A method for simultaneous determination of 21 kinds of Aconitum alkaloids(ATS)in biological specimens and infused liquor using ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)was developed.The biological samples were pretreated with methanol-acetonitrile(1∶2,V/V)for protein precipitation,while infused liquors were diluted 100-fold with acetonitrile,followed by centrifugation,and filtration by a 0.22-μm membrane.Chromatographic separation was carried out on an EC-C18 column using gradient elution with the mixture of 10 mmol/L ammonium acetate and 0.2%formic acid as mobile phase A and acetonitrile as mobile phase B.With this method,all the analytes were separated within 9.5 min.The samples were detected in positive ESI mode with dynamic multiple reaction monitoring(MRM)and quantified via external standard calibration.The results showed that the concentrations of the analytes in the range of 2-1000 ng/mL had excellent linearity(R2>0.9992)with the peak area.The developed method was successfully used for detection of 21 kinds of aconitum alkaloids,with limits of detection of 0.5-2 ng/mL,quantification limits of 2-6 ng/mL,intra/inter-day precision≤6.0%,spiked recoveries of 89.4%-100.9%,extraction recoveries of 74.2%-104.4%,and matrix effects ranging from-11.1%to 9.2%in blood/urine.The method was applied to detection of 12 samples from 4 fatal aconite poisoning cases,and all 21 kinds of ATS with total alkaloid concentrations of 0.04-4.18 μg/mL in blood and 154.96-422.83 μg/mL in medicinal liquors were detected.Tissue distribution revealed that the order of concentrations from highest to lowest is as follows:urine(157.22 μg/mL)>gastric contents(51.37 μg/mL)>kidney(21.6 μg/g)>whole blood(4.18 μg/mL)>liver(0.03 μg/g).This method showed many advantages such as simple pretreatment,low detection limits,accurate quantification,broad analyte coverage,and superior anti-interference capability in complex matrices,proving ideal for forensic and toxicological analysis of aconitum alkaloids.

ObjectiveTo investigate and analyze an outbreak of rotavirus infectious diarrhea in a prison in Chongqing Municipality, to provide a basis for adult rotavirus surveillance and prevention, and to explore the public health problems in special settings. MethodsA retrospective survey was conducted to collect and analyze data on individual cases with diarrheal disease on-site. The clinical characteristics, as well as the temporal, spatial and geographical distribution patterns of the epidemic were described. Multi-pathogen detection tests were conducted both on diarrhea cases and environmental samples, with viral genotyping performed on positive samples. A case-control analysis was performed to identify the causes of the outbreak, and an SEIR model was adopted to predict the outbreak trend and evaluate the effectiveness of interventions. ResultsA total of 65 cases were found among the inmates, with an attack rate of 2.03%. The predominant clinical manifestations included diarrhea (89.23%), watery stool (73.85%), and dehydration (18.46%). The epidemic curve indicated a “human-to-human” transmission pattern, with an average incubation period of 5‒6 days. The attack rates among chefs in the main canteen (80.00%, 8/10) and caterers (28.33%, 17/60) were significantly higher than those of other inmates （P<0.05）. Multi-pathogen polymerase chain reaction (PCR) testing detected positive for group A rotavirus, with the viral genotyping identified as G9P [8] strain. Factors such as unprotected "bare-handed" food distribution among cases with diarrhea (OR=9.512, 95%CI: 4.261‒21.234) and close contact with diarrhea cases (OR=3.656, 95%CI: 1.719‒7.778) were the possible cause of the outbreak. The SEIR model (r₀=5, α=0.3, β₁=0.08, β₂=0.04) was constructed using prison inmates as susceptible population, aiming at fitting the initial transmission trend of the outbreak, and the epidemic rate declined rapidly after intervention measures were implemented (r_t≈0). ConclusionThis rare rotavirus infection diarrhea outbreak among adults in confined settings suggests that the construction of public health prevention and control systems in prison may be overlooked. Cross infection during meal processing and distribution in the canteens of such settings is likely to be the cause of the outbreak. Given the potential neglect of public heath system construction in special settings, it is imperative to enhance the surveillance and monitoring of rotavirus and other intestinal multi-pathogens among adults, as well as the construction of public health prevention and control systems in these special settings.

OBJECTIVE: To assess the efficacy of Qingda Granule (QDG) in ameliorating hypertension-induced cardiac damage and investigate the underlying mechanisms involved. METHODS: Twenty spontaneously hypertensive rats (SHRs) were used to develope a hypertension-induced cardiac damage model. Another 10 Wistar Kyoto (WKY) rats were used as normotension group. Rats were administrated intragastrically QDG [0.9 g/(kg•d)] or an equivalent volume of pure water for 8 weeks. Blood pressure, histopathological changes, cardiac function, levels of oxidative stress and inflammatory response markers were measured. Furthermore, to gain insights into the potential mechanisms underlying the protective effects of QDG against hypertension-induced cardiac injury, a network pharmacology study was conducted. Predicted results were validated by Western blot, radioimmunoassay immunohistochemistry and quantitative polymerase chain reaction, respectively. RESULTS: The administration of QDG resulted in a significant decrease in blood pressure levels in SHRs (P<0.01). Histological examinations, including hematoxylin-eosin staining and Masson trichrome staining revealed that QDG effectively attenuated hypertension-induced cardiac damage. Furthermore, echocardiography demonstrated that QDG improved hypertension-associated cardiac dysfunction. Enzyme-linked immunosorbent assay and colorimetric method indicated that QDG significantly reduced oxidative stress and inflammatory response levels in both myocardial tissue and serum (P<0.01). CONCLUSIONS Both network pharmacology and experimental investigations confirmed that QDG exerted its beneficial effects in decreasing hypertension-induced cardiac damage by regulating the angiotensin converting enzyme (ACE)/angiotensin II (Ang II)/Ang II receptor type 1 axis and ACE/Ang II/Ang II receptor type 2 axis.
Animals ; Drugs, Chinese Herbal/therapeutic use* ; Hypertension/pathology* ; Renin-Angiotensin System/drug effects* ; Rats, Inbred SHR ; Oxidative Stress/drug effects* ; Male ; Rats, Inbred WKY ; Blood Pressure/drug effects* ; Myocardium/pathology* ; Rats ; Inflammation/pathology*

Purpose Based on multimodal imaging combined with a variety of histological techniques,to visually evaluate the changes of rats brain metabolic microenvironment after cerebral hypoxia and ischemia of prematurity,and to discuss the effects of abnormal lactate metabolism in the brain on oligodendrocyte precursor cells,so as to provide a basis for the early diagnosis and treatment of premature white matter injury(PWMI).Materials and Methods A total of 36 SPF-grade healthy 3-day-old Sprague-Dawley neonatal rats were randomly assigned to the sham surgery(Sham)group and the model(PWMI)group using a random number table method,with 18 rats in each group.A neonatal rat PWMI model was established by hypoxia-ischemia method.Twenty-four hours after modeling,laser speckle imaging was used to monitor cerebral blood flow and blood oxygen changes.Multimodal imaging was used to observe the changes in brain tissue microstructure and metabolism after PWMI.HE staining was used to observe the morphological changes of nerve cells in the white matter of the brain.Enzyme-linked immunosorbent assay was used to detect the changes of lactate content and lactate dehydrogenase activity in the white matter region of the brain after PWMI in neonatal rats.PDGFR-α immunofluorescence staining was used to observe the dynamic changes of the number of oligodendrocyte precursor cells in the subependymal zone after PWMI in neonatal rats.Results Twenty-four hours after modeling,the multimodal imaging results showed that the T2WI and diffusion-weighted imaging on the injured side of the PWMI group showed high intensity,and the relative cerebral blood flow,relative oxygen saturation,relative apparent diffusion coefficient and amide proton transfer(APT)Lorentzian difference value were lower than those in the Sham group(t=29.466,23.522,59.006,54.778,10.263,all P<0.001),and the lactate content was higher than that in the Sham group(t=-7.521,P<0.001).The results of HE staining and enzyme-linked immunosorbent assay showed that the arrangement of nerve cells in the white matter area of the injured side of the brain in the PWMI group was loose and disordered.The lactate content and lactate dehydrogenase activity were higher than those in the Sham group(t=-6.079,-10.548,both P<0.001).The results of immunofluorescence staining showed that the number of PDGFR-α+cells in the subependymal zone of the damaged side of the PWMI group was higher than that of the Sham group at 24 hours after modeling,and lower than that in the Sham group at 11 days after modeling(t=-8.386,6.676,both P<0.001).The correlation analysis between the lactate content and APT Lorentzian difference value in the brain and the number of oligodendrocyte precursor cells in the brain 11 days after modeling showed that the number of oligodendrocyte precursor cells in the subependymal zone was positively correlated with the APT Lorentzian difference value(r=0.821,P=0.001 1),and negatively correlated with the lactate content in the brain(r=-0.880,P=0.000 2).Conclusion Multimodal imaging can monitor the early brain metabolism changes of PWMI in neonatal rats,especially the changes of lactate,and provide a visual basis for their early diagnosis.The level of lactate in the brain increases after cerebral hypoxia and ischemia in prematurity,and oligodendrocyte precursor cells increase transiently and then decrease,resulting in PWMI.

Kv1.3,a voltage-gated potassium channel,is highly expressed in T lymphocytes,the nervous system,and vascular smooth muscle cells.It plays a critical role in membrane excitability and electrical signal transduction,serving as an important target for studying T-cell function and providing a promising direction for developing therapeutics against autoimmune and inflammatory diseases.Therefore,the de-velopment of specific inhibitors of Kv1.3 channel has emerged as a novel therapeutic strategy for these disorders.In this study,we isolated and purified a novel Kv1.3-inhibitory peptide toxin,LmKTx13,from the venom of the scorpion Lychas mucronatus using reversed-phase high-performance liquid chroma-tography(RP-HPLC).LmKTx13 consists of 38 amino acid residues,including six cysteines that form three disulfide bonds.Whole-cell patch-clamp recordings revealed that LmKTx13 potently inhibited Kv1.3 with an IC50 of 7.92±3.0 nmol/L.Selectivity analysis showed that 2 μmol/L LmKTx13 also in-hibited Kv1.2 and Kv1.7,but exhibited no significant effects on other potassium channel subtypes or voltage-gated sodium channels.Further investigation into the mechanism demonstrated that LmKTx13 acts as a pore-blocking inhibitor of Kv1.3.By analyzing the effects of LmKTx13 on Kv1.3 channel gating ki-netics and performing sequence alignment of the pore regions of Kv1.3 and Kv1.5,we constructed site-directed mutants and identified the pore region of Kv1.3 as the critical binding site for LmKTx13.Key residues involved in the interaction included T425,G427,and H451.In summary,we discovered a no-vel pore-blocking Kv1.3 inhibitor,LmKTx13,from L.mucronatus venom,which exhibits high affinity and selectivity for Kv1.3.These findings highlight its potential as a potential lead molecule for developing Kv1.3-targeted therapeutics.

Objective To investigate the effect of tissue-resident memory T cells(TRM)on imiquimod-induced psoriatic-like skin lesions in mice,and to elucidate the underlying mechanisms of TRM involvement in this process.Methods Forty female BALB/c mice were procured and randomly allocated into four groups:ten in the blank control group,and thirty for the establishment of a psoriasis mouse model.Following successful modeling,the thirty mice were further randomized into three groups:the model control group,the methotrexate-treated group,and the imiquimod-treated group,with ten mice in each group.Mice in the blank control group and model control group were uniformly treated with Vaseline for intervention.The methotrexate group and the imiquimod group were treated with 62.5mg of 5％imiquimod cream.The methotrexate group was administered by gavage at a dose of 1 mg/kg,and the gavage volume of each group was 10 mL/kg.The model control group,blank group and imiquimod group were gavaged with the same volume of normal saline.Treatment was conducted over six consecutive days.Subsequently,comparisons were made across groups regarding the psoriasis area and severity index(PASI),histopathological findings,inflammatory cytokine levels,and TRM cell levels.Results(1)The imiquimod group exhibited signifi-cantly lower scores for erythema(2.54±0.32),skin thickening(2.59±0.25),and scaling(2.52±0.29)compared to the methotrexate group,model control group,and blank control group(P<0.05).Additionally,the methotrexate group demonstrated reduced scores for erythema,skin thickening,and scaling compared to the model control group(P<0.05).(2)Hematoxylin-eosin(HE)staining revealed that the epidermis in the methotrexate group became thin-ner,with fewer parakeratotic cells and increased hair follicles.Conversely,the imiquimod group displayed abnor-mal cell morphology and relatively thicker white skin after modeling.(3)The imiquimod group showed significantly lower levels of TNF-α(51.63±4.39 pg/mL),IL-1β(35.53±4.15 pg/mL),IFN-γ(23.43±3.41 pg/mL),and IL-23(15.24±2.95 pg/mL)compared to the methotrexate and model control groups(P<0.05).Similarly,the methotrexate group exhibited reduced levels of TNF-α,IL-1β,IFN-γ,and IL-23 compared to the model control group(P<0.05).(4)The imiquimod group had significantly lower levels of CD8+CD103+cells(15.39±2.31)than the methotrexate and model control groups(P<0.05).Furthermore,the methotrexate group demonstrated lower levels of CD8+CD103+cells compared to the model control group(P<0.05).Conclusion Miquimod induces heavier skin lesions,faster response,and more epidermal thickening in psoriasis like mice.CD8+CD103+TRM cells and inflammatory factors may be involved in the recurrence of psoriasis.

Objective:To analyze the molecular characteristics of the virus in a local outbreak of dengue fever in Jiangsu province in 2023, and to provide a basis for the prevention and control of the outbreak.Methods:Serum samples were collected from suspected dengue patients in the acute phase of the outbreak for virus detection and serotyping by real-time fluorescence quantitative RT-PCR (RT-qPCR). Positive specimens were amplified with full-length genomic fragments and subjected to second-generation sequencing and related evolutionary analyses.Results:Four confirmed cases of dengue were found in Changzhou city, Jiangsu province, from October 18 to 21, 2023, with epidemiological association between the cases, which was recognized as a dengue outbreak. The serum RT-qPCR result of the four cases were all dengue type 1, and the whole genome sequences of three of the cases were obtained. The evolutionary tree of the E gene and the whole genome showed that the three sequences were located in the 3rd branch of the 1-I genotype, which is similar to the genotype 1-I. The genome-wide sequences of the E gene and the genome-wide evolution tree showed that the three sequences were located in the 3rd branch of the 1-I genotype, which is similar to the genome-wide genotype 1-Ⅰ. The E gene and the genome-wide evolutionary tree showed that all three sequences were located on branch 3 of genotype 1-Ⅰ, with high sequence similarity to the dengue virus epidemic strains in Guangdong and Yunnan provinces in 2023. Amino acid variant site analysis showed that there were 16 branch-specific amino acid site changes in the sequences of the three cases, among which the structural proteins, C protein and prM protein, had one variant site each, E protein had two, and the non-structural proteins had the largest number of NS5 variant sites (9).Conclusions:The local outbreak in Jiangsu was caused by dengue fever type 1 virus, with high nucleotide sequence similarity to strains from other regions of China, and amino acid site alterations.

Kv1.3,a voltage-gated potassium channel,is highly expressed in T lymphocytes,the nervous system,and vascular smooth muscle cells.It plays a critical role in membrane excitability and electrical signal transduction,serving as an important target for studying T-cell function and providing a promising direction for developing therapeutics against autoimmune and inflammatory diseases.Therefore,the de-velopment of specific inhibitors of Kv1.3 channel has emerged as a novel therapeutic strategy for these disorders.In this study,we isolated and purified a novel Kv1.3-inhibitory peptide toxin,LmKTx13,from the venom of the scorpion Lychas mucronatus using reversed-phase high-performance liquid chroma-tography(RP-HPLC).LmKTx13 consists of 38 amino acid residues,including six cysteines that form three disulfide bonds.Whole-cell patch-clamp recordings revealed that LmKTx13 potently inhibited Kv1.3 with an IC50 of 7.92±3.0 nmol/L.Selectivity analysis showed that 2 μmol/L LmKTx13 also in-hibited Kv1.2 and Kv1.7,but exhibited no significant effects on other potassium channel subtypes or voltage-gated sodium channels.Further investigation into the mechanism demonstrated that LmKTx13 acts as a pore-blocking inhibitor of Kv1.3.By analyzing the effects of LmKTx13 on Kv1.3 channel gating ki-netics and performing sequence alignment of the pore regions of Kv1.3 and Kv1.5,we constructed site-directed mutants and identified the pore region of Kv1.3 as the critical binding site for LmKTx13.Key residues involved in the interaction included T425,G427,and H451.In summary,we discovered a no-vel pore-blocking Kv1.3 inhibitor,LmKTx13,from L.mucronatus venom,which exhibits high affinity and selectivity for Kv1.3.These findings highlight its potential as a potential lead molecule for developing Kv1.3-targeted therapeutics.

Purpose Based on multimodal imaging combined with a variety of histological techniques,to visually evaluate the changes of rats brain metabolic microenvironment after cerebral hypoxia and ischemia of prematurity,and to discuss the effects of abnormal lactate metabolism in the brain on oligodendrocyte precursor cells,so as to provide a basis for the early diagnosis and treatment of premature white matter injury(PWMI).Materials and Methods A total of 36 SPF-grade healthy 3-day-old Sprague-Dawley neonatal rats were randomly assigned to the sham surgery(Sham)group and the model(PWMI)group using a random number table method,with 18 rats in each group.A neonatal rat PWMI model was established by hypoxia-ischemia method.Twenty-four hours after modeling,laser speckle imaging was used to monitor cerebral blood flow and blood oxygen changes.Multimodal imaging was used to observe the changes in brain tissue microstructure and metabolism after PWMI.HE staining was used to observe the morphological changes of nerve cells in the white matter of the brain.Enzyme-linked immunosorbent assay was used to detect the changes of lactate content and lactate dehydrogenase activity in the white matter region of the brain after PWMI in neonatal rats.PDGFR-α immunofluorescence staining was used to observe the dynamic changes of the number of oligodendrocyte precursor cells in the subependymal zone after PWMI in neonatal rats.Results Twenty-four hours after modeling,the multimodal imaging results showed that the T2WI and diffusion-weighted imaging on the injured side of the PWMI group showed high intensity,and the relative cerebral blood flow,relative oxygen saturation,relative apparent diffusion coefficient and amide proton transfer(APT)Lorentzian difference value were lower than those in the Sham group(t=29.466,23.522,59.006,54.778,10.263,all P<0.001),and the lactate content was higher than that in the Sham group(t=-7.521,P<0.001).The results of HE staining and enzyme-linked immunosorbent assay showed that the arrangement of nerve cells in the white matter area of the injured side of the brain in the PWMI group was loose and disordered.The lactate content and lactate dehydrogenase activity were higher than those in the Sham group(t=-6.079,-10.548,both P<0.001).The results of immunofluorescence staining showed that the number of PDGFR-α+cells in the subependymal zone of the damaged side of the PWMI group was higher than that of the Sham group at 24 hours after modeling,and lower than that in the Sham group at 11 days after modeling(t=-8.386,6.676,both P<0.001).The correlation analysis between the lactate content and APT Lorentzian difference value in the brain and the number of oligodendrocyte precursor cells in the brain 11 days after modeling showed that the number of oligodendrocyte precursor cells in the subependymal zone was positively correlated with the APT Lorentzian difference value(r=0.821,P=0.001 1),and negatively correlated with the lactate content in the brain(r=-0.880,P=0.000 2).Conclusion Multimodal imaging can monitor the early brain metabolism changes of PWMI in neonatal rats,especially the changes of lactate,and provide a visual basis for their early diagnosis.The level of lactate in the brain increases after cerebral hypoxia and ischemia in prematurity,and oligodendrocyte precursor cells increase transiently and then decrease,resulting in PWMI.

Objective To investigate the correlation between c-MYC expression and mitochondrial metabolism in malignant duct epithelial cells of pancreatic cancer patients.Methods GEPIA database was used to analyze the correlation between c-MYC expression and overall survival.The expression of c-MYC in tumor tissues was detected by immunohistochemical staining.The difference of DLAT and DLST gene expression between tumor and normal tis-sues was compared in GEPIA database.HP A database was used to analyze the correlation between c-MYC and DLAT,DLST expression in tumor tissues.The expression level of DLAT and DLST in tumor tissues was evaluated by immunofluorescence staining.Results The high expression of c-MYC gene was negatively correlated with overall survival(P＜0.01).The level of c-MYC protein was positively correlated with the pathological grade of PanIN.Compared with normal tissues,the expression of DLAT and DLST genes in pancreatic cancer cells was increased(P＜0.01).The protein level of c-MYC was positively correlated with those of DLAT and DLST(P＜0.01,P＜0.001).Conclusions The high expression of mitochondrial metabolic enzymes DLAT and DLST in pancreatic ductal adenocarcinoma cells is significantly correlated with the expression level of c-MYC,which increases with the progression of pancreatic cancer.