Cases of human infection with H7 subtype avian influenza virus(AIV)combined with five NA subtypes(N2,N3,N4,N7,and N9)have been reported.This study was aimed at establishing a method for simultaneous detection and dif-ferential diagnosis of H7 and five NA subtypes of AIV.Seven pairs of specific primers were designed according to the conserved sequences of the HA gene of H7 subtype AIV,the NA gene of five NA AIV subtypes,and the M gene of all AIV subtypes.A high-throughput GeXP typing method was established for simultaneous detection of the H7 subtype and the five NA subtypes of AIV by using GeXP multiple gene expression and capillary electrophoresis analysis technology.The specificity and sensitivity of the method were determined,and clinical samples were tested.The specificity results indicated that this method was able to simultaneously detect seven target genes in a single tube;each pair of specific primers was able to detect the corresponding AIV subtype,and the universal detection primers were able to detect all subtypes of AIV,with no cross-reaction with other common avian disease pathogens.Sensitivity results demonstrated that this method was able to simultaneously detect seven target genes with a threshold detection limit was 100 copies/μL.The detection results for 150 clinical samples were consistent with those of viral isolation and identification.The high-throughput GeXP method for simultaneous differential diagnosis of the H7 subtype and five subtypes of AIV established in this study has advantages of high specificity,high sensitivity,rapidity,and simplicity,thus providing a new detection method for the effective prevention and control of AIV.

Objective:To investigate the effect of GRIM-19 on the resistance of carcinoma cells to the chemotherapeutic agent docetaxel in the treatment of PCa.Methods:Using siRNA technology to interfere with the gene expression in PCa cells,we estab-lished a model of GRIM-19 overexpression/knockdown in PCa cells.We investigated the effect of different expression levels of GRIM-19 on docetaxel-induced death of the PCa cells by qPCR,Western blot and flow cytometry,and assessed the value of GRIM-19 in re-ducing the chemotherapy-resistance of PCa cells.Results:GRIM-19 was down-regulated in PCa tissues and cells.Knockout of GRIM-19 significantly decreased the expression of siGRIM19 in the PC-3 and LNCaP cells,and reduced their death rate when treated with docetaxel compared with the control group.The expressions of GRIM-19 mRNA and protein were remarkably upregulated after transfection with GRIM-19,and the overexpressed GRIM-19 promoted the death of the PC-3 and LNCaP cells treated with docetaxel in a dose-dependent manner.Flow cytometry analysis showed a lower apoptosis rate of PC-3-R cells than that of PC-3 cells at different time points of docetaxel-induction at different doses.Conclusion:GRIM-19 is a PCa suppressor gene with a significant facilitating effect on the apoptosis of PCa cells,and the overexpression of GRIM-19 promotes docetaxel-induced PCa cell death and improves the sensitivity of chemotherapy.

AIM To establish a UPLC-MS/MS method for the simultaneous content determination of pseudoginsenoside F11 and ginsenosides Rf,Rg1,Re,Rb1,Rb2,Rb3,Rg2,Rg3,Rc,Rd.METHODS The analysis was performed on a 40℃ thermostatic Phenomenex Kinetex F5 column(2.1 mm×100 mm,1.7 μm),with the mobile phase comprising of acetonitrile-2 mmol/L ammonium formate flowing at 0.3 mL/min in a gradient elution manner,and electron spray ionization source was adopted in negative ion scanning with multiple reaction monitoring mode.RESULTS Eleven ginsenosides showed good linear relationships within their own ranges(r>0.9900),whose average recoveries were 97.40％-103.74％with the RSDs of 1.76％-3.48％.CONCLUSION This sensitive,practical and reproducible method can be used for the quality control of Ginseng Radix et Rhizoma in Fukang Tablets.

AIM To study the chemical constituents from the leaves of Cyanocarya paliurus(Batalin)Iljinskaja and their α-glucosidase inhibitory activities.METHODS The 95%ethanol extract from the leaves of C.paliurus was isolated and purified by macroporous resin,silica gel,Sephadex LH-20,polyamide,C18 reversed-phase silica gel and semi-preparative HPLC,then the structures of obtained compounds were identified by physicochemical properties and spectral data.Their α-glucosidase inhibitory activities were evaluated by PNPG.RESULTS Fifteen compounds were isolated and identified as cyclopaloside C(1),cyclopaloside A(2),juglanosides E(3),vaccinin A(4),ent-murin A(5),kaempferol 3-O-α-L-rhamnopyranoside(6),kaempferol-3-O-β-D-glucopyranoside(7),kaempferol-3-O-β-D-glucuronide methyl ester(8),kaempferol-3-O-β-D-glucuronide ethyl ester(9),kaempferol-3-O-β-D-glucuronide butyl ester(10),quercetin-3-O-α-L-rhamnopyranoside(11)quercetin-3-O-β-D-glucopyranoside(12),quercetin-3-O-β-D-galactopyranoside(13),quercetin-3-O-β-D-glucuronide butyl ester(14),dihydrokaempferol(15).The IC50 value of total extracts ihibited α-glucosidase was(1.83±0.04)μg/mL,and the IC50 values of compounds 1,4-5 were(29.48±1.86),(0.50±0.07),(0.71±0.07)μmol/L,respectively.CONCLUSION Compound 1 is a new tetrahydronaphthalene glycoside.Compounds 4-5,8-10 and 14 are isolated from the leaves of C.paliurus for the first time.Compounds 4-5 are relatively rare flavonoid lignans with potential inhibitory activities against α-glucosidase.

Objective To observe the clinical efficacy of compound Wufengcao liquid combined with negative pressure wound therapy with instillation(NPWTi)for the treatment of stage Ⅲ-Ⅳ pressure injury(PI),and to preliminarily explore its action mechanism.Methods(1)Clinical research:from January 2019 to October 2022,60 PI patients who were admitted to the Scrofula Department and Wound Care Clinic at Nanjing Municipal Hospital of Traditional Chinese and Western Medicine were randomly divided into normal saline NPWTi group and compound Wufengcao liquid NPWTi group,with 30 cases in each group.Both groups underwent NPWTi under the premise of systemic basic treatment,before treatment,after removing the negative pressure device in the 1st,2nd and 3rd weeks of treatment,the pressure ulcer scale for healing(PUSH)score,the wound bacterial culture detection rate and the wound healing time were counted,and the vascular endothelial growth factor(VEGF)content of wound tissue was detected by ELISA method.(2)Animal experiments:24 SD rats were randomly divided into blank group,model group,normal saline NPWTi group and compound Wufengcao liquid NPWTi group,6 rats in each group.PI rat model was established by local tissue ischemia/reperfusion injury method,and the negative pressure device was removed at the end of each day of treatment.Before treatment and 3,7 and 10 days after treatment,the wound morphology of each group of rats was observed,the wound histopathology was observed by HE staining,the CD34 positive cells rate of wound tissue was detected by immunohistochemistry,and the expressions of p38 mitogen-activated protein kinase(p38 MAPK),nuclear factor-κB p65(NF-κB p65),inducible nitric oxide synthase(iNOS),tumor necrosis factor-α(TNF-α),arginase-1(Arg-1)and transforming growth factor-β(TGF-β)in rat blood and wound tissue were detected by ELISA and RT-qPCR.Results(1)Clinical research:Both groups could effectively reduce the PUSH score and the wound bacterial culture detection rate,shorten the wound healing time,and promote the expression of VEGF in wound tissue,the compound Wufengcao liquid NPWTi group was better than the normal saline NPWTi group(P<0.05).(2)Animal experiments:Compared with blank group,the rats in the model group showed obvious wound inflammatory response and tissue damage,and the CD34 positive cells rate,blood and wound tissue p38 MAPK,NF-κB p65,iNOS and TNF-α levels were significantly increased,Arg-1 and TGF-β level was significantly reduced(P<0.05);Compared with model group,after 7 days of treatment,the normal saline NPWTi group and the compound Wufengcao liquid NPWTi group significantly decreased the wound morphology score,the histopathological morphology was significantly improved,the CD34 positive cells rate was significantly increased(P<0.05),the levels of blood and wound tissue p38 MAPK,NF-κB p65,iNOS,and TNF-α were significantly reduced,and the levels of Arg-1 and TGF-β were significantly increased(P<0.05),and the compound Wufengcao liquid NPWTi group was better than that of the normal saline NPWTi group(P<0.05).Conclusion Compound Wufengcao liquid combined with NPWTi can effectively promote the healing of PI wounds,and its mechanism of action may be by inhibiting the activation and expression of p38 MAPK/NF-κB signaling pathway,thereby regulating the polarization balance of M1/M2 macrophages.

Objective To investigate the effect and mechanism of proteasome inhibitor MG132 on memory impairment induced by chronic hypoxia in mice.Methods(1)A hypoxic model of the mouse midbrain dopaminergic neuron cell line MN9D was established using a hypoxia workstation.To observe the effects of hypoxia on the expression of TH,Ub-K48 and Ub-K63,MN9D cells were divided into normoxia group and hypoxia(12 h,24 h and 48 h)groups.To observe the effects of MG132 on the expression of the above-mentioned proteins,MN9D cells were divided into normoxia group,hypoxia group and hypoxia + MG132(25,50,100,200 μmol/L)group.(2)A mouse model of memory impairment was established using a hypobaric chamber.To observe the effects of hypobaric hypoxia on the expression of TH,Ub-K48 and Ub-K63 in the substantia nigra compacta(SNc)of mice,thirty C57BL/6 mice were randomly and equally divided into normoxia group and hypobaric hypoxia(3 d and 21 d)groups,10 in each group.To observe the effects of MG132 on spatial memory impairment induced by hypobaric hypoxia,twenty-four C57BL/6 mice were randomly and equally divided into normoxia group,hypobaric hypoxia 21 d group and hypobaric hypoxia 21 d+MG132 group,8 in each group.(3)The protein expression levels of TH,Ub-K48,and Ub-K63 in MN9D cells which were either subjected to different durations of hypoxia treatment or pre-treated with MG132 prior to hypoxia treatment were detected using Western blotting(WB).The novel object recognition test was used to detect the memory function of mice.Immunofluorescence was used to detect the proportion of positive immunoreactive area of TH response in the SNc region.The expression levels of TH,Ub-K48,and Ub-K63 in the SNc region were detected by WB.Results(1)Compared with normoxia group,MN9D cells in hypoxia 24 h group showed increasing expression of Ub-K48 and Ub-K63(P<0.05),and decreasing expression of TH(P<0.05),and MN9D cells in all hypoxia groups showed increasing expression of Ub-K48/TH and Ub-K63/TH(P<0.05).Compared with hypoxia group,MN9D cells showed decreasing expression of Ub-K48/TH and Ub-K63/TH in hypoxia + MG132 100 umol/L group and hypoxia + MG132 200 umol/L group(P<0.05).(2)Compared with the mice in normoxia group,mice in 3 d and 21 d hypobaric hypoxia groups showed decreasing expression of TH(P<0.001),and increasing expression of Ub-K48/TH and Ub-K63/TH(P<0.05)in the SNc region.Compared with normoxia group,the mice in 21 d hypobaric hypoxia group showed a lower new object recognition index(P<0.01),and the proportion of positive immunoreactive area of TH response in the SNc region(P<0.05).Compared with 21 d hypobaric hypoxia group,the mice in hypobaric hypoxia 21 d+MG132 group showed a higher new object recognition index(P<0.01).Conclusion The proteasome inhibitor MG132 could alleviate the memory impairment induced by chronic hypoxia in mice,and its mechanism may be related to the inhibition of Ub-K63 and Ub-K48,which in turn upregulates expression of TH in dopaminergic neurons.

OBJECTIVE: To study effects of Shenmai Injection on hypertensive heart failure and its mechanism for inhibiting myocardial fibrosis. METHODS: Salt-sensitive (Dahl/SS) rats were fed with normal diet (0.3% NaCl) and the high-salt diet (8% NaCl) to observe the changes in blood pressure and heart function, as the control group and the model group. Salt-insensitive rats (SS-13BN) were fed with the high-salt diet (8% NaCl) as the negative control group. After modeling, the model rats were randomly divided into heart failure (HF) group, Shenmai Injection (SMI) group and pirfenidone (PFD) group by a random number table, with 6 rats in each group. They were given sterilized water, SMI and pirfenidone, respectively. Blood pressure, cardiac function, fibrosis and related molecular expression were detected by sphygmomanometer, echocardiogram, enzyme linked immunosorbent assay (ELISA), hematoxylin-eosin staining, Masson staining, immunofluorescence and qPCR analysis. RESULTS: After high-salt feeding, compared with the control and negative control group, in the model group the blood pressure increased significantly, the left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) were significantly reduced, and the serum NT-proBNP concentration increased significantly (all P<0.05); furthermore, the arrangement of myocardial cells was disordered, the edema was severe, and the degree of myocardial fibrosis was also significantly increased (P<0.05); the protein and mRNA expressions of collagen type I (Col I) were up-regulated (P<0.05), and the mRNA expressions of transforming growth factor β 1 (TGF- β 1), Smad2 and Smad3 were significantly up-regulated (P<0.05). Compared with HF group, after intervention of Shenmai Injection, LVEF and LVFS increased, myocardial morphology was improved, collagen volume fraction decreased significantly (P<0.05), and the mRNA expressions of Col I, TGF- β 1, Smad2 and Smad3, as well as Col I protein expression, were all significantly down-regulated (all P<0.05). CONCLUSION Myocardial fibrosis is the main pathological manifestation of hypertensive heart failure, and Shenmai Injection could inhibit myocardial fibrosis and effectively improve heart failure by regulating TGF-β 1/Smad signaling pathway.
Rats ; Animals ; Stroke Volume ; Sodium Chloride ; Rats, Inbred Dahl ; Ventricular Function, Left ; Heart Failure ; Transforming Growth Factor beta1/metabolism* ; Hypertension ; Fibrosis ; RNA, Messenger

In this study, the mechanism of Xiaoyan Lidan formula (XYLDF) against 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-collidine (DDC)-induced chronic intrahepatic cholestasis (CIHC) in mice was investigated based on metabolomics, molecular docking and pharmacological methods. In the pharmacodynamics study, a dosage of 5 g·kg^-1 (clinical equivalent) XYLDF was administered in DDC-induced mice, then the effect of XYLDF against CIHC was evaluated by measuring the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP) as well as total bilirubin (TBIL) in serum and observing liver histopathological changes. All experiments were approved by the Ethical Committee Experimental Animal Center of Guangzhou University of Chinese Medicine (ZYD-2021-001). The serum metabolites of mice in each group were detected and identified based on ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry, and the relevant biological pathways and molecular key targets were further enriched. Molecular docking technology was used to further evaluate the binding activity of the main active ingredients of XYLDF with potential targets. Subsequently, the in vitro experiment was conducted for the validation of the vital target. The results showed that compared with the model group, XYLDF significantly decreased the levels of ALT, AST, AKP and TBIL in the serum of CIHC mice, as well as alleviated inflammatory infiltration and hepatocyte necrosis in liver tissue. According to the metabonomic study, a total of 35 differential metabolites was identified as biomarkers associated with cholestasis, 12 of which were significantly recovered by XYLDF treatment. These biomarkers were involved in the pathways of primary bile acid biosynthesis and linoleic metabolism, which are closely related to the mechanism of XYLDF against CIHC. Protein-protein interaction network indicated that cytochrome P450 3A4 (CYP3A4) and cytochrome P450 1A1 (CYP1A1) are significant potential targets with good binding properties with six major active ingredients of XYLDF. Furthermore, it was found that 4-methoxy-5-hydroxycanthin-6-one, dehydroandrographolide and isodocarpin, three of the main active components in XYLDF, markedly induced the expression of CYP3A4 mRNA in vitro. This study revealed that XYLDF mainly mediates the biosynthesis of bile acids in CIHC mice to improve liver tissue lesions and bile efflux disorders, among which, CYP3A4 is the key target in the protection of XYLDF against CIHC. This research provides a reference for further elucidation of the pharmacological mechanism of XYLDF.

Objective: To explore the role of transient elastography technology in the assessment of disease staging and treatment in patients with chronic hepatitis B virus (HBV) infection. Methods: Patients who were clinically diagnosed with chronic HBV infection at Beijing Tsinghua Changgung Hospital from January 2018 to December 2021 was collected. Liver stiffness measurement (LSM) examination was performed more than once by transient elastography. The count data were expressed as cases (%) and the χ (2) test was made. Fisher's exact test was used with theoretical frequency less than 5. The measurement data between two groups was compared by t-test. Multiple groups were compared with an analysis of variance. Results: 1 055 patients were included in this study, including 669 (63.4%) males and 386 (36.6%) females. 757 (71.8%) patients were untreated. Among the untreated patients, the LSM value in the immune clearance (10.2 ± 3.8) kPa (187 cases, 40.4%), and the reactivation stages (9.1 ± 3.4) kPa (114 cases, 24.6%) was significantly higher than that in the immune tolerance (8.7 ± 3.6) kPa (78 cases, 16.8%) and immune control stages (8.4 ± 3.5) KPa (84 cases, 18.1%), and the difference between the four groups was statistically significant (F = 5.31 and P = 0.03). With ALT (male: 30 U/L, female: 19 U/L) as defined the normal value, the LSM value in the immune tolerance and the immune control stages were (5.8 ± 0.9) kPa and (7.1 ± 2.5) kPa, respectively, which were significantly lower than those of patients in the immune tolerance and immune control stages, and the difference was statistically significant (P < 0.01). There were 294 (38.8%) patients with uncertain period, excluding patients with fatty liver. Patients with uncertain periods were divided into four gray zone (GZ) groups: immune tolerance stage: LSM (5.1 ± 1.3) kPa was significantly lower than GZ-A (6.5 ± 2.4) kPa, t = 2.06, P = 0.03, and the difference was statistically significant; immune control stage: LSM was (5.6 ± 1.5) kPa, which was also lower than GZ-C (6.8 ± 1.3) kPa, t = 3.08, P = 0.02, and the difference was statistically significant; immune clearance stage: LSM > 8.0 kPa. LSM values showed a year-by-year reduction in patients with expanded indications who started antiviral treatment and were followed up for three years. Conclusion: The LSM value is significantly lower after the decrease of the defined high-normal ALT value in patients with the immune tolerance and immune control stages of chronic HBV infection. The LSM values of GZ-A and GZ-C in the uncertain periods of chronic HBV infection are higher than those of patients in the immune tolerance and immune control stages.
Humans ; Male ; Female ; Hepatitis B, Chronic/drug therapy* ; Liver Cirrhosis/pathology* ; Elasticity Imaging Techniques ; Antiviral Agents/therapeutic use* ; Liver/pathology*

OBJECTIVE: To analyze the immunological phenotype of chronic myeloid leukemia (CML), and explore its characteristics and significance. METHODS: The immunophenotypes of 40 CML children and 40 controls were analyzed by multicolor flow cytometry. CD45/SSC, as the basic gate, was used to delineate neutrophils. Then, the distribution of cluster differentiation (CD) molecules on the surface of granulocytes was analyzed in three ranges (≥1%, ≥5%, and ≥20%), and the expression rates of CD molecules (≥1% included in the statistical analysis) and the mean fluorescence intensity (MFI) were compared between the two groups. RESULTS: The proportion of granulocytes in the CML group was (82.1±6.4)%, which was significantly higher than (57.8±11.8)% in the control group (P <0.001). The expression rates of CD15/CD11b/CD33/CD13 in CML and control groups were high, and both distributed in the range of ≥20%. The differentiation trajectory of CD33/CD13 was normal and there were no significant differences in the expression rate and MFI between the two groups. However, both the expression rate of CD11b and CD15 MFI in the CML group were significantly lower than those in the control group (P <0.001). There were no significant differences in the expression rate and MFI of CD10 between the two groups, and the expression levels of CD10 between the two groups were consistent in different distributions. In the CML group, there was a large number of cases with abnormal high expression of CD56, 52.5% of the cases had a CD56 expression rate of ≥5%, and 42.5% had a CD56 expression rate of ≥20%, while the control group did not express CD56 (<1%). The expression distribution of CD117 was different between the two groups. In the range of expression rate ≥5%, there were 35.0% cases in the CML group, while only 2.5% in the control group. The expression rate of CD117 in the CML group was higher than that in the control group (P <0.001), though there was no significant difference in MFI. CONCLUSION The immunophenotyping of CML is characterized by increased proportion of mature neutrophils, decreased CD15 MFI, decreased proportion of CD11b and abnormal high expression of CD56 and CD117. Flow cytometric analysis of immunophenotype can effectively distinguish normal granulocytes from chronic granulocytes, and help in the diagnosis of CML.
Child ; Humans ; Flow Cytometry ; Leukemia, Myeloid ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics* ; Granulocytes ; Neutrophils ; Immunophenotyping