Specific DNA barcodes screening, germplasm resource identification, and genetic diversity analysis of Platycodon grandiflorum / 药学学报

Platycodonis Radix is the dry root of Platycodon grandiflorum of Campanulaceae, which has a variety of pharmacological effects and is a commonly used bulk Chinese medicine. In this study, the chloroplast genome sequences of six P. grandiflorum from different producing areas has been sequenced with Illumina HiSeq X Ten platform. The specific DNA barcodes were screened, and the germplasm resources and genetic diversity were analyzed according to the specific barcodes. The total length of the chloroplast genome of 6 P. grandiflorum samples was 172 260-172 275 bp, and all chloroplast genomes showed a typical circular tetrad structure and encoded 141 genes. The comparative genomics analysis and results of amplification efficiency demonstrated that trnG-UCC and ndhG_ndhF were the potential specific DNA barcodes for identification the germplasm resources of P. grandiflorum. A total of 305 P. grandiflorum samples were collected from 15 production areas in 9 provinces, for which the fragments of trnG-UCC and ndhG_ndhF were amplificated and the sequences were analyzed. The results showed that trnG-UCC and ndhG_ndhF have 5 and 11 mutation sites, respectively, and 5 and 7 haplotypes were identified, respectively. The combined analysis of the two sequences formed 13 haplotypes (named Hap1-Hap13), and Hap4 is the main genotype, followed by Hap1. The unique haplotypes possessed by the three producing areas can be used as DNA molecular tags in this area to distinguish from the germplasm resources of P. grandiflorum from other areas. The haplotype diversity, nucleotide diversity and genetic distance were 0.94, 4.79×10-3 and 0.000 0-0.020 3, respectively, suggesting that the genetic diversity was abundant and intraspecific kinship was relatively close. This study laid a foundation for the identification of P. grandiflorum, the protection and utilization of germplasm resources, and molecular breeding.

Research progress in quality research and evaluation of human mesenchymal stem cells / 国际生物医学工程杂志

As adult stem cells, human mesenchymal stem cells (hMSCs) have the potential for self-replication, renewal, and multidirection differentiation. Their unique biological function determines their wide clinical indications. Researchers can define the quality attributes of hMSCs according to clinical expectations. The quality study of hMSCs should consider microbiological safety, biological safety, cell biological properties, and biological effectiveness. Quality evaluation is a common physical, chemical, and biological evaluation method for hMSCs. Traditional product safety evaluation strategies cannot fully adapt to current technology and product usage characteristics. Researchers have developed new, effective evaluation methods based on current technology. In terms of product efficacy evaluation strategies, an efficacy evaluation system has been gradually established and standardized according to the intended clinical use and based on quality studies, which can enable researchers to evaluate hMSCs products more comprehensively at different stages and processes. In this paper, the progress of quality research and evaluation of human mesenchymal stem cells was reviewed to provide a reference for the utilization of stem cells in the field of regenerative medicine.

Clinicopathological features of Helicobacter pylori-negative early gastric cancer / 北京大学学报(医学版)

OBJECTIVE@#To investigate the clinicopathological features of Helicobacter pylori (Hp)-negative early gastric cancer.@*METHODS@#The clinicopathological data of 30 cases of Hp-negative early gastric cancer were collected retrospectively at Pingdingshan Medical District, 989 Hospital of PLA Joint Logistics Support Force, and Beijing Chaoyang Hospital, Capital Medical University, from 2009 to 2021, and the histomorphological characteristics and immunophenotype were observed, and combined with the literature to explore.@*RESULTS@#The median age of 30 patients was 58.5 years (range: 21-80 years), including 13 males and 17 females. The upper part of the stomach was 13 cases, the middle part of the sto-mach was 9 cases, and the lower part of the stomach was 8 cases. The median diameter of the tumor was 11 mm (range: 1-30 mm). According to the Paris classification, 9 cases were 0-Ⅱa, 7 cases were 0-Ⅱb, and 14 cases were 0-Ⅱc. Endoscopic examination showed that 18 cases of lesions were red, 12 cases of lesions were faded or white, and microvascular structures and microsurface structures were abnormal. In all the cases, collecting venules were regularly arranged in the gastric body and corner mucosa. There were 18 cases of well differentiated adenocarcinoma in the mucosa. The tumor presented glandular tubular-like and papillary structure, with dense glands and disordered arrangement; the cells were cuboidal or columnar, with increased nuclear chromatin and loss of nuclear polarity, and most of them expressed gastric mucin. Signet-ring cell carcinoma was found in 7 cases, all the cancer tissues were composed of signet-ring cells, and the cancer cells were mainly distributed in the middle layer to the surface layer of mucosa. Gastric oxyntic gland adenoma (gastric adenocarcinoma of the fundic gland type confined to mucosa) in 2 cases, gastric adenocarcinoma of the fundic gland type in 2 cases, and gastric adenocarcinoma of fundic gland mucosa type in 1 case. The tumor tissue was composed of branching tubular glands, except 1 case of mucosal surface epithelium was partially neoplastic, the other 4 cases of mucosal surface epi-thelium were all non-neoplastic; the cells were arranged in a single layer, and the nucleus was close to the basal side, and the nucleus was only slightly atypical. Pepsinogen I and H+/K+ ATPase were positive in 5 cases of gastric fundus gland type tumors, and 1 case of foveolar-type tumor cells at the surface and depth of mucosa showed MUC5AC positive. The gastric mucosa adjacent to cancer was generally normal in all cases, without atrophy, intestinal metaplasia and Hp.@*CONCLUSION@#Hp-negative early gastric cancer is a heterogeneous disease group with various histological types, and tubular adenocarcinoma and signet-ring cell carcinoma are common. Tubular adenocarcinoma mostly occurs in the elderly and the upper to middle part of the stomach, while signet-ring cell carcinoma mostly occurs in young and middle-aged people and the lower part of the stomach. Gastric neoplasm of the fundic gland type is relatively rare.

Pathogenesis of glucocorticoid-induced osteoporosis based on label-free mass proteomics / 中国骨伤

OBJECTIVE@#To explore pathogenesis of glucocortocoid-induced osteoporosis(GIOP) based on label-free mass proteomics.@*METHODS@#Twevle female Sprague-Dawley(SD) rats were randomly divided into two groups, named as sham group and GIOP group. After one-week adaptive feeding, the rats of GIOP group were administered with dexamethasone via intramuscular injection according to 2.5 mg/kg weighting, while the rats of sham group were administered with the same amount of saline, twice a week. The tibias of each group were collected after 8-week modeling and made pathological sections to confirm the success of modeling. Three samples of each group were picked up to perform label-free mass proteomics. After quality control, differentially expressed proteins were identified according to qualitative and quantitative analyses. Then gene ontology(GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, cluster analysis as well as protein-protein interaction analysis were performed using bioinformatics analysis.@*RESULTS@#Compared with sham group, the structure of bone trabecular in GIOP group showed abnormal arrangement, uneven distribution and obvious fragmentation, which could demonstrate successful modeling. A total of 47 differentially expressed proteins (DEPs) were identified including 20 up-regulated and 27 down-regulated proteins. The expression of protein nucleophosmin 1(NPM1), adipocyte plasma membrane associated protein (APMAP), cytochromec oxidase subunit 6A1 (COX6A1) and tartrate-resistant acid phosphatase (ACP5) showed a significant difference between two groups. KEGG results showed DEPs were enriched on metabolism-related pathways, immune-related pathways and AMP-activated kinase (AMPK) signaling pathway.@*CONCLUSION@#Protein NPM1, APMAP, COX6A1 and ACP5 showed a close relationship with pathogenesis of GIOP, which could serve as potential biomarkers of GIOP. AMPK signaling pathway played an important role in the occurrence and development of GIOP, which could be regarded as potential signaling pathway to treatment GIOP.

Distribution of memory B cell subsets in peripheral blood of children with frequently relapsing nephrotic syndrome / 中国当代儿科杂志

OBJECTIVES@#To investigate the change in the distribution of memory B cell subsets in children with frequently relapsing nephrotic syndrome (FRNS) during the course of the disease.@*METHODS@#A total of 35 children with primary nephrotic syndrome (PNS) who attended the Department of Pediatrics of the Affiliated Hospital of Xuzhou Medical University from October 2020 to October 2021 were enrolled as subjects in this prospective study. According to the response to glucocorticoid (GC) therapy and frequency of recurrence, the children were divided into two groups: FRNS (n=20) and non-FRNS (NFRNS; n=15). Fifteen children who underwent physical examination were enrolled as the control group. The change in memory B cells after GC therapy was compared between groups, and its correlation with clinical indicators was analyzed.@*RESULTS@#Before treatment, the FRNS and NFRNS groups had significantly increased percentages of total B cells, total memory B cells, IgD+ memory B cells, and IgE+ memory B cells compared with the control group, and the FRNS group had significantly greater increases than the NFRNS group (P<0.05); the FRNS group had a significantly lower percentage of class-switched memory B cells than the NFRNS and control groups (P<0.05). After treatment, the FRNS and NFRNS groups had significant reductions in the percentages of total B cells, total memory B cells, IgM+IgD+ memory B cells, IgM+ memory B cells, IgE+ memory B cells, IgD+ memory B cells, and IgG+ memory B cells (P<0.05) and a significant increase in the percentage of class-switched memory B cells (P<0.05). The FRNS group had a significantly higher urinary protein quantification than the NFRNS and control groups (P<0.05) and a significantly lower level of albumin than the control group (P<0.05). In the FRNS group, urinary protein quantification was negatively correlated with the percentage of class-switched memory B cells and was positively correlated with the percentage of IgE+ memory B cells (P<0.05).@*CONCLUSIONS@#Abnormal distribution of memory B cell subsets may be observed in children with FRNS, and the percentages of IgE+ memory B cells and class-switched memory B cells can be used as positive and negative correlation factors for predicting recurrence after GC therapy in these children.

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.

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.

Proteomics reveals the protective mechanism of salvianolate injection on early hyperacute myocardial infarction / 药学学报

The hyperacute stage of myocardial infarction refers to a period of time within 30 minutes after the occurrence of myocardial infarction, when the symptoms are not obvious and the diagnosis is difficult, and the related pathophysiological mechanism has received less attention. In this study, proteomics was used to investigate the pathological changes in the early hyperacute phase of myocardial infarction, aiming to provide experimental evidence for pathological mechanism of myocardial infarction hyperacute stage. Meanwhile, the intervention effect and related mechanism of salvianolate injection were discussed based on heat shock protein B6 (HSPB6), aiming to benefit the clinical rational use of salvianolate injection. The protein expression changes before and after myocardial infarction model establishment were detected by label-free proteomics via mass spectrometry and analyzed by bioinformatics method. Then the binding effect of salvianolate injection on the commonly differential protein HSPB6 was evaluated by molecular docking technology, which was finally verified by animal experiments. All animal experimental protocols were approved by the Ethics Committee of Xiyuan Hosptial (2022XLC041). The results of this study showed that a total of 2 166 proteins were quantified by lable-free proteomics, of which 194 shared differential proteins were involved in myocardial injury and body regulation in the hyperacute phase of myocardial infarction, mainly involving molecular functions such as protein homodimerization activity, oxygen binding and transport, and serine endopeptidase inhibitor activity. Among them, HSPB6 protein is involved in the regulation of myocardial function. Molecular docking results indicated that magnesium salvianolate acetate, which is the main component of salvianolate injection, had the lowest binding energy with HSPB6 protein: -14.53 kcal·mol-1. Animal experiments showed that compared with the Sham group, the model group had significantly lower ejection fraction (EF) and fractional shortening (FS) (P < 0.001), cardiac blood perfusion decreased significantly (P < 0.001). There were obvious pathological changes such as myocardial fiber disorder, cardiomyocyte edema and interstitial small blood vessel congestion; the injury of cardiac function of rats in the administration group was attenuated, and the FS of rats in the low-dose group was significantly improved (P < 0.05), the pathological injury of myocardial tissue was markedly mitigated, and the expression of HSPB6 protein was up-regulated to varying degrees (P < 0.01, P < 0.001). In conclusion, salvianolate injection could be able to improve the cardiac function and pathological morphology of rats in the early hyperacute stage of myocardial infarction, and its mechanism may be related to the promotion of expression of HSPB6.

The coronary heart disease of phlegm-stasis cementation syndrome in mini-swine based on platelet proteomics / 药学学报

Based on the technology of platelet proteomics, the key regulatory proteins and pathogenesis of coronary heart disease with phlegm and blood stasis syndrome were explored and analyzed. Based on the previous laboratory research, the model of coronary heart disease in mini-swine with phlegm-stasis cementation syndrome was duplicated. The model was judged by the changes in blood lipid and myocardial tissue characteristics. Furthermore, the platelet proteins were studied by quantitative proteomics, and the differentially expressed proteins were screened. The critical regulatory proteins and biological pathways of coronary heart disease with phlegm-stasis cementation syndrome were analyzed by bioinformatics. After ten weeks of modeling, the levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), very low density lipoprotein (VLDL-C), triglyceride (TG), creatine kinase (CK) and creatine kinase-MB (CK-MB) in the model group were significantly increased, reflecting the pathological changes such as increased blood lipid, abnormal coagulation function and myocardial ischemia in the model group. In addition, compared with the sham group, there were 26 up-regulated proteins and 8 down-regulated proteins in the platelets of the model group. Combined with bioinformatics analysis, it was found that differential proteins mainly involved in glycolysis/gluconeogenesis, pyruvate metabolism, lipid and atherosclerosis, Ras protein signal transduction. Among them, lactate dehydrogenase B (LDHB), alcohol dehydrogenase 5 (ADH5), neuroblastoma ratsarcoma viral oncogene homolog (NRAS) and Kirsten ratsarcoma viral oncogene homolog (KRAS) play a central role when interacting with other proteins and simultaneously participate in multiple action pathways. The results showed that LDHB, ADH5, NRAS, and KRAS may be the marker proteins in CHD with phlegm-stasis cementation syndrome by regulating glycolysis/gluconeogenesis, pyruvate metabolism, lipid and atherosclerosis, Ras protein signal transduction and other biological processes.

Lack of association between the VEGFA gene polymorphisms and preterm birth in Korean women

Preterm birth (PTB), a pregnancy-related disease, is defined as a birth before 37 weeks of gestation. It is a major cause of maternal mortality and morbidity worldwide, and its incidence rate is steadily increasing. Various genetic factors can contribute to the etiology of PTB. Vascular endothelial growth factor A (VEGFA) gene is an important angiogenic gene and its polymorphisms have been reported to be associated with PTB development. Therefore, we conducted a case-control study to evaluate the association between VEGFA rs699947, rs2010963, and rs3025039 polymorphisms and PTB in Korean women. A total of 271 subjects (116 patients with PTB and 155 women at ≥38 weeks of gestation) were analyzed in this study. The genotyping of VEGFA gene polymorphisms was performed using polymerase chain reaction– restriction fragment length polymorphism. No significant association between the patients with PTB and the control groups was confirmed. In the combination analysis, we found a significant association between PTB and VEGFA rs699947 CC-rs2010963 GG-rs3025039 CC combination (odds ratio, 3.77; 95% confidence interval, 1.091 to 13.032; p = 0.031). The VEGFA rs699947, rs2010963, and rs3025039 polymorphisms might have no genetic association with the pathogenesis of PTB in Korean women. However, the combination analysis indicates the possibility that VEGFA acts in PTB pathophysiology. Therefore, larger sample sets and replication studies are required to further elucidate our findings.

Comparison of biological characteristics of major epidemic strains of Clostridium difficile from nosocomial infectious diarrhea / 中华检验医学杂志

Objective:This work aims to investigate the virulence features, spore formation and the resistance mechanisms of major sequence types (STs) of clinical Clostridium difficile isolates from nosocomial infectious diarrhea. Methods:Clostridium difficile isolates were prospectively collected from 816 loose stool samples of in patients with antibiotic associated diarrhea at the Beijing Friendship Hospital of Capital Medical University from September 2017 to September 2019. The main ST types ST81 (26 strains), ST8 (15 strains) and ST42 (14 strains) of C. difficile were used as experimental strains. The polymerase chain reaction (PCR) and enzyme-linked immunoassay (ELISA) were performed to detect toxin genes and toxin production of different C. difficile ST types, respectively. The count of the colony forming units (CFU) of the strains as conducted by using the brain-heart infusion (BHI) agar plates. The antimicrobial resistance patterns of the strains to eleven kinds of antibiotics were determined by agar dilution method. The antimicrobial resistance genes: gyrA, gyrB and ermB were amplified and sequenced from the stains. Mutations in the resistance genes were analyzed by sequencing. Measure data was compared by Kruskal Wallis Test, differences in the resistance rates in three group were compared using Fisher exact test. Results:ST81 strains were identified as the tcdA-tcdB+/ cdtA-cdtB-toxin type, ST8 and ST42 strains belonged to tcdA+tcdB+/ cdtA-cdtB-toxin type. The toxin production of ST42 strains (41.9) were higher than ST8 (2.4) and ST81 groups (0.83) (all P<0.001). The number of spore quantities of ST81, ST8 and ST42 strains were 494×10 5CFU/ml, 160×10 5CFU/ml and 166×10 5CFU/ml, respectively. The spore quantities of ST81 strains were much higher than that of ST81 and ST42 strains (all P<0.001). From the in vitro susceptibility test, 100% (26/26) ST81 strains were featured as multi-drug resistant (MDR), and they were resistant to moxifloxacin, ceftriaxone, erythromycin and clindamycin. The resistance rates of ST8 strain to moxifloxacin, erythromycin and clindamycin were 9/15, 11/15 and 11/15, respectively. ST81 strains had higher resistance rates to moxifloxacin, clindamycin and erythromycin, compared to ST8 strains ( P=0.001, P=0.005 and P=0.005). All ST42 strains were susceptible to ceftriaxone and 3/14 ST42 strains were resistant to moxifloxacin. ST81 strains had higher resistance rates to ceftriaxone and moxifloxacin than the ST42 strains (both P<0.001). The positive rate of ermB in ST81 strains (100%, 26/26) were higher the ST8 strains (11/15) ( P<0.005). Amino acid mutation analysis showed that ST81and ST8 stains had one amino acid substitution in both GyrA and GyrB, but the amino acid substitutions were different in GyrB between two ST types. ST81 strains had two point-mutations: Thr82 replaced by Ile in GyrA, and Asp426 replaced by Val in GyrB. ST8 strains had point-mutation: Thr82 replaced by Ile in GyrA; Asp426 replaced by Asn in GyrB. For ST42 strains, Thr82 was replaced by Ile in GyrA. Conclusions:ST81 and ST42 strains were MDR. ST81 had higher spore ability, whereas ST42 strains had more virulence. ST81 strains and most of ST8 strains had high level of fluoroquinolones resistance. It is important to supervise persistently these three ST genotypes to prevent further dissemination.

Analysis of indicators related to visceral fat index based on the random forest model / 中华健康管理学杂志

Objective:To explore indicators related to visceral fat index by constructing a random forest model.Methods:In this cross-sectional study, the laboratory measures and body composition analysis records of 617 hospital employees (in-service and retired) who underwent physical examination in Heilongjiang Provincial Hospital Health Management Center from March to September 2021 were selected. The subjects were divided into a training set ( n=411) and a test set ( n=206) with the ratio of 2∶1. A total of 110 predictors were included in the model. The model was constructed with the training set and was evaluated with the test set. The optimal number of nodes and decision trees were selected to evaluate the prediction performance of the optimal model. And the top 10 relatively important factors were selected for further investigation. The 617 participants were further divided in to groups according to the visceral fat index: the normal or high visceral fat index group, and the differences of the top 10 relatively important factors were further compared between the two groups. Results:The optimal number of nodes of the final random forest model was 39 and the number of decision trees was 300. The accuracy, precision, sensitivity and specificity of the model was 83.3%, 73.9%, 89.4% and 78.7%, respectively. The area under the receiver operating characteristic curve and 95% confidence interval of the model was 0.881 (0.832-0.931). The top 10 relatively important factors in the model were body mass index, gender, age, serum uric acid, red blood cell count, monocyte cell count, C-peptide, carcinoembryonic antigen, glycosylated hemoglobin and glutamyl transpeptidase. There were significant differences in the up-mentioned 10 indicators between the subjects with normal and high visceral fat index (all P<0.05). Conclusions:The random forest model built in this study has good performance in predicting visceral fat index, and visceral fat is related with changes in liver function, pancreas function and immune function.

Effect of Buyang Huanwutang on Platelet Function and Inflammatory Cytokine in Rat Model of Acute Blood Stasis Induced by Multiple Factors / 中国实验方剂学杂志

ObjectiveTo explore the effect of Buyang Huanwutang （BYHWT） on platelet function and inflammatory cytokines in the rat model of acute blood stasis. MethodThe model of acute blood stasis was established with SD rats by ice water bath combined with injection of epinephrine. Rats were randomly assigned into four groups： normal group， model group， BYHWT （3.2 g·kg-1） group， and aspirin （60 mg·kg-1） group. The rats were injected with epinephrine hydrochloride on day 8 after 7 days of modeling. The macroscopic indexes of triditional Chinese medicine （TCM） syndrome including tongue manifestation and pulse manifestation were observed， while hemorheological indexes， blood coagulation， and platelet aggregation were detected. The serum levels of the inflammatory cytokine matrix metalloprotein-9 （MMP-9） and the adhesion factor intercellular adhesion molecule-1 （ICAM-1） and were determined by enzyme-linked immunosorbent assay （ELISA）. ResultThe pulse distention of rats in the model group was lower than that in the normal group （P<0.01）， while BYHWT improved the pulse distention of the rats with the syndrome of blood stasis （P<0.01）. In the model group， the tongue showed the characteristics of blood stasis syndrome， with dark purple veins at the tongue bottom and lower values of R， G， B on the tongue surface than those in the normal group （P<0.01）， which， however， can be recovered by BYHWT （P<0.01）. The blood viscosity at high， medium， and low shear stress and the plasma viscosity in the model group were higher than those in the normal group （P<0.01， P<0.05）. Compared with the model group， BYHWT restored the whole blood viscosity under high， medium and low shear stress and plasma viscosity （P<0.05，P<0.01）. The model group had shorter prothrombin time （PT）， shorter thrombin time （TT）， and higher fibrinogen （FIB） than the normal group （P<0.05， P<0.01）. BYHWT improved the TT and reduced the FIB in the rats with blood stasis syndrome （P<0.01）. The platelet aggregation rate induced by arachidonic acid （AA） and adenosine diphosphate （ADP） in the model group was higher than that in normal group （P<0.01） and BYHWT decreased the platelet aggregation rate of the rats with blood stasis syndrome （P<0.01）. The results of scanning electron microscopy showed that the model group exhibited excessive platelet activation， obvious pseudopodia， and increased aggregation of platelets compared with the normal group， while platelet activation and aggregation were rare in the BYHWT group. The serum levels of MMP-9 and ICAM-1 in the model group were higher than those in the normal group （P<0.01）， which were decreased in the BYHWT group （P<0.05， P<0.01）. ConclusionThe SD rats with the syndrome of acute blood stasis induced by ice water bath combined with injection of epinephrine demonstrate obvious changes in platelet function and morphology， inflammation， and abnormal cell adhesion. In the treatment of acute blood stasis in rats， BYHWT may reduce thrombosis and improve blood consistency and cohesion by mitigating inflammation， down-regulating cell adhesion factor overexpression， and improving platelet shape and function.

Clinicopathological features of very well-differentiated adenocarcinoma of the stomach / 中华病理学杂志

Objective: To investigate the clinicopathological features of very well-differentiated adenocarcinoma (VWDA) of the stomach. Methods: The clinicopathological data of 12 cases of VWDA of the stomach were collected retrospectively at the People's Liberation Army Joint Logistics Support Force 989 Hospital (formerly 152 Hospital), Pingdingshan, China, from January 2013 to May 2021. The histological characteristics and immunophenotypes were observed and analyzed with review of current literature. Results: There were 8 males and 4 females with a median age of 63 years (range 47 to 80 years). The tumor involved in the upper part of the stomach in 6 cases, the middle part in 2 cases, and the lower part in 4 cases. The median diameter of the tumors was 17 mm (range 5-65 mm). The tumor cells were similar to absorbent cells, Paneth cells, foveolar epithelial cells, and goblet cells. The cells were arranged in a single layer, and the nuclei were slightly enlarged and located at the base. The nuclei were fusiform to slightly irregular, with loss of nuclear polarity. Early tubular VWDA was found in 9 cases, and the tumor glands were similar to intestinal metaplasia. In two cases the tumors infiltrated into the submucosa. The lesions in the mucosa and submucosa showed the glands with cystic expansion, bending, branching, spiky and abortive growth pattern. One case of early papillary tubular VWDA was confined to the mucosal layer and composed of foveolar-type epithelial cells. There were two cases of advanced papillary tubular VWDA, which consisted of foveolar-type epithelial, pyloric glands, or mucinous neck cells and were associated with intra-lymphatic cancer embolus and lymph node metastases. Background mucosal atrophy and intestinal metaplasia were observed in all cases. Immunohistochemical staining showed intestinal type VWDA in 1 case, mixed gastrointestinal type VWDA in 9 cases, and gastric type VWDA in 2 cases. The Ki-67 proliferation index of 8 cases limited to the mucosa was 40%-70%, 2 cases of infiltration into the submucosa and 2 cases of advanced carcinoma was 10%-25%. All the tumors showed a wild type of p53 protein expression pattern and negative HER2. Adenocarcinoma or high-grade dysplasia was diagnosed on preoperative biopsy in 5 cases, and chronic atrophic gastritis with intestinal metaplasia in 7 cases. The median follow-up time was 28 months (range 12-72 months). No recurrence was found in the 10 patients with early cancer. Of the two patients with advanced carcinoma, one patient had lung metastases and the other died. Conclusions: Gastric VWDA is a rare low-grade malignancy with structural features of highly differentiated adenocarcinoma and extremely low cytological atypia. The diagnostic value of structural abnormality is significantly greater than cytological atypia. The invasive growth of irregular glands in the deep mucosa and submucosa is reliable evidence for diagnosis. The diagnosis of intramucosal VWDA is challenging and very difficult in some biopsy specimens.

Expression of silent information regulator 1 and 3 and hypoxia-inducible factor 1α in cutaneous squamous cell carcinoma tissues and cells / 中华皮肤科杂志

Objective:To determine the expression of silent information regulator 1 (Sirt1) , Sirt3 and hypoxia-inducible factor 1α (HIF-1α) in cutaneous squamous cell carcinoma (CSCC) tissues and cells, and to explore their role in the occurrence and development of CSCC.Methods:From January 2019 to December 2020, 30 lesional skin tissues were obtained from patients with histopathologically confirmed poorly-, moderately- or well-differentiated CSCC, and 30 normal skin tissues were obtained from patients with non-cancerous diseases in Department of Dermatology, General Hospital of Ningxia Medical University. A CSCC cell line A431 and a human keratinocyte cell line HaCaT were cultured. Immunohistochemical study, Western blot analysis and real-time quantitative PCR (RT-PCR) were performed to determine the protein and mRNA expression of Sirt1, Sirt3 and HIF-1α in CSCC tissues of different grades of differentiation and normal skin tissues, cytochemical and immunofluorescence staining and RT-PCR were conducted to determine the protein and mRNA expression of Sirt1, Sirt3 and HIF-1α in A431 and HaCaT cells, respectively. Comparisons of measurement data among multiple groups were performed by using one-way analysis of variance, and comparisons between two groups by using t test. Results:Immunohistochemical study showed that the expression level of Sirt3 (expressed as the average optical density) was 100 ± 12.12, 117.72 ± 26.23, 127.32 ± 24.45, 132.71 ± 31.61 in the normal skin tissues and well-, moderately- and poorly-differentiated CSCC tissues respectively, and there was a significant difference among these groups ( F = 20.14, P < 0.001) ; the expression of Sirt1 and HIF-1α increased in turn from the normal skin tissues to the well-, moderately- and poorly-differentiated CSCC tissues, and significantly differred in these groups ( F = 174.50, 225.00, respectively, both P < 0.001) . As Western blot analysis revealed, the expression level of Sirt3 significantly differed among the normal skin tissues, well-, moderately- and poorly-differentiated CSCC tissues (expressed as relative gray value: 1.000 ± 0.132, 1.403 ± 0.411, 1.387 ± 0.393, 1.677 ± 0.683, respectively; F = 34.97, P < 0.001) , and so did the expression levels of Sirt1 and HIF-1α ( F = 69.29, 199.90, respectively, both P < 0.00l) , with a gradually increasing trend in their expression levels from the the normal skin tissues to well-, moderately- and poorly-differentiated CSCC tissues. RT-PCR showed that the mRNA expression of Sirt3, Sirt1 and HIF-1α was sequentially increased from the normal skin tissues to well-, moderately- and poorly-differentiated CSCC tissues, and significant differences were observed among these groups ( F = 113.00, 174.50, 50.33, respectively, all P < 0.001) . The protein expression levels of Sirt3, Sirt1 and HIF-1α were significantly higher in the A431 cells than in the HaCaT cells ( t = 16.75, 18.34, 27.76, respectively, all P < 0.001) , and so were their mRNA expression levels ( t= 14.22, 9.62, 16.86, respectively, all P < 0.001) . Conclusion:Increased expression of Sirt3, Sirt1 and HIF-1α was observed in CSCC tissues and cells, which may promote the occurrence and development of CSCC.

Establishment and Evaluation of Rat Model of Acute Coronary Syndrome with Combined Blood Stasis and Poison / 中国实验方剂学杂志

ObjectiveTo compare the feasibility of establishing the rat model of acute coronary syndrome with combined blood stasis and poison by lipopolysacharide （LPS） injection， ligation of coronary artery and different combinations of the two methods. MethodA total of 225 male SD rats were randomly divided into sham operation group， simple coronary artery ligation group， first injected LPS group ［LPS（5 mg·kg）injection 24 h before coronary artery ligation］ and follow injected LPS group ［LPS（5 mg·kg）injection 10 min after coronary artery ligation］. The indexes of each group were detected at 3， 24， 72 h after modeling， and the model was comprehensively evaluated. The general state and macroscopic evaluation indexes of traditional Chinese medicine （TCM） syndrome （tongue and pulse） of rats in each group were observed. ECG and echocardiography were used to evaluate cardiac function， and the myocardial ischemia and infarction areas were measured by Evans blue/2，3，5-triphenyltetrazolium chloride （TTC） staining. The content of creatine kinase isoenzyme （CK-MB）， lactate dehydrogenase （LDH）， creatine kinase （CK）， and troponin T （cTnT） in serum as well as interleukin-1 β （IL-1β） and IL-6 changes were determined by biochemical method or enzyme-linked immunosorbent assay （ELISA）. Hematology analyzer was adopted to determine the white blood cell （WBC） count， and the four coagulation indexes， platelet aggregation rate， hemorheology and other coagulation evaluation indexes were also detected. The myocardial tissue was observed by hematoxylin-eosin（HE）staining. ResultAfter 3 h of modeling， compared with the conditions in sham operation group， the R， G and B values of tongue of rats （P<0.01）， pulse amplitude （P<0.01）， and cardiac function in simple coronary artery ligation group were decreased， and the color of hypoglossal veins became purple（P<0.01）. The content of CK， LDH， cTnT， IL-1β and IL-6 in serum（P<0.05）， myocardial infarction area（P<0.01）， and total number of WBCs （P<0.05）were increased. Compared with simple coronary artery ligation group， first injected LPS group and follow injected LPS group had increased hypoglossal veins， decreased R value of tongue and elevated cTnT content （P<0.01）， while follow injected LPS group had reduced B value of tongue， decreased cardiac output （CO）（P<0.05）， increased IL-1β content， and thinned left ventricular anterior walls at end-systole （LVAWs）（P<0.01）. After 24 h of modeling， compared with sham operation group， simple coronary artery ligation group presented significantly decreased R， G and B values of tongue， lengthened purplish dark hypoglossal veins （P<0.01）， reduced pulse amplitude（P<0.01） and cardiac function， enlarged myocardial infarction area（P<0.01）， increased whole blood viscosity， platelet aggregation rate， fibrinogen （FIB）， shortened prothrombin time （PT） and thrombin time （TT）（P<0.01）， and elevated total number of WBCs （P<0.01）and content of CK， LDH， cTnT and IL-6 in serum（P<0.05）. Compared with the conditions in simple coronary artery ligation group， the pulse amplitude， R， G and B values of tongue （P<0.01）， and ejection fraction （EF） and fractional shortening （FS） scores （P<0.05）dropped， and hypoglossal veins were deepened and lengthened（P<0.05）， and cTnT content was increased（P<0.01）in first injected LPS group and follow injected LPS group. However， follow injected LPS group had thinned LVPWs， increased LDH content， platelet aggregation rate（P<0.05）， myocardial infarction area， and total number of WBC， level of IL-1β（P<0.05）， and shortened TT（P<0.01）. Additionally， 72 h after modeling， compared with sham operation group， simple coronary artery ligation group showed significantly reduced pulse amplitude， lowered R， G and B values of tongue， thickened and lengthened hypoglossal veins（P<0.01）， decreased cardiac function， and increased content of cTnT， FIB， whole blood viscosity（P<0.01），platelet aggregation rate， level of IL-6 and IL-1β（P<0.05）. Compared with the conditions in simple coronary artery ligation group， the hypoglossal veins of the first injected LPS group and the follow injected LPS group were more purple， and the content of cTnT was boosted（P<0.01）， whereas follow injected LPS group had decreased pulse amplitude， R， G and B values of tongue， EF and FS scores （P<0.05）， and enlarged myocardial infarction area（P<0.01）. ConclusionCompared with the other modeling methods and models at different modeling time， the established model by LPS injection 10 min after coronary artery ligation for 24 h was more consistent with the clinical characteristics of acute coronary syndrome with combined blood stasis and poison.

Establishment and Evaluation of Rat Model of Acute Coronary Syndrome with Combined Blood Stasis and Poison / 中国实验方剂学杂志

ObjectiveTo compare the feasibility of establishing the rat model of acute coronary syndrome with combined blood stasis and poison by lipopolysacharide （LPS） injection， ligation of coronary artery and different combinations of the two methods. MethodA total of 225 male SD rats were randomly divided into sham operation group， simple coronary artery ligation group， first injected LPS group ［LPS（5 mg·kg）injection 24 h before coronary artery ligation］ and follow injected LPS group ［LPS（5 mg·kg）injection 10 min after coronary artery ligation］. The indexes of each group were detected at 3， 24， 72 h after modeling， and the model was comprehensively evaluated. The general state and macroscopic evaluation indexes of traditional Chinese medicine （TCM） syndrome （tongue and pulse） of rats in each group were observed. ECG and echocardiography were used to evaluate cardiac function， and the myocardial ischemia and infarction areas were measured by Evans blue/2，3，5-triphenyltetrazolium chloride （TTC） staining. The content of creatine kinase isoenzyme （CK-MB）， lactate dehydrogenase （LDH）， creatine kinase （CK）， and troponin T （cTnT） in serum as well as interleukin-1 β （IL-1β） and IL-6 changes were determined by biochemical method or enzyme-linked immunosorbent assay （ELISA）. Hematology analyzer was adopted to determine the white blood cell （WBC） count， and the four coagulation indexes， platelet aggregation rate， hemorheology and other coagulation evaluation indexes were also detected. The myocardial tissue was observed by hematoxylin-eosin（HE）staining. ResultAfter 3 h of modeling， compared with the conditions in sham operation group， the R， G and B values of tongue of rats （P<0.01）， pulse amplitude （P<0.01）， and cardiac function in simple coronary artery ligation group were decreased， and the color of hypoglossal veins became purple（P<0.01）. The content of CK， LDH， cTnT， IL-1β and IL-6 in serum（P<0.05）， myocardial infarction area（P<0.01）， and total number of WBCs （P<0.05）were increased. Compared with simple coronary artery ligation group， first injected LPS group and follow injected LPS group had increased hypoglossal veins， decreased R value of tongue and elevated cTnT content （P<0.01）， while follow injected LPS group had reduced B value of tongue， decreased cardiac output （CO）（P<0.05）， increased IL-1β content， and thinned left ventricular anterior walls at end-systole （LVAWs）（P<0.01）. After 24 h of modeling， compared with sham operation group， simple coronary artery ligation group presented significantly decreased R， G and B values of tongue， lengthened purplish dark hypoglossal veins （P<0.01）， reduced pulse amplitude（P<0.01） and cardiac function， enlarged myocardial infarction area（P<0.01）， increased whole blood viscosity， platelet aggregation rate， fibrinogen （FIB）， shortened prothrombin time （PT） and thrombin time （TT）（P<0.01）， and elevated total number of WBCs （P<0.01）and content of CK， LDH， cTnT and IL-6 in serum（P<0.05）. Compared with the conditions in simple coronary artery ligation group， the pulse amplitude， R， G and B values of tongue （P<0.01）， and ejection fraction （EF） and fractional shortening （FS） scores （P<0.05）dropped， and hypoglossal veins were deepened and lengthened（P<0.05）， and cTnT content was increased（P<0.01）in first injected LPS group and follow injected LPS group. However， follow injected LPS group had thinned LVPWs， increased LDH content， platelet aggregation rate（P<0.05）， myocardial infarction area， and total number of WBC， level of IL-1β（P<0.05）， and shortened TT（P<0.01）. Additionally， 72 h after modeling， compared with sham operation group， simple coronary artery ligation group showed significantly reduced pulse amplitude， lowered R， G and B values of tongue， thickened and lengthened hypoglossal veins（P<0.01）， decreased cardiac function， and increased content of cTnT， FIB， whole blood viscosity（P<0.01），platelet aggregation rate， level of IL-6 and IL-1β（P<0.05）. Compared with the conditions in simple coronary artery ligation group， the hypoglossal veins of the first injected LPS group and the follow injected LPS group were more purple， and the content of cTnT was boosted（P<0.01）， whereas follow injected LPS group had decreased pulse amplitude， R， G and B values of tongue， EF and FS scores （P<0.05）， and enlarged myocardial infarction area（P<0.01）. ConclusionCompared with the other modeling methods and models at different modeling time， the established model by LPS injection 10 min after coronary artery ligation for 24 h was more consistent with the clinical characteristics of acute coronary syndrome with combined blood stasis and poison.

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.