ObjectiveThis study aimed to investigate the intervention effect of Renqing Mangjue on the malignant transformation of gastric mucosal epithelial cells induced by N-methyl-N′-nitro-N-nitrosoguanidine （MNNG） and to explore its molecular mechanism in preventing precancerous lesions of gastric cancer based on the cyclic guanosine monophosphate （cGMP）/protein kinase G （PKG）/mitogen-activated protein kinase （MEK）/extracellular signal-regulated kinase （ERK） signaling pathway. MethodsHuman gastric mucosal epithelial cells （GES-1） were initially induced by MNNG to establish a precancerous cell model （MC cells）. The effective concentration of MNNG for inducing malignant transformation in GES-1 cells was screened using the cell proliferation activity decection （CCK-8） assay， and the effective concentration of Renqing Mangjue for inhibiting the proliferation of transformed GES-1 cells was also determined. GES-1 cells were divided into a blank control group， a model group， and treatment groups with Renqing Mangjue at concentrations of 1， 3， 10， and 30 mg·L^-1. Furthermore， the effects of Renqing Mangjue on the migratory ability and epithelial-mesenchymal transition （EMT） characteristics of GES-1 malignant transformed cells were evaluated using Transwell migration assays， wound healing assays， and real-time quantitative reverse transcription polymerase chain reaction （Real-time PCR）. Additionally， candidate chemical components and target sites of Renqing Mangjue were obtained from the TCMIP v2.0 database， and disease targets at various stages of gastric cancer precursors were sourced from the Gene Expression Omnibus （GEO） database. Pathway enrichment analysis was performed using the Metascape database to predict the potential mechanisms of action of Renqing Mangjue. Finally， the protective mechanism of Renqing Mangjue against gastric cancer precursors was validated through Western blot analysis. ResultsAt a concentration of 20 μmol·L^-1， MNNG exhibited an inhibition rate of approximately 50% on GES-1 cells （P<0.01）， and at this concentration， the GES-1 cells displayed biological characteristics indicative of malignant transformation. In contrast， Renqing Mangjue had no significant effect on the proliferation of normal GES-1 cells， but significantly inhibited the proliferation of MC cells （P<0.01） and markedly reduced their migratory capacity （P<0.01）. Moreover， it also increased the mRNA expression level of E-cadherin during the EMT process （P<0.05）， while inhibiting the expression of both N-cadherin and the transcription factor Snail mRNA （P<0.05， P<0.01）. Network predictions suggested that Renqing Mangjue may prevent gastric cancer precursors through modulating the cGMP/PKG and MAPK/ERK signaling pathways. Furthermore， Western blot results indicated that Renqing Mangjue upregulated the expression of PKG and NPRB （B-type natriuretic peptide receptor） proteins in the cGMP/PKG pathway （P<0.01）， while downregulating the expression of the downstream proteins MEK and ERK （P<0.05， P<0.01）. ConclusionIn summary， Renqing Mangjue can prevent gastric cancer precursors by inhibiting the proliferation and migration of malignant transformed GES-1 cells， thereby delaying the EMT process. The underlying mechanisms may be related to the activation of the cGMP/PKG pathway and the inhibition of the MEK/ERK signaling pathway.

ObjectiveTo identify the pharmacodynamic material basis of Ruyi Zhenbaowan in relieving neuropathic pain by integrating the calculation of biological network proximity and pharmacokinetic characterization. MethodThe interaction network of "drug candidate target-related gene of disease" was constructed by Cytoscape 3.8.2， and the average shortest path value of each drug putative target acting on neuropathic pain-related genes in this network was calculated by Pesca 3.8.0 tool so as to evaluate the network proximity between them， and screen prescription candidate targets with strong intervention efficiency and their corresponding potential effect components. After that， plasma and cerebrospinal fluid samples were collected from rats after administration of Ruyi Zhenbaowan at set time points， and the contents of potential effect components in samples was quantified by ultra performance liquid chromatography-quadrupole-ion trap mass spectrometry（UPLC-Q-TRAP/MS）， and drug concentration-time curves were plotted， then the pharmacokinetic parameters were calculated by DAS 2.1.1. ResultBy evaluating the network proximity between candidate targets and neuropathic pain-related genes in the interaction network， a total of 40 putative targets of Ruyi Zhenbaowan with strong intervention effects on neuropathic pain-related genes， such as estrogen receptor 1（ESR1）， cyclic adenosine monophosphate（cAMP）-dependent protein kinase catalytic subunit alpha（PRKACA） and protein kinase B1 （Akt1）， and 10 corresponding potential effect components， such as glycyrrhizic acid and betulinic acid， were obtained. Pharmacokinetic characterization showed that among the 10 potential effect components， gallic acid， apigenin-7-O-glucuronide， glycyrrhizic acid and apigenin were well absorbed and metabolized in plasma and cerebrospinal fluid， with long onset time and good bioavailability. ConclusionFrom the perspective of efficacy-target-constituent-pharmacokinetic， this study analyzes the main effective materials of Ruyi Zhenbaowan， such as glycyrrhizic acid， gallic acid， apigenin-7-O-glucuronide and apigenin， which have a high exposure in plasma or cerebrospinal fluid and have a strong intervention effect on neuropathic pain. The related results provide reliable experimental evidences for clarifying the material basis and developing quality standards of Ruyi Zhenbaowan.

Objective:To explore the feasibility and influencing factors of E-cervix cervical elasticity analysis technology in analyzing normal cervical function during non pregnancy.Methods:213 women who underwent vaginal ultrasound examination in the Ultrasound Department of Foshan Maternal and Child Health Hospital from May 2019 to November 2019 were selected as the research objects. Taking the median sagittal section of the cervix as the initial section, the E-cervix technology software package was started to automatically obtain the elastic contrast index (ECI), hardness ratio (HR), cervical strain rate (IOS), cervical strain rate (EOS), cervical strain ratio (IOS/EOS) and cervical length (CL). The relationship among age, menstrual cycle, BMI index, birth history, delivery mode and elastic parameters were compared.Results:There was no correlation between the elastic parameters and age, and there was no significant difference among different age groups ( P>0.05); there was no significant difference in the elastic parameters of cervical tissue in menstrual period, proliferative period and secretory period ( P>0.05); there was no significant difference in the elastic parameters of underweight, normal and overweight ( P>0.05); CL was positively correlated with body mass index (BMI) ( r=0.225, P<0.05), there was no correlation between other parameters and BMI ( P>0.05); there was no significant difference between the elastic parameters of patients with and without birth history ( P>0.05); the CL of women with cesarean section [(34.22±4.96)mm] was higher than that of women with natural birth [(29.03±4.14)mm] ( P<0.05), and the other parameters had no statistical significance ( P>0.05). Conclusions:The elastic parameters of cervix obtained by E-cervix technique are not affected by age, BMI, menstrual period, reproductive history and delivery mode, and can be used for quantitative evaluation of cervical function.

Objective To study the histomorphology structure of the femur in adult horses and adults, analyze the histological features and establish the method of identifying the species between humans and horses. Methods The 4 cm mid-diaphyseal segment of the right femur from adult human at autopsy was obtained. At the same time, the right femur of the horse was collected and the middle section was obtained about 4cm. After decalcification, a bone tissue section about 25 μm in thickness was obtained. Observe under an optical microscope, images under a microscope were input into a computer, and 25 indicators were selected for stepwise discriminant analysis. Results Significant differences between horses and human were observed on 13 indicators such as number of Haversian system and Haversian system diameter. Mathematical model for species identification was established based on these indicators. After a blind test,the discrimination reaches 99.6%. Conclusion Horse and human femur histological structure have obvious species characteristics and the established discriminant equation can effectively identify horses and human femur fragments.

Objective The content of urea and amino acids in sweat fingerprints is examined by ultraviolet spectrophotometry at ambient temperature of 25 and relative humidity of 65%. Methods In the fingerprint of sweat, urea and amino acids are chemically reacted to produce blue and blue purple matter.Their concentrations are calculated by ultraviolet spectrophotometerand the two ratios are calculated to infer the remaining time. Results Left time of sweat fingerprints shows a linear correlation with urea and amino acid ratio, the linear regression equation is Y=-3.227X+6.706, the female is Y=-3.672X+6.546, the regression equation is Y=-3.276X+6.638.Conclusion The remaining time of human sweat fingerprints is linearly related to the ratio of urea to amino acidsand the regression relation can be used to infer the time of fingerprint retention.

Objective To investigate personal identification of mixed seminal stain of two individuals, we combined the detection of genotyping autosomal, Y and X STR and sequencing mtDNA hypervariable Ⅰ (HV Ⅰ ) region. Methods We analyzed autosomal, Y and X STR with commercial kit and separating and sequencing HVⅠfragments of mixed seminal stain from two males by SSCP electrophoresis. Results Four genetic markers of the high amount sample can be obtained when mixed ratio is more than 1:10. When the proportion of two samples is close, the suspect could be excluded or, to some extent, identified by comparing with our results. Conclusion The combined detection of four genetic marker systems can, to some degree, solve the personal identification from mixed seminal stain of two individuals.

Objective To compare the PCR effect of rapid PCR instrument and common PCR instrument. Methods The concentration of 9947A standard was diluted to 0.1, 0.05, 0.025, 0.0125, 0.0063 ng/μL, 100 samples from each concentration group to establish PCR reaction system with Identifiler? Plus PCR Amplification Kit, 50 samples of them test with rapid PCR instrument (Speed cycler2 thermocycler), the other 50 samples test with common PCR instrument (9700 thermocycler). Detection of PCR product with 3500xL Genetic Analyser, the STR typing of both groups of each concentration group should be compared. Results The success rate of both thermocyclers have no significant difference (P>0.05); The success number of STR typing of common PCR instrument (13.7±1.0; 11.3±1.5) were higher than rapid PCR instrument (13.1±1.3; 9.9±1.9) when the concentration of 9947A were 0.0125, 0.0063ng/μL (P=0.029; P<0.001); The peak height of DNA typing map obtained from common PCR instrument (18931±4625;13437±3165; 5752±1344) were higher than rapid PCR instrument (16929±4034; 11815±4120; 4865±1401) when the concentration of 9947A were 0.1, 0.05, 0.025ng/μL (P=0.023; P=0.030; P=0.002). Conclusions The rapid PCR instrument could achieve the equal success rate to the common PCR instrument with less time, which revealed that the rapid PCR instrument was suitable for application in practical cases; The quality of STR typing from common PCR instrument may be more higher.

Objective To discuss the effect of DNA extraction of bloodstain on the filter paper with four methods of solid phase absorption.Methods 180 bloodstain samples on the iflter paper, each one contains 1 microlitre anticoagulation peripheral venous blood, divided into 4 groups with 45 samples, respectively. All samples were treated with four methods of solid phase absorption, i.e. DNA IQ? System, D-shield sensitive DNA Extraction Kit, High efficiency Silica Bead DNA Extraction Kit and Conventional silica bead method. The concentration of DNA and the results of STR typing of four groups were compared each other.Results The concentration of DNA was 3.764±1.790μg/mL and 3.634±1.112μg/mL by using D-shield sensitive DNA Extraction Kit and High efifciency Silica Bead DNA Extraction Kit, respectively. However, the concentration of DNA by using Conventional silica bead method group (3.350±1.250) was not signiifcantly different from each other (P<0.05), while the concentration of DNA extracted with above three methods were higher than by using DNA IQ? System (1.864±1.207)(P<0.001); Signiifcant differences of peak height existed between DNA IQ? System and other three methods (P<0.001); As the same time, the peak height of samples by using High efficiency Silica Bead DNA Extraction Kit and Conventional silica bead method were signiifcantly different from D-shield sensitive DNA Extraction Kit (P<0.01).Conclusions The DNA extracted in bloodstain on the iflter paper by using D-shield sensitive DNA Extraction Kit, High efifciency Silica Bead DNA Extraction Kit and Conventional silica bead method was more than DNA IQ? System. Meanwhile, the quality of DNA using High efifciency Silica Bead DNA Extraction Kit and Conventional silica bead method may be higher.

OBJECTIVEThis paper aimed to determine the mRNA expression of osteoclast-related factors interleukin-6 (IL-6), interleukin-12 (IL-12) p35, IL-12p40, matrix metalloproteinase-9 (MMP-9), nuclear factor of activated T-cells cytoplasmic 1 (NFATcl), receptor activator of nuclear factor-KB (RANK), and tumor necrosis factor-α (TNF-α) mRNA in murine macrophages infected by a periodontitis patient's own tissue nucleic acid. Another aim was to investigate the effects of a periodontitis patient's own tissue nucleic acid on the differentiation of macrophages into osteoclasts.

METHODSInflammatory periodontal tissue samples of chronic periodontitis patients were taken during periodontal flap surgery, and healthy gingival tissue samples were taken from orthodontic patients during tooth extractions. Total RNA from periodontal tissue was extracted and reversely transcribed into cDNA and then cryo-preserved until further use. First, specific sequence oligodeoxynucleotide MT0I at a concentration of 1 µg · mL⁻¹ was added in murine macrophage RAW264.7, and the cells were incubated for 3 hours. Cells with PBS (1 µg · mL⁻¹) were used as negative controls. The inflammatory periodontal tissue cDNA and healthy periodontal tissue cDNA (1 µg · mL⁻¹) was added subsequently. There were four experimental groups: healthy periodontal tissue cDNA+ RAW264.7, inflammatory periodontal tissue cDNA+RAW264.7, MT01+healthy periodontal tissue cDNA+RAW264.7, and MT01+inflammatory periodontal tissue cDNA+RAW264.7. Real-time quantitative polymerase chain reaction was used to detect the mRNA expression of osteoclast-related factors IL-6, IL-12p35, IL-12p4O, MMP-9, NFATcl, RANK, and TNF-α mRNA after 3, 6, 12, and 24-hours.

RESULTSThe mRNA levels of osteoclast-related factors NFATc1, MMP-9, TNF-a, IL-6, IL-12p40, IL-12p35, and RANK in RAW264.7 were markedly upregulated with the treatment of periodontitis patient's own tissue nucleic acid. However, the mRNA expression of osteoclast-related factors was inhibited by use of an immunosuppressant MT01.

CONCLUSIONThe periodontitis patient's own tissue nucleic acid could promote the differentiation of murine macrophage into osteoclasts.

Animals ; Cell Differentiation ; Cytokines ; metabolism ; Gene Expression ; Gingiva ; Humans ; Interleukin-12 Subunit p40 ; Interleukin-6 ; Macrophages ; Matrix Metalloproteinase 9 ; Mice ; Osteoclasts ; metabolism ; Periodontitis ; RNA, Messenger ; Tumor Necrosis Factor-alpha

Objective To study the morphological characteristics of femurs of adult human and 11 kinds of adult animals fromcattle, horses, pigs, goats, sheep, dogs, cats, rabbits, geese, ducks, chickens, and to establish an effective species identification method among various species. Methods The 4 cmmid-dia-physeal segment of the femur fromadult human (older than 20 years old) at autopsy w as obtained. Addi-tionally, the 4 cmones from11 kinds of adult animals w ere obtained. After decalcification, all femurs w ere made into slices, and then w ere observed by optical microscope. The 25 indexes w ere selected and analyzed by step discriminant analysis according to differences betw een human and mammal, human and poultry, and human and 11 kinds of animals. Results The histological structure of bone mineral density of middle part of femur had obvious characteristics among the species. And the morphology and number of osteon show ed the trend of obvious biological evolution. There w ere 11 indexes w ith significant differ-ences betw een human and 11 kinds of animals to establish some mathematical models to discriminate all species. The correct discrimination rate w as 96.3% betw een human and mammal. The correct discrimina-tion rate w as up to 100% betw een human and poultry, and w as 89.4% among human, mammal and poultry. Conclusion The mathematical models have good correct discrimination rate among human and the other animals, w hich could be applied in the practical species identification cases.