DNA barcodes were screened and the germplasm resources of A. chinensis samples from different producing areas and the genetic diversity of the population were analyzed basing on the specific barcodes. The whole chloroplast genomes of the four A. chinensis samples had a typical cyclic tetrad structure, with 112 genes annotated. The comparative genomics results indicated that ccsA and trnC-GCA_petN were potential specific DNA barcodes for intraspecific identification of A. chinensis. Polymerase chain reaction (PCR) analysis of ccsA and trnC-GCA_petN was performed on 256 samples from 14 areas in 9 provinces, and the amplification efficiency was 100%. Sequence analysis showed that ccsA and trnC-GCA_petN had 11 and 22 variant positions, which could identify 16 and 22 haplotypes, respectively. The combined sequence analysis identified 39 haplotypes, named Hap1-Hap39, of which the most abundant and widely distributed genotype was Hap9. Haplotype diversity (Hd) = 0.896 and nucleotide diversity (Pi) = 0.002 22 indicated high genetic diversity at the species level in A. chinensis. The genetic distances of the haplotypes were 0.000 00-0.004 88, indicating that there were small genetic differences among the haplotypes. The results of phylogenetic tree analysis showed that 39 haplotypes had very close genetic relationship, and formed two obvious branches with other groups of the same genus except Atractylodes macrocephala. This study plays an important role in the identification of the origin of A. chinensis and the protection and breeding of germplasm resources.

Specific DNA barcodes, germplasm resources, and genetic diversity of Eleutherococcus senticosus / 中国中药杂志

Eleutherococcus senticosus is one of the Dao-di herbs in northeast China. In this study, the chloroplast genomes of three E. senticosus samples from different genuine producing areas were sequenced and then used for the screening of specific DNA barcodes. The germplasm resources and genetic diversity of E. senticosus were analyzed basing on the specific DNA barcodes. The chloroplast genomes of E. senticosus from different genuine producing areas showed the total length of 156 779-156 781 bp and a typical tetrad structure. Each of the chloroplast genomes carried 132 genes, including 87 protein-coding genes, 37 tRNAs, and 8 rRNAs. The chloroplast genomes were relatively conserved. Sequence analysis of the three chloroplast genomes indicated that atpI, ndhA, ycf1, atpB-rbcL, ndhF-rpl32, petA-psbJ, psbM-psbD, and rps16-psbK can be used as specific DNA barcodes of E. senticosus. In this study, we selected atpI and atpB-rbcL which were 700-800 bp and easy to be amplified for the identification of 184 E. senticosus samples from 13 genuine producing areas. The results demonstrated that 9 and 10 genotypes were identified based on atpI and atpB-rbcL sequences, respectively. Furthermore, the two barcodes identified 23 genotypes which were named H1-H23. The haplotype with the highest proportion and widest distribution was H10, followed by H2. The haplotype diversity and nucleotide diversity were 0.94 and 1.82×10~(-3), respectively, suggesting the high genetic diversity of E. senticosus. The results of the median-joining network analysis showed that the 23 genotypes could be classified into 4 categories. H2 was the oldest haplotype, and it served as the center of the network characterized by starlike radiation, which suggested that population expansion of E. senticosus occurred in the genuine producing areas. This study lays a foundation for the research on the genetic quality and chloroplast genetic engineering of E. senticosus and further research on the genetic mechanism of its population, providing new ideas for studying the genetic evolution of E. senticosus.

Potential mechanism of NVP-LDE225 combined with radiotherapy for melanoma / 中华放射肿瘤学杂志

Objective:To explore the potential mechanism and feasibility of sonic hedgehog (SHH) signal pathway inhibitor NVP-LDE225 combined with radiotherapy in the treatment of melanoma.Methods:The Gli1 mRNA expression of the melanoma cells (Melan-A and SK-MEL-2) was detected by qPCR. After treatment with NVP-LDE225, GDC-0449 or combined with radiation for 24 hours, the number and activity of Melan-A and SK-MEL-2 were detected by cell counting and CCK-8 kit. The survival and proliferation ratio of Melan-A and SK-MEL-2 were detected by cell cloning. The changes of cell cycle of melanoma Melan-A cells were determined by flow cytometry. The levels of the Bax and Caspase3 of Melan-A apoptosis protein in melanoma cells were detected by Western blot.Results:NVP-LDE225, an inhibitor of Smo, decreased the mRNA expression of Gli1 in the melanoma cells in a dose-dependent manner, inhibited the proliferation ratio of the melanoma cells, induced apoptosis, and arrested melanoma cells in G 0 / G 1 phase. Gamma ray irradiation after NVP-LDE225 treatment further inhibited the SHH signal pathway, arrested the melanoma cells in G 2 / M phase, and further increased the inhibitory effect on melanoma cell proliferation. Conclusions:NVP-LDE225, an inhibitor of Smo, can inhibit SHH signal pathway in melanocytes, suppress cell proliferation, and further increases the effect of inhibiting cell proliferation after combining with irradiation. It can be used as a potential drug in combination with radiotherapy in the treatment of melanoma, which is worthy of further study.

Cloning and subcellular location of the PnMYB1R1 gene from Panax notoginseng and expression analysis with different stresses / 药学学报

MYB transcription factors, one of the largest transcription factor families in plants, play an important role in signal transduction, plant growth and plant resistance. In this study a full-length cDNA of the PnMYB1R1 gene was cloned from Panax notoginseng. Sequence analysis, prokaryotic expression and purification, subcellular location, transcriptional activity analysis, tissue-specific analysis and expression analysis under different abiotic stresses was performed. The open reading frame (ORF) of PnMYB1R gene was 738 bp, encoding a protein of 245 amino acids with a predicted molecular mass (MW) of 27.0 kD. The sequence analysis and polygenetic analysis indicated that the PnMYB1R1 protein contains a conserved R3 domain, belonging to TRF-like protein in 1R-MYB-type transcription factors. The recombinant PnMYB1R1 protein was expressed in Escherichia coli BL21(DE3) cells using the prokaryotic expression vector pET28a-PnMYB1R1 and was purified. Subcellular localization analysis showed that PnMYB1R1 was localized in the nucleus. Transcriptional activity analysis indicated that the PnMYB1R1 transcription factor has transcriptional activation activity. Expression analysis indicated that PnMYB1R1 was primarily expressed in roots, followed by stems and leaves, and then rootlets. The expression level of PnMYB1R1 in root, stems, leaves and rootlets was influenced by salt, low temperature and drought treatment, while the abundance of PnMYB1R1 was significantly induced by salt stress in these tissues. These results provide valuable insights into the role of 1R-MYB transcription factors in plant defense.

Molecular identification and quality analysis of Rhei Rhizoma based on chloroplast matK gene and UPLC / 药学学报

Rhei Rhizoma is commonly used as a traditional Chinese medicine with multiple botanical origins. Different botanical sources may have different pharmacological activities. The germplasm resources of commercial Rhei Rhizoma were determined based on the chloroplast gene matK, and the anthraquinone and free anthraquinone content was determined by UPLC to analyze quality of commercial Rhei Rhizoma. Eighty-nine commercial Rhei Rhizoma samples were collected from 40 cities in 27 provinces. DNA was extracted and the matK gene was amplified by PCR. Results indicated that the collected samples were from the same botanical origin, Rheum palmatum, and 8 genotypes were identified, including Rp1, Rp2, Rp3, Rp4, Rp5, Rp6, Rp10 and Rp12. Rp4 and Rp6, cultivated in Gansu, Sichuan and Yunnan provinces were the main circulating genotypes, representing 40.45% and 37.08% of the total samples, respectively. Phylogenetic tree analysis showed that the eight genotypes were mainly divided into two branches, of which the main genotypes Rp4 and Rp6 were in one branch. Genetic distance analysis indicated that the genetic separation of the eight genotypes was between 0.001 and 0.010. UPLC analysis indicated that 93.26% of the samples met the Pharmacopoeia standards. There were significant differences in the content of total anthraquinone and free anthraquinone among the samples, in which the difference in free anthraquinone was 1.01% and the difference in total anthraquinone content was 3.79%, indicating that the quality of commercial Rhei Rhizoma samples varies considerably. There was no significant difference in the content of total anthraquinone and free anthraquinone in commercial Rhei Rhizoma among different collection provinces and genotypes. This study will help guide the circulation of Rhei Rhizoma in the market and provides valuable insights for molecular identification and quality analysis of other traditional Chinese medicines.

Identification and quality evaluation of germplasm resources of commercial Scutellaria baicalensis based on DNA barcode and HPLC / 中国中药杂志

Scutellaria baicalensis is a commonly used Chinese medicinal herb. In this study, we identified the germplasm resources of commercial S. baicalensis samples based on trnH-psbA, petA-psbJ, and ycf4-cemA sequences according to the available chloroplast genome sequencing results, and measured the content of baicalin by HPLC. Through the above means we determined the best DNA barcode that can be used to detect the germplasm resources and evaluate the quality of commercial S. baicalensis samples. A total of 104 samples were collected from 24 provinces, from which DNA was extracted for PCR amplification. The amplification efficiencies of trnH-psbA, petA-psbJ, and ycf4-cemA sequences were 100%, 59.62%, and 25.96%, respectively. The results of sequence analysis showed that 5, 4, and 2 haplotypes were identified based on trnH-psbA, petA-psbJ, and ycf4-cemA sequences, respectively. However, the sequences of haplotypes in commercial samples were different from that of the wild type, and the joint analysis of three fragments of S. baicalensis only identified 6 haplotypes. Furthermore, the phylogenetic analysis and genetic distance analysis indicated that trnH-psbA could be used to identify S. baicalensis from adulterants. The above analysis showed that trnH-psbA was the best fragment for identifying the germplasm resources of commercial S. baicalensis samples. We then analyzed the haplotypes(THap1-THap5) of commercial S. baicalensis samples based on trnH-psbA and found that THap2 was the main circulating haplotype of the commercial samples, accounting for 86.55% of the total samples, which indicated the scarce germplasm resources of commercial S. baicalensis samples. The content of baicalin in all the collected commercial S. baicalensis samples exceeded the standard in Chinese Pharmacopoeia and had significant differences(maximum of 12.21%) among samples, suggesting that the quality of commercial S. baicalensis samples varied considerably. However, there was no significant difference in baicalin content between different provinces or between different haplotypes. This study facilitates the establishment of the standard identification system for S. baicalensis, and can guide the commercial circulation and reasonable medication of S. baicalensis.

Study on the effect of optimized dosing regimen of atropine on the treatment of myopia in guinea pigs / 药学实践杂志

Objective To study the effect of optimized atropine administration regimen on myopia in guinea pigs. Methods Forty six 21-day old guinea pigs were used for this study. Six were randomly selected as blank control, and the remaining 40 were randomly divided into 5 intervention groups: 1% atropine group, 0.01% atropine group, optimized group 1, optimized group 2, and saline group. One eye of the guinea pig in the intervention groups was randomly selected as the model eye and given form deprivation, and the contralateral eye was the self-control. The duration of intervention was 4 weeks. The diopter and axial length of guinea pig eyes were measured before the experiment and at each weekend. Choroid and sclera were measured after the experiment. Results The diopter of the model eyes in the 0.01% atropine group decreased rapidly. There was a significant difference before and after the experiment [(2.82±1.35)D vs (−0.64±0.20)D, P<0.01]. The diopter of model eyes decreased in 1% atropine group and optimized group 1, and the difference was statistically significant [(3. 50±1.14)D vs (1.38±1.15)D, P<0.05; (3.55±1.85)D vs (0.95±1.90)D, P<0.01]. In optimized group 2, the diopter of model eyes decreased, and there was no significant difference before and after the experiment [(1.36±1.61)D vs (2.93±1.42)D, P>0.05). After form deprivation, the axial length in 1% atropine group did not change significantly (P>0.05). The axial length in other intervention groups was extended to varying degrees. The thickness of choroid and sclera in 1% atropine group, optimized group 1 and optimized group 2 were greater than that in 0.01% atropine group. Conclusion The two optimized dosing regimens worked better than 0.01% atropine in inhibiting myopia in guinea pigs with form deprivation, and were similar to 1% atropine.

A study on social support and associated factors among HIV/AIDS patients in Liangshan / 中华疾病控制杂志

Objective To assess the social support of PLWHA in Liangshan and explore the associated factors. Methods A total of PLWHA 322 was selected by two-stage sampling. The questionnaire consisted of basic characteristics,simplified Chinese medical outcomes study social support survey and family concern index questionnaire. Results The social support score of PLWHA was 46.09（32.49，61.59）in Liangshan, which was lower than other regions of China. Scores of four dimensions were listed as follows: tangible support 56.25（29.69，75.00）, affectionate support 50.00（33.33，75.00）, positive social interaction 46.88（31.25，56.25）, informational and emotional support 43.75（25.00，56.25）. Multiple stepwise regression analysis suggested that the older PLWHA had lower sores of the social support（β=-1.997，P=0.037）; PLWHA who were living alone got lower sores of the social support（β=8.127, P=0.008）; PLWHA who were more satisfied with family function was associated with higher scores of social support（β=13.809,P<0.001）. Conclusions The status of social support among PLWHA was poor in Liangshan, which needed urgent improvement. Meanwhile, PLWHA who were older and living alone needed special attention. Health care workers should help PLWHA build a comprehensive family support system.

Central obesity and metabolic risk factors in middle-aged Chinese / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Central obesity is considered to be a central component of metabolic syndrome. Waist circumference (WC) has been widely used as a simple indicator of central obesity. This study is aimed to evaluate the sensitivity of WC cut-off values for predicting metabolic risk factors in middle-aged Chinese.</p><p><b>METHODS</b>The study involved 923 subjects aged 40-65 years. The metabolic risk factors were defined according to the Chinese Joint Committee for Developing Chinese Guidelines on Prevention and Treatment of Dyslipidemia in Adults. WC cut-off 85-90 cm and ⋝90 cm were used as cut-off values of central pre-obesity and central obesity in males, respectively, while WC 80-85 cm and ⋝85 cm were used as cut-off values of central pre-obesity and central obesity in females.</p><p><b>RESULTS</b>First, WC values corresponding to body mass index (BMI) 24 kg/m2 and visceral fat area (VFA) 80 cm2 were 88.55 cm and 88.51 cm in males, and 81.46 cm and 82.51 cm in females respectively. Second, receiver operating characteristic curves showed that the optimal WC cut-off of value was 88.75 cm in males, higher than that in females (81.75 cm). Third, the subjects with higher WC values were more likely to have accumulating metabolic risk factors. The prevalence of metabolic risk factors increased linearly and significantly in relation to WC levels.</p><p><b>CONCLUSION</b>WC cut-off values of central pre-/central obesity are optimal to predict multiple metabolic risk factors.</p>

Real-time quantitative PCR for evaluating murine thymic function / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a real-time quantitative PCR method for detecting the levels of the signal joint T cell receptor excision circles (sjTRECs) in murine thymocytes and spleen lymphocytes for determining the amount of naive T cells and evaluating the thymic function.</p><p><b>METHODS</b>The genomic DNA was extracted from murine thymocytes and splenocytes for PCR amplification of the target fragments. After purification of the PCR product, the recombination-activating gene 2 (RAG(2)) fragment was cloned into pGEMT-Easy vector to construct the standard plasmid. After PCR optimization, the standard curve was obtained and the samples (thymocytes and splenocytes of BALB/c and C(57)BL/6 mice) were detected for sjTRECs by real-time quantitative PCR.</p><p><b>RESULTS</b>The standard plasmid was correctly constructed, and the standard curve with high reliability was obtained. No statistical difference was observed in sjTREC contents in the T lymphocytes between the two mouse strains.</p><p><b>CONCLUSIONS</b>Real-time quantitative PCR for sjTREC analysis is established successfully, which offers an important means for thymic function analysis and a reliable model establishment for study the thymus.</p>

Effect of Weikangfu granule on the physiopathologic figure of precancerosis of gastric mucosa in patients of chronic gastritis with Pi-deficiency syndrome / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the physiopathologic basis of Weikangfu Granule (WKFG) in treating precancerosis of gastric mucosa in patients of chronic gastritis with Pi-deficiency syndrome (CG-PDS).</p><p><b>METHODS</b>One hundred and fifteen patients of CG-PDS who suffered from intestinal metaplasia (IM) and atypical hyperplasia (ATHP) of gastric mucosa, were divided into two groups. The treated group (n = 61) was treated by WKFG with its ingredients modified according to the syndrome type of patients. The control group (n = 54) was treated with Weishu granule. The histopathological and subcellular ultrastructural changes were detected by optical microscope, screening electronic microscope, transmission electronic microscope and histochemical staining; the nuclear and mitochondrial ultrastructure of gastric mucosa were analyzed with energy dispersion X-ray analyser and image analysis system. And the changes of cAMP, lipid peroxide (LPO), superoxide dismutase (SOD) before and after treatment in the treated group were measured and compared with those of the health control group consisting of 15 volunteers.</p><p><b>RESULTS</b>The symptomatic and pathological therapeutic effect in the treated group were significantly superior to those in the control group (P < 0.05). The contents of Zn, Cu, cAMP, SOD and (3)H-TdR LCT in gastric mucosa of the treated group before treatment were all lower than those of the healthy control group, yet all these indexes markedly increased after treatment, while serum LPO level, which increased before treatment was lowered after treatment. All the changes showed statistical significance (P < 0.05 or P < 0.01).</p><p><b>CONCLUSION</b>WKFG can reverse IM and ATHP in patients of CG-PDS, and the effect may be realized by way of increasing the level of Zn, Cu, cAMP and SOD in gastric mucosa, promoting cell differentiation, enhancing cellular immunity and reducing oxygen free radicals and lipid peroxidation.</p>

Study on the pathophysiologic basis of classification of 'spleen' deficiency in chronic gastritis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Most of the studies on traditional Chinese medicine (TCM) 'spleen' deficiency syndrome in the recent 30 years were conducted only on the basis of single functional index, neglecting the study on the pathophysiologic internal relationship between spleen deficiency syndrome and gastric diseases in modern medicine. But it was at the subcellular molecular biological level that we explored the pathophysiologic basis of classification of spleen deficiency in chronic gastritis by detecting the bioactive substances in gastric mucosa nuclei and mitochondria.</p><p><b>METHODS</b>By means of optical microscope, scanning electron microscope (SEM), transmission electron microscopy (TEM) and histochemical staining, we conducted histopathological, subcellular ultrastructural analysis and nuclei and mitochondrial ultrastructural analysis of gastric mucosa of 188 spleen deficiency patients and of 42 voluntary blood donors. At the same time, bioactive substances were measured by means of X-ray energy dispersive analysis system (EDAX) image analysis system, radioimmunoassay method and chemiluminescence method.</p><p><b>RESULTS</b>The content of cAMP, superoxide dismutase (SOD), Zn and Cu in gastric mucosa, and the content of Zn and Cu in mitochondria decreased progressively in order of groups: healthy control (HC), spleen Qi deficiency without organic lesion (F-SQD), spleen Yang deficiency without organic lesion (F-SyangD), disease without symptoms group, spleen Qi deficiency with organic lesion (G-SQD), spleen Yang deficiency with organic lesion (G-SyangD), spleen Yin deficiency (SyinD) and spleen deficiency with Qi stagnation (SDQS), chronic spleen deficiency gastritis (CSG) and chronic atrophic gastritis (CAG); decreased in order of HC, intestinal metaplasia (IM)Ia, IMIb, IMIIa and IMIIb, P < 0.05. The content of DNA, Zn and Cu in nuclei progressively increased in order mentioned above, P < 0.05.</p><p><b>CONCLUSIONS</b>The quantitative changes of gastric mucosal cAMP, SOD, Zn, Cu, of mitochondrial Zn, Cu and of nuclear DNA, Zn and Cu are not only the substance base on which the lesion of gastric mucosa tissue structure occurs, but also the substance base on which spleen deficiency is classified. G-SQD and G-SyangD were more likely to be found in low-grade or middle-grade CSG and CAG, while SyinD and SDQS in middle-grade or high-grade CSG, CAG and IMIIb.</p>

Study on the pathological basis of classification of spleen deficiency in chronic gastritis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Spleen in Traditional Chinese Medicine (TCM) is not actually the spleen in the anatomic sense designated in western medicine because its functions basically belong to the physiological category of digestive system in modern medicine, and it represents a macroscopic concept of digestion, absorption and nutrition metabolism. Spleen deficiency syndrome refers to the clinical phenomena such as hypofunction of digestion, absorption and nutrition metabolism. By integrating TCM with modern medicine, this paper is intended to explore the pathological basis of classification of spleen deficiency in chronic gastritis.</p><p><b>METHOD</b>By means of optical microscope, scanning electron microscope (SEM), transmission electron microscope (TEM) and histochemical staining, we conducted histopathological and subcellular ultrastructural (nuclei and mitochondrial) analysis of gastric mucosa of 188 patients of spleen deficiency, and that of 42 voluntary blood donors without clinical symptoms.</p><p><b>RESULTS</b>The gastric mucosa of patients with spleen Qi deficiency (SQD) and spleen yang deficiency (SyangD) could either be affected by organic lesion (type G-occurring on the basis of chronic superficial gastritis (CSG), chronic atrophic gastritis (CAG)) or unaffected (type F-chiefly belonging to functional indigestion); spleen yin deficiency (SyinD) and spleen deficiency with Qi stagnation (SDQS) both occurred on the basis of CSG and CAG; and the degree of mucosa inflammatory cells infiltration, the degree of decrease in glands propria, and the incidence of IMIIb in CSG and CAG were more serious than those of G-SQD and G-SyangD, P < 0.05 - 0.01.</p><p><b>CONCLUSION</b>Spleen deficiency syndrome is likely to occur on the basis of organic lesion of gastric mucosa (disease with symptoms of both CSG or CAG and spleen deficiency symptoms), as well as on the basis of inorganic lesion of gastric mucosa (nondisease with symptoms, which is, despite spleen deficiency symptoms, there is no CSG or CAG). Besides, the clinical phenomenon of disease without symptoms (despite CSG or CAG, there is no spleen deficiency symptoms) occurres because of such factors as genetic diathesis and compensation. The lesion degree of CSG or CAG and the incidence of IMIIb of SyinD and SDQS are more serious than those CSG and CAG of G-SQD and G-SyangD.</p>