ObjectiveTo decipher the mechanism by which Zuoguiwan （ZGW） treat hyperthyroidism in rats with kidney-Yin deficiency based on the dopamine receptor D4 （DRD4）/nicotinamide adenine dinucleotide phosphate （NADPH） oxidase 4 （NOX4） signaling pathway. MethodsThe rat model of kidney-Yin deficiency was induced by unilateral intramuscular injection of dexamethasone （0.35 mg·kg^-1）. After successful modeling， the rats were randomized into model， methimazole （positive control， 5 mg·kg^-1）， low-， medium-， and high-dose （1.85， 3.70， 7.40 g·kg^-1， respectively） ZGW， and normal control groups. After 21 days of continuous gavage， the behavioral indexes and body weight changes of rats were evaluated. The pathological changes of the renal tissue were observed by hematoxylin-eosin staining. The serum levels of thyroid hormones ［triiodothyronine （T₃）， thyroxine （T₄）， thyroid-stimulating hormone （TSH）］， renal function indexes ［serum creatine （Scr） and blood urea nitrogen （BUN）］， energy metabolism markers ［cyclic adenosine monophosphate （cAMP） and cyclic guanosine monophosphate （cGMP）］， and oxidative stress-related factors ［superoxide dismutase （SOD）， malondialdehyde （MDA）， and NADPH）］ were measured by enzyme-linked immunosorbent assay （ELISA）. Western blot was employed to analyze the expression of DRD4， NOX4， mitochondrial respiratory chain complex proteins ［NADH：ubiquinone oxidoreductase subunit S4 （NDUFS4） and cytochrome C oxidase subunit 4 （COX4）］， and inflammation-related protein ［tumor necrosis factor-alpha （TNF-α）， interleukin-6 （IL-6）， p38 mitogen-activated protein kinase （MAPK）］ pathway in the renal tissue. ResultsCompared with the normal group， the model group showed mental malaise， body weight decreases （P<0.01）， inflammatory cell infiltration in the renal tissue， a few residual parotid glands in the thyroid， elevations in serum levels of T₃， T₄， Scr， BUN， cAMP， cAMP/cGMP， MDA， and NADPH （P<0.01）， down-regulation in protein levels of TSH， SOD， and DRD4 （P<0.05， P<0.01）， and up-regulation in expression of NOX4， p-p38 MAPK/p38 MAPK， and inflammatory factors （P<0.01）. Compared with the model group， ZGW increased the body weight （P<0.05， P<0.01）， reduced the infiltration of renal interstitial inflammatory cells， restored the thyroid structure and follicle size， lowered the serum levels of T₃， T₄， Scr， BUN， cAMP， cAMP/cGMP， MDA and NADPH （P<0.05， P<0.01）， up-regulated the expression of TSH， SOD and DRD4 （P<0.05， P<0.01）， and down-regulated the expression of NOX4， p-p38 MAPK/p38 MAPK， and inflammatory factors （P<0.05， P<0.01）. Moreover， high-dose ZGW outperformed methimazole （P<0.05）. ConclusionBy activating DRD4， ZGW can inhibit the expression of NOX4 mediated by the p38 MAPK pathway， reduce oxidative stress and inflammatory response， thereby ameliorating the pathological state of hyperthyroidism due to kidney-Yin deficiency. This study provides new molecular mechanism support for the clinical application of ZGW.

To formulate an expert consensus on the principles of preoperative hair removal and provide scientific guidance for standardized removal of hair before surgical procedures so as to reduce the incidence of surgical site infections.METHODS Led by the Hospital Management Institute of National Health Commission of the People's Republic of China,this consensus was reached with the joint efforts from the expects of relevant fields such as surgeries,interventional therapies,nursing,and infection prevention and control.The consensus facilitates the classification and evaluation of literatures by following the evidence grade formulated by Oxford Evidence-based Medicine Center and focuses on the association of preoperative hair removal with surgical site infection,it reaches the evidence grade of expert consensus and recommendation intensity by integrating with discussions on meetings and clinical experience of the expects from relevant fields.RESULTS A total of 6 items of consensus were reached by summarizing the latest evidence on the aspects including the indications for preoperative hair removal,tools,range,timing and places.CONCLUSION The consensus,to some extent,make supplements to and complete the exiting regulations and standards.It provides guidance for the medical institutions to carry out the preoperative hair removal.

Objective To simulate modern social stress using a pre-pregnancy chronic mild stress(CMS)model and to explore the mechanisms of emotional,behavioral,and neurodevelopmental changes in male offspring of pre-pregnancy liver qi stagnation female mice through corticosterone(CORT)-brain-derived neurotrophic factor(BDNF)extracellular signal-regulated kinase(ERK)1/2 signaling cascade-mediated hippocampal injury.This study aimed to elucidate the impact of negative life events on offspring and the interventional mechanism of Shuyu Capsules.Methods CMS stress was used to induce pre-pregnancy depression in female rats(liver qi stagnation state),followed by intervention with Shuyu and fluoxetine capsules.After screening,male rats were mated and 12 male offspring from each group were selected for behavioral testing and detection of serum CORT levels by enzyme-linked immunosorbent assay.BDNF,ERK1/2,phospho(p)-ERK1/2,cAMP-response element binding protein(CREB),and p-CREB protein levels in the hippocampus were detected by Western Blot,and BDNF,ERK1,ERK2,and CREB mRNA levels in the hippocampus were detected by reverse transcription-polymerase chain reaction(RT-PCR),to verify the effects of pre-pregnancy CMS on the BDNF-ERK1/2-CREB signaling pathway and to investigate the key micro-mechanisms of Shuyu Capsules on emotional and learning memory-related behaviors of male offspring of females with pre-pregnancy liver qi stagnation syndrome.Results The distance,number of entries,and duration of stay in the central area in open-field experiments were significantly reduced in offspring in the model group(all P＜0.05).The escape latency during the exploration period of the water-maze experiment was significantly prolonged(P＜0.05)and the swimming distance,duration of the target quadrant,and number of platform crossings were significantly reduced(P＜0.05,P＜0.05,P＜0.01),the suspension time and frequency in the forced-swimming experiment were increased(P＜0.05,P＜0.01),and the incubation period was shortened(P＜0.05)in offspring in the model group.Prophylactic treatment with Shuyu Capsules and fluoxetine improved the depression-like behavior and cognitive impairment in the offspring in the model group.Biochemical tests showed that CORT levels were increased in the CMS model group(P＜0.05),BDNF,p-ERK1/2,and p-CREB protein levels in the hippocampus were decreased(all P＜0.05),and BDNF,ERK1,ERK2,and CREB mRNA levels were significantly reduced(P＜0.01,P＜0.05,P＜0.01,P＜0.05).Treatment with Shuyu Capsules and fluoxetine increased the CORT content and BDNF,ERK1/2,and CREB protein and mRNA levels in male offspring to varying degrees.Conclusions High levels of CORT in offspring act selectively on the hippocampus,exerting adverse effects on the emotional and learning memory functions of rats by downregulating the BDNF-ERK1/2 signaling cascade.The Chinese medicine Shuyu Capsules can reduce the impact of an adverse intrauterine environment on offspring development by correcting abnormal levels and pathways of glucocorticoids.

The receptor-binding region(RBD)of the spike protein(S)on the surface of porcine epi-demic diarrhea virus(PEDV)is a critical structural domain mediating viral invasion of host cells and serves as a key target for inducing neutralizing antibodies.In order to prepare antibodies that can be used to study the biological function of PEDV RBD and develop novel diagnostic and thera-peutic methods,recombinant RBD protein expressed in Sf9 insect cells was utilized as an immuno-gen to immunize BALB/c mice.Monoclonal antibodies(mAbs)were generated via hybridoma tech-nology,and positive hybridoma clones were screened using indirect ELISA.The reactivity of the mAbs was subsequently characterized.The results of ELISA,Western blot,and indirect immuno-fluorescence assay(IFA)showed that three monoclonal antibodies screened(3E5,4F9 and 5A5)had good reactivity with the virus and RBD protein.Antibody subtype results showed that 3E5 and 4F9 were of IgG1 subtypes and 5A5 was of IgM subtype.Neutralization assay further revealed that 3E5 monoclonal antibody had viral neutralizing activity.In this study,three monoclonal antibodies against PEDV RBD proteins were successfully prepared,providing the basis for the study of the bi-ological function of RBD proteins,PEDV serologic detection and vaccine development.

The receptor-binding region(RBD)of the spike protein(S)on the surface of porcine epi-demic diarrhea virus(PEDV)is a critical structural domain mediating viral invasion of host cells and serves as a key target for inducing neutralizing antibodies.In order to prepare antibodies that can be used to study the biological function of PEDV RBD and develop novel diagnostic and thera-peutic methods,recombinant RBD protein expressed in Sf9 insect cells was utilized as an immuno-gen to immunize BALB/c mice.Monoclonal antibodies(mAbs)were generated via hybridoma tech-nology,and positive hybridoma clones were screened using indirect ELISA.The reactivity of the mAbs was subsequently characterized.The results of ELISA,Western blot,and indirect immuno-fluorescence assay(IFA)showed that three monoclonal antibodies screened(3E5,4F9 and 5A5)had good reactivity with the virus and RBD protein.Antibody subtype results showed that 3E5 and 4F9 were of IgG1 subtypes and 5A5 was of IgM subtype.Neutralization assay further revealed that 3E5 monoclonal antibody had viral neutralizing activity.In this study,three monoclonal antibodies against PEDV RBD proteins were successfully prepared,providing the basis for the study of the bi-ological function of RBD proteins,PEDV serologic detection and vaccine development.

To identify clinical viral diseases characterized by reproductive disorders and abortion,a quadruple qPCR method was established for simultaneous detection of PRV,PPV,PCV2 and AS-FV.Four pairs of specific primers and probes were designed according to the conserved genes of four viruses in the NCBI gene bank.The annealing temperature,primer concentration and probe concentration of the reaction were optimized,and the specificity,sensitivity and repeatability of the method were tested.The results showed that the method could not detect other pathogens except the target ones.The minimum detection limit of PRV,PPV,PCV2 and ASFV was 10 copies.Intra-group and inter-group repeatability tests showed that the coefficient of variation of C,values be-tween different batches was less than 3％,indicating that the method was highly specific,sensitive and stable.Establishment of an efficient and sensitive quadruple qPCR method provides technical reference for the clinical prevention and control of porcine pseudorabies virus disease,porcine circo-virus disease,porcine parvovirus disease and African swine fever.

This study investigates the effects of pseudorabies virus(PRV)infection on the antiviral immune signaling pathways and type Ⅰ interferon factors in mouse trigeminal ganglion(TG)cells.In this experiment,primary TG cells were infected with PRV at a multiplicity of infection(MOI)of 1,while mice were infected via a drop-nose method using 106,29 TCID50 of PRV.Real-time fluorescence quantitative PCR(qPCR),Western blot and ELISA were used to assess gene tran-scription,protein expression,and the secretion of IFN-α and IFN-β.The results indicated that PRV infection of mouse TG primary cells led to alterations in the gene and protein expression of RIG-Ⅰ,MAVS,and IRF3,as well as the phosphorylation of IRF3 and IKBα both in vivo and ex vivo.ELISA results showed that PRV infection could regulate the secretion of IFN-α and IFN-β in mouse primary TG cells and mouse TGs.The results of RIG-Ⅰ signaling pathway-related proteins and the secretion of IFN-a and IFN-β were analyzed using Western blot after using siRNA to interfere with RIG-Ⅰ expression in TG cells.The results showed that siRIG-Ⅰ successfully inter-fered with RIG-Ⅰ protein expression in TG cells and caused changes in the expression of down-stream proteins such as MAVS and IRF3,and also regulated the secretion of IFN-α and IFN-β in TG cells.Furthermore,the results indicated that PRV infection induced the expression of RIG-Ⅰ in mouse TG progenitor cells,regulating the antiviral immune response of type Ⅰ interferon factors in TG cells through the RIG-Ⅰ-MAVS-IRF3 signaling axis.Notably,PRV inhibited the expression of IRF3 in TG cells while significantly upregulating the expression of IFN-β during the later stages of infection,which may be an important factor in the important reason for the rapid mortality ob-served in mice during the late stages of PRV infection.This experiment elucidates part of the anti-viral immune mechanism mediated by the RIG-Ⅰ-MAVS-IRF3 signaling pathway in regulating type Ⅰ interferon factor after PRV infection of mouse TG cells,as well as the discovery of differ-ent trends of IRF3 protein changes in vivo and ex vivo,laying the groundwork for future in-depth studies.

BACKGROUND:Mongolian medicine Echinops sphaerocephalus L.is a commonly used medicine for bone injury in Mongolian medicine.It is effective for tendon injury,fracture,bone nonunion,bone fever,tingling,sore and other diseases.Our previous studies have confirmed that Mongolian medicine Echinops sphaerocephalus L.can promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells,but its effect on angiogenesis in the process of bone defect repair is unknown.OBJECTIVE:To investigate the effect of Echinops sphaerocephalus L.on in vitro angiogenesis in human umbilical vein vascular endothelial cells and to explore the angiogenesis-promoting active ingredients and their mechanisms of action of Echinops sphaerocephalus L.using network pharmacology technology.METHODS:The ethanol extract of Echinops sphaerocephalus L.was prepared and preserved by freeze-drying.The proliferation,migration,chemotaxis and angiogenesis of human umbilical vein endothelial cells were observed after treatment with different concentrations(1 000,100,and 10 μg/mL)of Echinops sphaerocephalus L.The active components and possible signaling pathways that promoted angiogenesis were enriched and analyzed by network pharmacology.RESULTS AND CONCLUSION:(1)The effect of Echinops sphaerocephalus L.on angiogenesis was regulated by its mass concentration:at low mass concentration(10 μg/mL),Echinops sphaerocephalus L.could promote the proliferation,migration,chemotaxis and angiogenesis of human umbilical vein vascular endothelial cells;on the contrary,Echinops sphaerocephalus L.inhibited the proliferation,migration,and chemotaxis of human umbilical vein endothelial cells at high mass concentration(1 000 μg/mL).However,the inhibitory effect of Echinops sphaerocephalus L.on angiogenesis was not significant at high mass concentration due to the limitation of experimental time.10 μg/mL Echinops sphaerocephalus L.could up-regulate the mRNA expression of angiogenesis-associated factors,including kinase insert domain receptor,vascular endothelial growth factor A,and hypoxia-inducible factor α,and thereby influenced angiogenesis during bone repair.(2)Network pharmacological analyses indicated that Echinops sphaerocephalus L.may bind to eight core targets(TGFB1,TNF,IL-6,STAT3,CTNNB1,IL-1B,AKT1,and HIF-1A)through four core active components(apigenin,caffeic acid,quercetin,and chlorogenic acid)to exert an effect on angiogenesis,atherosclerosis,multiple viral infections,and tumor angiogenesis-related signaling pathways.

To establish a TaqMan-based multiplex RT-qPCR method for the identification of Japa-nese encephalitis virus(JEV),Porcine reproductive and respiratory syndrome virus(PRRSV),and Classical swine fever virus(CSFV),this study designed and synthesized three pairs of specific primers and probes based on the conserved sequences of JEV E,PRRSV ORF6,and CSFV E2 a-vailable in the NCBI GenBank.By optimizing the reaction system and protocol,a multiplex RT-qPCR method for detecting these three viruses was developed and applied to the detection of clini-cal samples.The results showed that the established TaqMan multiplex RT-qPCR specifically am-plified the gene fragments of JEV,PRRSV,and CSFV,and did not amplify other non-target genes,indicating good specificity of the method.Intra-assay and inter-assay repeatability tests showed that the coefficient of variation(Cv)values were all below 3％,demonstrating that the method has ex-cellent repeatability.Sensitivity tests revealed that the minimum detectable amount for the recom-binant plasmids of the three viruses was 100 copies/pL.Using the established method,a total of 969 samples,including blood,aborted fetuses,semen,and deceased pigs,from 26 pig farms in Guizhou Province were tested.The detection rates were 34.3％(332/969)for JEV,28.3％(274/969)for PRRSV,and 19.8％(192/969)for CSFV.The co-infection rates were 10.1％(98/969)for JEV and PRRSV,12.1％(117/969)for JEV and CSFV,and 14.6％(141/969)for CSFV and PRRSV.Additionally,the triple co-infection rate of JEV,PRRSV,and CSFV was 7.9％(77/969).These results indicate that the TaqMan multiplex RT-qPCR method developed in this study is ef-fective for detecting these three viruses in pig farms,providing technical support for identifying vi-ral causes of reproductive disorders.

To establish a TaqMan-based multiplex RT-qPCR method for the identification of Japa-nese encephalitis virus(JEV),Porcine reproductive and respiratory syndrome virus(PRRSV),and Classical swine fever virus(CSFV),this study designed and synthesized three pairs of specific primers and probes based on the conserved sequences of JEV E,PRRSV ORF6,and CSFV E2 a-vailable in the NCBI GenBank.By optimizing the reaction system and protocol,a multiplex RT-qPCR method for detecting these three viruses was developed and applied to the detection of clini-cal samples.The results showed that the established TaqMan multiplex RT-qPCR specifically am-plified the gene fragments of JEV,PRRSV,and CSFV,and did not amplify other non-target genes,indicating good specificity of the method.Intra-assay and inter-assay repeatability tests showed that the coefficient of variation(Cv)values were all below 3％,demonstrating that the method has ex-cellent repeatability.Sensitivity tests revealed that the minimum detectable amount for the recom-binant plasmids of the three viruses was 100 copies/pL.Using the established method,a total of 969 samples,including blood,aborted fetuses,semen,and deceased pigs,from 26 pig farms in Guizhou Province were tested.The detection rates were 34.3％(332/969)for JEV,28.3％(274/969)for PRRSV,and 19.8％(192/969)for CSFV.The co-infection rates were 10.1％(98/969)for JEV and PRRSV,12.1％(117/969)for JEV and CSFV,and 14.6％(141/969)for CSFV and PRRSV.Additionally,the triple co-infection rate of JEV,PRRSV,and CSFV was 7.9％(77/969).These results indicate that the TaqMan multiplex RT-qPCR method developed in this study is ef-fective for detecting these three viruses in pig farms,providing technical support for identifying vi-ral causes of reproductive disorders.