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ObjectiveTo investigate the pharmacodynamic characteristics and explore the molecular mechanism of Honghua oral liquid （HOL） in relieving neuropathic pain （NP）. MethodHealthy male SD rats were randomly assigned into sham group， model group， low-， medium-， high-dose （0.5， 1.0， 2.0 mL·kg-1·d-1， respectively） HOL groups， and a positive drug （pregabalin， 25 mg·kg-1·d-1） group， with 6 rats in each group. Spinal nerve ligation （SNL） of L5 was conducted in other groups except the sham group. Drug administration was performed 3 days after the SNL surgery for 2 consecutive weeks， and samples were collected after the end of the administration. During the treatment period， the mechanical pain threshold and cold pain threshold were determined to measure the pain-relieving effect of HOL. Transcriptome sequencing was performed on hippocampal tissue samples from the sham， model， and high-dose HOL groups， and differentially expressed genes between the sham group and the model group as well as the model group and HOL high-dose group were obtained. After pathway enrichment analysis， we selected the targets which were closely related to neuroinflammation for validation， and predicted the specific binding sites of the major active components in HOL with the targets through molecular docking. In addition， the serum levels of tumor necrosis factor-α （TNF-α） and interleukin-10 （IL-10） were determined by enzyme-linked immunosorbent assay （ELISA） to evaluate the effect of HOL on neuroinflammation in NP rats. ResultCompared with the sham group， SNL decreased the mechanical pain threshold and cold pain threshold （P<0.05）. Compared with the model group， HOL recovered the mechanical pain threshold and cold pain threshold （P<0.05）. The transcriptome data showed that 376 differentially expressed genes （DEGs） were identified between the model group and the sham group， including 124 upregulated genes and 252 downregulated genes， and 194 DEGs between the model group and the high-dose HOL group， including 33 upregulated genes and 161 downregulated genes. Among them， insulin-like growth factor 1（IGF1）， matrix metallopeptidase-2 （MMP-2）， matrix metallopeptidase-14 （MMP-14）， erb-B2 receptor tyrosine kinase 2 （ERBB2）， and integrin subunit alpha 5 （ITGA5） associated with NP were selected for further validation. The Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） results showed that compared with the sham group， the modeling up-gurelated the mRNA levels of the above five molecules in the hippocampus （P<0.01）. Compared with model group， HOL down-regulated the mRNA levels of these molecules （P<0.01）. The molecular docking results showed that the main active components of safflower， hydroxysafflor yellow A， kaempferol， and quercetin， formed stable hydrogen bonds with the amino acid residues of IGF1， MMP-2， MMP-14， ERBB2， and ITGA5. The enzyme-linked immunosorbent assay（ELISA） results showed that compared with those in the sham group， the serum levels of TNF-α and IL-10 were out of balance in the model rats （P<0.01）. Compared with the model group， HOL lowered the level of the pro-inflammatory cytokine TNF-α （P<0.01） and elevated that of the anti-inflammatory cytokine IL-10 （P<0.05）. ConclusionHOL exerts analgesic effect on SNL rats by inhibiting neuroinflammation.
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ObjectiveTo study the in vitro kinetics of Jiaojiang cataplasms and evaluate its pharmacodynamics， so as to provide a feasible basis for the development of this preparation. MethodThe improved Franz diffusion cell was used for the in vitro release in semipermeable membrane and transdermal absorption in in vitro mouse skins. The contents of hydroxy-α-sanshool， 6-gingerol， ginsenoside Rb1 were determined by high performance liquid chromatography （HPLC）， to evaluate the in vitro release and transdermal absorption of Jiaojiang cataplasms. The mobile phase of 6-gingerol and hydroxy-α-sanshool was water-acetonitrile-methanol （2∶1∶1） with the detection wavelength of 280 nm. The mobile phase of ginsenoside Rb1 was acetonitrile-0.1% phosphoric acid aqueous solution （31∶69） with the detection wavelength of 203 nm. A mouse intestinal paralysis model was established， and mice were randomly divided into five groups， namely sham operation group， model group， domperidone group （3.9 mg·kg-1） and high- and low-dose groups of Jiaojiang cataplasms （6.2， 3.1 g·kg-1， measured by crude drug dosage）， to observe the effect of this preparation on gastrointestinal propulsion function. ResultAverage release rates of hydroxy-α-sanshool， 6-gingerol and ginsenoside Rb1 at 24 h were 16.41， 4.23， 4.15 μg∙cm-2∙h-1， the average transdermal rates of them at 24 h were 2.31， 0.64， 0.29 μg∙cm-2∙h-1， their skin retention values were 19.56， 3.59， 1.61 μg， respectively. According to the Ritger-Peppas equation， the release of hydroxy-α-sanshool， 6-gingerol， ginsenoside Rb1 was non-Fick diffusion. The high-dose group of Jiaojiang cataplasms could improve intestinal function of model mice after small intestinal friction injury， and promote intestinal peristalsis and small intestinal propulsion rate （P<0.05）. ConclusionJiaojiang cataplasms has in vitro release and transdermal properties， the in vitro release conforms to Higuchi equation， and transdermal absorption behavior conforms to zero-order kinetic equation， which can improve the postoperative function of the small intestine and the propulsion function of small intestine. It preliminarily indicates that the preparation has certain clinical development value.
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Objective:To analyze the clinical and pathological features and gene mutations of pancreatic acinar cell carcinoma (PACC).Methods:Clinical data of 34 patients with PACC admitted to the Department of Pancreatic Surgery of the First Affiliated Hospital of Naval Medical University from December 2009 to July 2018 were retrospectively analyzed to summarize its clinical characteristics, and the expressions of α1-ACT, CaM5.2, Syn and CgA in pancreatic tumor tissues were detected by immunohistochemistry. Next-generation gene sequencing technology was used to detect gene mutations in tumor specimens.Results:Among the 34 PACC patients, 23(68%) were males and 11(32%) were females; the age ranged from 25 to 75 years, with an average age of 54 years. The first symptom was abdominal pain or distension in 21 cases (62%), skin or scleral yellow staining in 4 cases(12%), and 9 cases(26%) were found in routine physical examination. BMI was 17.6-34.0 kg/m 2, of which 3 cases (9%) were <18.5 kg/m 2, 23 cases (68%) were 18.5-24.0 kg/m 2, and 8 cases (23%) were >24.0 kg/m 2. Preoperative examination showed elevated CA19-9 in 7 cases (20.6%), elevated CEA in 3 cases (8.8%), and elevated AFP in 7 cases (20.6%). Blood amylase was 16-247 U/L, with an average of 80 U/L. Enhanced CT showed that the lesion was irregular in shape, showing inhomogeneity and slightly low density, with areas of cystic degeneration and necrosis. The tumor was located in the head of the pancreas in 14 cases (41%), the body and tail of the pancreas in 19 cases (56%), and the neck of the pancreas in 1 case (3%). The largest tumor diameter was 1.5-15.5 cm, with an average of 5.4 cm. Postoperative pathologic stage I was confirmed in 4 cases (12%), stage Ⅱ in 14 cases (41%), stage Ⅲ in 14 cases (41%) and stage Ⅳ in 2 cases (6%). Immunohistochemical results showed that both α1-ACT and CaM5.2 were positively expressed (100%). Syn was positive in 8 cases (23.5%) and CgA was positive in 6 cases (17.6%). Ki-67 index was from 9% to 70%, with an average of 41%. Gene sequencing of pancreatic tumor tissue from 6 patients showed BRCA2 mutation in 2 patients (7155C>G), K-ras mutation in 1 patient (35G>T), RET mutation in 1 patient (200G>A), and LKB1 mutation (234G>T) in 1 patient, and one double mutation of K-ras and RET (35G>A, 1 798C>T). 30 patients were followed up, and the median survival was 38.3 months. Conclusions:PACC was a rare pancreatic tumor with no specific clinical manifestations. The positive expression rates of α1-ACT and CAM5.2 in tumor tissues were 100%. BRCA2, K-ras, RET and LKB1 were common gene mutations.
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On the basis of the qualitative preparation quality markers of Yulian Decoction, we screened out the quantitative markers and explored a general strategy for analyzing the component migration in Chinese herbal pieces, preparations, and plasma. A method capable of simultaneously determining 28 chemical components in Yulian Decoction was established based on HPLC-MS/MS. This method was used to determine the migrated components in herbal pieces-lyophilized powder preparations-rat plasma after administration of Yulian Decoction. Liquid chromatography was performed under the following conditions: C_(18)-reversed phase chromatographic column(2.1 mm × 100 mm, 1.8 μm); acetonitrile-water(containing 0.1% formic acid) as the mobile phase for gradient elution; the flow rate of 0.2 mL·min~(-1). Electrospray ionization source was adopted for mass spectrometry detection, in which positive and negative ion modes and multiple reaction monitoring were applied. Confirmed by the methodological investigation in linear range, recovery(95.48%-103.4%), precision(RSD, 0.45%-3.8%), stability, and repeatability(RSD, 5.6%-14%), the established method was suitable for the detection and quantification of the components in Yulian Decoction. The results showed that in the lyophilized powder of Yulian Decoction, berberine was greater than 5% in mass fraction, magnoflorine, epiberberine, coptisine, palmatine, and limonin in the range of 1%-5%, and dehydroevodiamine, evodiamine, rutaecarpine, costunolide, and dehydrocostus lactone in the range of 0.002%-1%. Of the 28 components detected in pieces, 27 were found to migrate to the lyophilized powder, and 11 were detected in rat plasma. Fifteen components were preliminarily determined as quantitative preparation quality markers for Yulian Decoction, including berberine, epiberberine, coptisine, palmatine, evodiamine, rutaecarpine, limonin, costunolide, dehydrocostus lactone, magnoflorine, jatrorrhizine, columbamine, groenlandicine, chlorogenic acid, and neochlorogenic acid. In conclusion, the HPLC-MS/MS general strategy was established for analyzing the migration of multiple components in Chinese herbal pieces, preparations, and plasma, which can provide the basis for the screening of quantitative preparation quality markers and multi-index quality control of Yulian Decoction.
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Animals , Rats , Chromatography, High Pressure Liquid , Chromatography, Liquid , Drugs, Chinese Herbal , Spectrometry, Mass, Electrospray Ionization , Tandem Mass SpectrometryABSTRACT
Objective:To analyze the molecular characteristics of Echovirus 11 (Echo11) strains isolated in Xiangyang, Hubei Province from 2016 to 2017 based on the sequences of VP1 gene.Methods:Rectal and throat swab specimens were collected from children with hand, foot and mouth disease (HFMD) in Xiangyang from 2016 to 2017. Echo11 strains were detected by real-time reverse transcriptase PCR (RT-PCR) and isolated after cultured in human rhabdosarcoma (RD) cells. The VP1 regions of Echo11 strains isolated from RD cells and the whole genomes of three representative Echo11 strains were amplified by conventional RT-PCR and the sequences were analyzed. DNAStar7.0 (MegAlign) and MEGA6.0 (Data) were used to analyze the homology and mutation sites in nucleotide and amino acid sequences. Neighbor-joining method was used to construct phylogenetic trees. Recombination analysis was performed with SimPlot software (BootScanning).Results:A total of 11 Echo11 strains were isolated from 3 494 HFMD cases, accounting for 0.31%. They were highly homologous in the VP1 gene. These strains shared 98.4%-100.0% homology in nucleotide sequences and 98.3%-100.0% homology in amino acid sequences. The homology between the 11 Echo11 strains and the prototype strain (Echo11/Gregory, X80059) was 73.9%-74.8% in nucleotide sequences and 87.7%-88.7% in amino acid sequences. All of the Echo11 strains circulating in Xiangyang were classified into lineage D, having a similarity to the strains circulating in some regions of mainland China since 2013. In multiple regions of the genome, the Echo11 strains isolated in Xiangyang were highly similar to the Henan Echo1 strains in 2010 and the Hubei Echo6 strains in 2015, suggesting there was recombination within the genome of Echo11 strains in Xiangyang.Conclusions:The Echo11 strains circulating in Xiangyang from 2016 to 2017 belonged to lineage D and were recombinant strains.
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Objective:To investigate the effect of the production process of Zushima Guanjie Zhitong Gao from solvent method to hot-pressed method on <italic>in vitro</italic> kinetic behavior of this preparation. Method:Solvent and hot-pressed methods were used to prepare three batches of samples above pilot scale， and <italic>in vitro</italic> release and percutaneous penetration of the index components （7，8-dihydroxycoumarin and methyl salicylate） in Zushima Guanjie Zhitong Gao were investigated by modified Franz diffusing cells. Result:The contents of 7，8-dihydroxycoumarin and methyl salicylate in Zushima Guanjie Zhitong Gao prepared by solvent method were 73.72， 494.67 μg/patch， and their contents in hot-pressed method samples were 159.21， 2 638.99 μg/patch， respectively. In the solvent method samples， the average cumulative release amounts of 7，8-dihydroxycoumarin and methyl salicylate in 24 h were 2.04， 12.21 μg， and their average cumulative release amounts in 24 h of hot-pressed method samples were 2.16， 36.24 μg， respectively. In the solvent method samples， the average cumulative permeation amounts of 7，8-dihydroxycoumarin and methyl salicylate in 24 h were 0.38， 2.79 μg， and they were 0.40， 7.49 μg in hot-pressed method samples. The cumulative release and permeation amounts in 24 h of 7，8-dihydroxycoumarin in the hot-pressed method samples were basically the same as those of the solvent method samples， but the cumulative release and permeation amounts in 24 h of methyl salicylate in the hot-pressed method samples were significantly higher than those of the solvent method samples （<italic>P</italic><0.05）. Conclusion:The retention of 7，8-dihydroxycoumarin and methyl salicylate by hot-pressed method is better than that of the solvent method. The process change has no significant effect on the <italic>in vitro</italic> kinetics of 7，8-dihydroxycoumarin in Zushima Guanjie Zhitong Gao， however， after the change from the solvent method to the hot-pressed method， the methyl salicylate in this preparation has a higher cumulative release and permeation amounts.
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Objective:To identify the transdermal constituents of Euodiae Fructus and predict its molecular mechanism in treating diarrhea by transdermal drug delivery. Method:Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS） and integrated pharmacology methods were used. The rapid identification of transdermal constituents of Euodiae Fructus was realized by the means of comparison of reference substances， analysis of UNIFI system and mass spectrometry. On this basis， Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine （TCMIP） v2.0， SymMap， DisGeNET databases and literature were used to collected potential targets of transdermal constituents of Euodiae Fructus and targets for diarrhea-related diseases. The disease targets and drug targets were topologically analyzed to obtain the core targets， which were used for the Gene Ontology （GO） function and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis. Finally， Cytoscape 3.6.0 was used to build up a network of transdermal constituents-core targets-key pathways. Result:A total of 19 chemical constituents were speculatively identified from Euodiae Fructus extract， including quinolone alkaloids， limonins， indole alkaloids， organic acids and sterols. A total of 174 core targets of Euodiae Fructus for treating diarrhea were obtained by a topology analysis， signaling pathways of inflammatory response， cell proliferation， nutrient regulation and energy metabolism， signal transduction， bacterial infection were obtained through the analysis of KEGG enrichment. Conclusion:In this study， the transdermal constituents of Euodiae Fructus are identified for the first time， they can participate in the regulation of intestinal inflammation， maintain the integrity of intestinal mucosa， repaire and adjust the metabolism of the body by acting on Rac protein family， phosphatidylinositol 3-kinase， cytochrome P450 enzymes and aldo-keto reductase， respectively. In general， the molecular mechanism of Euodiae Fructus in the treatment of diarrhea is preliminarily elucidated.
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Objective:To screen qualitative preparation quality markers of Yuliantang， in order to provide data support for the selection of indicator components， and establish the direct connection between indicator components and efficacy （Xiehuo Zhitong） for achieving the quantity-effect combination. Method:The stability of preparation process of Yuliantang lyophilized powder was investigated by HPLC fingerprint technology， then， the components in Yuliantang lyophilized powder were identified by UHPLC-LTQ-Orbitrap-MS. By referring to the relevant literature， the pharmacological activities of these identified compounds were compared with the pharmacological effects corresponding to the efficacy of Yuliantang， and the composition of the qualitative preparation quality markers of Yuliantang lyophilized powder was determined. Result:The similarities between HPLC fingerprint of 10 batches of Yuliantang lyophilized powder and the control fingerprint were >0.9， indicating that the preparation process was stable and feasible. A total of 29 components were identified from Yuliantang， of which 23 alkaloids， 3 phenylpropanoids， 2 sesquiterpenoids and 1 limonoid， and there were 15 ingredients of<italic> </italic>Coptidis Rhizoma， 12 ingredients of<italic> </italic>Euodiae Fructus， and 2 ingredients of<italic> </italic>Aucklandiae Radix. The composition of the qualitative preparation quality markers of Yuliantang was initially determined as magnoflorine or 10-hydroxy-2，3，9-trimethoxyberberine， phellodendrine， menisperine， thalifendine， groenlandicine， dehydroevodiamine， coptisine， jatrorrhizine， columbamine， methylcoptisine， berberine， epiberberine， palmatine， evodiamine， rutaecarpine， neochlorogenic acid， cryptochlorogenic acid， chlorogenic acid， limonin， costunolide， dehydrocostus lactone. Conclusion:The method for researching and screening the preparation quality markers in Yuliantang lyophilized powder is scientific， reasonable and feasible， it can provide reference for the determination of component indicators in the process research of Yuliantang and qualitative and quantitative indexes in its quality standard.
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As a new direction of pancreatic cancer treatment, neoadjuvant therapy for pancreatic cancer has been confirmed to be able to improve the prognosis of the patients. Under multidisciplinary treatment (MDT) mode, neoadjuvant therapy combines multidisciplinary advantages to solve patients′ problems of diagnosis and treatment, provides accurate, comprehensive and individual treatments, and maximizes the clinical benefit for patients. In this article, we summarize the present problems of neoadjuvant therapy for pancreatic cancer in patient selection, treatment regimen selection, treatment response evaluation and surgical selection, and explore the direction of clinical research and neoadjuvant therapy for pancreatic cancer under MDT mode.
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Objective::To study on the feasibility of a new method for determination of astragaloside Ⅳ in Astragali Radix. Method::By summarizing the literatures about the method for determining the content of astragaloside Ⅳ in Astragali Radix and analyzing the results of the preliminary test, a new method for preparing a test solution of astragaloside was established, named " Reflow alkalization derivatization method" . The content determination test was carried out, and the content data of astragaloside Ⅳ in different batches of Astragali Radix measured by the pharmacopoeia method were compared with that by the Reflow alkalization derivatization method. Result::The new method for the determination of astragaloside Ⅳ conformed to the corresponding regulations. The content of astragaloside Ⅳ in astragali radix determined by the new method was higher than that by the pharmacopoeia method. The standard curve was Y=1.315X+ 6.311 2(r=0.999 7, n=6, linear range is 0.044 6-8.92 μg). The RSDs of intraday precision and daytime precision were 0.5% and 0.6%, respectively. The RSD of the repetitive experiment was 1.2%. The RSD of the 48 h stability test was 2.1%, and the RSD of the recovery test was 2.0%. The contents of astragaloside Ⅳ in 16 batches of Astragali Radix determined by Reflow alkalization derivatization method and pharmacopoeia method were 0.371%, 0.203%, 0.315%, 0.218%, 0.386%, 0.221%, 0.353%, 0.192%, 0.303%, 0.197%, 0.373%, 0.188%, 0.361%, 0.114%, 0.349%, 0.112%; 0.243%, 0.152%, 0.214%, 0.168%, 0.274%, 0.157%, 0.221%, 0.133%, 0.203%, 0.141%, 0.257%, 0.132%, 0.238%, 0.084%, 0.242%, and 0.096%. Conclusion::The Reflow alkalization derivatization method can be used to determine the content of astragaloside Ⅳ in Astragali Radix. This method is simpler to operate than the pharmacopoeia method, and the conversion efficiency of astragalus glycosides derivatives is better and reproducible. This method can provide reference for the formation of a fast, scientific and accurate method for the determination of astragalus Ⅳ.
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Objective: To study the chemical constituents of Lianhua Qingwen Capsule and illuminate its substance foundation. Methods: The compounds were isolated and purified by LPLC and preparative HPLC from the 30% ethanol fraction of Lianhua Qingwen Capsule macroporous resin column chromatography. Their chemical structures were elucidated by the spectral analyses. Results: 18 compounds were isolated and identified as forsythoside A (1), forsythoside I (2), forsythoside H (3), lugrandoside (4), isolugrandoside (5), ferruginoside A (6), lianqiaoxinoside C (7), calceolarioside C (8), forsythoside E (9), ferruginoside B (10), D-amygdaloside (11), L-amygdaloside (12), sambunigrin (13), cornoside (14), 4-hydroxy-4-methylenecarbomethoxy-cyclohexa- 2,5-dienone (15), liriodendrin (16), liquiritigenin-7-O-β-D-glucopyranoside (17), and 3,4-dihydroxy benzaldehyde (18). Conclusion: Compounds 2-8, 10, and 13-18 are isolated from Lianhua Qingwen Capsule for the first time, and compounds 4-6, 10, 15, and 16 are isolated from single herb in Lianhua Qingwen Capsule compound for the first time. The spectral data in DMSO-d6 solution of compound 8 are reported firstly with 2D NMR spectral data. The above results show the high polar chemical constitutions of Lianhua Qingwen Capsule, which provides more chemical information of Lianhua Qingwen Capsule.
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Objective: In this paper,the effect of microemulsion in Chuanqi ophthalmic microemulsion in situ gel was investigated. Method: The effect of microemulsion was confirmed by the parallel comparison between the Chuanqi microemulsion in situ gel and normal in situ gel,including study of pharmaceutical characterization and tissue distribution. Result: The average particle sizes of Chuanqi microemulsion in situ gel and normal in situ gel were (38.20±0.13) nm and (985±37) nm,respectively.Microemulsion could maintain the properties of nanocarrier in a microemulsion in situ gel composite system.The result of tissue distribution study showed that only ligustilide could be detected.This was related to the nature of these three indicator components(ligustrazine,ligustilide and astragaloside A).The common logarithm of oil and water partition coefficient of ligustilide(lgP) was 2.87,which was consistent with the range of lgP of ideal ophthalmic drugs(lgP=2.0-3.0).The ligustilide from Chuanqi microemulsion in situ gel could be detected in the cornea,vitreous body and retina,and this compound from normal in situ gel could only be detected in the cornea with low content.At the same time,microemulsion could increase the content of ligustilide in corneal tissues. Conclusion: The characteristics of microemulsion nanocarriers can increase the solubility of ligustilide,compared with normal in situ gel,it can be better distributed in the tears outside the corneal,it reaches the cornea with a higher concentration,and forms a corneal concentration gradient,and ligustilide is transported from the anterior ocular region to the posterior ocular region through the transocular barrier.
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Objective@#To detect norovirus (NoV) GⅠ.1- and GⅡ.4-specific IgG, IgA and histo-blood group antigen (HBGA)-blocking antibodies in healthy populations of all age groups in China for better understanding the epidemiological features of norovirus in China from a serological point of view and providing basic data for vaccine development and clinical trial design.@*Methods@#Indirect ELISA and HBGA-blocking assay were used to detect NoV-specific IgG, IgA and HBGA-blocking antibodies in serum samples collected from healthy natural populations (n=839, aged from six months to 88 years old) in Guangzhou, Fuyang and Yantai. The results were statistically analyzed.@*Results@#The total positive rates of NoV GⅠ.1- and GⅡ.4-specific IgG antibodies were 91.9% and 93.0%. The positive rates of GⅠ.1- and GⅡ.4-specific IgA antibodies were 48.6% and 75.6%, and the titers of HBGA-blocking antibodies to GⅠ.1 and GⅡ.4 norovirus were 5.04 (95%CI: 4.63-5.49) and 18.15 (95%CI: 16.11-20.44). The positive rates of IgG and IgA antibodies generally showed an increasing trend with age. The positive rates of GⅠ.1- and GⅡ.4-specific IgG antibodies ranged from 79.2% to 100.0% and 76.7% to 100.0% in different age groups. They were 81.7% and 85.0% in the age group of 0.5-<1 year, 79.2% and 76.7% in the age group of 1-<2 years, and 98.1% and 96.3% in the age group of 12-<18 years, and maintained at 96% and 98% in the older age groups. The positive rates of GⅠ.1-specific IgA antibody ranged from 11.7% to 93.8% in different age groups and rapidly increased with age. It was 11.7% in the age group of 0.5-<1 year, and reached 93.3% in people aged 45-<60 years and 93.8% in people aged ≥60 years. The positive rates of GⅡ.4-specific IgA antibody ranged from 50.8% to 88.8% in different age groups with 50.8% in people aged 0.5-<1 year, and 86.7%-90.7% in people aged 12-<18 years and older. The titer of GⅠ.1 HBGA-blocking antibody generally increased with age. The antibody titer in populations aged 0.5-<12 years old was lower than that in those aged 18 years and above (GMT: 2.98-4.07 vs 8.21-11.62, P<0.001), and the titer in people of 12-<18 years old was lower than that in those of 45 years old and above (GMT: 5.21 vs 11.03-11.62, P<0.05). No obvious change with age was observed in the titer of GⅡ.4 HBGA-blocking antibody excepting the significant difference between populations of 2-<5 and 22-<45 years old (GMT: 26.73 vs 11.87, P<0.01).@*Conclusions@#This study revealed the characteristics of serum NoV GⅠ.1- and GⅡ.4-specific IgG, IgA and HBGA blocking antibodies in populations of different age groups in central and eastern China through analyzing their positive rates and titers and provided preliminary seroepidemiological data for the development of NoV vaccines in China.
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Objective@#To examine the prognostic value of four important driver gene mutations in patients with radical resection of pancreatic cancer.@*Methods@#The clinical data and follow-up data of pancreatic cancer patients undergoing radical pancreatectomy and targeted sequencing from January 2016 to March 2018 at Department of Hepato-Biliary-Pancreatic Surgery, Changhai Hospital were retrospectively analyzed.There were 159 males and 88 females,aged of (60.8±8.7)years(range:33-83 years) and preoperative CA19-9 of (492.4±496.6)kU/L(range: 2-1 200 kU/L). One hundred and fifty nine cases of tumors were located in the head and 88 cases in the body and tail of the pancreas. After univariate analysis of clinical pathological factors (including gender, age, preoperative CA19-9, tumor location, tumor differentiation, pathological T and N stage, Micr. perineural invasion, Micr. lympho-vascular invasion, resection margin), the variable whose P<0.1 was included in COX regression model with four important driver gene mutations to find which mutation was related to prognosis independently. The number of gene mutations and KRAS subgroups were analyzed by Kaplan-Meier curve.@*Results@#Among 247 patients,the number of KRAS,TP53, SMAD4 and CDKN2A mutations was 212 cases(85.8%), 160 cases(64.8%), 66 cases(26.7%) and 44 cases(17.8%),respectively.KRAS mutation was correlated with the tumor differentiation and pathological T stage (χ2=24.570/6.690, P=0.000/0.035), TP53 mutation was correlated with the tumor differentiation and the resected margin(χ2=5.500/4.620, P=0.019/0.032), and CDKN2A mutation was correlated with gender(χ2=16.574, P=0.000).COX regression model analysis showed that only KRAS mutation was an independent risk factor for disease free survival and overall survival(HR=1.776, 95%CI: 1.079-2.923, P=0.024; HR=1.923, 95%CI: 1.016-3.639, P=0.045); KRASG12D mutation was associated with shorter OS(P=0.007).@*Conclusion@#KRAS and its subgroup KRASG12D mutation can be used as a prognostic index for patients with radical resection of pancreatic cancer.
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Objective To detect and analyze enteroviruses causing suspected aseptic meningitis in a kindergarten in Jinhua City, Zhejiang Province. Methods Viral RNA was extracted from samples and cDNA was prepared by reverse transcription. PCR was performed to amplify the partial sequences of the 5′-untranslated region ( UTR) and VP1 gene of enteroviruses. Serotypes of the viruses were determined by com-paring the homology between the partial sequences of VP1 gene. Phylogenetic tree of the partial VP1 se-quences was constructed using MEGA6. Results This study included seven patients and twenty-six asymp-tomatic students. Coxsakievirus A10 (CV-A10) was detected in 48. 5％ of the students and echovirus 6 (Echo 6) in 21. 2％. Besides, 12. 1％ of the students might be co-infected by the two viruses. Among the seven patients, six were infected by CV-A10 and the other one might have co-infection. According to the phylogenetic analysis, CV-A10 strains detected in this study were closely related to those isolated in China in recent years, including the strains isolated in Xiamen in 2015 and Yunnan in 2017, while the Echo 6 strains were phylogenetically related to those isolated in Yunnan, Guangzhou and Shandong in 2014. Conclusions CV-A10 and Echo 6 were detected in the cases with suspected aseptic meningitis and had close phylogenetic relationships to the strains appeared in China in recent years.
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Objective In this paper, the extraction process of Xiaozhong-Zhitong granule (XZG) was investigated. Methods In Process 1, the Panax notoginseng (Burk.) F.H.Chen was extracted with other drugs, and in the process 2, the Panax notoginseng (Burk.) F.H.Chen was crushed and directly used as medicine. Rats were randomly divided into blank group, process 1 group, process 2 group and mixture group.The sample concentration of the process 1 group 1 for the stomach was 2.07 g/ml, and the sample concentration of the process 2 group 1 for the stomach was 1.06 g/ml, and the sample concentration of the mixture group for the stomach was 0.015 g/ml, and blank group gavaged equal volume saline. Anti-inflammatory and analgesic effects of acetic acid writhing test, hot plate test, auricle swelling test and egg white-induced plantar swelling test were evaluated. Results Compared with the blank group after 1, 2, 3, and 4 h after administration, the average writhing number of the mice in the process group 1 and the process 2 group (35.50％ ± 8.06％, 35.00％ ± 7.63％vs. 47.00％ ± 1.45％) significantly decreased (P<0.01), and the auricular swelling degree (46.31％ ± 22.25％, 45.41％ ± 21.43％ vs. 73.89％ ± 15.55％) significantly decreased. And compared with the blank group after 0.5, 1, 2, and 4 h after inflammation, the percentage of pain threshold in the first group and the second group increased (P<0.01). While there was no significant difference in the inhibition rate of paw swelling in each group (P>0.05). Conclusions The design of extraction process of XZG was reasonable; when treating swelling and pain, it was better to put the powder of Panax notoginseng (Burk.) F.H.Chen in XZG.
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Objective@#To construct the prokaryotic plasmid expressing the recombinant protein Coxsackie virus A6 VP1 or VP2 and prepare the antiserum of rabbit anti-CVA6 VP1 or anti-CVA6 VP2.@*Methods@#CVA6 VP1and VP2 gene fragments were amplified by reverse transcription PCR and inserted into the prokaryotic expression vectors. The recombinant plasmids were expressed in E. coli BL21 (DE3). After induction with Isopropyl β-D-Thiogalactoside (IPTG), the fusion proteins were obtained, and then purified by SDS-PAGE electrophoresis and gel extraction. The polyclonal antibodies were prepared by immunizing rabbits with the fusion proteins and analyzed with Western blot(WB) and indirect immunofluorescence assay(IFA).@*Results@#The prokaryotic expression vector of CVA6 VP1 or VP2 was confirmed by PCR, double enzyme digestion and sequencing. CVA6 VP1 and VP2 fusion proteins with high purity were obtained. WB and IFA were used to identify polyclonal antibodies.@*Conclusions@#The CVA6 VP1 and VP2 prokaryotic expression vectors were successfully constructed, and the recombinant CVA6 VP1 and VP2 proteins and their corresponding polyclonal antibodies were obtained.
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Dengue virus ( DENV) threatens the lives of hundreds of millions of people every year. Animal model is an effective tool for virus pathogenesis research and vaccine evaluation. At present, the de-velopment of animal models of DENV infection and disease has been challenging, as epidemic DENV does not naturally infect non-human species. Animal models of dengue virus infection are mainly non-human pri-mate ( NHP) model and mouse model. NHPs have been used to study dengue infection and candidate vac-cines because they can sustain viral replication in relevant cell types and develop a robust immune response, but they do not develop overt disease. However, NHP models are not suitable for early preclinical studies be-cause the cost of using these animals is high. In contrast, mouse models that are established mainly by using wild-type and immunodeficient mice can be more economical for preclinical evaluation of dengue vaccines. Overall, every model has its advantages and disadvantages and is differentially suited for studies of dengue pathogenesis and immunopathogenesis and/or preclinical testing of antiviral drugs.
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Dengue virus ( DENV) threatens the lives of hundreds of millions of people every year. Animal model is an effective tool for virus pathogenesis research and vaccine evaluation. At present, the de-velopment of animal models of DENV infection and disease has been challenging, as epidemic DENV does not naturally infect non-human species. Animal models of dengue virus infection are mainly non-human pri-mate ( NHP) model and mouse model. NHPs have been used to study dengue infection and candidate vac-cines because they can sustain viral replication in relevant cell types and develop a robust immune response, but they do not develop overt disease. However, NHP models are not suitable for early preclinical studies be-cause the cost of using these animals is high. In contrast, mouse models that are established mainly by using wild-type and immunodeficient mice can be more economical for preclinical evaluation of dengue vaccines. Overall, every model has its advantages and disadvantages and is differentially suited for studies of dengue pathogenesis and immunopathogenesis and/or preclinical testing of antiviral drugs.
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Objective To optimize refinement of water extract from Bushen Yangxue Granules by chitosan flocculation.Methods According to the content of icariin detected by HPLC, the waters amount, extraction time and extraction times were evaluated by orthogonal design. The effects of the solution concentration, clarifying temperature and the amount of clarifying agent on the flocculation clarification processes were optimized with the content of icariin and polysaccharides.Results The optimum water extraction processes A2B1C3 were follows: 10 times amount of water, three times extraction and 1 h for each extraction process. The optimized flocculation clarification processes A1B2C3 were as follows: solution concentration was 0.4 g/mL, the clarifying temperature was 40℃ and the addition of chitosan was 0.1%.Conclusion The optimized refining process is stable and feasible.